B cell depletion can augment or inhibit auto- and allo- immune responses in humans and mice without altering Ig levels. This is likely due to the presence both regulatory B cells (Bregs) and effector B cells (Beff) expressing anti- or pro- inflammatory cytokines, respectively. While TIM-1 is an inclusive marker for IL-10+ Bregs, the phenotype of Beff cells is completely unknown, hampering study of their in vivo role and therapeutic targeting.

We now show that TIM-4 and TIM-1 are expressed by distinct B cell subsets. While TIM-1⁺ B cells are enriched for IL-10, TIM-4⁺ B cells are enriched for IFN-γ. Transfer of TIM-1⁺ B cells prolongs allograft survival (GS) in B-deficient mice, while TIM-4⁺ B cells accelerate rejection in an IFN-g-dependent manner. TIM-4⁺ B cells promote pro-inflammatory Th differentiation in vivo (↑IFN-g and ↓IL-4, IL-10, and Foxp3). Thus, TIM-4 identifies proinflammatory Beff cells. We hypothesized that targeting these cells could prolong GS. Indeed α-TIM-4 (RMT4-53; 250 ug ip d1-,0,5) induced long-term GS in BALB/c recipients of B6 islets (MST >100d). Moreover, α-TIM-4 ↑'s CD4+ T cell IL-4, IL-10 and Foxp3, and ↓'s IFN-γ and CD4 proliferation. However, these α-TIM-4-mediated changes and long-term GS were completely B cell dependent. To address whether α-TIM-4 directly targeted TIM-4 on Beff cells, we showed that adoptive transfer of WT, but not TIM-4^-/- B cells, into [mu]MT recipients, reconstitutes α-TIM-4-mediated long-term GS. Similar results were obtained in mixed BM chimeras where only B cells lack TIM-4 (uMT + TIM-4-/- marrow at 10:1 ratio). While α-TIM-4 treatment has no effect on TIM-4+ or TIM-1+ B cell frequency in vivo, it ↓'s B cell IFNγ (by ~55%) and ↑'s B cell IL-10 (by 60%). Next, purified TIM-4⁺ B cells were stimulated in vitro +/- α-TIM-4. Surprisingly, α-TIM-4 dramatically inhibited IFNg (↓4X) by TIM-4+ B cells.

Thus, TIM-4 ligation with α-TIM-4 can act directly on TIM-4+ B cells to suppress inflammatory cytokine production, rather than acting indirectly – for example by blocking interaction between TIM-4 and its ligands. This was surprising because TIM-4 has no known signaling motifs and it has not been previously thought to only signal in “trans”. We believe decreased TIM-4 B cell IFNg augments IL-10 expressed by TIM-1+ B cells. Taken together, our data reveal that targeting TIM-4 enhances IL-10 expressing Bregs and also reduces inflammatory Beff cells to strongly enhance allograft tolerance.

CITATION INFORMATION: Ding Q, Mohib K, Xiao S, Kuchroo V, Rothstein D. Targeting IFN-g-Expressing TIM-4⁺ B Effector (Beff) Cells as a Novel Strategy to Prevent Allograft Rejection. Am J Transplant. 2016;16 (suppl 3).

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