T cell function is tightly regulated by a fine balance of coinhibitory signals, including those transduced by 2B4 (SLAMf4, CD244), an immunoglobulin-superfamily member constitutively expressed on NK cells and induced on some T cell subsets. Recent studies from our group suggest that 2B4 plays a functional role in the inhibition of donor-reactive CD8⁺ T cell responses in vivo in the setting of selective CD28 blockade. These findings raise the possibility that 2B4 itself may be a therapeutic target to attenuate allograft rejection. Thus, we hypothesized that augmenting 2B4 signaling would attenuate graft-specific CD8+ T cell responses following transplantation. To test this, we created 2B4 retrogenic (2B4rg) donor-reactive CD8+ OT-I T cells which ectopically express 2B4. 2B4 retrogenic Thy1.1+ CD8+ T cells (or empty vector-transduced controls) were adoptively transferred into naïve animals. Mice then received an OVA-expressing skin graft and were sacrificed ten days later. Data show that constitutive 2B4 expression results in significantly reduced accumulation of antigen-specific CD8+ T cells in the spleen 10 days post-transplantation as compared to wild-type pMY controls (1.23×10⁶ +/- 4.27×10⁵ vs. 6.51×10⁶ +/- 2.81×10⁶, respectively, p=0.0267). This was not due to differences in expression of the 2B4 ligand CD48 or the T cell activation or exhaustion markers CD44, KLRG-1, CD127, TIM-1, PD-1, and LAG-3. Further, the differences in donor-reactive CD8+ T cell accumulation were not explained by increased cell death, as we observed no difference in frequencies of Annexin V+ 7-AAD+ apoptotic cells between 2B4rg donor-reactive CD8+ T cells and empty vector controls. Instead, we observed a marked reduction in the proliferation of the CD8+ Thy1.1+ 2B4rg cells when compared to controls as measured by CellTrace Violet (CTV) analysis. Specifically, the 2B4rg CD8+ Thy1.1+ population contained a higher frequency of CTV^hi undivided cells as compared to non-retrogenic controls (13.62% +/- 0.5476 vs. 2.642% +/- 1.167, respectively, p=0.0022), suggesting that overexpression of 2B4 on donor-reactive CD8+ T cells results in reduced recruitment into the response. Interestingly, 2B4rg cells still differentiated into cytokine-producing effectors despite their inability to divide normally. These findings suggest that engaging the 2B4 coinhibitory pathway could represent a novel therapeutic strategy to prevent the expansion of alloreactive CD8+ T cells following transplantation.

CITATION INFORMATION: Laurie S, Liu D, Ford M. 2B4 Over-Expression Impairs Donor-Reactive CD8+ T Cell Proliferation and Accumulation Following Transplantation. Am J Transplant. 2016;16 (suppl 3).

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