B-cell antibody class switchings are pressuromodulated events: Part II, gene recombination

Abstract

Background

The esebssiwaagoT_Q method is applicable for the of study cell gene recombination events as the esebssiwaagoT_Q is a measure of the intracellular pressure required to establish a horizontal reading frame for alignment of a gene and its intergene bases for maximal transcription and recombination enzyme activity.

B-cell differentiation stages have recently been studied by gene esebssiwaagoT_Q-based pressuromodulation mapping. In this study, the B-cell differentiation stage pressuromodulation map is utilized as a template to simulate B-cell immunoglobulin locus recombination events that take place in the pressuromodulated state in vivo.

Methods

Chromosome 14 (−) strand location 105,566,277 and 106,879,844 germline genes were recombined after determination of gene esebssiwaagoT_Qs with respect to the germline, and then recombined genes were recombined further after determination of gene esebssiwaagoT_Qs with respect to rearranged configurations. For both alleles, first, IGHD_-_ to IGHJ_ was performed, and then IGHV_-_ to IGHD_-_-IGHJ_ was performed. For Allele 1 (IGHM), internal consensus recognition sequence (iCSR) and further CSR isotype switchings were performed; and for Allele 2 (IGHD), homologous recombination was performed and initial allelic exclusion determined.

Results

First, the esebssiwaagoT_Q of a joining (J_) and diversity (D_-_) gene in its native germline configuration is the basis for predictable subsequent gene rearrangement. Second, D_-_ to J_ gene recombination events are bi-allelic and mutually exclusive. Third, the entire process from beginning to end depends on the grade of the pressuromodulation effect, and as per the classical pathway it is an antigen presenting cell (APC)-dependent CD4R+ T-cell-mediated B-cell polarization process. Fourth, CD4R+ T-cells are positively pressuromodulated, while B-cells are subject to the effect of both positive and negative forms of antigen pressuromodulation. And fifth, B-cell to plasma cell transformation and the extra-nodal periphery/tissue nidus phase take place in the presence of antigen load and either positive or negative pressuromodulation of the cell to its recombined antibody gene expression intracellular pressure.

Conclusions

B-cell gene recombination rearrangement events can be predicted with a reasonable degree of certainty.

It is envisioned that further esebssiwaagoT_Q-based study of the remaining B-cell variability gene recombinations isotype switching events will further our understanding of pressuromodulated basis for antigen selection including the evolutionary underpinnings of.

Background

Gene transcription is a pressuromodulated process, pressurotopic [1,2,3]. B-cell maturation has recently been studied by gene esebssiwaagoT_Q-based pressuromodulation mapping of B-cell differentiation stage markers [4]. The esebssiwaagoT_Q-based pressuromodulation map of B-cell differentiation stage genes accurately simulates B-cell maturation in vivo and applies to both the classical B-cell maturation pathway (2-allele T-cell mediated pressuromodulation effect pathway) and the parallel alternative non-classical B-cell maturation pathway (1-allele T-cell independent antigen-mediated pressuromodulation effect pathway).

The classical 2-allele T-cell dependent B-cell maturation pathway involves three phases.

The first phase is the antigen presenting cell (APC)-more primed CD4R+ CD40LG T-cell-mediated CD40R B-cell cell membrane (CM) polarization effect or less primed T-cell-mediated polarization effect myeloid bone marrow phase until the Immature B-cell stage (CM IgM+), during which there is Allele 1 (IGHM) VDJ and internal consensus sequence recognition (iCSR) (CM IgM+) and after which there is Allele 2 (IGHD) VDJ.

The second phase is the CD4R+ CD40LG T-cell-mediated CD40R B-cell polarization effect lymph node phase until either the Mature naïve B-cell (CM IgM+ IgD+ or IgM+ IgM+) or Evolved mature naïve B-cell (CM IgG_ + IgG_+) stage, during which there is Allele 2 (IGHD) post-V(D)J homologous recombination (HR) or initial allelic exclusion followed by delayed IgM iCSR and primary, secondary or further CSR isotype switching [5].

And, the third phase is the T-cell independent B-cell CM receptor antigenic pressuromodulation effect-mediated extra-nodal periphery/tissue nidus secretory antibody phase upon exposure to either positive pressuromodulator antigens or negative pressuromodulator antigens, during which there is transformation from B-cell to B-pre-plasma cell followed by B-plasma cell node exiting diapedesis as a secretory long-lived B-plasma cell/plasmablast [6, 7].

The parallel alternate non-classical 1-allele T-cell independent B-cell maturation pathway involves two phases, the myeloid bone marrow phase during acute exposure to antigenic positve pressuromodulators, when the T-cell is out of intracellular pressure window for CD40LG expression, making the myeloid phase a T-cell independent process; and then the extra-nodal periphery/tissue nidus secretory anibody B-plasma cell phase, which is also a T-cell independent pressuromodulator antigen dependent process.

The non-classical pathway myeloid bone marrow phase involves B-cell CM toll-like receptor (TLR)-mediated endocytosis for example, which substitutes for the T-cell-mediated B-cell polarization pressuromodulation effect in oscillating B-cell intracellular pressure in the marrow [⊂ 0.26 esebssiwaagoT_Q units (CD40) ⟶ 0.36–0.41 esebssiwaagoT_Q units (PRDM1–0.41) ⟶ 0.10–0.14 esebssiwaagoT_Q units ⊃] [4] through the Mature naïve B-cell stage (CM IgM+ IgD-). Thus, during the marrow phase, there is Allele 1 (IGHM) VDJ, iCSR (CM IgM+) and Allele 2 (IGHD) VDJ, which results in a circulating Allele 1 IgM+ only Mature (naïve) B-cell [8].

And, the non-classical pathway T-cell independent extra-marrow periphery/tissue nidus phase results in the IgM+ only (IgM+ IgD-) secretory B-plasma cell or in a further CSRing B-cell and a Ig_+ only (Ig_+ IgD-) secretory B-plasma cell, both short-lived B-plasma cells/plasmablasts [6, 9].

The general intervals of B-cell gene recombination events are shown on the recently developed B-cell differentiation stage pressuromodulation map [4], as are general intervals of internal consensus sequence recognition (iCSR), homologous recombination and further CSR antibody isotype switchings. Thus, the B-cell differentiation stage pressuromodulation map serves as a template for predicting B-cell gene rearrangement events.

B-cell gene recombination events can be predicted for both B-cell differentiation pathways when the following points are considered:

• (1) The discordant mechanisms of the recombination enzyme actions on DNA segments, (a) RAG1 and RAG2 recombinases facilitate the excision of intervening variability (V_-_), diversity (D_-_) and joining (J_) genes, where V(D)J recombination does not require an esebssiwaagoT_Q match [4] because the mechanism is as such [10], while (b) AICDA and APOBEC3A-G cytidine deaminases facilitate the excision of intervening heavy chain (IGH_) genes, where iCSR [10, 11], CSR recombination [10, 12, 13] and B-cell homologous recombination [14] do require an esebssiwaagoT_Q match [4];

• (2) B-cell gene recombination begins at greater intracellular pressure on Allele 1 (IGHM) and results in earlier assembly and presentation of IgM on the cell membrane (CM) as compared to Allele 2 (IGHD);

• (3) D_-_ to J_ gene rearrangement process of the VDJ [10, 15,16,17] is a limited step process for each allele and the number of steps depend on the grade of the positive antigen pressuromodulation effect;

• (4) non-functional D_-_ genes are not present in the VDJ [18], as they serve only the purpose of being stepping stone recombination genes for 2-step D_-_ to J_, and include IGHD1–20 (nf), IGHD4–11 (nf) and IGHD5–18 (nf) with the exception of IGHD7–27 (nf), which does not participate; and

• (5) the percentage of V(D)J IGHJ_ genes [18] for Allele 1 (IGHM) 2-step D_-_ to J_ is 40% (IGHJ6) and Allele 1 (IGHM) 1-step D_-_ to J_ is 10% (IGHJ5), and for Allele 2 (IGHD) 2-step D_-_ to J_ is 32% (IGHJ4) and Allele 2 (IGHD) 1-step D_-_ to J_ is 8.5% (IGHJ3), ~ 1.5% (IGHJ2) and 8.5% (IGHJ1).

In this study, B-cell gene recombination is studied by determining germline gene esebssiwaagoT_Qs, and re-arranging germline genes to simulate actual pressuromodulated in vivo gene recombination events including (1) IGHD_-_ to IGHJ_, (2) IGHV_-_ to IGHD_-_-IGHJ_, (3) internal consensus sequence recognition (iCSR) for Allele 1 (IGHM) and homologous recombination or initial allelic exclusion for Allele 2 (IGHD); and (4) further CSR isotype switchings for Allele 1 (IGHM) or for both alleles.

Methods

Data mining

Locations of germline Ig heavy chain locus genes between chromosome 14 (−) strand location 105,566,277 and 106,879,844 [19], as well as locations of downstream and upstream genes were mined at GeneCards (https://www.genecards.org/) genomic neighborhood GeneLoc genome locator database and at LNCipedia.org database (http://www.lncipedia.org/), pseudogenes included and enhancers excluded [2, 4] (Additional file 1: Table S1).

The 5′ - > 3′ direction episodic sub-episode sums split-integrated weighted average-averaged gene overexpression tropy quotient (esebssiwaagoT _Q) method and overall approach to gene rearrangement

The downstream and upstream intergene base distances were tabulated, and then the final 5′- > 3′ esebssiwaagoT_Q (fract) for each gene was calculated in upstream anisotropic, upstream mesotropic, downstream anisotropic and downstream mesotropic parts. First, the 3′- > 5′ and 5′- > 3′ direction paired point tropy quotients (prpT_Qs; fract) were determined. Second, initial anisotropic and mesotropic sub-episode blocks (SEB; ASEB, MSEB) were determined, which are constant per episode where the number of initial SEBs for establishing a gene category with 100% sensitivity and 100% specificity (100% accuracy) are 5 initial SEBs for an Episode 2 category gene, 7 initial SEBs for an Episode 3 category gene, 9 initial SEBs for an Episode 4 gene, 11 initial SEBs for an Episode 5 gene, 13 initial SEBs for an Episode 6 gene. Third, on the basis of the initial SEBs, the final anisotropic and mesotropic sub-episode blocks (SEB; ASEB, MSEB) were determined, which are variable. And fourth, the 5′- > 3′ direction esebssiwaagoT_Qs to the final esebssiwaagoT_Q were determined, where a gene with an anisotropic final esebssiwaagoT_Q for < 0.25 is an infra-pressuromodulated gene (Infra gene), and where a gene with a mesotropic final esebssiwaagoT_Q ≥ 0.25 < 0.75 is a supra-pressuromodulated gene (Supra gene). The detailed esebssiwaagoT_Q method is in references [2, 4].

Germline genes were recombined after determination of gene esebssiwaagoT_Qs with respect to the germline, and recombined genes were recombined further after determination of gene esebssiwaagoT_Qs with respect to the rearranged configuration. For both alleles, first, IGHD_-_ to IGHJ_ recombination was performed, and then IGHV_-_ to IGHD_-_-IGHJ_ recombination was performed. For Allele 1 (IGHM), internal consensus sequence recognition (iCSR) and further CSR isotype switchings were performed; and for Allele 2 (IGHD), homologous recombination was performed and initial allelic exclusion determined.

Gene esebssiwaagoT _Q-based simulation of 2-step and 1-step IGHD_-_ and IGHJ_ recombinations for allele 1 (IGHM) and allele 2 (IGHD)

For both alleles, 2-step (1, 2a, 2b) and 1-step D_-_↔ J_ recombination simulations were performed.

For the 2-step D_-_↔ J_ stimulation: first, the non-functional (nf) germline genes were determined (Step 1 of 2) and the IGHJ_ gene was determined (Step 1 of 2) to yield the recombined gene, IGHD_-_ (nf)-IGHJ_; second, the esebssiwaagoT_Qs for the remaining IGHD_-_ and IGHJ_ genes with respect to IGHD_-_ (nf)-IGHJ_ were determined; and third, these genes with respect to IGHD_-_ (nf)-IGHJ_, the remaining IGHD_-_ with respect to IGHD_-_ (nf)-IGHJ_ (Step 2a of 2) and IGHJ_ genes with respect to IGHD_-_ (nf)-IGHJ_ (Step 2a of 2) were recombined to yield the final step recombined gene, IGHD_-_ -IGHJ_ (Step 2b of 2), ready for IGHV_-_ ↔ IGHD_-_-IGHJ_.

For the 1-step D_-_↔ J_ stimulation, the germline IGHD_-_ and IGHJ_ genes were recombined to yield the final recombined gene, IGHD_-_ -IGHJ_ (Step 1of 1), ready for IGHV_-_ ↔ IGHD_-_-IGHJ_.

Gene esebssiwaagoT _Q-based simulation of IGHV_-_ and IGHD_-_-IGHJ_ recombinations for allele 1 (IGHM) and allele 2 (IGHD)

The most common variable genes were sampled. These genes included IGHV1–3, IGHV3–23, IGHV4–28, IGHV3–48, IGHV4–59 and IGHV4–61 with the exception of IGHV5–51 [18]. The IGHV_-_ to IGHD_-_-IGHJ_ recombination events through further CSR isotype switching recombinations were performed for V1–3, V3–23, and V5–51.

Gene esebssiwaagoT _Q-based simulation of internal consensus recognition sequence (iCSR) recombination for allele 1 (IGHM)

For Allele 1 (IGHM) [11], internal consensus sequence recognition (iCSR) recombination was performed between MIR4537, MIR4507/MIR4538 and MIR4539 with respect to IGHV_-_-IGHD_-_-(IGHJ5)-IGHJ6. The complete recombined V(D)J gene is VDJ6 irrespective of whether it is the 1-step recombined IGHV_-_ -D_-_-J5 gene or the 2-step recombined IGHV_-_ -D_-_-J5-J6 gene.

Gene esebssiwaagoT _Q-based simulation of homologous recombination for allele 2 (IGHD) and determination of initial allelic exclusion

For Allele 2 (IGHD) [14], homologous recombination (HR) was performed between IGHV_-_-IGHD_-_-(J1)–(J2)-(J3)-J4-(J5)-J6 (VDJ6) and IGHD with respect to IGHV_-_ -IGHD_-_-(J1)–(J2)-(J3)-J4-(J5)-J6. If there was no esebssiwaagoT_Q match (gene esebssiwaagoT_Q ± 0.015 units) for homologous recombination, then further CSR was performed (as in Allele 1).

Gene esebssiwaagoT _Q-based simulation of consensus recognition sequence (CSR) antibody isotype switchings for allele 1 (IGHM)

For Allele 1 (IGHM) isotype switching [12, 13], consensus sequence recognition (CSR) recombination was performed if there was an esebssiwaagoT_Q match between a downstream Ig heavy chain gene IGHG3, IGHG1, IGHA1, IGHG4/IGHG2, IGHE and IGHA2 with respect to VDJ6-IGHM (± 0.015 units) and VDJ6-IGHM (± 0.015 units). If there was an esebssiwaagoT_Q match with a downstream gene for example with IGHG3, then the esebssiwaagoT_Qs for each remaining downstream Ig heavy chain gene IGHG1, IGHA1, IGHG4/IGHG2, IGHE and IGHA2 with respect to VDJ6-IGHG3 were determined, after which further esebssiwaagoT_Q matches were determined and remaining downstream gene recombinations simulated.

Results

Germline IGH_ genes

IGHA2 is a 3 episode, 7 initial SEB and final SEB gene that begins with a mesotropic SEB. IGHA2 has one instance of non-contributory anisotropy. IGHA2 is a 3 M (7) NCA gene with a final esebssiwaagoT_Q of 0.10 (0.099).

IGHE is a 3 episode, 7 initial SEB and final SEB gene that begins with an anisotropic SEB. IGHE is a 3 A (7) gene with a final esebssiwaagoT_Q of 0.11 (0.111).

IGHG4/IGHG2 is a 2 episode, 5 initial SEB and final SEB gene that begins with a mesotropic SEB. IGHG4/IGHG2 has one instance of non-contributory anisotropy. IGHG4/IGHG2 is a 2 M (5) NCA gene with a final esebssiwaagoT_Q of 0.16 (0.163).

IGHA1 is a 3 episode, 7 initial SEB and final SEB gene that begins with a mesotropic SEB. IGHA1 is a 3 M (7) gene with a final esebssiwaagoT_Q of 0.13 (0.131).

IGHG1 is a 3 episode, 7 initial SEB and final SEB gene that begins with an anisotropic SEB. IGHG1 is a 3 A (7) gene with a final esebssiwaagoT_Q of 0.12 (0.120).

IGHG3 is a 2 episode, 5 initial SEB and final SEB gene that begins with a anisotropic SEB. IGHG3 is a 2 A (5) gene with a final esebssiwaagoT_Q of 0.08 (0.075).

IGHD is a 3 episode, 7 initial SEB and final SEB gene that begins with a mesotropic SEB. IGHD is a 3 M (7) gene with a final esebssiwaagoT_Q of 0.09 (0.092).

IGHM is a 3 episode, 7 initial SEB and final SEB gene that begins with anisotropic SEB. IGHM is a 3 A (7) gene with a final esebssiwaagoT_Q of 0.09 (0.088).

MIR4539 is a 3 episode, 7 initial SEB and final SEB gene that begins with a mesotropic SEB. MIR4539 has one instance of non-contributory anisotropy. MIR4539 is a 3 M (7) NCA gene with a final esebssiwaagoT_Q of 0.08 (0.076).

MIR4507/MIR4538 is a 3 episode, 7 initial SEB and final SEB gene that begins with an anisotropic SEB. MIR4507/MIR4538 has one instance of non-contributory anisotropy. MIR4507/MIR4538 is a 3 A (7) NCA gene with a final esebssiwaagoT_Q of 0.08 (0.081).

MIR4537 is a 3 episode, 7 initial SEB and final SEB gene that begins with a mesotropic SEB. MIR4537 has one instance of non-contributory anisotropy. MIR4537 is a 3 M (7) NCA gene with a final esebssiwaagoT_Q of 0.06 (0.064) (Table 1, Additional file 2: Table S2).

Table 1 Chromosome 14 (−) strand chromatin Ig heavy chain locus immunoglobin gene esebssiwaagoT_Qs for germline genes in native 5′- > 3′ chronology before gene rearrangement

Germline IGHJ_ genes

IGHJ6 is a 3 episode, 7 initial SEB and final SEB gene that begins with an anisotropic SEB. IGHJ6 is a 3 A (7) gene with a final esebssiwaagoT_Q of 0.10 (0.097).

IGHJ5 is a 3 episode, 7 initial SEB and final SEB gene that begins with an anisotropic SEB. IGHJ5 is a 3 A (7) gene with a final esebssiwaagoT_Q of 0.24 (0.235).

IGHJ4 is a 3 episode, 7 initial SEB and final SEB gene that begins with a mesotropic SEB. IGHJ4 has one instance of non-contributory anisotropy. IGHJ4 is a 3 M (7) NCA gene with a final esebssiwaagoT_Q of 0.11 (0.110).

IGHJ3 is a 3 episode, 7 initial SEB and final SEB gene that begins with a mesotropic SEB. IGHJ3 is a 3 M (7) gene with a final esebssiwaagoT_Q of 0.11 (0.112).

IGHJ2 is a 3 episode, 7 initial SEB and final SEB gene that begins with a mesotropic SEB. IGHJ2 is a 3 M (7) gene with a final esebssiwaagoT_Q of 0.11 (0.114).

IGHJ1 is a 3 episode, 7 initial SEB and final SEB gene that begins with a mesotropic SEB. IGHJ1 has one instance of non-contributory anisotropy. IGHJ1 is a 3 M (7) NCA gene with a final esebssiwaagoT_Q of 0.12 (0.116) (Table 2, Additional file 3: Table S3).

Table 2 Chromosome 14 (−) strand chromatin Ig heavy chain locus joining gene esebssiwaagoT_Qs for germline genes in native 5′- > 3′ chronology before gene rearrangement

Germline non-functional IGHD1–20 (nf) gene for allele 1 (IGHM) 2-step (1 of 2) recombination

IGHD1–20 (nf) is a 3 episode, 7 initial SEB and 7* final SEB gene that begins with an anisotropic SEB. IGHD1–20 (nf) has one instance of non-contributory anisotropy at the ending. IGHD1–20 (nf) is a 3 A [7 (+ 2): 7*] NCA* gene with a final esebssiwaagoT_Q of 0.41 (0.406) (Table 3, Additional file 4: Table S4; Table 4).

Table 3 Chromosome 14 (−) strand chromatin Ig heavy chain locus diversity gene esebssiwaagoT_Qs for germline genes in native 5′- > 3′ chronology before gene rearrangement

Table 4 Chromosome 14 (−) strand chromatin Ig heavy chain locus diversity (D)-to-joining (J) recombination sequence for allele 1 (IGHM) before VDJ

Germline functional IGHD_-_ genes un-involved in the 1st step of 2-step allele 1 (IGHM) recombination

IGHD6–19 is a 3 episode, 7 initial and 5 final SEB gene that begins with a mesotropic SEB. IGHD6–19 has one instance of anisotropy converted-to-mesotropy, and one instance of indirect stIsotropy for mesotropy. IGHD6–19 is a 3 M [7(− 2): 5] ACM stIMfA gene with a final esebssiwaagoT_Q of 0.31 (0.309).

IGHD4–17 is a 3 episode, 7 initial and 9 final SEB gene that begins with an anisotropic SEB. IGHD4–17 has one instance of anisotropy converted-to-mesotropy. IGHD4–17 is a 3 A [7 (+ 2): 9] ACM with a final esebssiwaagoT_Q of 0.31 (0.310).

IGHD3–16 is a 3 episode, 7 initial and final SEB gene that begins with a mesotropic SEB. IGHD3–16 has one instance of non-contributory anisotropy. IGHD3–16 is a 3 M (7) NCA gene with a final esebssiwaagoT_Q of 0.31 (0.308).

IGHD3–10 is a 3 episode, 7 initial and final SEB gene that begins with an anisotropic SEB. IGHD3–10 has one instance of anisotropy converted-to-mesotropy, and non-contributory reverse/stIsotropy. IGHD3–10 is a 3 A (7) ACM NCstI gene with a final esebssiwaagoT_Q of 0.34 (0.342).

IGHD2–8 is a 3 episode, 7 initial and final SEB gene that begins with a mesotropic SEB. IGHD2–8 has one instance of non-contributory reverse/ stIsotropy. IGHD2–8 is a 3 M (7) NCstI gene with a final esebssiwaagoT_Q of 0.30. (0.295).

IGHD2–2 is a 3 episode, 7 initial and final SEB gene that begins with a mesotropic SEB. IGHD2–2 has one instance of non-contributory reverse/ stIsotropy. IGHD2–2 is a 3 M (7) NCstI gene with a final esebssiwaagoT_Q of 0.30 (0.301) (Table 3, Additional file 4: Table S4; Table 4).

Germline non-functional IGHD4–11 (nf) gene and IGHD5–8 (nf) for allele 2 (IGHD) 2-step (1 of 2) recombination

IGHD4–11(nf) is a 3 episode, 7 initial SEB and final SEB gene that begins with an anisotropic SEB. IGHD4–11(nf) has one instance of anisotropy converted-to-mesotropy IGHD4–11(nf) is a 3 A (7) ACM gene with a final esebssiwaagoT_Q of 0.29 (0.293).

IGHD5–18 (nf) is a 3 episode, 7 initial and 6 final SEB gene that begins with a mesotropic SEB. IGHD5–18 (nf) has one instance of a non-contributory anisotropy, and one instance of indirect stIsotropy for anisotropy. IGHD5–18 (nf) is a 3 M [7 (− 3): 4] NCA stIMfA gene with a final esebssiwaagoT_Q 0.25 (0.254) (Table 3, Additional file 4: Table S4; Table 5).

Table 5 Chromosome 14 (−) strand chromatin Ig heavy chain locus diversity (D)-to-joining (J) recombination sequence for allele 2 (IGHD) before VDJ

Germline functional IGHD_-_ genes un-involved in the 1st step of 2-step allele 2 (IGHD) recombination

IGHD3–9 is a 3 episode, 7 initial and 3 final SEB gene that begins with a mesotropic SEB. IGHD3–9 has one instance of anisotropy converted-to-mesotropy, one instance of non-contributory anisotropy, and one instance of non-contributory reverse/stIsotropy. IGHD3–9 is a 3 M [7 (− 4): 3] ACM NCA NCstI gene with a final esebssiwaagoT_Q of 0.24 (0.239).

IGHD1–7 is a 3 episode, 7 initial and 5 final SEB gene that begins with an anisotropic SEB. IGHD1–7 has one instance of anisotropy converted-to-mesotropy, one instance of non-contributory anisotropy, and non-contributory reverse/stIsotropy. IGHD1–7 is a 3 A [7 (− 2): 5] ACM NCA NCstI gene with a final esebssiwaagoT_Q of 0.28 (0.276).

IGHD6–6 is a 3 episode, 7 initial and final SEB gene that begins with a mesotropic SEB. IGHD6–6 has one instance of indirect stIsotropy for mesotropy. IGHD6–19 is a 3 M (7) stIMfM gene with a final esebssiwaagoT_Q of 0.27 (0.275).

IGHD5–5 is a 3 episode, 7 initial and final SEB gene that begins with a mesotropic SEB. IGHD5–5 has one instance of non-contributory reverse/stIsotropy. IGHD5–5 is a 3 M (7) NCstI gene with a final esebssiwaagoT_Q of 0.23 (0.233).

IGHD4–4 is a 3 episode, 7 initial and final SEB gene that begins with a mesotropic SEB. IGHD4–4 has two instances of non-contributory reverse/stIsotropy. IGHD4–4 is a 3 M (7) NCstI × 2 gene with a final esebssiwaagoT_Q of 0.26 (0.258).

IGHD3–3 is a 3 episode, 7 initial and 9 final SEB gene that begins with a mesotropic SEB. IGHD3–3 has one instance of non-contributory anisotropy, and one instance of non-contributory reverse/stIsotropy. IGHD3–3 is a 3 M [7 (+ 2): 9] NCA NCstI gene with a final esebssiwaagoT_Q of 0.24 (0.243).

IGHD1–1 is a 3 episode, 7 initial SEB and 7* final SEB gene that begins with a mesotropic SEB. IGHD1–1 has one instance of anisotropy converted-to-mesotropy preceding ending confirmation (anisotropic SEB no. 8), and three instances of non-contributory reverse/stIsotropy. IGHD1–1 is a 3 M [7 (+ 1): 7*] ACM* NCstI gene with a final esebssiwaagoT_Q of 0.23 (0.233) (Table 3, Additional file 4: Table S4; Table 5).

Germline non-functional IGHD_-_ gene

IGHD7–27^† is a 3 episode, 7 initial SEB and final SEB gene that begins with a mesotropic SEB. IGHD7–27^† is a 3 M (7) gene with a final esebssiwaagoT_Q of. 0.17 (0.165)^† (Table 3, Additional file 4: Table S4).

Germline IGHJ5 gene for allele 1 (IGHM) 2-step (1 of 2) recombination [and for allele 1 (IGHM) 1-step recombination]

IGHJ5 is a 3 A (7) gene with a final esebssiwaagoT_Q of 0.24 (0.235) (Table 2, Additional file 3: Table S3; Table 4).

Germline non-functional IGHD1–20 (nf) gene for allele 1 (IGHM) 2-step (1 of 2) recombination

IGHD1–20 (nf) is a 3 A [7 (+ 2): 7*] NCA* gene with a final esebssiwaagoT_Q of 0.41 (0.406) (Table 3, Additional file 4: Table S4; Table 4).

IGHJ6 gene with respect to IGHD1–20 (nf)-IGHJ5 for allele 1 (IGHM) 2-step (2a of 2) recombination

IGHJ6 with respect to IGHD1–20-IGHJ5 is a 3 episode, 7 initial and final SEB gene that begins with an anisotropic SEB. IGHJ6 with respect to IGHD1–20-IGHJ5 is a 3 A (7) gene with a final esebssiwaagoT_Q of 0.10 (0.101) (Table 4, Additional file 5: Table S5).

IGHD_-_ genes with respect to IGHD1–20 (nf)-IGHJ5 for allele 1 (IGHM) 2-step (2a of 2) recombination

IGHD3–10 with respect to IGHD1–20-IGHJ5 is a 3 episode, 7 initial and 5 final SEB gene that begins with an anisotropic SEB. IGHD3–10 with respect to IGHD1–20-IGHJ5 has one instance of non-contributory anisotropy, and one instance of non-contributory reverse/stIsotropy. IGHD3–10 with respect to IGHD1–20-IGHJ5 is a 3 A [7(− 2): 5] NCA NCstI gene with a final esebssiwaagoT_Q of 0.40 (0.402).

IGHD4–17 with respect to IGHD1–20-IGHJ5 is a 3 episode, 7 initial and final SEB gene that begins with a mesotropic SEB. IGHD4–17 with respect to IGHD1–20-IGHJ5 has one instance of non-contributory anisotropy. IGHD4–17 with respect to IGHD1–20-IGHJ5 is a 3 M (7) NCA gene with a final esebssiwaagoT_Q of 0.15 (0.150).

IGHD6–19 with respect to IGHD1–20-IGHJ5 is a 3 episode, 7 initial and 3 final SEB gene that begins with a mesotropic SEB. IGHD6–19 with respect to IGHD1–20-IGHJ5 is a 3 M (7) gene with a final esebssiwaagoT_Q of 0.23 (0.233).

IGHD3–16 with respect to IGHD1–20-IGHJ5 is a 3 episode, 7 initial and 5 final SEB gene that begins with a mesotropic SEB. IGHD3–16 with respect to IGHD1–20-IGHJ5 has one instance of anisotropy converted-to-mesotropy. IGHD3–16 with respect to IGHD1–20-IGHJ5 is a 3 M [7(− 2): 5] ACM gene with a final esebssiwaagoT_Q of 0.18 (0.177).

IGHD2–2 with respect to IGHD1–20-IGHJ5 is a 3 episode, 7 initial and 3 final SEB gene that begins with a mesotropic SEB. IGHD2–2 with respect to IGHD1–20-IGHJ5 has one instance of non-contributory reverse/stIsotropy. IGHD3–9 is a 3 M (7) NCstI gene with a final esebssiwaagoT_Q of 0.30 (0.296).

IGHD2–8 with respect to IGHD1–20-IGHJ5 is a 3 episode, 7 initial and 3 final SEB gene that begins with a mesotropic SEB. IGHD2–8 with respect to IGHD1–20-IGHJ5 has one instance of non-contributory anisotropy, and one instance of non-contributory reverse/stIsotropy. IGHD2–8 with respect to IGHD1–20-IGHJ5 is a 3 M (7) NCA NCstI gene with a final esebssiwaagoT_Q of 0.24 (0.238) (Table 4, Additional file 5: Table S5).

IGHD_-_-IGHJ_ genes for allele 1 (IGHM) 2-step (2b of 2) recombination

IGHD3–10-IGHJ6 is a 3 episode, 7 initial and 5 final SEB gene that begins with an anisotropic SEB. IGHD3–10-IGHJ6 has one instance of anisotropy converted-to-mesotropy. IGHD3–10-IGHJ6 is a 3 A [7(− 2): 5] ACM gene with a final esebssiwaagoT_Q of 0.23 (0.231).

IGHD4–17-IGHJ6 is a 3 episode, 7 initial and final SEB gene that begins with a mesotropic SEB. IGHD4–17-IGHJ6 has one instance of non-contributory anisotropy. IGHD4–17-IGHJ6 is a 3 M (7) NCA gene with a final esebssiwaagoT_Q of 0.13 (0.130).

IGHD6–19-IGHJ6 is a 3 episode, 7 initial and 5 final SEB gene that begins with a mesotropic SEB. IGHD6–19-IGHJ6 has one instance of anisotropy converted-to-mesotropy. IGHD6–19-IGHJ6 is a 3 M [7(− 2): 5] ACM gene with a final esebssiwaagoT_Q of 0.19 (0.194).

IGHD3–16-IGHJ6 is a 3 episode, 7 initial SEB and final SEB gene that begins with a mesotropic SEB. IGHD3–16-IGHJ6 is a 3 M (7) gene with a final esebssiwaagoT_Q of 0.23 (0.233).

IGHD2–2-IGHJ6 is a 3 episode, 7 initial and 3 final SEB gene that begins with a mesotropic SEB. IGHD2–2-IGHJ6 has one instance of non-contributory anisotropy, and one instance of indirect stIsotropy for anisotropy. IGHD2–2-IGHJ6 is a 3 M [7(− 4): 3] NCA stIMfA gene with a final esebssiwaagoT_Q of 0.30 (0.297).

IGHD2–8-IGHJ6 is a 3 episode, 7 initial SEB and final SEB gene that begins with a mesotropic SEB. IGHD2–8-IGHJ6 is a 3 M (7) gene with a final esebssiwaagoT_Q of 0.20 (0.196) (Table 4, Additional file 5: Table S5).

Germline IGHJ2 gene for allele 2 (IGHD) 2-step (1 of 2) [and for allele 2 (IGHD) 1-step recombination]

IGHJ2 is a 3 M (7) gene with a final esebssiwaagoT_Q of 0.11 (0.114) (Table 2, Additional file 3: Table S3; Table 5).

Germline non-functional IGHD_-_ (nf) genes for allele 2 (IGHD) 2-step (1 of 2) recombination

IGHD4–11(nf) is a 3 A (7) ACM gene with a final esebssiwaagoT_Q of 0.29 (0.293).

IGHD5–18 (nf) is a 3 M [7 (− 3): 4] NCA stIMfA gene with a final esebssiwaagoT_Q 0.25 (0.254) (Table 3, Additional file 4: Table S4; Table 5).

IGHJ4 gene with respect to IGHD4–11 (nf)-IGHJ2 and IGHJ4 gene with respect to IGHD5–18 (nf)-IGHJ2 for allele 2 (IGHD) 2-step (2a of 2) recombination

IGHJ4 with respect to IGHD4–11-IGHJ2 is a 3 episode, 7 initial and 9 final SEB gene that begins with an mesotropic SEB. IGHJ4 with respect to IGHD4–11-IGHJ2 has one instance of anisotropy converted-to-mesotropy. IGHJ4 with respect to IGHD4–11-IGHJ2 is a 3 M [7(+ 2): 9] ACM with a final esebssiwaagoT_Q 0.19 (0.193).

IGHJ4 with respect to IGHD5–18-IGHJ4 is a 3 episode, 7 initial SEB and final SEB gene that begins with a mesotropic SEB. IGHJ4 with respect to IGHD5–18-IGHJ4 is a 3 M (7) gene with a final esebssiwaagoT_Q of 0.17 (0.165) (Table 5, Additional file 6: Table S6).

IGHD_-_ genes with respect to IGHD_-_ (nf)-IGHJ2 for allele 2 (IGHD) 2-step (2a of 2) recombination

IGHD1–7 with respect to IGHD4–11-IGHJ2 is a 3 episode, 7 initial and final SEB gene that begins with an anisotropic SEB. IGHD1–7 with respect to IGHD4–11-IGHJ2 has one instance of non-contributory reverse/stIsotropy. IGHD1–7 with respect to IGHD4–11-IGHJ2 is a 3 A (7) NCstI gene with a final esebssiwaagoT_Q of 0.31 (0.311).

IGHD6–6 with respect to IGHD4–11-IGHJ2 is a 3 episode, 7 initial and 5 final SEB gene that begins with a mesotropic SEB. IGHD6–6 with respect to IGHD4–11-IGHJ2 has one instance of anisotropy converted-to-mesotropy, one instance of non-contributory anisotropy, and one instance of non-contributory reverse/stIsotropy. IGHD6–6 with respect to IGHD4–11-IGHJ2 is a 3 M [7(− 2): 5] ACM NCA NCstI gene with a final esebssiwaagoT_Q of 0.27 (0.266).

IGHD4–4 with respect to IGHD4–11-IGHJ2 is a 3 episode, 7 initial and final SEB gene that begins with a mesotropic SEB. IGHD4–4 with respect to IGHD4–11-IGHJ2 has one instance of non-contributory reverse/stIsotropy. IGHD4–4 with respect to IGHD4–11-IGHJ2 is a 3 M (7) NCstI gene with a final esebssiwaagoT_Q of 0.27 (0.268).

IGHD3–3 with respect to IGHD4–11-IGHJ2 is a 3 episode, 7 initial and final SEB gene that begins with a mesotropic SEB. IGHD3–3 with respect to IGHD4–11-IGHJ2 has one instance of non-contributory reverse/stIsotropy. IGHD3–3 with respect to IGHD4–11-IGHJ2 is a 3 M (7) NCstI gene with a final esebssiwaagoT_Q of 0.16 (0.156).

IGHD3–9 with respect to IGHD4–11-IGHJ2 is a 3 episode, 7 initial and final SEB gene that begins with a mesotropic SEB. IGHD3–9 with respect to IGHD4–11-IGHJ2 has one instance of anisotropy converted-to-mesotropy. IGHD3–9 with respect to IGHD4–11-IGHJ2 a 3 M (7) ACM gene with a final esebssiwaagoT_Q of 0.33 (0.332).

IGHD5–5 with respect to IGHD5–18-IGHJ2 is a 3 episode, 7 initial and final SEB gene that begins with a mesotropic SEB. IGHD5–5 with respect to IGHD5–18-IGHJ2 has one instance of anisotropy converted-to-mesotropy, and one instance of non-contributory reverse/stIsotropy. IGHD5–5 with respect to IGHD5–18-IGHJ2 is a 3 M (7) ACM NCstI gene with a final esebssiwaagoT_Q of 0.31 (0.308).

IGHD1–1 with respect to IGHD5–18-IGHJ2 is a 3 episode, 7 initial and final SEB gene that begins with a mesotropic SEB. IGHD1–1 with respect to IGHD5–18-IGHJ2 has one instance of non-contributory reverse/stIsotropy, and one instance of indirect stIsotropy for mesotropy. IGHD1–1 with respect to IGHD5–18-IGHJ2 is a 3 M (7) NCstI stIMfM gene with a final esebssiwaagoT_Q of 0.25 (0.250) (Table 5, Additional file 6: Table S6).

IGHD_-_ -IGHJ_ genes for allele 2 (IGHD) 2-step (2b of 2) recombination

IGHD1–7-IGHJ4 is a 3 episode, 7 initial and 5 final SEB gene that begins with an anisotropic SEB. IGHD1–7-IGHJ4 has one instance of anisotropy converted-to-mesotropy, and one instance of indirect stIsotropy for mesotropy. IGHD1–7-IGHJ4 is a 3 A [7(− 2): 5] ACM stIMfM gene with a final esebssiwaagoT_Q of 0.35 (0.350).

IGHD6–6-IGHJ4 is a 3 episode, 7 initial and 9 final SEB gene that begins with a mesotropic SEB. IGHD6–6-IGHJ4 has one instance of anisotropy converted-to-mesotropy. IGHD6–6-IGHJ4 is a 3 M [7(+ 2): 9] ACM gene with a final esebssiwaagoT_Q of 0.25 (0.249).

IGHD4–4-IGHJ4 is a 3 episode, 7 initial and final SEB gene that begins with anisotropic SEB. IGHD4–4-IGHJ4 has one instance of anisotropy converted-to-mesotropy. IGHD4–4-IGHJ4 is a 3 A (7) ACM gene with a final esebssiwaagoT_Q of 0.33 (0.332).

IGHD3–3-IGHJ4 is a 3 episode, 7 initial and 9 final SEB gene that begins with a mesotropic SEB. IGHD3–3-IGHJ4 has one instance of anisotropy converted-to-mesotropy. IGHD3–3-IGHJ4 is a 3 M [7(+ 2): 9] ACM gene with a final esebssiwaagoT_Q of 0.26 (0.258).

IGHD3–9-IGHJ4 is a 3 episode, 7 initial and final SEB gene that begins with a mesotropic SEB. IGHD3–3-IGHJ4 is a 3 M (7) gene with a final esebssiwaagoT_Q of 0.32 (0.324).

IGHD5–5-IGHJ4 is a 3 episode, 7 initial and 3 final SEB gene that begins with a mesotropic SEB. IGHD5–5-IGHJ4 has one instance of a non-contributory anisotropy, and one instance of indirect stIsotropy for anisotropy. IGHD5–5-IGHJ4 is a 3 M [7(− 4): 3] NCA stIMfA gene with a final esebssiwaagoT_Q of 0.30 (0.297).

IGHD1–1-IGHJ4 is a 3 episode, 7 initial and 6 final SEB gene that begins with an anisotropic SEB. IGHD1–1-IGHJ4 has one instance of a non-contributory anisotropy. IGHD1–1-IGHJ4 is a 3 A [7(− 1): 6] NCA gene with a final esebssiwaagoT_Q of 0.34 (0.340) (Table 5, Additional file 6: Table S6).

Germline IGHJ5 gene for allele 1 (IGHM) allele 1 (IGHM) 1-step recombination

IGHJ5 is a 3 A (7) gene with a final esebssiwaagoT_Q of 0.24 (0.235 (Table 2, Additional file 3: Table S3; Table 4).

Germline functional IGHD_-_ genes for allele 1 (IGHM) 1-step recombination

IGHD2–15 is a 3 episode, 7 initial SEB and final SEB gene that begins with a mesotropic SEB. IGHD2–15 has one instance of non-contributory anisotropy. IGHD2–15 is a 3 M (7) NCA gene with a final esebssiwaagoT_Q of 0.29 (0.294).

IGHD1–14 is a 3 episode, 7 initial and final SEB gene that begins with an anisotropic SEB. IGHD1–14 has two instances of anisotropy converted-to-mesotropy, and one instance of non-contributory anisotropy. IGHD1–14 is a 3 A (7) ACM × 2 NCA with a final esebssiwaagoT_Q of 0.29 (0.292).

IGHD6–13 is a 3 episode, 7 initial and final SEB gene that begins with a mesotropic SEB. IGHD6–13 has one instance of non-contributory anisotropy, and one instance of non-contributory reverse stIsotropy. IGHD6–13 is a 3 M (7) NCA NCstI gene with a final esebssiwaagoT_Q of 0.29 (0.286) (Table 3, Additional file 4: Table S4; Table 4).

IGHD_-_-IGHJ5 genes for allele 1 (IGHM) V-to-DJ after 1-step recombination

IGHD2–15-IGHJ5 is a 3 episode, 7 initial SEB and final SEB gene that begins with a mesotropic SEB. IGHD2–15-IGHJ5 is a 3 M (7) gene with a final esebssiwaagoT_Q of 0.25 (0.251).

IGHD1–14-IGHJ5 is a 3 episode, 7 initial and 3 final SEB gene that begins with a mesotropic SEB. IGHD1–14-IGHJ5 has one instance of anisotropy converted-to-mesotropy, and one instance of non-contributory anisotropy. IGHD1–14-IGHJ5 is a 3 M (7) ACM NCA gene with a final esebssiwaagoT_Q of 0.15 (0.149).

IGHD6–13-IGHJ5 is a 3 episode, 7 initial and 9 final SEB gene that begins with an anisotropic SEB. IGHD6–13-IGHJ5 has one instance of anisotropy converted-to-mesotropy, and one instance of non-contributory anisotropy. IGHD6–13- IGHJ5 is a 3 A [7(+ 2): 9] ACM NCA gene with a final esebssiwaagoT_Q of 0.20 (0.205) (Table 4, Additional file 5: Table S5).

Germline functional IGHJ3, IGHJ2 and IGHJ1 genes for allele 2 (IGHD) 1-step recombination

IGHJ3 is a 3 M (7) gene with a final esebssiwaagoT_Q of 0.11 (0.112).

IGHJ2 is a 3 M (7) gene with a final esebssiwaagoT_Q of 0.11 (0.114).

IGHJ1 is a 3 M (7) NCA gene with a final esebssiwaagoT_Q of 0.12 (0.116) (Table 2, Additional file 3: Table S3; Table 5).

Germline functional IGHD_-_ genes for allele 2 (IGHD) 1-step recombination

IGHD1–26 is a 3 episode, 7 initial and final SEB gene that begins with an anisotropic SEB. IGHD1–26 has two instances of non-contributory anisotropy. IGHD1–26 is a 3 A (7) NCA × 2gene with a final esebssiwaagoT_Q of 0.22 (0.217).

IGHD6–25 is a 3 episode, 7 initial and final SEB gene that begins with an anisotropic SEB. IGHD1–14 IGHD6–25 has one instance of anisotropy converted-to-mesotropy, and two instances of non-contributory anisotropy. IGHD6–25 is a 3 A (7) ACM NCA × 2 with a final esebssiwaagoT_Q 0.17 (0.172).

IGHD5–24/IGHD4–23/IGHD3–22 is a 3 episode, 7 initial and 5 final SEB gene that begins with an anisotropic SEB. IGHD5–24/IGHD4–23/IGHD3–22 has one instance of anisotropy converted-to-mesotropy, and one instance of non-contributory anisotropy. IGHD5–24/IGHD4–23/IGHD3–22 is a 3 A [(7(− 2): 5] ACM NCA with a final esebssiwaagoT_Q of 0.22 (0.216).

IGHD2–21 is a 3 episode, 7 initial and final SEB gene that begins with a mesotropic SEB. IGHD2–21 has one instance of non-contributory anisotropy. IGHD2–21 is a 3 M (7) NCA with a final esebssiwaagoT_Q of 0.20 (0.205).

IGHD5–12 is a 3 episode, 7 initial SEB and final SEB gene that begins with a mesotropic SEB. IGHJ3 IGHD5–12 is a 3 M (7) gene with a final esebssiwaagoT_Q of 0.22 (0.218) (Table 3, Additional file 4: Table S4; Table 5).

IGHD_-_-IGHJ_ genes for allele 2 (IGHD) V-to-DJ after 1-step recombination

IGHD5–24/IGHD4–23/IGHD3–22-IGHJ1 is a 3 episode, 7 initial and final SEB gene that begins with a mesotropic SEB. IGHD5–24/IGHD4–23/IGHD3–22-IGHJ1 has one instance of indirect stIsotropy for mesotropy. IGHD5–24/IGHD4–23/IGHD3–22-IGHJ1 is a 3 M (7) stIMfM gene with a final esebssiwaagoT_Q of 0.16 (0.161).

IGHD1–26-IGHJ1 is a 3 episode, 7 initial SEB and final SEB gene that begins with a mesotropic SEB. IGHD1–26-IGHJ1 is a 3 M (7) gene with a final esebssiwaagoT_Q of 0.13 (0.130).

IGHD2–21-IGHJ1 is a 3 episode, 7 initial and 6 final SEB gene that begins with a mesotropic SEB. IGHD2–21- IGHJ1 has two instances of non-contributory anisotropy. IGHD2–21- IGHJ1 is a 3 M [7(− 1): 6] NCA × 2 gene with a final esebssiwaagoT_Q of 0.21 (0.206).

IGHD6–25-IGHJ1 is a 3 episode, 7 initial SEB and final SEB gene that begins with a mesotropic SEB. IGHD6–25-IGHJ1 is a 3 M (7) gene with a final esebssiwaagoT_Q of 0.23 (0.233).

IGHD5–12-IGHJ2 is a 3 episode, 7 initial and 5 final SEB gene that begins with a mesotropic SEB. IGHD5–12-IGHJ2 has one instance of non-contributory anisotropy. IGHD5–12-IGHJ2 is a 3 M [7(− 2): 5] NCA gene with a final esebssiwaagoT_Q of 0.35 (0.347).

IGHD5–24/IGHD4–23/IGHD3–22-IGHJ3 is a 3 episode, 7 initial SEB and final SEB gene that begins with a mesotropic SEB. IGHD5–24/IGHD4–23/IGHD3–22-IGHJ3 is a 3 M (7) gene with a final esebssiwaagoT_Q of 0.15 (0.147).

IGHD1–26-IGHJ3 is a 3 episode, 7 initial and 6 final SEB gene that begins with a mesotropic SEB. IGHD1–26-IGHJ3 has one instance of non-contributory anisotropy. IGHD1–14-IGHJ5 is a 3 M [7(− 1): 6] NCA gene with a final esebssiwaagoT_Q of 0.11 (0.114).

IGHD2–21- IGHJ3 is a 3 episode, 7 initial SEB and final SEB gene that begins with a mesotropic SEB. IGHD2–21- IGHJ3 is a 3 M (7) gene with a final esebssiwaagoT_Q of 0.13 (0.130).

IGHD6–25-IGHJ3 is a 3 episode, 7 initial and 5 final SEB gene that begins with an mesotropic SEB. IGHD6–25-IGHJ3 has one instance of anisotropy converted-to-mesotropy. IGHD6–25-IGHJ3 is a 3 M [7(− 2): 5] ACM with a final esebssiwaagoT_Q of 0.20 (0.195) (Table 5, Additional file 6: Table S6).

Germline IGHV_ genes

IGHV1–3/IGHV4–4§ with respect to IGHJ_-IGHD_-_ is a 3 episode, 7 initial and variable final SEB gene that begins with either an anisotropic SEB or a mesotropic SEB. The final esebssiwaagoT_Q is variable.

IGHV3–23 is a 3 episode, 7 initial SEB and final SEB gene that begins with an anisotropic SEB. IGHV3–23 is a 3 A (7) gene with a final esebssiwaagoT_Q of 0.33 (0.332).

IGHV4–28 is a 3 episode, 7 initial and 5 final SEB gene that begins with a mesotropic SEB. IGHV4–28 has one instance of anisotropy converted-to-mesotropy, and one instance of indirect stIsotropy for mesotropy. IGHV4–28 is a 3 M [7 (− 2): 5] ACM stIMfM gene with a final esebssiwaagoT_Q of 0.41 (0.415).

IGHV3–48 is a 3 episode, 7 initial SEB and final SEB gene that begins with an anisotropic SEB. IGHV3–48 is a 3 A (7) gene with a final esebssiwaagoT_Q of 0.27 (0.274).

IGHV5–51/IGHV3–53 is a 2 episode, 5 initial SEB and final SEB gene that begins with an anisotropic SEB. IGHV5–51/IGHV3–53 has one instance of non-contributory anisotropy. IGHV5–51/IGHV3–53 is a 2 A (5) NCA gene with a final esebssiwaagoT_Q of 0.25 (0.245).

IGHV4–59 is a 3 episode, 7 initial SEB and final SEB gene that begins with a mesotropic SEB. IGHV4–59 is a 3 M (7) gene with a final esebssiwaagoT_Q of. 0.34 (0.336).

IGHV4–61 is a 3 episode, 7 initial SEB and final SEB gene that begins with a mesotropic SEB. IGHV4–61 is a 3 M (7) gene with a final esebssiwaagoT_Q of. 0.26 (0.258) (Table 6, Additional file 7: Table S7).

Table 6 Chromosome 14 (−) strand chromatin Ig heavy chain locus variability gene esebssiwaagoT_Qs for germline genes in native 5′- > 3′ chronology

Ig heavy chain genes before iCSR and homologous recombination after IGHV1–3-IGHD_-_-IGHJ6

IGHV1–3- IGHD_-_-IGHJ6 is a 3 episode, 7 initial and 9* final SEB gene that begins with a mesotropic SEB. IGHV1–3- IGHD_-_-IGHJ6 has one instance of anisotropy converted-to-mesotropy, and one instance of indirect stIsotropy for anisotropy. IGHV1–3- IGHD_-_-IGHJ6 is a 3 M [7(+ 2)(+ 1):9*] ACM stIMfA* gene with a final esebssiwaagoT_Q 0.23 (0.226).

MIR4537 with respect to V1–3- D_-_-J6 is a 3 episode, 7 initial and final SEB gene that begins with an anisotropic SEB. MIR4537 with respect to V1–3-D_-_-J6 is a 3 A (7) gene with a final esebssiwaagoT_Q 0.25 (0.251).

MIR4507/MIR4538 with respect to V1–3-D_-_-J6 is a 3 episode, 7 initial and final SEB gene that begins with a mesotropic SEB. MIR4507/MIR4538 with respect to V1–3-D_-_-J6 is a 3 M (7) gene with a final esebssiwaagoT_Q of 0.26 (0.260).

MIR4539 with respect to V1–3-D_-_-J6 is a 3 episode, 7 initial and final SEB gene that begins with a mesotropic SEB. MIR4539 with respect to V1–3-D_-_-J6 is a 3 M (7) gene with a final esebssiwaagoT_Q of 0.27 (0.268).

IGHD with respect to V1–3- D_-_-J6 is a 3 episode, 7 initial and final SEB gene that begins with an anisotropic SEB. IGHD with respect to V1–3-D_-_-J6 is a 3 A (7) gene with a final esebssiwaagoT_Q of 0.20 (0.198) (Table 7, Additional file 8: Table S8).

Table 7 Chromosome 14 (−) strand chromatin Ig heavy chain locus recombination sequence for both alleles after IGHV1-3-IGHD_-_-IGHJ6

Ig heavy chain genes after iCSR, homologous recombination and further CSRs after IGHV1–3-IGHD_-_-IGHJ6

V1–3-D_-_-J6-IGHM is a 2 episode, 5 initial and final SEB gene that begins with a mesotropic SEB. V1–3-D_-_-J6-IGHM is a 2 M (5) gene with a final esebssiwaagoT_Q of 0.27 (0.275).

V1–3-D_-_-IGHD is a 2 episode, 5 initial and final SEB gene that begins with a mesotropic SEB. V1–3-D_-_-IGHD is a 2 M (5) gene with a final esebssiwaagoT_Q of 0.32 (0.320).

V1–3-D_-_-IGHG3 is a 2 episode, 5 initial and 4 final SEB gene that begins with a mesotropic SEB. V1–3-D_-_-IGHG3 has one instance of anisotropy converted-to-mesotropy. V1–3-D_-_ -IGHG3 is a 2 M [5(− 1): 4] ACM gene with a final esebssiwaagoT_Q of 0.31 (0.306).

V1–3-D_-_-IGHG4 is a 2 episode, 5 initial and 5* final SEB gene that begins with an anisotropic SEB. V1–3-D_-_-IGHG4 has one instance of anisotropy converted-to-mesotropy. V1–3-D_-_-IGHG4 is 2 A [5(+ 1): 5] ACM* gene with a final esebssiwaagoT_Q of 0.24 (0.237).

V1–3-D_-_-IGHA2 is a 2 episode, 5 initial and 7 final SEB gene that begins with a mesotropic SEB. V1–3-D_-_-IGHA2 has one instance of anisotropy converted-to-mesotropy. V1–3-D_-_ -IGHA2 s a 3 M [5(+ 2): 7] ACM gene with a final esebssiwaagoT_Q of 0.18 (0.185) (Table 7, Additional file 8: Table S8).

See Table 7 and Additional file 8: Table S8 for with respect to V1–3-D_-_-IGHM, with respect to V1–3-D_-_-IGHD, with respect to V1–3-D_-_-IGHG3, with respect to V1–3-D_-_-IGHG4, and with respect to V1–3-D_-_-IGHA2 genes.

Ig heavy chain genes before iCSR and initial allelic exclusion after IGHV3–23-IGHD_-_-IGHJ6

IGHV3–23- IGHD_-_-IGHJ6 is a 3 episode, 7 initial and final SEB gene that begins with a mesotropic SEB. IGHV3–23- IGHD_-_-IGHJ6 is a 3 M (7) gene with a final esebssiwaagoT_Q of 0.29 (0.285).

MIR4537 with respect to V3–23-D_-_-J6 is a 3 episode, 7 initial and final SEB gene that begins with an anisotropic SEB. MIR4537 with respect to V3–23-D_-_-J6 is a 3 A (7) gene with a final esebssiwaagoT_Q of 0.27 (0.272).

MIR4507/MIR4538 with respect to V3–23-D_-_-J6 is a 3 episode, 7 initial and final SEB gene that begins with a mesotropic SEB. MIR4507/MIR4538 with respect to V3–23-D_-_-J6 is a 3 M (7) gene with a final esebssiwaagoT_Q of 0.28 (0.277).

MIR4539 with respect to V3–23-D_-_-J6 is a 3 episode, 7 initial and 5 final SEB gene that begins with a mesotropic SEB. MIR4539 with respect to V3–23-D_-_-J6 has one instance of anisotropy converted-to-mesotropy. MIR4539 with respect to V3–23-D_-_-J6 is a 3 M [7(− 2): 5] ACM gene with a final esebssiwaagoT_Q of 0.23 (0.232).

IGHD with respect to V3–23-D_-_-J6 is a 3 episode, 7 initial and 8 final SEB gene that begins with an anisotropic SEB. IGHD with respect to V3–23-D_-_-J6 has one instance of anisotropy converted-to-mesotropy. IGHD with respect to V3–23-D_-_-J6 is a 3 A [7 (+ 1): 8] ACM 3 A (7) gene with a final esebssiwaagoT_Q of 0.22 (0.218) (Table 8, Additional file 9: Table S9).

Table 8 Chromosome 14 (−) strand chromatin Ig heavy chain locus recombination sequence for both alleles after IGHV3–23-IGHD_-_-IGHJ6

Ig heavy chain genes after iCSR and further CSRs following IGHV3–23-IGHD_-_-IGHJ6

V3–23-D_-_-J6--IGHM is a 3 episode, 7 initial and final SEB gene that begins with a mesotropic SEB. V3–23-D_-_-J6-IGHM is a 3 M (7) gene with a final esebssiwaagoT_Q of. 0.28 (0.277).

V3–23-D_-_-J6-IGHG1 is a 2 episode, 5 initial and final SEB gene that begins with a mesotropic SEB. V3–23-D_-_-J6-IGHG1 has one instance of anisotropy converted-to-mesotropy. V3–23-D_-_-J6-IGHG1 is 2 M (5) ACM gene with a final esebssiwaagoT_Q of 0.26 (0.256).

V3–23-D_-_-J6-IGHA1 is a 3 episode, 7 initial and final SEB gene that begins with a mesotropic SEB. V3–23-D_-_-J6-IGHA1 has one instance of anisotropy converted-to-mesotropy. V3–23-D_-_-J6-IGHA1 is 3 M (7) ACM gene with a final esebssiwaagoT_Q of 0.17 (0.171).

V3–23-D_-_-J6-IGHE is a 3 episode, 7 initial and final SEB gene that begins with a mesotropic SEB. V3–23-D_-_-J6-IGHE is a 3 M (7)) gene with a final esebssiwaagoT_Q of. 0.21 (0.205).

V3–23-D_-_-J6-IGHA2 is a 2 episode, 5 initial and 7 final SEB gene that begins with an anisotropic SEB. V3–23-D_-_-J6-IGHA2 s a 3 A (7) gene with a final esebssiwaagoT_Q of 0.21 (0.208) (Table 8, Additional file 9: Table S9).

See Table 8 and Additional file 9: Table S9 for with respect to V3–23-D_-_-J6-IGHM, with respect to V3–23-D_-_-J6-IGHG1, with respect to V3–23-D_-_-J6-IGHA1, with respect to V3–23-D_-_-J6-IGHE, and with respect to V3–23-D_-_-J6-IGHA2 genes.

Ig heavy chain genes before iCSR and initial allelic exclusion after IGHV5–51-IGHD_-_-IGHJ6

IGHV5–51-IGHD_-_-IGHJ6 is a 2 episode, 5 initial and final SEB gene that begins with a mesotropic SEB. IGHV5–51-IGHD_-_-IGHJ6 is a 2 M (5) gene with a final esebssiwaagoT_Q of 0.23 (0.233).

MIR4537 with respect to V5–51-D_-_-J6 is a 3 episode, 7 initial and final SEB gene that begins with a mesotropic SEB. MIR4537 with respect to V5–51- D_-_-J6 is a 3 M (7) gene with a final esebssiwaagoT_Q of 0.15 (0.152).

MIR4507/MIR4538 with respect to V5–51-D_-_-J6 is a 3 episode, 7 initial and final SEB gene that begins with an anisotropic SEB. MIR4507/MIR4538 with respect to V5–51-D_-_-J6 is a 3 A (7) gene with a final esebssiwaagoT_Q of 0.15 (0.155).

MIR4539 with respect to V5–51-D_-_-J6 is a 3 episode, 7 initial and final SEB gene that begins with a mesotropic SEB. MIR4539 with respect to V5–51-D_-_-J6 is a 3 M (7) gene with a final esebssiwaagoT_Q of 0.16 (0.159).

IGHD with respect to V5–51-D_-_-J6 is a 3 episode, 7 initial and 9 final SEB gene that begins with an anisotropic SEB. IGHD with respect to V5–51-D_-_-J6 has one instance of anisotropy converted-to-mesotropy. IGHD with respect to V3–51-D_-_-J6 is a 3 A [7(+ 2): 9] ACM gene with a final esebssiwaagoT_Q of 0.17 (0.169) (Table 9, Additional file 10: Table S10).

Table 9 Chromosome 14 (−) strand chromatin Ig heavy chain locus recombination sequence for both alleles after IGHV5–51-IGHD_-_-IGHJ6

Ig heavy chain genes after iCSR and further CSRs following IGHV5–51-IGHD_-_-IGHJ6

V5–51-D_-_-J6-IGHM is a 2 episode, 5 initial and final SEB gene that begins with an anisotropic SEB. V5–51-D_-_-J6-IGHM is a 2 A (5) gene with a final esebssiwaagoT_Q of.0.16 (0.165).

V5–51-D_-_-J6-IGHG1 is a 2 episode, 5 initial and final SEB gene that begins with a mesotropic SEB. V5–51-D_-_-J6-IGHG1 has one instance of anisotropy converted-to-mesotropy. V5–51-D_-_-J6-IGHG1 is 2 M (5) ACM gene with a final esebssiwaagoT_Q of 0.15 (0.153).

V5–51-D_-_-J6-IGHA1 is a is a 2 episode, 5 initial and final SEB gene that begins with a mesotropic SEB. V5–51-D_-_-J6-IGHA1 is 2 M (5) gene with a final esebssiwaagoT_Q of 0.13 (0.134).

V5–51-D_-_-J6-IGHG4 is a is a 2 episode, 5 initial and final SEB gene that begins with a mesotropic SEB. V5–51-D_-_-J6-IGHG4 is a 2 M (5) gene with a final esebssiwaagoT_Q of 0.18 (0.184).

V5–51-D_-_-J6-IGHE is a 2 episode, 5 initial and 6* final SEB gene that begins with an anisotropic SEB. V5–51-D_-_-J6-IGHE has one instance of anisotropy converted-to-mesotropy. V5–51-D_-_-J6-IGHE is 2 A [5(+ 1): 6*] ACM* gene with a final esebssiwaagoT_Q of 0.15 (0.152).

V5–51-D_-_-J6-IGHA2 is a 2 episode, 5 initial and final SEB gene that begins with a mesotropic SEB. V5–51-D_-_-J_-J6-IGHA2 has one instance of indirect stIsotropy for anisotropy. V5–51-D_-_-J6-IGHA2 s a 2 M (5) stIMfA gene with a final esebssiwaagoT_Q of 0.15 (0.148) (Table 9, Additional file 10: Table S10).

See Table 9 and Additional file 10: Table S10 for with respect to V5–51-D_-_-J6-IGHM, with respect to V5–51-D_-_-J6-IGHG1, with respect to V5–51-D_-_-J6-IGHA1, with respect to V5–51-D_-_-J6-IGHG4, with respect to V5–51-D_-_-J6-IGHE, and with respect to V5–51-D_-_-J6-IGHA2 genes.

Discussion

The intracellular pressure required to establish a horizontal reading frame for recombination of joining and diversity genes in native germline arrangement is the basis for predictable gene rearrangement

Variability-to-diversity-to-joining gene recombination is close to perfect when it completes in the pressuromodulated state in vivo [18]. As true allelic exclusion of the non-classical pathway in every case is due to failure of Allele 2 (IGHD) homologous recombination and not a failure of VDJ, Allele 1 (IGHM) accounts for 50% of VDJs while Allele 2 (IGHD) accounts for the other 50% of VDJs in vivo. The frequencies of diversity (D), joining (J) and variability (V) gene distribution is known, that of IGHJ6 is 40% (esebssiwaagoT_Q: 0.097), that of IGHJ5 is 10% (esebssiwaagoT_Q: 0.235), that of IGHJ4 is 32% (esebssiwaagoT_Q: 0.110), that of IGHJ3 is ~ 8.5% (esebssiwaagoT_Q: 0.112), that of IGHJ2 is ~ 1.5% (esebssiwaagoT_Q: 0.114) and IGHJ1 accounts for ~ 8.5% (esebssiwaagoT_Q: 0.116).

Allele 1 (IGHM) gene recombination begins first, when B-cell intracellular pressure is in the supra-pressuromodulated gene expression range [4]. Only IGHJ5 that expresses at an esebssiwaagoT_Q of 0.235 units can be the 1^st step candidate gene for Allele 1, which only leaves 1 other step for Allele 1 J ↔ D recombination. Thus, IGHJ5 is a 1-step J_ gene and recipient of 10% of Allele 1 one-step D_-_ genes 10% of the time, while it is a 2-step stepping stone J_ gene for Allele 1 non-functional (nf) gene IGHD1–20 (nf) (esebssiwaagoT_Q: 0.406) the rest of the time, making IGHJ6 the recipient gene for 40% of the D_-_ genes involved in the 2-step of Allele 1 two-step.

Allele 2 (IGHD) gene recombination follows that of Allele 1, when B-cell intracellular pressure is in the infra-pressuromodulated range, IGHJ1 through IGHJ4 all express in the 0.110 to 0.116 esebssiwaagoT_Q units range; of these, the first J_ gene IGHJ1 and the third one IGHJ3 are each present at a frequency of ~ 8.5% and are Allele 2 1-step J_ genes. IGHJ2 is present at a frequency of ~ 1.5% and is also an Allele 2 1-step J_ gene. As IGHJ2 is present at the lowest frequency, this implies that it is the 2-step stepping stone J_ gene for Allele 2 non-functional genes IGHD4–11 (nf) (esebssiwaagoT_Q: 0.293) and IGHD5–18 (nf) (esebssiwaagoT_Q: 0.254) the rest of the time, making IGHJ2 the recipient of 32% of Allele 2 two-step D_-_ genes.

Germline functional diversity genes in their native configuration and destined to participate in 2-step Allele 1 (IGHM) recombination have esebssiwaagoT_Qs in the 0.342 to 0.295 units range, which is approximately 40% of D_-_ genes, while those germline functional diversity genes that participate in 1-step Allele 1 recombination have esebssiwaagoT_Qs in the 0.294 to 0.286 units range, which is the remaining 10% of D_-_ genes.

Germline functional diversity genes in their native configuration and destined to participate in 2-step Allele 2 (IGHD) recombination have esebssiwaagoT_Qs in the 0.276 to 0.233 units range, which is approximately 32% of D _-_ genes, while those germline functional diversity genes that participate in 1-step Allele 2 recombination have esebssiwaagoT_Q in the 0.218 to 0.172 units range, which is the remaining 18% of D_-_ genes.

Germline non-functional diversity gene IGHD7–27 that does not participate in D to J recombination has an esebssiwaagoT_Q of 0.165.

Therefore, the intracellular pressure required to establish a horizontal reading frame for efficient RAG1 and RAG2 recombinase activity [4] is the basis for predictable B-cell joining and diversity gene recombination in the pressuromodulated state in vivo, of which the gene esebssiwaagoT_Q is the measure as it is a property of the gene.

The 2-step and 1-step D to J recombination processes are mutually exclusive

The 2-step Allele 1 (IGHM) gene recombination process involves a more primed CD4R+ CD40LG T-cell-mediated CD40R B-cell CM polarization pressuromodulation effect as an uphill intracellular pressure of 0.41 esebssiwaagoT_Q units is required for IGHJ5 (esebssiwaagoT_Q: 0.235) to IGHD1–20 (nf) (esebssiwaagoT_Q: 0.406) gene recombination (J5 ⟶ D1–20 (nf)).

The 1-step Allele 1 recombination process involves a less primed CD4R+ CD40LG T-cell-mediated CD40R B-cell CM polarization pressuromodulation effect when an intracellular pressure of 0.36 units is achieved, which is sufficient for PRDM1 expression at 0.36 units and B-cell cyclic pressure oscillation, however Allele 1 two-step does not take place. Instead, 1-step Allele 1 recombination takes place as an intracellular pressure of 0.30 esebssiwaagoT_Q units is achieved when IGHJ5 (esebssiwaagoT_Q: 0.235) to IGHD2–1 (esebssiwaagoT_Q: 0.296) recombination takes place (J5 ⟶ D2–1). The 2-step Allele 1 (IGHD) gene recombination process can also take place as it requires a maximum intracellular pressure of 0.293 esebssiwaagoT_Q units to engage IGHD4–11 (nf) for recombination with IGHJ2 (esebssiwaagoT_Q: 0.114) (D4–11(nf) ⟶ J2).

Therefore, when the Allele 1 (IGHM) recombination process is a 2-step process [J → D (D → J)], then the Allele 2 (IGHD) recombination process is a 1-step process (D → J); and when the Allele 1 (IGHM) recombination process is a 1-step process (J → D), then the Allele 2 (IGHD) recombination process is a 2-step process [D → J (D → J)].

The above deductions are supported by the literature as D_-_ gene, IGHD4–17, must be the Allele 1 (IGHM) Step 2b of 2 recombination gene in which case the D_-_ gene, IGHD1–26, must be the Allele 2 (IGHD) Step 1 of 1 recombination gene [20].

Allele 1 (IGHM) 2-step recombination involves germline IGHJ5 and IGHD1–20 (nf) recombination (step 1), IGHD_-_ and IGHJ6 gene with respect to D1–20 (nf)-J5 recombination (step 2a) that results in IGHD_-_-IGHJ6 (step 2b) and then IGHV_-_ to IGHD_-_-IGHJ6 recombination that results in V_-_-DJ6

Step 1 of the Allele 1 two-step recombination process begins after PRDM1 expression at 0.36 esebssiwaagoT_Q units, when B-cell intracellular pressure decreases to just below the CD40 gene expression intracellular pressure of 0.26 esebssiwaagoT_Q units to around 0.24 units and RAG2 engages the upstream handle of IGHJ5 at an esebssiwaagoT_Q of 0.235 units. At around 0.24 units there is an increase in cell pressure back to around 0.26 units as the PRDM1 effect wanes, when the 1st maximum polarization period begins with increasing cell pressure to 0.41 units when RAG2 engages the downstream handle of IGHD1–20 (nf), which results in IGHD1–20 (nf)-IGHJ5 (Fig. 1).

Fig. 1

Allele 1 (IGHM) 2-step and 1-step Ig heavy chain locus gene rearrangement recombination steps superimposed on the pressuromodulation map of B-cell differentiation stages. The Allele 1 two-step recombination steps are IGHD1–20 (nf) and IGHJ5 recombination (step 1 of 2), IGHD_-_ genes with respect to D1–20(nf)-J5 (step 2a of 2) and IGHJ6 with respect to D1–20(nf)-J5 (step 2a of 2) recombination that results that results in IGHD_-_-IGHJ6 (step 2b of 2) through the final IGHV_-_ and IGHD_-_-IGHJ6 recombination that results in V_-_-DJ6. The Allele 1 one-step recombination step is IGHD_-_ and IGHJ5 recombination that results in IGHD_-_-IGHJ5 (step 1 of 1) through the final IGHV_-_ and IGHD_-_-J5 recombination that results in V_-_-DJ6. Note: Allele 1 locus rearrangement recombination steps complete to the point of the CM IgM+ B-cell before Allele 2 VDJ completes. †, upper esebssiwaagoT_Q units range, 0.41–0.36. Black, CD40 at maximum cell polarization potential. Dark blue, CD40 at half-refractory. Light blue, CD40 at full-refractory. Text boxes with complete borders, 2-step. Text boxes with dashed borders, 1-step. Large rectangular box with complete borders, extra-nodal secretory antibody phase

The step 2a of the process is downhill and begins after RAG2 engages IGHD3–10 with respect to D1–20 (nf)-J5, which is the upper limit D_-_ gene with respect to D1–20 (nf)-J5 with an esebssiwaagoT_Q of 0.402 units. Then, cell pressure decreases back down into the PRDM1 maximum expression range at 0.36 units, and thereafter, at a sufficient enough rate through the CD40 expression cell pressure of 0.26 units into peri-nadir at 0.15 plus minus 0.05 units, which defines four fifths of one limb of 1^st fully refractory period. In the downhill limb of the fully refractory period, RAG1 engages IGHD4–17 with respect to D1–20 (nf)-J5 at 0.15 (0.150) units, which is lower limit for D_-_ genes with respect to D1–20 (nf)-J5. The step 2a stage completes after RAG1 association with IGHJ6 with respect to D1–20-J5 at 0.101 units of the period nadir, when the two free ends of DNA come together to result in IGHD_-_-IGHJ6 (step 2b) (Fig. 1).

The IGHD_-_-IGHJ6 recombined gene esebssiwaagoT_Q range is 0.13 (0.133) to 0.30 (0.297) units and the IGHV_-_ gene esebssiwaagoT_Q range with respect to IGHD_-_-IGHJ6 is an unestablished lower limit to 0.41 (0.415) units. The VDJ step of the process completes between the 1^st fully refractory pre-nadir period and the uphill limb of 2^nd maximum polarization period. The lower limit of the IGHV_ gene esebssiwaagoT_Q range is unestablished as more variability genes need to be sampled (Fig. 1).

Allele 1 recombination is a 2-step process when a more primed CD4R+ T-cell is involved and the greater magnitude of the B-cell polarization pressuromodulation effect. This implies that during a more robust pressuromodulation effect:

(1) there is deceleration during the 2-step Allele 1 step 1 downhill limb around an intracellular pressure of 0.25 esebssiwaagoT_Q units because it is a function of PRDM1 expression that results in full expression of CD40 at 0.26 units, after which there is maximum B-cell polarization and acceleration in the opposite direction with an uphill increase in cell pressure back to 0.41 units, then down into the PRDM1 expression pressure of 0.36 units;

and (2) there is acceleration during the step 2a downhill limb through the CD40 expression intracellular pressure into the fully refractory period because it is a function of preceding maximum CD40 expression followed by PRDM1 expression in series that then results in non-expression of CD40 at 0.26 units, after which there is maintained acceleration downhill into the peri-nadir due to the PRDM1 C-MYC gene antagonism effect.

Allele 2 (IGHD) 1-step recombination step involves germline IGHD_-_ and IGHJ1, IGHJ2 or IGHJ3 recombination that results in IGHD_-_-IGHJ1 -J2 or -J3 (step1) and then IGHV_-_ to IGHD_-_-J1-J4, IGHD_-_-J2-J4 or IGHD_-_-J3-J4 recombination that results in V_-_-DJ6

Step 1 of the Allele 2 one-step recombination process begins during the downhill limb of the 1^st refractory period in between 0.22 (0.218) and 0.17 (0.172) esebssiwaagoT_Q units at which the IGHD_-_ genes are substrates for RAG. Step 1 completes to the point of IGHD_-_-IGHJ1, −J2 or -J3 during peri-nadir before the uphill limb in between 0.12 (0.116) and 0.11 (0.112) units at which the 1-step IGHJ_ genes, IGHJ1 (~ 8.5%), IGHJ2 (~ 1.5%) or IGHJ3 (~ 8.5%) are substrates for the same (Fig. 2).

Fig. 2

Allele 2 (IGHD) 1-step and 2-step Ig heavy chain locus gene rearrangement recombination steps superimposed on the pressuromodulation map of B-cell differentiation stages. The Allele 2 one-step recombination step is IGHD_-_ and IGHJ1, IGHJ2 or IGHJ3 recombination that results in IGHD_-_-IGHJ1 -J2 or -J3 (step 1 of 1) through the final IGHV_-_ and IGHD_-_-IGHJ1, IGHD_-_-IGHJ2 or IGHD_-_-IGHJ3 recombination that results in V_-_-DJ6. The Allele 2 two-step recombination steps are IGHD1–14 (nf) or IGHD5–18 (nf) and IGHJ2 recombination (step 1 of 2), the IGHD_-_ genes with respect to D1–14 (nf)-J2 or D5–18 (nf)-J2 (step 2a of 2) and the IGHJ4 with respect to D1–14 (nf)-J2 or D5–18 (nf)-J2 (step 2a of 2) recombination that results in IGHD_-_-IGHJ4 (step 2b of 2) through the final IGHV_-_ and IGHD_-_-IGHJ4 recombination that results in V_-_-DJ6. Note: Allele 2 Ig locus rearrangement recombination follows that of Allele 1 and always completes to the point of VDJ rearrangement in the marrow. In the classical pathway, homologous recombination or initial allelic exclusion followed by delayed Allele 2 iCSR (i.e. CM IgM+ IgD+) are the rule in the lymph node. In the non-classical pathway, Allele 2 locus recombination completes to the point of VDJ rearrangement (i.e CM IgM+ only). †, upper esebssiwaagoT_Q units range, 0.41–0.36. Black, CD40 at maximum cell polarization potential. Dark blue, CD40 at half-refractory. Light blue, CD40 at full-refractory. Text boxes with complete borders, 2-step. Text boxes with dashed borders, 1-step. Large rectangular box with complete borders, extra-nodal secretory antibody phase

The step 1 IGHD_-_-IGHJ1, −J2 or -J3 recombined gene esebssiwaagoT_Q range is 0.11 (0.114) to 0.35 (0.347) units and the IGHV_-_ gene with respect to IGHD_-_-IGHJ12, −J and -J3 esebssiwaagoT_Q range is an unestablished lower limit to 0.41 (0.415) units. The VDJ step of the Allele 2 one-step process completes during the uphill limb of the 1^st fully refractory period into the 2^nd maximum polarization period (Fig. 2).

The Allele 2 recombination process is a 1-step process during the more primed CD4R+ T-cell-mediated polarization effect and follows Allele 2 two-step after CD40 non-expression as Allele 1 one-step begins during the downhill limb of the 1^st fully refractory period.

Allele 2 (IGHD) 2-step recombination involves germline IGHJ2 and IGHD4–11 (nf) or IGHD5–18 (nf) recombination (step 1), IGHD_-_ and IGHJ4 with respect to D4–11(nf)-J2 and D5–18(nf)-J2 gene recombination (step 2a) that results in IGHD_-_-IGHJ4 (step 2b) and then IGHV_-_ to IGHD_-_-IGHJ4 recombination that results in V_-_-DJ6

Step 1 of the Allele 2 two-step recombination process begins during the second downhill limb at an intracellular pressure of 0.29 esebssiwaagoT_Q units with RAG association with either IGHD4–11 (nf) (esebssiwaagoT_Q: 0.293) or IGHD5–18 (nf) (esebssiwaagoT_Q: 0.254) followed by IGHJ2 association at 0.114 units during the 1^st peri-nadir, which results in IGHD4–11 (nf)-IGHJ2 or in IGHD5–18 (nf)-IGHJ2 (Fig. 2).

The step 2a of 2 of the process begins in the peri-nadir at 0.16 (0.156) esebssiwaagoT_Q units and continues units into the uphill limb of the 2^nd three-fourths maximum polarization period when intracellular pressure increases to 0.33 (0.332) units, which is the cell pressure range for IGHD_-_ genes with respect to D1–14 (nf)-J2 and D5–18 (nf)-J2. Then, IGHD_-_ with respect to D1–14 (nf)-J2 or D5–18 (nf)-J2, and IGHJ4 with respect to D1–14 (nf)-J2 or IGHJ4 with respect to D5–18 (nf)-J2 gene recombination follows in the 3^rd three-fourths full-refractory period when the B-cell pressure is in the 0.19 (0.193) to 0.17 (0.165) esebssiwaagoT_Q units range, and ends in IGHD_-_-IGHJ4 when step 2b is complete. There is no IGHD_-_ to IGHJ4 gene recombination during the first 3/8^ths refractory period in between the 2^nd three-fourths maximum polarization period and the 3^rd three-fourths full-refractory period, when cell division takes place (Fig. 2).

The 2-step Allele 2 VDJ step begins with the 3rd three-fourths maximum polarization period in the IGHD_-_-IGHJ4 recombined gene intracellular pressure range between 0.26 (0.258) and 0.35 (0.350) esebssiwaagoT_Q units, and completes in the IGHV_-_ gene with respect to IGHD_-_-IGHJ4 cell pressure range between an unestablished lower limit and the PRDM1 expression pressure of 0.36 units as the upper limit (Fig. 2) due to a less pressuromodulated B-cell.

Allele 2 recombination is a 2-step process when Allele 1 recombination is a 1-step process as a less primed CD4R+ T-cell is involved, which results in lesser grade pressuromodulation of the B-cell. This implies that during a less robust pressuromodulation effect: (1) there is ¾^ths of maximum polarization during the maximum polarization periods but sufficient enough for full PRDM1 expression at 0.36 esebssiwaagoT_Q units, and (2) there is ¾^ths of full refractoriness during the fully refractory periods during which there is only transient CD40 expression comparable to non-expression.

Allele 1 (IGHM) 1-step recombination step involves germline IGHD_-_ and IGHJ5 recombination that results in IGHD_-_-IGHJ5 (step1) and then IGHV_-_ and IGHD_-_-J5 recombination that results in V_-_-DJ6

The Allele 1 one-step recombination process begins during the downhill limb after the 1st three-fourths maximal polarization period in between 0.29 (0.294) and 0.29 (0.286) esebssiwaagoT_Q units range when the genes destined for Allele 1 one-step are horizontal, and completes to the point of IGHD_-_-IGHJ5 at 0.24 (0.235) esebssiwaagoT_Q units just as the 1^st refractory period begins at which the 1-step IGHJ_ gene, IGHJ5 expresses (Fig. 1).

The step 1 IGHD_-_-IGHJ5 recombined gene B-cell pressure range is 0.15 (0.149) to 0.25 (0.251) and the IGHV_-_ gene with respect to IGHD_-_-IGHJ5 esebssiwaagoT_Q range is the unestablished lower limit to 0.36 esebssiwaagoT_Q units. The 1-step Allele 1 VDJ begins in the peri-nadir and completes during the 2^nd three-fourths maximum polarization period (Fig. 1).

The Allele 1 recombination process is a 1-step process during the less primed CD4R+ T-cell-mediated B-cell polarization effect. The Allele 1 one-step process can begin after the 1^st three-fourths maximal polarization period during the downhill limb as the intracellular pressure decreases at a slower rate compared to the Allele 2 two-step process into the three-fourths refractory CD40 expression period.

Variability gene esebssiwaagoT _Qs can be constant or variable

The variability genes that were sampled from 5′ to 3′ include, IGHV4–61, IGHV4–59, IGHV5–51, IGHV3–48, IGHV4–28, IGHV3–23 and IGHV1–3. All of these except IGHV1–3 have constant esebssiwaagoT_Qs with respect to germline IGHD_-_ genes.

The esebssiwaagoT_Q for IGHV1–3 varies depending on the downstream location of IGHD_-_-IGHJ6 in Allele 1 two-step or IGHD_-_-IGHJ4, IGHJ5 and IGHJ6 in series in Allele 2 two-step for example since the split integration includes the intergene distances downstream of these genes with respect to IGHV1–3.

IGHV_-_ to IGHDJ_ recombination results in VDJ6 for both alleles

Variability gene to DJ_ gene recombination results in VDJ6 for both Allele 1 (IGHM) 1-step D_-_-J5 genes and for Allele 2 (IGHD) 1-step D_-_-J1, −J2, −J3 and 2-step D_-_-J4 genes.

After IGHV_-_ to IGHD_-_-IGHJ1 for example, the V_-_-D_-_-J1 becomes a V_-_-D_-_-J1-J6 gene as the VDJ promoters offer sufficient transcription factor-bound anchoring stability for RNA polymerase to be able to transcribe the entire V-to-J6 complex. The VDJ promoters however do not offer enough stability for transcription further downstream to immunoglobulin heavy chain M (IGHM), which requires internal consensus sequence recognition (iCSR) excision of intervening bases around the MIR genes for VDJ6-IGHM. Analogously, homologous recombination displacement removal of intervening bases results in VDJ6-IGHD, which transcribes in toto as one gene.

This observation forms the basis for determining the gene esebssiwaagoT_Q with respect to the correct VDJ, which is VDJ6.

CD4R+ T-cells are positively pressuromodulated

Antigen presenting cells (APC) scavenge endocytic antigens and re-present endocytic antigen fragments to the T-cell receptor (TCR) [21, 22] and other T-cell receptors [22]. Therefore, APC-mediated positive pressuromodulator antigen presentation to CD4R+ T-cells results in endocytic positive pressuromodulation of the CD4R+ T-cell.

More primed CD4R+ T-cells are subject to higher grades of positive pressuromodulation that results in maximal CD40LG expression and CD40LG R+ T-cell-mediated CD40R+ B-cell polarization effect that increases B-cell pressure to 0.41 esebssiwaagoT_Q units (Allele 1 two-step/Allele 2 one-step), while less primed T-cells are subject to lower grades of positive pressuromodulation, which only increases B-cell pressure into the PRDM1 expression range of 0.36 units (Allele 2 two-step/Allele 1 one-step).

Antigen presenting cell-dependent T-cell-mediated B-cell polarization is the primary mode of increasing B-cell pressure in the classical maturation pathway, where vaccines serve the purpose of boosting the response [23].

B-cells are subject to the effect of both positive and negative forms of antigen pressuromodulation

The effects of positive pressuromodulation out-weigh those of mixed or negative forms during B-cell maturation from the VDJ pro-B-cell stage to the consensus sequence recognition (CSR) isotype switching Evolved mature B-cell stage, as it is during oscillating positive pressuromodulation [4].

In the classical pathway the dominant form of pressuromodulation is T-cell-mediated B-cell polarization via CD40R, while in the non-classical pathway the dominant form is positive pressuromodulator antigen-mediated B-cell pressuromodulation such as toll-like receptor (TLR) [24]-mediated endocytic via high isoelectric point basic peptides such as profilin II [25] during acute infection for example.

The B-cell to plasma cell transformation takes place in the presence of an antigen load. The majority of antigens such as virus capsid peptide and microbe cell wall or membrane fragments [26] are positive pressuromodulators of cell surface receptors [1], which increase and maintain B-plasma cell pressure in the supra-pressuromodulated range (≥ 0.25 esebssiwaagoT_Q units) [2]. A minority of antigens such as phospholipases and proteases [27] are negative pressuromodulators of cell membranes [1], which decrease and maintain B-plasma cell pressure in the infra-pressuromodulated range (< 0.25 esebssiwaagoT_Q units) [2].

IGHV1–3 antibody heavy chain recombination sequence after IGHV1–3-IGHD_-_-IGHJ6 is iCSR followed by further CSRs for allele 1 (IGHM) and homologous recombination for allele 2 (IGHD)

For Allele 1 (IGHM), there is internal CSR (iCSR) [11] between the switch sequence region intergene bases of MIR4507 with respect to V1–3DJ6 (esebssiwaagoT_Q: 0.260) and the sequence intergene bases of MIR4539 with respect to V1–3DJ6 (esebssiwaagoT_Q: 0.268), which is the closest esebssiwaagoT_Q match between the candidate MIR_ genes of the IGHM switch region. The resultant recombined gene is V1–3-D_-_-J6-remaining MIR/MIRs-IGHM with an esebssiwaagoT_Q of 0.275 (Table 7), and the cell a CM IgM+ Mature naïve B-cell.

For Allele 2 (IGHD), there is homologous recombination displacement replacement of 442 intergene bases downstream of IGHJ6 of V1–3-D_-_-IGHJ6 (esebssiwaagoT_Q: 0.226) by 443 intergene bases upstream of IGHD with respect to V1–3-D_-_-IGHJ6 (esebssiwaagoT_Q: 0.198) [14] due to an esebssiwaagoT_Q match between the two genes, at minus 0.014 units for the former (IGHD) and plus 0.014 units for the latter (V1–2-DJ6), which meets the esebssiwaagoT_Q match criterion of ±0.015 units with reference to the gene as it is the point of convergence. The resultant gene is V1–3D_-_-J6-IGHD with an esebssiwaagoT_Q of 0.320 (Table 7).

For Allele 1 (IGHM), the downstream genes for further CSR to V1–3DJ6-IGHM (esebssiwaagoT_Q: 0.275) are IGHG3 (esebssiwaagoT_Q: 0.271) and IGHA2 (esebssiwaagoT_Q: 0.258), but not IGHG1, IGHA1, IGHG4/IGHG2 and IGHE. The esebssiwaagoT_Q match for IGHG3 with respect to V1–3DJ-IGHM is plus minus 0.002 units and that for IGHA2 is plus minus 0.0085 units. The former will be a primary IGHG3 switch-to-IGHM switch region CSR resulting in V1–3DJ6-IGHG3 with an esebssiwaagoT_Q of 0.306, and the latter will be a direct primary IGHA2 switch-to-IGHM switch region CSR resulting in V1–3DJ6-IGHA2 with an esebssiwaagoT_Q of 0.258 (Table 7).

After primary CSR, the CD4R+ T-cell polarized 1^st generation Evolved mature naïve nodal B-cell will express both V1–3DJ6-IGHG3 (esebssiwaagoT_Q: 0.306) and V1–3DJ6-IGHD (esebssiwaagoT_Q: 0.320) simultaneously when the B-cell intracellular pressure oscillates to 0.313 esebssiwaagoT_Q units as V1–3DJ6-IGHG3 (+ 0.007) and V1–3DJ6-IGHD (− 0.007) are within 0.014 units of each other. Likewise, an antigen stimulated extra-nodal B-plasma cell will secrete both IgG3 and IgD (see Fig. 1 or Fig. 2 periphery/tissue nidus secretory phase).

The only secondary CSR for a CM IgG3+/IgD+ 1^st generation Evolved mature naïve nodal B-cell is IGHG4 with respect to V1–3DJ6-IGHG3 (esebssiwaagoT_Q: 0.325) at plus minus 0.0095 units, which will result in V1–3DJ6-IGHG4 with an esebssiwaagoT_Q of 0.237 (Table 7).

The further CSR for the CM IgG4+ 2^nd generation nodal cell will be a tertiary CSR to IGHA2 with respect to V1–3DJ6-IGHG4 (esebssiwaagoT_Q of 0.226), where there is a match at plus minus 0.0055 units, which results in CM IgHA+ 3^rd generation nodal cell. After both direct primary CSR between IGHA2 and V1–3DJ6-IGHM and indirect tertiary CSR between IGHA2 and V1–3DJ6-IGHG4, the resultant gene is the same, V1–3DJ6-IGHA2 expressed at 0.185 units, one expressed by a 1^st generation cell and the other by a 3^rd generation cell.

IGHV3–23 antibody heavy chain recombination sequence after IGHV3–23-IGHD_-_-IGHJ_-J6 is iCSR followed by further CSRs for allele 1 (IGHM) and initial allelic exclusion for allele 2 (IGHD)

For Allele 1 (IGHM), there is internal CSR between the sequence intergene bases of MIR4537 with respect to V3–23DJ6 (esebssiwaagoT_Q: 0.272) and the intergene bases of MIR4539 with respect to V3–23DJ6 (esebssiwaagoT_Q: 0.277), which is the closest esebssiwaagoT_Q match and results in V3–23-D_-_-J6-remaining MIR/MIRs-IGHM with an esebssiwaagoT_Q of 0.277 (Table 8).

For Allele 2 (IGHD), homologous recombination does not take place between IGHD (esebssiwaagoT_Q: 0.208) and V3–23-D_-_-J6 (esebssiwaagoT_Q: 0.285) as there is no esebssiwaagoT_Q match (Table 8). Instead, delayed internal CSR will follow, which will result in Allele 2 V3–23-D_-_-J6-remaining MIR/MIRs-IGHM and in further CSRs analogous to Allele 1.

For both alleles, the primary CSR esebssiwaagoT_Q match genes for V3–23DJ6--IGHM (esebssiwaagoT_Q: 0.277) are IGHG1 (esebssiwaagoT_Q: 0.271) and IGHA2 (esebssiwaagoT_Q: 0.258), respectively. A primary direct IGHG1-to-IGHM CSR at plus minus 0.003 units results in V3–23DJ6-IGHG1 that expresses at an esebssiwaagoT_Q of 0.256, and a primary direct IGHA2-to-IGHM CSR at plus minus 0.0095 units results in V3–23DJ6-IGHA2 that expresses at an esebssiwaagoT_Q of 0.208 (Table 8).

The CM IgG1+/IgG1+ 1^st generation Evolved mature naïve B-cell secondary CSRs with IGHA1 with respect to V3–23DJ6-IGHG1 with an esebssiwaagoT_Q of 0.263 that is sufficiently horizontal at minus 0.0035 units in reference to V3–23DJ6-IGHG1, which is the only potential CSR for V3–23DJ6-IGHG1 with a downstream gene and results in V3-23DJ6-IGHA1 with an esebssiwaagoT_Q of 0.171 (Table 8).

For the population of isotype switching B-cells, there are two potential tertiary CSRs for a CM IgA1+/IgA1+ 2^nd generation-Evolved mature naïve B-cell with an esebssiwaagoT_Q of 0.171 units, one with IGHE with respect to V3–23DJ6-IGHA1 at 0.167 units (±0.002), and the other with IGHA2 with respect to V3–23DJ6-IGHA1 at 0.179 units (±0.004). The further quaternary CSR for CM IgE+/IgE+ 3^rd generation-Evolved mature naïve B-cell at 0.205 esebssiwaagoT_Q units is with IGHA2 with respect to V3–23DJ6-IGHE at an esebssiwaagoT_Q of 0.183 units (± 0.011) (Table 8).

The higher generation-Evolved mature naïve cells in reference to V3–23DJ6-IGHM are more somatically mutated B-cells to the point of B-cell to B-plasma cell transformation as they are the product of multiple CSRs [28], particularly when CSRing around the intracellular pressures at which the somatic hypermutation (SHM) cytidine deaminases (CDA) are maximally expressed and available [4].

IGHV5–51 antibody heavy chain recombination sequence after IGHV5–51-IGHD_-_-IGHJ_-J6 is iCSR followed by further CSRs for allele 1 (IGHM) and initial allelic exclusion for allele 2 (IGHD)

For Allele 1 (IGHM), there is internal CSR between the sequence intergene bases of MIR4537 with respect to V5–51DJ6 (esebssiwaagoT_Q: 0.152) and those of MIR4507/MIR4538 with respect to V5–51DJ6 (esebssiwaagoT_Q: 0.155), which is the closest esebssiwaagoT_Q match and results in V5–51-D_-_-J6-remaining MIR/MIRs-IGHM with an esebssiwaagoT_Q of 0.165 (Table 9).

For Allele 2 (IGHD), homologous recombination does not take place between IGHD (esebssiwaagoT_Q: 0.169) and V5–51-D_-_-J6 (esebssiwaagoT_Q: 0.233), internal CSR takes place on both alleles (Table 9).

There are four potential primary CSR esebssiwaagoT_Q match genes for V5–51DJ6-IGHM (esebssiwaagoT_Q: 0.165), IGHG1 that is horizontal at 0.160 units (±0.0025), IGHA1 that is horizontal at 0.180 units (±0.0075), IGHG4 that is horizontal at 0.189 units (±0.012), and IGHA2 that is horizontal at 0.175 units (±0.005), where the choice of further CSR depends on the B-cell pressure as it oscillates with cyclic periodicity in context of the local milieu. Primary CSR with V5–51DJ6--IGHM will result in either V5–51DJ6-IGHG1 (esebssiwaagoT_Q: 0.153), V5–51DJ6-IGHA1 (esebssiwaagoT_Q: 0.134), V5–51DJ6-IGHG4 (esebssiwaagoT_Q: 0.184) or V5–51DJ6-IGHA2 (esebssiwaagoT_Q: 0.148), and in a 1^st generation evolved B-cell (Table 9).

There are also four potential downstream secondary CSR esebssiwaagoT_Q match genes for V5–51DJ6-IGHG1 (esebssiwaagoT_Q: 0.153), IGHA1 at 0.166 units (±0.0065), IGHG4 at 0.137 units (±0.008), IGHE at 0.145 units (±0.004), and IGHA2 at 0.157 units (±0.002). Secondary CSR with V5–51DJ6-IGHG1 will result in the recombined genes as before expressing at the their respective esebssiwaagoT_Qs, in either V5–51DJ6-IGHA1, V5–51DJ6-IGHG4, or V5–51DJ6-IGHA2 and in addition, V5–51DJ6-IGHE that expresses at 0.152 units, and in a 2^nd generation evolved B-cell (Table 9).

There are three potential downstream tertiary CSR esebssiwaagoT_Q match genes for V5–51DJ6-IGHA1 (esebssiwaagoT_Q: 0.134), IGHG4 at 0.106 units (±0.014), IGHE at 0.153 units (±0.0095), and IGHA2 at 0.163 units (±0.00145). Tertiary CSR with V5–51DJ6-IGHA1 will result in the recombined genes as after the prior isotype switch, in either V5–51DJ6-IGHG4, V5–51DJ6-IGHE or V5–51DJ6-IGHA2, and in a 3^rd generation evolved B-cell (Table 9). Thus, tertiary CSRed 3^rd generation evolved B-cell plasma cells will express and secrete antibody that is somatically hypermutated, for example IgE that is the product of three sequential CSRs (V5–51DJ6-IGHM ⟶ V5–51DJ6-IGHA1 ⟶ V5–51DJ6-IGHG1 ⟶ V5–51DJ6-IGHE) [29].

There is no potential for a further quaternary CSR from V5–51DJ6-IGHG4 (esebssiwaagoT_Q: 0.184), as there is no match with downstream genes, IGHE has an esebssiwaagoT_Q of 0.146, while IGHA2 has an esebssiwaagoT_Q of 0.153, both being outside of the match range, IGHE at plus minus 0.019 units and IGHA2 at plus minus 0.0155 units when neither DNA segment is horizontal enough for stable CSR by cytidine deaminases [4] (Table 9).

There is the potential for a quaternary CSR between IGHA2 with respect to V5–51DJ6-IGHE (esebssiwaagoT_Q: 0.129) and upstream V5–51DJ6-IGHE (esebssiwaagoT_Q: 0.152) as its plus minus 0.0115 units at the point of gene esebssiwaagoT_Q convergence (Table 9),

During the secretory phase, B-plasma cells with recombined V5–51 antibody heavy chain genes will express the V5–51DJ6-IGH_ antibody gene in response to negative pressuromodulation antigens of the cell membrane such as lipases and proteases.

Clinical correlation with allergen-induced immunogenicity

Allergic disease states such as rhinitis, asthma and venom sensitivity are subsets of IgE-mediated Type I hypersensitivity, the former two caused by mucosal exposure to plant pollen (i.e. Amb a series) and arthropod excreta (Der p) for example [27, 30], and the latter one caused by intravascular exposure to hymenoptera venoms (bee, Api m; jacket, Ves g; wasp, Pol a) [31].

Pollen and mite excreta are contaminated with endotoxins such as bacterial lipopolysaccharide (LPS) [32] and fungal profilin II [24, 25] and proteases [33]; while, honey bee venom apitoxin contains gland melittin (Api m III) [34], mast cell degranulating peptide 401 [35] and enzymes hyaluronidase (Api m II), phospholipase A2 (Api m 1), acid phosphatase (Api m IV) and dipeptidyl peptidase 4 (Api m V) [31, 36, 37].

In the classical pathway, antigen presenting cells (APC) scavenge and endocytose agglomerated nanoparticulates such as pollen-coated with lipopolysaccharide [38, 39] and melittin peptide lysed-cell membrane complexes [34] and re-present previously encountered endocytic positive pressuromodulator antigen fragments to CD4R+ T-cells for subsequent T-cell--mediated B-cell polarization pressuromodulation; while, in the non-classical pathway, dissoluted monomeric forms of LPS and profilin II positively pressuromodulate B-cells via endocytosis [40], as does melittin [41] however in concentrated form it is cell lytic as it is a very basic peptide [42].

In atopic allergic disease, antigen-specific serum IgE antibody is present; it is a product of multiple sequential indirect CSRs at the intracellular pressures at which there is maximal expression of somatic hypermutation (SHM) enzymes [4], as is the case in atopic rhinitis [20, 43], asthma [44, 45] and venom sensitivity [46, 47].

In bee venom sensitization and asthma for example, over time there is a shift from somatically hypermutated specific IgE to specific IgG4 [45,46,47]. The IgG4 is less mutated than IgE which CSR equivalently, probably since the PRDM1-induced drop in B-cell pressure to between 0.10 and 0.12 units is transient during the peri-nadir when SHM enzyme APOBEC3H is expressed [4]. The basis for the shift from IgE to IgG4 is probably a shift in B-cell pressure from a higher pressure at around 0.144 esebssiwaagoT_Q units required for CSR to IGHE to a lower pressure at around 0.124 esebssiwaagoT_Q units required for CSR to IGHG4 (i.e. V5–51), due to the exposure of local lymph node B-cells to similar concentrations of negative antigen pressuromodulator enzymes such as phospholipase A2 during each successive sting, but to decreasing concentrations of positive antigen pressuromodulators (i.e. LPS) due to increasing endocytic efficiency of scavenging cells.

Conclusions

In this study, the recently developed gene esebssiwaagoT_Q-based B-cell maturation stage gene overexpression pressuromodulation map [4] has been utilized as a template to stimulate B-cell immunoglobulin locus recombination events that take place in the pressuromodulated state in vivo. Germline joining-to-diversity gene rearrangements have been performed with respect to the germline followed by variability-to-diversityjoining gene recombinations through further consensus sequence recognition (CSR) isotype switching recombinations with respect to their recombined position.

Based on the findings of this study the following inferences can be made: (1) the esebssiwaagoT_Q of a joining (J_) and diversity (D_-_) gene in its native germline configuration is the basis for predictable subsequent gene rearrangement; (2) D_-_ to J_ gene recombination events are bi-allelic and mutually exclusive; (3) the entire process from beginning to end depends on the grade of the pressuromodulation effect, and as per the classical pathway it is an antigen presenting cell (APC)-dependent CD4R+ T-cell-mediated B-cell polarization process; (4) CD4R+ T-cells are positively pressuromodulated, while B-cells are subject to the effect of both positive and negative forms of antigen pressuromodulation; and (5) the B-cell to plasma cell transformation and the extra-nodal periphery/tissue nidus phase take place in the presence of antigen load and either positive or negative pressuromodulation of the cell to its recombined antibody gene expression intracellular pressure.

B-cell gene recombination rearrangement events can be predicted with a reasonable degree of certainty. It is envisioned that further esebssiwaagoT_Q-based study of the remaining B-cell variability gene recombinations isotype switching events will further our understanding of pressuromodulated basis for antigen selection including the evolutionary underpinnings of.