Antigen-specific precursor frequency impacts T cell proliferation, differentiation, and requirement for costimulation

After a brief period of antigenic stimulation, T cells become committed to a program of autonomous expansion and differentiation. We investigated the role of antigen-specific T cell precursor frequency as a possible cell-extrinsic factor impacting T cell programming in a model of allogeneic tissue transplantation. Using an adoptive transfer system to incrementally raise the precursor frequency of antigen-specific CD8+ T cells, we found that donor-reactive T cells primed at low frequency exhibited increased cellular division, decreased development of multifunctional effector activity, and an increased requirement for CD28- and CD154-mediated costimulation relative to those primed at high frequency. The results demonstrated that recipients with low CD4+ and CD8+ donor-reactive T cell frequencies exhibited long-term skin graft survival upon CD28/CD154 blockade, whereas simultaneously raising the frequency of CD4+ T cells to ∼0.5% and CD8+ T cells to ∼5% precipitated graft rejection despite CD28/CD154 blockade. Antigenic rechallenge of equal numbers of cells stimulated at high or low frequency revealed that cells retained an imprint of the frequency at which they were primed. These results demonstrate a critical role for initial precursor frequency in determining the CD8+ T cell requirement for CD28- and CD154-mediated costimulatory signals during graft rejection

CD154 blockade alters innate immune cell recruitment and programs alloreactive CD8+ T cells into KLRG-1(high) short-lived effector T cells.

CD154/CD40 blockade combined with donor specific transfusion remains one of the most effective therapies in prolonging allograft survival. Despite this, the mechanisms by which these pathways synergize to prevent rejection are not completely understood. Utilizing a BALB/c (H2-K(d)) to B6 (H2-K(b)) fully allogeneic skin transplant model system, we performed a detailed longitudinal analysis of the kinetics and magnitude of CD8(+) T cell expansion and differentiation in the presence of CD154/CD40 pathway blockade. Results demonstrated that treatment with anti-CD154 vs. DST had distinct and opposing effects on activated CD44(high) CD62L(low) CD8(+) T cells in skin graft recipients. Specifically, CD154 blockade delayed alloreactive CD8(+) T cell responses, while DST accelerated them. DST inhibited the differentiation of alloreactive CD8(+) T cells into multi-cytokine producing effectors, while CD40/CD154 blockade led to the diminution of the KLRG-1(low) long-lived memory precursor population compared with either untreated or DST treated animals. Moreover, only CD154 blockade effectively inhibited CXCL1 expression and neutrophil recruitment into the graft. When combined, anti-CD154 and DST acted synergistically to profoundly diminish the absolute number of IFN-γ producing alloreactive CD8(+) T cells, and intra-graft expression of inflammatory chemokines. These findings demonstrate that the previously described ability of anti-CD154 and DST to result in alloreactive T cell deletion involves both delayed kinetics of T cell expansion and differentiation and inhibited development of KLRG-1(low) memory precursor cells

Anti-CD154 and DST independently alter the expansion kinetics of activated CD44^high CD62L^low CD8⁺ T cells.

<p>B6-Ly5.2/Cr mice were transplanted with BALB/c skin grafts and were treated with 10⁷ BALB/c DST and/ or anti-CD154 mAb, where indicated. A. Representative flow plots of CD44^high and CD62L^low CD8⁺ T cells isolated from spleens of mice on day 7 post-transplantation. B. Expansion kinetics of activated CD8⁺ T cells after allo-transplantation. C. Accumulation of CD44^high CD62L^low CD8⁺ T cells on day 10, 14, and 50 post-transplantation. Data are summary of two experiments with three mice per group. Values are mean ± SEM. *p<0.05, **p<0.01, ***p<0.001.</p

Anti-CD154 and DST interact to protect allogeneic grafts from rejection.

<p>A. B6-Ly5.2/Cr mice were transplanted with BALB/c skin grafts and were treated with 10⁷ BALB/c splenocytes (DST) and/ or anti-CD154 monoclonal antibody (500 μg on D0, 2, 4, 6), where indicated. B. Allo-skin grafts in untreated mice had an MST of 13 days. Monotherapy with either CD40/CD154 pathway blockade or DST led to rapid rejection of the allograft with MSTs of 17.5d (p = 0.039) and 13d (p = n.s.), respectively. Anti-CD154 and DST combined treated significantly prolonged allograft survival to 50 days (p = 0.0002). Data are summary of two experiments of 4–5 mice per group. *p<0.05, ***p<0.001.</p

Anti-CD154 treatment increases the frequency of KLRG-1^high short-lived CD8⁺ effectors.

<p>B6-Ly5.2/Cr mice were transplanted with BALB/c skin grafts and were treated with 10⁷ BALB/c DST and/ or anti-CD154 mAb, where indicated. A. Flow plots of KLRG-1 expression on antigen experienced CD44^high CD62L^low CD8⁺ T cells at day 7 post-transplantation. B. Frequency of KLRG-1^high antigen experienced CD44^high CD62L^low CD8⁺ T cells on day 7. C. Absolute count of alloreactive CD4⁺ T cells producing IFN-γ on day 7. Data are summary of two experiments with three mice per group. Values are mean ± SEM. *p<0.05, **p<0.01, ***p<0.001.</p

Anti-CD154 and DST distinctly alter alloreactive CD8⁺ T cell programming into cytokine-producing effector cells.

<p>B6-Ly5.2/Cr mice were transplanted with BALB/c skin grafts and were treated with 10⁷ BALB/c DST and/ or anti-CD154 mAb, where indicated. A. Representative flow plots of TNF and IFN-γ producing CD8⁺ T cells after <i>ex vivo</i> restimulation with BALB/c splenocytes, isolated from spleens of mice at day 7 post-transplantation. B. Absolute count of total IFN-γ producing CD8⁺ T cells in the spleen over time following <i>ex vivo</i> restimulation. C. Pie charts represent total activated CD44^high CD62L^low CD8⁺ T cells. The black wedges represent the frequency of activated CD44^high CD62L^low CD8⁺ T cells that produce IFN-γ on day 7 post-transplantation (p<0.05). D. Pie charts represent all IFN-γ producing CD8⁺ T cells. The striped wedges represent the IFN-γ-only producing population and black segments represent the TNF/IFN-γ double producing population in untreated vs. DST treated mice on day 7 (p = 0.0028). Data are summary of two experiments with three mice per group. Values are mean ± SEM. *p<0.05, **p<0.01, ***p<0.001.</p

CD154 blockade decreases CXCL1, CCL3, and CCL5 expression in allografts.

<p>B6.SJL mice were transplanted with BALB/c skin grafts and were treated with 10⁷ BALB/c DST and/ or anti-CD154 mAb, where indicated. On day 7, skin grafts were explanted and processed for RNA extraction. Real time PCRs for chemokines CXCL1/ KC, CCL3/ MIP-1α, and CCL5/ RANTES were performed from cDNA. Data are summary of two experiments with three mice per group. Values are mean ± SEM. **p<0.01, ***p<0.0001.</p

IFN- dictates allograft fate via opposing effects on the graft and on recipient CD8 T cell responses

CD8 T cells are necessary for costimulation blockade-resistant rejection. However, the mechanism by which CD8 T cells mediate rejection in the absence of major costimulatory signals is poorly understood. IFN-γ promotes CD8 T cell-mediated immune responses, but IFN-γ-deficient mice show early graft loss despite costimulation blockade. In contrast, we found that IFN-γ receptor knockout mice show dramatically prolonged graft survival under costimulation blockade. To investigate this paradox, we addressed the effects of IFN-γ on T cell alloresponses in vivo independent of the effects of IFN-γ on graft survival. We identified a donor-specific CD8 T cell breakthrough response temporally correlated with costimulation blockade-resistant rejection. Neither IFN-γ receptor knockout recipients nor IFN-γ-deficient recipients showed a CD8 breakthrough response. Graft death on IFN-γ-deficient recipients despite costimulation blockade could be explained by the lack of IFN-γ available to act on the graft. Indeed, the presence of IFN-γ was necessary for graft survival on IFN-γ receptor knockout recipients, as either IFN-γ neutralization or the lack of the IFN-γ receptor on the graft precipitated early graft loss. Thus, IFN-γ is required both for the recipient to mount a donor-specific CD8 T cell response under costimulation blockade as well as for the graft to survive after allotransplantation