*Purpose: Kidney transplant (KTx) into CCR5 KO mice is an excellent model to study AMR immunobiology due to high alloantibody (alloAb) production, rapid allograft rejection, and pathology consistent with human AMR. A proposed mechanism for this heightened humoral response is impaired CD4⁺T_reg cell trafficking to the allograft. We have previously observed that adoptive cell therapy (ACT) with CXCR5⁺CD8⁺T cells mediates significant reduction of alloAb titer after KTx in CCR5 KO mice. We hypothesized that CXCR5⁺CD8⁺T cells are more potent regulators of alloAb production compared to CD4⁺T_regs.

*Methods: WT and KO mice (H-2^b) underwent allogeneic KTx (A/J; H-2^a). Recipients underwent ACT with 2*10⁶ CXCR5⁺CD8⁺T cells or CD25⁺CD4⁺Tregs. Cohorts of WT mice were treated with either anti-CD8 or anti-CD25 mAbs. AlloAb titer and immune cell subsets were analyzed by flow cytometry on postTx day 14.

*Results: CCR5 KO recipients have significantly higher alloAb titer (5951±630) compared to WT (1508±145; p<0.001). KO recipients have significantly fewer CXCR5⁺CD8⁺T cells (782±183 vs 2058±167 cells/mm³; p<0.0001) and graft-infiltrating CD4⁺T_reg cells (CD25⁺FoxP3⁺; 12.9±8.8 vs 56.2±8.8 cells/mm³; p=0.003) compared to WT recipients. KO compared to WT recipients have significantly greater quantity of splenic IL-21⁺CXCR5⁺PD1⁺CD4⁺T_FH cells (331±34 vs 36±44 cells/mm³) and germinal center (GC; GL7⁺Fas⁺B220⁺) B cells (5814±436 vs 671±342 cells/mm³; both p<0.0001). AlloAb titer in KO recipients is significantly reduced following ACT with alloprimed CXCR5⁺CD8⁺T cells (1259±436 vs 5951±404 in untreated controls; p<0.0001) but not following CD4⁺ACT (5688±436; p=ns). ACT with CXCR5⁺CD8⁺T cells reduced the quantity of splenic IL-21⁺T_FH cells (from 331±34 in untreated controls to 66±31 cells/mm³; p<0.0001) and GC B cells (from 5813±589 in untreated controls to 4087±537; p=0.002), while CD4⁺T_reg ACT had no impact (IL-21⁺T_FH: 241±36 cells and GC B cells: 6916±538/mm³; p=ns for both). Compared to untreated controls, CD8-depletion in WT recipients led to significant increase in alloAb titer (1508±338 vs 6078±436; p<0.001) and GC B cells (671±436 vs 7761±430; p<0.0001). CD4⁺T_reg depletion resulted in increased alloAb titer (3180±436; p=0.004) and GC B cell quantity (4911±429; p<0.0001), however, these increases were less pronounced than in the CD8-depleted group (p<0.001 for both).

*Conclusions: CCR5 KO KTx recipients are deficient in CXCR5⁺CD8⁺T cells and graft-infiltrating CD4⁺T_reg cells, which is associated with heightened humoral alloimmunity. This is rescued following ACT of CXCR5⁺CD8⁺ T cells but not CD4⁺Tregs, suggesting that Ab-suppressor CXCR5⁺CD8⁺ T cells are more potent regulators of antibody production than CD4⁺Tregs.

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