*Purpose: Antibody-mediated lymphocyte depletion is used in solid organ and stem cell transplantation. However, T cell reconstitution after lymphoablation may lead to acute rejection and poor graft outcome. Using a mouse model of heart transplantation we previously showed that CD8 T cell recovery after murine anti-thymocyte globulin (mATG) depletion is dependent on B cells. The goal of this study was to investigate the mechanisms by which B cells mediate T cell reconstitution.

*Methods: Mouse model of heart transplantation, anti-thymocyte globulin depletion, Flow Cytometry, NanoString, cytokine neutralization

*Results: B cell depletion with anti-mouse CD20 mAb significantly diminished T cell reconstitution in B6 (H-2b) recipients of BALB/c (H-2d) heart allografts treated with mATG (0.25 mg i.p. on days 0, 4). We previously showed that cognate T/B cell interactions are dispensable for T cell recovery after mATG treatment suggesting the potential role for B cell derived cytokines. The NanoString gene expression analysis of B cells isolated from recipients on day 12 posttransplant showed that mATG treatment upregulated IL-1β and IL-6 expression and that upregulation was dependent on CD4 T cells. Intracellular flow cytometry confirmed that B cells are the major source of IL-1β and IL-6 following mATG treatment. The proportion of IL-1β secreting B cells was increased in mATG treated compared to untreated recipients (<5% B cells produced IL-1β in naive mice, 20% after transplantation, and more than 30% after transplantation + mATG), whereas IL-6 producing B cells were not significantly affected by lymphoablation. In vivo neutralization of either IL-1β or IL-6 with mAb significantly decreased T cell recovery and extended BALB/c heart allograft survival in mATG treated B6 recipients (MST of 18 d in mATG + anti-IL-1β, and 19 d in mATG + anti-IL-6 vs 11 d in mATG alone). To further test the role of B cell derived IL-1β, we injected 30 x 10⁶ either WT or Caspase-1-/- B cells (do not secrete mature IL-1β) into groups of B cell deficient µMT mice followed by BALB/c heart transplantation and mATG treatment. By day 28 posttransplant, CD8 T cell numbers were significantly higher in recipients injected with WT but not Caspase-1-/- B cells (65,000 vs 39,000 cells/100 µl blood, p = 0.047, n=3/group; 22,000 cells/100 µl blood in µMT mice without B cell transfer).

*Conclusions: Our results show the novel function of IL-1β and IL-6 in regulating homeostatic T cell recovery following mATG treatment and suggest that targeting IL-1β and IL-6 can be a promising strategy for enhancing the efficacy of mATG lymphoablation in transplant recipients.

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