Increased mortality in CD43-deficient mice during sepsis.

CD43 is a large transmembrane protein involved in T cell activation. Previous studies of CD43-/- mice in viral models have demonstrated a role for CD43 in Th1/Th2 skewing, activation of Foxp3+ Treg, and T cell apoptosis. However, the role of CD43 during sepsis has never been tested. Thus, we interrogated the role of CD43 during sepsis using a murine cecal ligation and puncture (CLP) model, and found that CD43-/- mice demonstrated significantly worsened mortality compared to B6 mice following CLP. Phenotypic analysis of splenocytes isolated 24 h after septic insult revealed significantly increased apoptosis of central memory cells in both CD4+ and CD8+ T cell compartments in CD43-/- septic mice compared to WT septic mice. Furthermore, CD43-/-septic mice exhibited a prominent Th2 skewing following sepsis relative to WT septic mice, as evidenced by a significant decrease in the frequency of IL-2+ CXCR3+ TH1 cells as a significant increase in the frequency of IL-4+ CCR4+ TH2 cells. Finally, septic CD43-/- animals contained significantly fewer CD25+ Foxp3+ TReg cells as compared to WT septic animals. Importantly, depleting CD25+ Treg eliminated the increased mortality observed in CD43-/- mice. Taken together, these data demonstrate an important role of CD43 in modulating immune dysregulation and mortality following sepsis

CXCR4 blockade decreases CD4+ T cell exhaustion and improves survival in a murine model of polymicrobial sepsis.

Sepsis is a dysregulated systemic response to infection involving many inflammatory pathways and the induction of counter-regulatory anti-inflammatory processes that results in a state of immune incompetence and can lead to multi-organ failure. CXCR4 is a chemokine receptor that, following ligation by CXCL12, directs cells to bone marrow niches and also plays an important role in T cell cosignaling and formation of the immunological synapse. Here, we investigated the expression and function of CXCR4 in a murine model of polymicrobial sepsis. Results indicate that CXCR4 is selectively upregulated on naïve CD4+ and CD8+ T cells and CD4+ central memory T cells following the induction of sepsis, and that CXCR4 antagonism resulted in a significant decrease in sepsis-induced mortality. We probed the mechanistic basis for these findings and found that CXCR4 antagonism significantly increased the number of peripheral CD4+ and CD8+ T cells following sepsis. Moreover, mice treated with the CXCR4 antagonist contained fewer PD-1+ LAG-3+ 2B4+ cells, suggesting that blockade of CXCR4 mitigates CD4+ T cell exhaustion during sepsis. Taken together, these results characterize CXCR4 as an important pathway that modulates immune dysfunction and mortality following sepsis, which may hold promise as a target for future therapeutic intervention in septic patients

CXCR4 blockade did not affect levels of circulating inflammatory cytokines in sepsis.

<p>Multiple pro-inflammatory (<b>A</b>) and anti-inflammatory (<b>B</b>) cytokine levels were measured in the serum of sham, septic control and plerixafor treated septic mice. No statistically significant differences were observed between septic control mice and septic mice treated with plerixafor. n = 4/group. Representative of 2 independent experiments with a total of 8 mice/group.</p

CXCR4 blockade decreased the percentage of PD-1 expressing adaptive immune cells in sepsis.

<p><b>(A-B)</b> Representative flow plots (gated on CD3 cells) demonstrating PD-1 expression in CD4⁺ T cells and CD8⁺ T cells. (<b>C</b>) The frequency of PD-1⁺ CD4⁺ T cells was significantly increased in septic mice compared to sham mice (28.3% vs. 16.8%; p = 0.002). When septic mice were treated with plerixafor, the frequency of PD-1⁺ CD4⁺ T cells was significantly decreased compared to septic control mice (21.1% vs. 28.3%; p = 0.0156). (<b>D</b>) In septic mice treated with plerixafor, the per-cell expression of PD-1 on CD4⁺ T cells, as measured by MFI, was significantly decreased compared to septic control mice (66.1 vs. 76.8; p = 0.033). (<b>E-F</b>) There was no statistically significant difference in frequency (<b>E</b>) or MFI (<b>F</b>) of PD-1⁺ CD8⁺ T cells in septic mice treated with plerixafor compared to septic control mice (21.5% vs. 30.0%; p = 0.169 and 58.0 vs. 68.3; p = 0.327, respectively). N = 4–8 mice/group. Representative of 3 independent experiments with a total of 12–24 mice/group.</p

Plerixafor decreased the frequency of 2B4⁺ CD4⁺ T cells in septic mice.

<p><b>(A</b>) Representative flow plots (gated on CD3⁺ cells) demonstrating 2B4 expression on CD4⁺ T cells. <b>(B)</b> Septic mice exhibited a significant increase in frequency of 2B4⁺ CD4⁺ T cells compared to sham mice (7.79% vs. 4.3%; p = 0.0421). When septic mice were treated with plerixafor, the frequency of 2B4⁺ CD4⁺ T cells was significantly decreased compared to septic control mice (3.4% vs. 7.79%; p = 0.0108). (<b>C</b>) Septic control mice exhibited an increase in the per-cell expression of 2B4 on CD4⁺ T cells compared to sham mice, as measured by the MFI (101.4 vs. 80.9; p = 0.068). Septic mice treated with plerixafor exhibited a significant decrease in the expression of 2B4 on CD4⁺ T cells compared to septic control mice (80.6 vs. 101.4; p = 0.05). N = 3–5 mice/group. Representative of 3 independent experiments with a total of 9–15 mice/group.</p

CXCR4 was upregulated on less differentiated T cell subsets during sepsis.

<p>Representative histograms demonstrating the expression of CXCR4 on <b>(A)</b> CD4⁺ T cells and <b>(B)</b> CD8⁺ T cells. <b>(C)</b> The frequency of CXCR4 expression was increased in septic mice compared to sham mice on total CD4⁺ T cells in the spleen (22% vs 17.8%; p = 0.002). When the CD4⁺ T cell compartment was further analyzed, the increase in CXCR4 expression on CD4 T cells was limited to (<b>D</b>) naïve CD4⁺ T cells (21.7% vs. 15.6%; p = 0.001) and (<b>E</b>) central memory CD4⁺ T cells (24.1% vs. 16.5%; p = 0.0002). (<b>F</b>) There was no difference in the frequency of CXCR4 expression on effector memory CD4⁺ T cells in septic mice compared to sham mice (32% vs. 31.8%; p = 0.86). (<b>G</b>) There was no difference in the frequency of CXCR4⁺ total CD8⁺ T cells in septic mice compared to sham mice (28.8% vs. 27.9%; p = 0.4136). However, the frequency of CXCR4⁺ naïve CD8⁺ T cells (<b>H</b>) was significantly increased in septic mice compared to sham mice (22% vs. 16.6%; p = 0.0003) but there were no differences in the frequency of CXCR4⁺ central memory CD8⁺ T cells (<b>I</b>; 17.8% vs. 15.7%; p = 0.115) or effector memory CD8⁺ T cells (<b>J</b>; 50.8% vs. 50.9%; p = 0.970). Data shown are n = 5/group, representative of a total of 3 independent experiments with a total of n = 15/group.</p

Plerixafor treatment abrogates the loss of peripheral T cells in sepsis.

<p>(A) Sepsis results in a decrease in the absolute counts of CD4+ T cells in the spleen at 24 hours post sepsis compared to sham mice (p = 0.0012). Septic mice treated with plerixafor have a trend toward increased absolute counts of CD4+ T cells compared to septic control mice (p = 0.055). (B) Similarly, septic mice have a decrease in the absolute number of circulating CD8+ T cells in the blood compared to sham mice (p = 0.0004) and septic mice treated with plerixafor have a trend toward an increase in circulating CD8+ T cells compared to septic control mice (p = 0.054). n = 4-6/group. Representative of 2 independent experiments with a total of n = 8-10/group.</p

Cytokine production by CD4+ and CD8+ T cells is similar in plerixafor-treated septic animals as compared to control septic animals.

<p>Control or plerixafor-treated septic animals were sacrificed at 24h post-CLP and splenocytes were restimulated ex vivo with PMA/ionomycin for 4 h. Cells were fixed, permeabilized, and frequencies of IL-2 (A-B), IFN-g (C-D), and TNF (E-F) secreting CD4+ (A, C, E) and CD8+ (B, D, F) T cells were assessed by flow cytometry. Data shown are cumulative from two independent experiments (n = 4-8/group).</p

Plerixafor decreased the frequency of LAG-3+ CD4+ T cells in septic mice.

<p>(<b>A</b>) Representative flow plots (gated on CD3⁺ cells) demonstrating LAG-3 expression on CD4⁺ T cells. <b>(B)</b> Septic mice exhibited a significant increase in frequency of LAG-3⁺ CD4⁺ T cells compared to sham mice (17.7% vs. 8.5%; p = 0.046). When septic mice were treated with plerixafor, the frequency of LAG-3⁺ CD4⁺ T cells was significantly decreased compared to septic control mice (6.9% vs. 17.7%; p = 0.0173). (<b>C</b>) Septic mice exhibited a significant increase in the per-cell expression of LAG-3 on CD4⁺ T cells compared to sham mice (MFI 104.4 vs. 64.6; p = 0.025) and septic mice treated with plerixafor exhibited a significant decrease in the expression of LAG-3 on CD4⁺ T cells compared to septic control mice (71.9 vs. 104.4; p = 0.0461). N = 3–5 mice/group. Representative of 3 independent experiments with a total of 12 mice/group.</p