Lymphocyte subsets in the peripheral blood are disturbed in systemic sclerosis patients and can be changed by immunosuppressive medication

Systemic sclerosis (SSc) is a severe chronic disease with a broad spectrum of clinical manifestations. SSc displays disturbed lymphocyte homeostasis. Immunosuppressive medications targeting T or B cells can improve disease manifestations. SSc clinical manifestations and immunosuppressive medication in itself can cause changes in lymphocyte subsets. The aim of this study was to investigate peripheral lymphocyte homeostasis in SSc with regards to the immunosuppression and to major organ involvement. 44 SSc patients and 19 healthy donors (HD) were included. Immunophenotyping of peripheral whole blood by fluorescence-activated cell sorting was performed. Cytokine secretions of stimulated B cell cultures were measured. SSc patients without immunosuppression compared to HD displayed lower γδ T cells, lower T helper cells (CD3+/CD4+), lower transitional B cells (CD19+/CD38++/CD10+/IgD+), lower pre-switched memory B cells (CD19+/CD27+/IgD+), and lower post-switched memory B cells (CD19+/CD27+/IgD−). There was no difference in the cytokine production of whole B cell cultures between SSc and HD. Within the SSc cohort, mycophenolate intake was associated with lower T helper cells and lower NK cells (CD56+/CD3−). The described differences in peripheral lymphocyte subsets between SSc and HD generate further insight in SSc pathogenesis. Lymphocyte changes under effective immunosuppression indicate how lymphocyte homeostasis in SSc might be restored. Supplementary Information The online version contains supplementary material available at 10.1007/s00296-021-05034-8.

the T cell compartment, lymphocytes expressing the γδ T cell receptor (γδ T cells) are widely investigated in autoimmune diseases and are disturbed in SSc [7]. γδ T cells participate in innate and acquired immune functions: As part of the first line of defense, they expand in the initial phase of infections. They produce interleukin (IL)-17 and thereby attract neutrophils [8][9][10]. Also, γδ T cells support B cell antibody class switching and dendritic cell maturation [11][12][13]. In the later immune response, γδ T cells downregulate activated macrophages and other T cells and promote tissue repair [11,14,15].
Within the B cell compartment, memory B cells are of special interest in autoimmune diseases. Memory B cells in the peripheral blood are mostly characterized by cluster of differentiation (CD) 27-expression. They can be categorized in pre-and post-switched memory B cells according to their immunoglobulin (Ig) D-expression (CD19 + / CD27 + /IgD + and CD19 + /CD27 + /IgD − , respectively) and show hypermutation in the immunoglobulin variableregion genes [16,17]. Double negative (DN) B cells (CD19 + /CD27 − /IgD − ), although lacking CD27-expression, show somatic hypermutation of the B cell receptor and are, therefore, attributed to the memory compartment [18]. DN B cells play a pathogenic role in autoimmune diseases, as they are enhanced and activated in systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) [18][19][20].
Both, the T cell and the B cell compartment are targeted effectively by immunosuppressive medications in SSc. However, data on immunosuppressive treatment in SSc is limited. The European League against Rheumatism (EULAR) recommendations for the treatment of SSc [21] include methotrexate in early disease stages [22]. Cyclophosphamide [23], rituximab [24,25], and mycophenolate [26] showed efficacy in SSc interstitial lung disease. Autologous hematopoietic stem cell transplantation (aHSCT) has shown superiority compared to cyclophosphamide in three randomized controlled trials (ASSIST [27], ASTIS [28] and SCOT [29]).
It is known that immunosuppressive medication changes lymphocyte subsets in SSc [30,31]. Conversely, alterations in lymphocyte subsets influence effectiveness and adverse event rates of immunosuppressive medication [32]. Furthermore, disease stage influences the lymphocyte composition in SSc [33]. Therefore, the mode of immunosuppressive medication and major organ involvement has to be taken into account, when lymphocytes of SSc patients are analyzed.
The aim of the present study was to characterize differences in lymphocyte subsets and B cell function of SSc patients compared to healthy controls taking the immunosuppressive medication and major organ involvement into consideration. 44 patients fulfilling the American College of Rheumatology (ACR)/EULAR criteria [34] for SSc and 19 healthy donors (HD) were included between March 2015 and September 2018 in this study. Clinical data was collected in electronic files (EMIL by itc-ms.de, Marburg, Germany and SAP SE, Walldorf, Germany). SSc patients (whole cohort = SSc total ) were grouped (a) according to their immunosuppressive (immunomodulatory) medication at the time of blood collection (SSc patients without immunosuppressive medication (SSc noIS ), intake of methotrexate, azathioprine, hydroxychloroquine, or mycophenolate (SSc +MMF )), (b) according to the extent of skin involvement (limited cutaneous form (lcSSc) or diffuse cutaneous form (dcSSc)), (c) according to SSc disease duration (under (or equal to) 5 years or over 5 years), and (d) according to presence of lung fibrosis (lung fibrosis on computed tomography present or not).

Differential blood count to calculate absolute lymphocyte numbers
25 µl of EDTA-anticoagulated whole blood were measured in a XN-550 automated hematology analyzer (Sysmex, Kobe, Japan) to determine the absolute lymphocyte numbers (i.e., lymphocytes per microliter whole blood). The lymphocyte number was multiplied with the respective cell percentages obtained in the FACS analyses to calculate absolute numbers of lymphocyte subsets.

Peripheral blood mononuclear cells (PBMCs) preparation and B cell enrichment
Ficoll-Paque Plus separation (GE Healthcare, Munich, Germany) was used according to the manufacturer's instructions to isolate PBMCs out of 15-20 ml peripheral blood from EDTA-tubes. PBMCs were stored in liquid nitrogen before further processing. Magnetic cell sorting (MACS) with CD19 monoclonal antibody-coupled microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) was performed. PBMCs were twice positively selected (i.e., two consecutive columns) for CD19 to achieve a purity of more than 87% of B cells for the B cell cultures.

Statistical analysis
Shapiro-Wilk tests were used to test for normal distribution. As normal distribution was mostly absent, medians with interquartile ranges (IQR) were shown. For continuous variables Wilcoxon signed-rank tests were performed to  44 SSc patients participated in this study. 36 patients (81.8%) were female, mean age was 58.1 years (range 22-82 years). Mean disease duration before blood collection was 9.  Table 1). Each immunosuppressive medication was taken at least for 3 months before blood collection. 19 healthy persons (HD) donated blood to serve as comparison group, 13 of them (68.4%) were female and their mean age was 49.7 years (range 23-79 years).

Altered memory B cells, transitional B cells, T helper cells, and γδ T cells in SSc compared to HD
To exclude the influence of immunosuppressive medication on lymphocyte subsets, SSc patients without immunosuppressive medication (SSc noIS , n = 17) were compared to HD. Within the B cell compartment SSc patients had lower pre-switched memory B cell numbers compared to HD  Table 2 shows the lymphocyte numbers (and the lymphocyte percentages obtained from FACS to calculate lymphocyte numbers). Figure 2 shows significant differences.

Disease characteristics do not alter lymphocyte subsets in SSc
To analyze the effect of skin involvement on lymphocytes, lcSSc vs dcSSc was compared. Differences in the numbers of the lymphocyte subsets were not detected. Also no differences were seen when SSc patients with lung fibrosis were compared to patients without lung fibrosis. Patients with a long-lasting disease (over 5 years) did not have differences in their lymphocyte subsets comparted to patients with shorter disease duration.

Mycophenolate intake correlates with lower T helper cells and NK cells in SSc
To analyze the effect of mycophenolate on lymphocyte subsets SSc patients without any immunosuppressive medication (SSc noIS = 17) were compared to SSc patients receiving mycophenolate (SSc +MMF , n = 12  (Fig. 3). Disease characteristics might  influence the prescription of mycophenolate. In our cohort no differences in the prevalence of mycophenolate intake were seen between lsSSc vs dcSSc (P = 0.139) and between disease duration under (or equal to) 5 years vs over 5 years (P = 0.102). Patients with lung fibrosis took more often mycophenolate (11/27) compared to patients without lung fibrosis (1/16, P = 0.015). No differences in the lymphocyte subsets were seen in SSc patients taking methotrexate or hydroxychloroquine compared to SSc noIS .

Comparison of cytokine productions in B cell cultures between HD and SSc
Cytokine concentrations in CpG-stimulated B cell cultures were detectable for IL-6, IL-10, IL-1β, tumor necrosis factor (TNF)-α and IL12/23(p40). Cytokine secretions were inducible upon CpG stimulation (Supplementary Figure S1). No differences in the median cytokine productions were seen comparing B cell cultures of SSc noIS patients vs HD (IL-6:  Figure S2). Also no differences in the cytokine secretion in B cell cultures could be detected when immunosuppressive medication, extent of skin involvement, disease duration, or lung fibrosis were regarded for analysis.

Discussion
In this study, we characterized the lymphocyte subsets of 44 SSc patients, with respect to their immunosuppressive medication and to major organ involvement in comparison to healthy controls. The reduction of γδ T cells, reduction of transitional B cells, reduction of pre-switched memory B cells and reduction of IgA + and IgG + post-switched memory B cells can be attributed to SSc per se, whereas the reduction of T helper cells and NK cells might be explained by mycophenolate intake. Within the T cell compartment reduced γδ T cells in PBMCs of SSc (compared to HD) [33,35] and in the peripheral blood [36] have been reported. In contrast, γδ T cells are elevated in skin lesions of SSc [37]. The lowest γδ T cells in SSc patients were reported when Scl-70 antibodies were present, a diffuse cutaneous form was present and in patients with short disease duration (less than 3 years) [7]. It seems that lower γδ T cells are associated with more aggressive and early forms of SSc.
We investigated the effect of immunosuppressive medications on lymphocyte subsets in SSc patients and could show that patients taking mycophenolate therapy had significant lower T helper cell and NK cell numbers. Among our SSc patients, those taking mycophenolate were the largest group (n = 12 of 44) and they were balanced concerning their extent of skin involvement (7 were lcSSc and 5 were dcSSc). This might be one possible reason for the detection of significant changes. All other immunosuppressive medications such as methotrexate or hydroxychloroquine were taken by fewer patients. Our findings match the expected mode of action of mycophenolate as it inhibits lymphocyte proliferation [38]. Inhibition of T helper cells in SSc patients seems reasonable, as T helper cells are known to participate in the pathogenesis of SSc [39,40].
B cells in SSc exhibit an activated status, and contribute to SSc pathogenesis by production of profibrotic cytokines and autoantibodies [3]. So far there is little data available about the role of memory B cells in SSc pathogenesis. Other findings support our results, showing reduced total memory B cells (CD19 + /CD27 + ) in SSc though activated with overexpression of CD19 and CD95 [41]. We detected reduced memory B cells in SSc with a significant reduction of pre-switched memory B cells and post-switched memory B cells compared to HD. Examination of IgA + and IgG + subgroups on post-switched memory B cells revealed significant reductions in both populations. Thereby an IgG/IgA ratio of 1.4 on post-switched memory B cells in HD is not different in SSc. In a cohort of RA patients the IgG/IgA ratio of post-switched memory B cells was similar to our SSc patients and not changed by immunosuppressive medication [19]. The reduced numbers of IgA + and IgG + post-switched memory B cells indicate an impaired adaptive immune response in SSc, the class switch process of B cells, however, seems to be intact. Another explanation for reduced memory B cells in SSc patients could be an augmented apoptosis of memory B cells [41].
The disturbance of memory B cell homeostasis in SSc is different from diseases, thought to be mainly antibody driven, such as RA and SLE. Patients with these diseases exhibit elevated DN memory B cells [18, 19, whereas in SSc DN memory B cell counts are not different to HD. DN B cells of our SSc patients exhibited an IgG/IgA ratio of 2.0 and HD of 3.1. A similar high IgG/IgA ratio is reported in RA [19]. The low switch to IgA may be inherent in DN B cells and might reflect their putative abortive or inadequate differentiation niche in autoimmune diseases as well as in healthy individuals. In concordance with this finding, CD27 − memory B cells have a lower rate of somatic hypermutation compared to CD27 + memory B cells [42]. It has been postulated that impaired germinal center reactions or extra-follicular reactions might be the reason for a suboptimal DN memory B cell maturation [43]. IgG + DN memory B cells are increased in elderly people indicating they might be late memory or exhausted memory B cells [44].
Our results of unaltered cytokine productions in whole B cell cultures fit to the findings in the immunophenotyping, where no differences in whole B cell percentages and B cell numbers between SSc and HD were detected. To investigate the functional relevance of the alterations that were found by immunophenotyping in the pre-switched and post-switched memory B cell compartment, these cells have to be investigated separately in cell cultures. This could be addressed in future studies.
The described disturbances in B cell subsets may reflect pathogenic factors for SSc and might be one reason why targeting B cells is an effective treatment in SSc: Application of rituximab showed improvements of skin and lung manifestations of SSc [25, [45]. Rituximab causes a longlasting depletion of the memory B cell compartment [46]. B cell depletion also causes changes in the T cell compartment with reduction of memory T helper cells [47]. But none of our SSc patients received rituximab.
Limitations of our study are the single center design and the singular blood collection per patient with a lack of follow-up values.

Conclusion
SSc patients display a disturbed lymphocyte homeostasis, both, within the B cell compartment (with reduced preswitched memory B cells and reduced IgA and IgG expressing post-switched memory B cells) and within the T cell compartment (with reduced γδ T cells and T helper cells). These changes may reflect SSc pathogenesis and might offer future therapeutic options.
Funding Open Access funding enabled and organized by Projekt DEAL.
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