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Quantitation of lymphatic transport mechanism and barrier influences on lymph node-resident leukocyte access to lymph-borne macromolecules and drug delivery systems

Abstract

Lymph nodes (LNs) are tissues of the immune system that house leukocytes, making them targets of interest for a variety of therapeutic immunomodulation applications. However, achieving accumulation of a therapeutic in the LN does not guarantee equal access to all leukocyte subsets. LNs are structured to enable sampling of lymph draining from peripheral tissues in a highly spatiotemporally regulated fashion in order to facilitate optimal adaptive immune responses. This structure results in restricted nanoscale drug delivery carrier access to specific leukocyte targets within the LN parenchyma. Herein, a framework is presented to assess the manner in which lymph-derived macromolecules and particles are sampled in the LN to reveal new insights into how therapeutic strategies or drug delivery systems may be designed to improve access to dLN-resident leukocytes. This summary analysis of previous reports from our group assesses model nanoscale fluorescent tracer association with various leukocyte populations across relevant time periods post administration, studies the effects of bioactive molecule NO on access of lymph-borne solutes to dLN leukocytes, and illustrates the benefits to leukocyte access afforded by lymphatic-targeted multistage drug delivery systems. Results reveal trends consistent with the consensus view of how lymph is sampled by LN leukocytes resulting from tissue structural barriers that regulate inter-LN transport and demonstrate how novel, engineered delivery systems may be designed to overcome these barriers to unlock the therapeutic potential of LN-resident cells as drug delivery targets.

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References

  1. 1.

    Gasteiger G, Ataide M, Kastenmüller W. Lymph node—an organ for T-cell activation and pathogen defense. Immunol Rev. 2016;271:200–20. https://doi.org/10.1111/imr.12399.

    CAS  Article  PubMed  Google Scholar 

  2. 2.

    Moussion C, Girard JP. Dendritic cells control lymphocyte entry to lymph nodes through high endothelial venules. Nature. 2011;479:542–6. https://doi.org/10.1038/nature10540.

    CAS  Article  PubMed  Google Scholar 

  3. 3.

    Schudel A, Francis DM, Thomas SN. Material design for lymph node drug delivery. Nat Rev Mater. 2019;4. https://doi.org/10.1038/s41578-019-0110-7.

  4. 4.

    Ryan GM, Kaminskas LM, Porter CJH. Nano-chemotherapeutics: maximising lymphatic drug exposure to improve the treatment of lymph-metastatic cancers. J Control Release. 2014;193:241–56. https://doi.org/10.1016/j.jconrel.2014.04.051.

    CAS  Article  PubMed  Google Scholar 

  5. 5.

    Irvine DJ, Dane EL. Enhancing cancer immunotherapy with nanomedicine. Nat Rev Immunol. 2020;20:321–34. https://doi.org/10.1038/s41577-019-0269-6.Enhancing.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Thomas SN, Rohner NA, Edwards EE. Implications of lymphatic transport to lymph nodes in immunity and immunotherapy. Annu Rev Biomed Eng. 2016;18:207–33. https://doi.org/10.1146/annurev-bioeng-101515-014413.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Zhang YN, Poon W, Sefton E, Chan WCW. Suppressing subcapsular sinus macrophages enhances transport of nanovaccines to lymph node follicles for robust humoral immunity. ACS Nano. 2020;14:9478–90. https://doi.org/10.1021/acsnano.0c02240.

    CAS  Article  PubMed  Google Scholar 

  8. 8.

    Gerner MY, Torabi-Parizi P, Germain RN. Strategically localized dendritic cells promote rapid T cell responses to lymph-borne particulate antigens. Immunity. 2015;42:172–85. https://doi.org/10.1016/j.immuni.2014.12.024.

    CAS  Article  PubMed  Google Scholar 

  9. 9.

    Swartz MA. The physiology of the lymphatic system. Adv Drug Deliv Rev. 2001;50:3–20. https://doi.org/10.1016/S0169-409X(01)00150-8.

    CAS  Article  PubMed  Google Scholar 

  10. 10.

    Gause KT, Wheatley AK, Cui J, Yan Y, Kent SJ, Caruso F. Immunological principles guiding the rational design of particles for vaccine delivery. ACS Nano. 2017;11:54–68. https://doi.org/10.1021/acsnano.6b07343.

    CAS  Article  PubMed  Google Scholar 

  11. 11.

    Moynihan KD, Holden RL, Mehta NK, Wang C, Karver MR, Dinter J, et al. Enhancement of peptide vaccine immunogenicity by increasing lymphatic drainage and boosting serum stability. Cancer Immunol Res. 2018;6:1025–38. https://doi.org/10.1158/2326-6066.CIR-17-0607.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  12. 12.

    Reddy ST, Van Der Vlies AJ, Simeoni E, Angeli V, Randolph GJ, O’Neil CP, et al. Exploiting lymphatic transport and complement activation in nanoparticle vaccines. Nat Biotechnol. 2007;25:1159–64. https://doi.org/10.1038/nbt1332.

    CAS  Article  PubMed  Google Scholar 

  13. 13.

    Grant SM, Lou M, Yao L, Germain RN, Radtke AJ. The lymph node at a glance—how spatial organization optimizes the immune response. J Cell Sci. 2020;133:1–7. https://doi.org/10.1242/jcs.241828.

    CAS  Article  Google Scholar 

  14. 14.

    O’Melia MJ, Rohner NA, Manspeaker MP, Francis DM, Kissick HT, Thomas SN. Quality of CD8+ T cell immunity evoked in lymph nodes is compartmentalized by route of antigen transport and functional in tumor context. Sci Adv. 2020;6:1–17. https://doi.org/10.1126/sciadv.abd7134.

    CAS  Article  Google Scholar 

  15. 15.

    Sestito LF, Thomas SN. Lymph-directed nitric oxide increases immune cell access to lymph-borne nanoscale solutes. Biomaterials. 2020;265: 120411. https://doi.org/10.1016/j.biomaterials.2020.120411.

    CAS  Article  PubMed  Google Scholar 

  16. 16.

    Schudel A, Chapman AP, Yau MK, Higginson CJ, Francis DM, Manspeaker MP, et al. Programmable multistage drug delivery to lymph nodes. Nat Nanotechnol. 2020;15. https://doi.org/10.1038/s41565-020-0679-4.

  17. 17.

    Thomas SN, Vokali E, Lund AW, Hubbell JA, Swartz MA. Targeting the tumor-draining lymph node with adjuvanted nanoparticles reshapes the anti-tumor immune response. Biomaterials. 2014;35:814–24. https://doi.org/10.1016/j.biomaterials.2013.10.003.

    CAS  Article  PubMed  Google Scholar 

  18. 18.

    Schudel A, Sestito LF, Thomas SN. Winner of the society for biomaterials young investigator award for the annual meeting of the society for biomaterials, April 11–14, 2018, Atlanta, GA: S-nitrosated poly(propylene sulfide) nanoparticles for enhanced nitric oxide delivery to lymphatic tiss. J Biomed Mater Res - Part A. 2018;106:1463–75. https://doi.org/10.1002/jbm.a.36348.

    CAS  Article  Google Scholar 

  19. 19.

    Jalkanen S, Salmi M. Lymphatic endothelial cells of the lymph node. Nat Rev Immunol. 2020;20:566–78. https://doi.org/10.1038/s41577-020-0281-x.

    CAS  Article  PubMed  Google Scholar 

  20. 20.

    Moran I, Grootveld AK, Nguyen A, Phan TG. Subcapsular sinus macrophages: the seat of innate and adaptive memory in murine lymph nodes. Trends Immunol. 2019;40:35–48. https://doi.org/10.1016/j.it.2018.11.004.

    CAS  Article  PubMed  Google Scholar 

  21. 21.

    Cyster JG. B cell follicles and antigen encounters of the third kind. Nat Immunol. 2010;11:989–96. https://doi.org/10.1038/ni.1946.

    CAS  Article  PubMed  Google Scholar 

  22. 22.

    Qi H, Kastenmüller W, Germain RN. Spatiotemporal Basis of Innate and Adaptive Immunity in Secondary Lymphoid Tissue. Annu Rev Cell Dev Biol. 2014;30:141–67. https://doi.org/10.1146/annurev-cellbio-100913-013254.

    CAS  Article  PubMed  Google Scholar 

  23. 23.

    Gretz JE, Norbury CC, Anderson AO, Proudfoot AEI, Shaw S. Lymph-borne chemokines and other low molecular weight molecules reach high endothelial venules via specialized conduits while a functional barrier limits access to the lymphocyte microenvironments in lymph node cortex. J Exp Med. 2000;192:1425–39. https://doi.org/10.1084/jem.192.10.1425.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Gerner M, Kastenmuller W, Ifrim I, Kabat J, Germain R. Histo-Cytometry: in situ multiplex cell phenotyping, quantification, and spatial analysis applied to dendritic cell subset micro-anatomy in lymph nodes. Immunity. 2012;37:364–76. https://doi.org/10.1016/j.immuni.2012.07.011.Histo-Cytometry.

  25. 25.

    Roozendaal R, Mempel TR, Pitcher LA, Gonzalez SF, Verschoor A, Mebius RE, et al. Conduits mediate transport of low-molecular-weight antigen to lymph node follicles. Immunity. 2009;30:264–76. https://doi.org/10.1016/j.immuni.2008.12.014.Conduits.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Sixt M, Kanazawa N, Selg M, Samson T, Roos G, Reinhardt DP, et al. The conduit system transports soluble antigens from the afferent lymph to resident dendritic cells in the T cell area of the lymph node. Immunity. 2005;22:19–29. https://doi.org/10.1016/j.immuni.2004.11.013.

    CAS  Article  PubMed  Google Scholar 

  27. 27.

    Rohner NA, Thomas SN. Melanoma growth effects on molecular clearance from tumors and biodistribution into systemic tissues versus draining lymph nodes. J Control Release. 2016;223:99–108. https://doi.org/10.1016/j.jconrel.2015.12.027.

    CAS  Article  PubMed  Google Scholar 

  28. 28.

    Schudel A, Chapman AP, Yau MK, Higginson CJ, Francis DM, Manspeaker MP, et al. Programmable multistage drug delivery to lymph nodes. Nat Nanotechnol. 2020;15:491–9. https://doi.org/10.1038/s41565-020-0679-4.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  29. 29.

    Ferrari SLP, Cribari-Neto F. Beta regression for modelling rates and proportions. J Appl Stat. 2004;31:799–815. https://doi.org/10.1080/0266476042000214501.

    Article  Google Scholar 

  30. 30.

    Schudel A, Sestito LF, Thomas SN. Nitric oxide delivery to lymphatic tissues. 2019;106:1463–75. https://doi.org/10.1002/jbm.a.36348.S-nitrosated.

  31. 31.

    Weiler M, Kassis T, Dixon JB. Sensitivity analysis of near-infrared functional lymphatic imaging. J Biomed Opt. 2012;17: 066019. https://doi.org/10.1117/1.jbo.17.6.066019.

    Article  PubMed  PubMed Central  Google Scholar 

  32. 32.

    Bohlen HG, Gasheva OY, Zawieja DC. Nitric oxide formation by lymphatic bulb and valves is a major regulatory component of lymphatic pumping. Am J Physiol - Hear Circ Physiol. 2011;301:1897–906. https://doi.org/10.1152/ajpheart.00260.2011.

    CAS  Article  Google Scholar 

  33. 33.

    Liao S, Cheng G, Conner DA, Huang Y, Kucherlapati RS, Munn LL, et al. Impaired lymphatic contraction associated with immunosuppression. Proc Natl Acad Sci USA. 2016;113:E5992. https://doi.org/10.1073/pnas.1614689113.

    CAS  Article  Google Scholar 

  34. 34.

    Durán WN, Beuve AV, Sánchez FA. Nitric oxide, S-Nitrosation, and endothelial permeability. IUBMB Life. 2013;65:819–26. https://doi.org/10.1002/iub.1204.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Thibeault S, Rautureau Y, Oubaha M, Faubert D, Wilkes BC, Delisle C, et al. S-nitrosylation of β-catenin by eNOS-derived NO promotes VEGF-induced endothelial cell permeability. Mol Cell. 2010;39:468–76. https://doi.org/10.1016/j.molcel.2010.07.013.

    CAS  Article  PubMed  Google Scholar 

  36. 36.

    Scallan JP, Hill MA, Davis MJ. Lymphatic vascular integrity is disrupted in type 2 diabetes due to impaired nitric oxide signalling. Cardiovasc Res. 2015;107:89–97. https://doi.org/10.1093/cvr/cvv117.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Lukacs-Kornek V, Malhotra D, Fletcher AL, Acton SE, Elpek KG, Tayalia P, et al. Regulated release of nitric oxide by nonhematopoietic stroma controls expansion of the activated T cell pool in lymph nodes. Nat Immunol. 2011;12:1096–104. https://doi.org/10.1038/ni.2112.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  38. 38.

    Kislukhin AA, Higginson CJ, Hong VP, Finn MG. Degradable conjugates from oxanorbornadiene reagents. J Am Chem Soc. 2012;134:6491–7. https://doi.org/10.1021/ja301491h.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  39. 39.

    Kislukhin AA, Higginson CJ, Finn MG. Aqueous-phase deactivation and intramolecular [2 + 2 + 2] cycloaddition of oxanorbornadiene esters. Org Lett. 2011;13:1832–5. https://doi.org/10.1021/ol103153f.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  40. 40.

    Bellomo A, Gentek R, Bajénoff M, Baratin M. Lymph node macrophages: scavengers, immune sentinels and trophic effectors. Cell Immunol. 2018;330:168–74. https://doi.org/10.1016/j.cellimm.2018.01.010.

    CAS  Article  PubMed  Google Scholar 

  41. 41.

    Savina A, Amigorena S. Phagocytosis and antigen presentation in dendritic cells. Immunol Rev. 2007;219:143–56. https://doi.org/10.1111/j.1600-065X.2007.00552.x.

    CAS  Article  PubMed  Google Scholar 

  42. 42.

    Martínez‐Riaño A, Bovolenta ER, Mendoza P, Oeste CL, Martín‐Bermejo MJ, Bovolenta P, et al. Antigen phagocytosis by B cells is required for a potent humoral response. EMBO Rep. 2018;19:1–15. https://doi.org/10.15252/embr.201846016.

  43. 43.

    Rohner NA, Thomas SN. Flexible macromolecule versus rigid particle retention in the injected skin and accumulation in draining lymph nodes are differentially influenced by hydrodynamic size. ACS Biomater Sci Eng. 2017;3:153–9. https://doi.org/10.1021/acsbiomaterials.6b00438.

    CAS  Article  PubMed  Google Scholar 

  44. 44.

    Sestito LF, Thomas SN. Biomaterials for modulating lymphatic function in immunoengineering. ACS Pharmacol Transl Sci. 2019;2:293–310. https://doi.org/10.1021/acsptsci.9b00047.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  45. 45.

    Kuai R, Ochyl LJ, Bahjat KS, Schwendeman A, Moon JJ. Designer vaccine nanodiscs for personalized cancer immunotherapy. Nat Mater. 2017;16:489–98. https://doi.org/10.1038/NMAT4822.

    CAS  Article  PubMed  Google Scholar 

  46. 46.

    Kim SY, Noh YW, Kang TH, Kim JE, Kim S, Um SH, et al. Synthetic vaccine nanoparticles target to lymph node triggering enhanced innate and adaptive antitumor immunity. Biomaterials. 2017;130:56–66. https://doi.org/10.1016/j.biomaterials.2017.03.034.

    CAS  Article  PubMed  Google Scholar 

  47. 47.

    Jeanbart L, Ballester M, De Titta A, Corthésy P, Romero P, Hubbell JA, et al. Enhancing efficacy of anticancer vaccines by targeted delivery to tumor-draining lymph nodes. Cancer Immunol Res. 2014;2:436–47. https://doi.org/10.1158/2326-6066.CIR-14-0019-T.

    CAS  Article  PubMed  Google Scholar 

  48. 48.

    Reddy ST, Rehor A, Schmoekel HG, Hubbell JA, Swartz MA. In vivo targeting of dendritic cells in lymph nodes with poly(propylene sulfide) nanoparticles. J Control Release. 2006;112:26–34. https://doi.org/10.1016/j.jconrel.2006.01.006.

    CAS  Article  PubMed  Google Scholar 

  49. 49.

    Azzi J, Yin Q, Uehara M, Ohori S, Abdi R. Targeted delivery of immunomodulators to lymph nodes. 2016;46:1247–62. https://doi.org/10.1002/jmri.25711.PET/MRI.

    Article  Google Scholar 

  50. 50.

    Mchugh MD, Park J, Uhrich R, Gao W, Horwitz DA, Fahmy TM. Biomaterials Paracrine co-delivery of TGF- b and IL-2 using CD4-targeted nanoparticles for induction and maintenance of regulatory T cells. Biomaterials. 2015;59:172–81. https://doi.org/10.1016/j.biomaterials.2015.04.003.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  51. 51.

    Zhang YN, Lazarovits J, Poon W, Ouyang B, Nguyen LNM, Kingston BR, et al. Nanoparticle size influences antigen retention and presentation in lymph node follicles for humoral immunity. Nano Lett. 2019;19:7226–35. https://doi.org/10.1021/acs.nanolett.9b02834.

    CAS  Article  PubMed  Google Scholar 

  52. 52.

    Cyster JG, Allen CDC. B Cell Responses: Cell interaction dynamics and decisions. Cell. 2019;177:524–40. https://doi.org/10.1016/j.cell.2019.03.016.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  53. 53.

    Tokatlian T, Read BJ, Jones CA, Kulp DW, Menis S, Chang JYH, et al. Innate immune recognition of glycans targets HIV nanoparticle immunogens to germinal centers. Science. 2019;363:649–54. https://doi.org/10.1126/science.aat9120.

  54. 54.

    Ke X, Howard GP, Tang H, Cheng B, Saung MT, Santos JL, et al. Physical and chemical profiles of nanoparticles for lymphatic targeting. Adv Drug Deliv Rev. 2019. https://doi.org/10.1016/j.addr.2019.09.005.

    Article  PubMed  Google Scholar 

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Acknowledgements

LFS and AS were American Heart Association Pre-doctoral Fellows. We thank Jared P. Beyersdorf for technical assistance.

Funding

This work was supported by National Institutes of Health (NIH) Grants R01CA207619 (SNT), R01CA247484 (SNT), U01CA214354 (SNT), T32GM008433 (MJO, NAR), and T32EB006343 (LFS) and the Curci Foundation (SNT). M.P.M. was supported by a National Science Foundation Graduate Research Fellowship.

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Correspondence to Susan N. Thomas.

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13346_2021_1015_MOESM1_ESM.pdf

Supplementary file1: Supplementary Fig. 1 Regression coefficients, datadistributions and regression fits of tracer frequency data sets. a) Linear regression coefficientsrepresenting the influence of increasing quantity of tracer within the dLN ontracer association with each marker-expressing cell subset. Coefficients arepresented as mean ± standard error in confidence interval format. * indicatessignificant difference from zero (* and *** indicate p < 0.05 and p <0.001, respectively). # indicates significant difference between coefficientsas determined by unpaired two-sample t test with Bonferroni correction, p <0.05. b) Histograms displaying distribution of the frequency of tracer+ signalin fractional form in each marker expressing cell subset. c) Q-Q plots fromlinear regression on each cell subset displaying deviation of standardizedlinear regression residuals from normality. d) Half normal plots from betaregression displaying adherence of standardized weighted beta regressionresiduals to confidence bands indicative of quality fit. (PDF 414 KB)

13346_2021_1015_MOESM2_ESM.pdf

Supplementary file2: Supplementary Fig. 2 Immune cell association with tracerafter in vitro co-incubation. a)Influence of co-incubated tracer amount on frequency of tracer association withvarious marker-expressing cells isolated from murine LNs incubated at 37C for 4h. Data are presented as mean ± standard error from n = 3 replicates. b) Betaregression coefficients quantifying the relationship between increasingincubated tracer quantity and tracer association with each cell type in vitro.*** indicates coefficient significantly different from zero, p < 0.001.Coefficients are presented as mean ± standard error in confidence intervalformat. (PDF 278 KB)

13346_2021_1015_MOESM3_ESM.pdf

Supplementary file3: Supplementary Fig. 3 Nitric oxide treatment influences onextent of tracer association with cells and tracer accumulation within thelymph node per tissue weight. Extentof tracer association with various lymph node leukocyte subtypes versus theirmeasured quantity within the dLN 72 h post i.d. co-injection with vehiclecontrols or NO donors. Data are presented as mean ± standard error from n = 12animals.[F0341][F0341]SPI:Please capture Supplementary information section accordingly. (PDF 67 KB)

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Archer, P.A., Sestito, L.F., Manspeaker, M.P. et al. Quantitation of lymphatic transport mechanism and barrier influences on lymph node-resident leukocyte access to lymph-borne macromolecules and drug delivery systems. Drug Deliv. and Transl. Res. 11, 2328–2343 (2021). https://doi.org/10.1007/s13346-021-01015-3

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Keywords

  • Lymph node
  • Lymphatic system
  • Transport barrier
  • Transport mechanism
  • Drug delivery