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

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 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

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

CD8+ T cell frequencies and counts.

<p>A) There were no relevant differences in CD8 frequencies or counts specifically due to sepsis or ethanol alone at 24h. B) At 72h, there was a strong trend toward a decrease in absolute count due to sepsis in both the water and alcohol fed groups. n = 6-9/group.</p

Alcohol delays increase in the CD69+CD43+ population in naïve and memory CD4+ T cells.

<p>A) Representative flow plot for frequency of CD69+CD43+ of CD4+ CD44^hi at 24h. B) Representative flow plot for frequency of CD69+CD43+ of CD4+ CD44^hi at 72h. C) By 24h, there was significant increase in CD69+ CD43+ population in naïve CD4s in water sepsis (H2O sham 0.4±0.04% vs H2O CLP 1.6±0.4%, p = 0.03). This population was not increased in alcohol sepsis. By 72h, both water septic and alcohol septic groups showed significant increase in the CD69+ CD43+ population (H2O sham 0.3±0.08% vs H2O CLP2.5±0.4%, p = 0.02; EtOH sham 0.3±0.05% vs EtOH CLP 3.5±0.5%, p = 0.01). D) In memory CD4s, at 24h there was a significant increase in CD69+ CD43+ population in water sepsis (H2O sham 6.2±0.7% vs H2O CLP 23.7±4.2%, p = 0.02) but not alcohol sepsis. By 72h, both water and alcohol septic groups show significant increase in this population above sham controls (H2O sham 11.4±-.2% vs H2O CLP 30.8±1.8%, p = 0.04; EtOH sham 10.3±0.9% vs EtOH CLP 32.9±2.6%, p = 0.005). n = 4-8/group.</p

Serum cytokine suggests Th2 skewing in EtOH-fed septic relative to water-fed septic animals.

<p>A) Chronic alcohol ingestion induced elevated baseline levels of serum IL1β over water-feeding in sham animals (906.5±187.7 vs 1883±444.6, p = 0.01). B) Alcohol ingestion increased baseline serum IL-4 concentration in sham animals (19322±473.2 vs 26584±2516, p = 0.02), with a concurrent strong trend toward increase in septic animals (19629±523 vs 24433±1686, p = 0.05). C) Chronic alcohol ingestion increased baseline serum IL12 concentration in sham animals (1073±421.5 vs 35.9±3.1, p = 0.04). D) Alcohol ingestion increased baseline serum TNF concentration in sham animals (1093±79.9 vs 823.8±34.7, p = 0.005). E) Serum IL-6 was increased in alcohol sepsis over water sepsis (58697±27081 vs 896.7±356, p = 0.02). F) Serum IL-10 concentration was increased is alcohol sepsis over water sepsis (10057±4412 vs 979.7±896.2, p = 0.01). n = 8/group.</p

CD4+ T cell frequencies and counts.

<p>A) Representative flow plots and gating strategy. B) There were no differences in CD4 frequencies or counts at 24h. C) At 72h, there were no relevant differences in CD4 frequency. A significant decrease in cell count was seen between alcohol sham and alcohol CLP (8.7x10⁷±1.3x10⁷ vs 4.1x10⁷±3.8x10⁶, p = 0.03). n = 6-9/group.</p

IL-2 (but not IFN-γ or TNF) production by CD4+ T cells is decreased 72h following CLP in alcohol-fed animals.

<p>Representative flow plot and summary data 72h following sepsis, demonstrating a trend toward decreased IL-2 production due to alcohol alone between sham groups which did not reach significance, but there was a significant decrease in IL-2 in alcohol septic animals compared with water septic (16.15±1.7% vs 26.7±1.74%, p = 0.004). There were no differences in the frequencies of IFN-γ or TNF- producing CD4+ T cells between any of the groups. n = 7-12/group.</p