Antibody-suppressor CXCR5+CD8+ T cells preferentially mediate the cytotoxic killing of alloprimed germinal center B cells in an antigen-specific manner
Date
2022-05
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The Ohio State University
Abstract
Background – Antibody-mediated rejection (AMR) is a significant contributor to transplant rejection. Our prior work identified a novel subset of murine CXCR5+CD8+ T cells (CD8+ TAb-supp cells) that suppresses alloantibody production by killing IgG+ alloantibody-producing B cells, resulting in prolonged transplant survival. The purpose of this investigation was to determine the susceptibility of specific cellular subsets known to contribute to the development of humoral immunity to CD8+ TAb-supp cell-mediated cytotoxicity. As CXCR5 is a key molecule involved with lymphocyte trafficking to germinal centers (GCs), it was hypothesized that CD8+ TAb-supp cells mediate the cytotoxic killing of alloprimed (IgG+) GC B cells, resulting in suppressed alloantibody production.
Methods – C57BL/6 (wild-type, WT) and CD8 KO (both H-2b) mice were stimulated with FVB/N (H-2q) allogeneic lysate (2mg bulk protein). Alloprimed CXCR5+CD8+ T cells were isolated from splenocytes of C57BL/6 mice 7 days after allo-lysate stimulation using CD8 StemCell isolation and flow-sorting. Various B cell subsets (IgG+ StemCell isolation kits and/or flow sort of splenocytes), CD4+ T cells subsets (CD4+CD25+ StemCell isolation or CD4 StemCell isolation and flow sort), macrophages (peritoneal lavage), and dendritic cells (density gradient centrifugation and culture plate non-adherence of low-density splenocytes) were isolated from CD8 KO mice 7 days after allo-lysate stimulation. Cells isolated from CD8 KO mice were tested as targets in co-cultures with effector CD8+ T cell populations to evaluate their susceptibility to CXCR5+CD8+ T cell-mediated in vitro cytotoxicity. Cell purity was greater than 70% for all subsets.
Results – CXCR5+CD8+ T cells exhibited significantly higher in vitro cytotoxicity towards alloprimed GC B cells (IgG+GL-7+Fas+; 11.4±1.0%) than towards other antibody-producing B cell subsets, including alloprimed extrafollicular plasmablasts (IgG+CD138+; 0.9±0.3%) and plasma cells (IgG-CD138+; 1.6±0.5%; p<0.0001 for both). In addition, CXCR5+CD8+ T cells did not mediate cytotoxicity towards other alloprimed antigen presentation cells like dendritic cells (MHCII+CD11c+; 1.5±0.8%) and macrophages (F4/80+; 1.7±0.4%). No significant cytotoxicity was observed by CXCR5+CD8+ T cells towards CD4+ T follicular helper (CD4+ TFH) cells (CXCR5+CD4+; 1.2±0.3%) or CD4+ T regulatory (CD4+ Treg) cells (CD25+CD4+; 1.20±0.6%) as well. The CXCR5+CD8-mediated cytotoxic killing of GC B cells was allospecific and required CXCR5 expression as alloprimed CXCR5+CD8+ T cells mediated in vitro cytotoxicity towards alloprimed GC B cells (11.4±1.0%) whereas third party primed CXCR5+CD8+ T cells (1.1±0.2%)
and alloprimed CXCR5-CD8+ T cells did not (0.6±0.3%; p<0.0001 for both compared to alloprimed CXCR5+CD8+ T cells). No significant cytotoxicity was observed in the control coculture using naïve CD8+ T cells (1.2±0.3%). Finally, cytokine production by CD4+ TFH and CD4+ Treg cells was not altered in co-cultures with CXCR5+CD8+ T cells. The percent of CD4+ TFH cells that produce IFN-γ, IL-4, and IL-21 cytokines did not differ in cellular-co-cultures with (48±3.4%, 39±2.3%, and 57±1.3% respectively) or without (47±4.5%, 38±3.0%, and 55±1.7% respectively; p=NS for all) CXCR5+CD8+ T cells. The percent of CD4+ Treg cells that produce IL-10 remained similar in cellular co-cultures with (37±3.8%) or without (32±3.8%; p=NS) CXCR5+CD8+ T cells.
Conclusions –This data suggests that the mechanism for antibody reduction is through CD8+ TAbsupp cell-mediated cytotoxic killing of allogeneic germinal center B cells.
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Keywords
Transplant Immunology, B Cells, T Cells, Antibody-Mediated Rejection