Background Inhibition of IL-6/IL-6R signaling using anti-IL6R monoclonal antibody is effective in attenuating donor-specific antibody (DSA) responses to skin allografts in a mouse model. Here, we analyzed the splenic B-cell subpopulations involved in the primary humoral response and assessed the effects of anti-IL6R treatment.
Methods Splenic lymphocytes were obtained from C57BL/6 mice bearing a HLA.A2+ skin graft (SG) with or without anti-IL6R treatment at designated time points post-transplant. B-cell subsets were identified by phenotyping using multi-parameter flow cytometry. Serum anti-HLA.A2 IgM titers were qualified in an antibody binding assay.
Results In naÏve spleens B220+ cells accounted for 47.8±2.5% splenic lymphocytes, including subsets of CD5+ B-1a (17.01±6.2% of total B-cells), CD11b+ CD5–B-1b (7.7±2.1%), and B220+CD5–CD11b– conventional B-2 cells (90.6±3%). Dynamic changes in B-cell subset composition occurred 24-hour post-transplant which were featured by a 2-fold expansion of CD5+ B-1a cells (38.02±2.37%, p=0.004 vs. Day 0) in the spleens. These CD5+ B-1a cells expressed a phenotype of B220+sIgMhisIgD–CD5+CD11b+/-. Anti-IL6R significantly reduced the B-1a subset (18.75+7.7% at Day 1 post-transplant, p=0.014 vs. control Day 1). Suppression of B-1a cells was accompanied by reduced membrane-bound IL-6RΑ expression on the B cells. On the other hand, the CD11b–CD5–B220+ B-2 cells were reduced in relative population in sensitized spleens (57.7±0.97% vs. 91.4±2.1% in naÏve spleens, p= 1.27849E-05). Anti-IL6R was able to normalize the B-2 cell relative population in the spleens (86.2±6.7% vs. Day 0, p=0.243). The dynamic changes observed in splenic B-cell populations in anti-IL6R treated mice were accompanied with significant reduction of donor-specific IgM response to HLA.A2 (mMR16-1 vs. control: 12.7±2.3 MFI vs. 22.9+3.2 MFI at day 14, p=0.00022).
Conclusion Primary IgM response to HLA.A2 alloantigen is likely generated by B-1a cells dependent of IL-6. Suppression of B-1a cells by anti-IL6R is one of the postulated immunosuppressive mechanism(s) by which IL6R blockage by therapeutic antibody moderates alloimmunity in transplant rejection.
To cite this abstract in AMA style:Wu G, Chai N, Kim I, Klein A, Jordan S. Anti-IL6R Suppresses Primary IgM Response and CD5+ B-1a Cell Subset Stimulated by HLA.A2+ Skin Allografts [abstract]. Am J Transplant. 2013; 13 (suppl 5). https://atcmeetingabstracts.com/abstract/anti-il6r-suppresses-primary-igm-response-and-cd5-b-1a-cell-subset-stimulated-by-hla-a2-skin-allografts/. Accessed October 27, 2020.
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