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.

Sepsis is a leading cause of death in the U.S. but the mechanisms underlying sepsis-induced immune dysregulation remain poorly understood. 2B4 (CD244, SLAM4) is a cosignaling molecule expressed predominantly on NK cells and memory CD8+ T cells that has been shown to regulate T cell function in models of viral infection and autoimmunity. Here we show that 2B4 signaling mediates sepsis lymphocyte dysfunction and mortality. 2B4 expression is increased on CD4+ T cells in both septic animals and human patients at early time points. Importantly, genetic loss or pharmacologic inhibition of 2B4 each significantly increased survival in a murine cecal ligation and puncture (CLP) model. Further, CD4-specific conditional knockouts showed that 2B4 functions on CD4+ T cell populations in a cell-intrinsic manner and modulates both adaptive and innate immune responses during sepsis. Our results illuminate a novel role for 2B4 coinhibitory signaling on CD4+ T cells in mediating immune dysregulation.

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Epstein–Barr virus (EBV) reactivation commonly occurs following sepsis, but the mechanisms underlying this are unknown. We utilized a murine EBV homolog (gHV) and the cecal ligation and puncture model of polymicrobial sepsis to study the impact of sepsis on gHV reactivation and CD8+ T cell immune surveillance following a septic insult. We observed a significant increase in the frequency of gHV‐infected germinal center B cells on day 7 following sepsis. This increase in viral load was associated with a concomitant significant decrease in the frequencies of gHV‐specific CD8+ T cells, as measured by class I MHC tetramers corresponding to the immunodominant viral epitopes. Phenotypic analysis revealed an increased frequency of gHV‐specific CD8+ T cells expressing the 2B4 coinhibitory receptor in septic animals compared with sham controls. We sought to interrogate the role of 2B4 in modulating the gHV‐specific CD8+ T cell response during sepsis. Results indicated that in the absence of 2B4, gHV‐specific CD8+ T cell populations were maintained during sepsis, and gHV viral load was unchanged in 2B4−/− septic animals relative to 2B4−/− sham controls. WT CD8+ T cells upregulated PD‐1 during sepsis, whereas 2B4−/− CD8+ T cells did not. Finally, adoptive transfer studies revealed a T cell‐intrinsic effect of 2B4 coinhibition on virus‐specific CD8+ T cells and gHV viral load during sepsis. These data demonstrate that sepsis‐induced immune dysregulation erodes antigen‐specific CD8+ responses against a latent viral infection and suggest that blockade of 2B4 may better maintain protective immunity against EBV in the context of sepsis.

The presence of pre-existing malignancy in murine hosts results in increased immune dysregulation and risk of mortality following a septic insult. Based on the known systemic immunologic changes that occur in cancer hosts, we hypothesized that the presence of pre-existing malignancy would result in phenotypic and functional changes in CD4+ T cell responses following sepsis. In order to conduct a non-biased, unsupervised analysis of phenotypic differences between CD4+ T cell compartments, cohorts of mice were injected with LLC1 tumor cells and tumors were allowed to grow for 3 weeks. These cancer hosts and age-matched non-cancer controls were then subjected to CLP. Splenocytes were harvested at 24h post CLP and flow cytometry and SPADE (Spanning-tree Progression Analysis of Density-normalized Events) were used to analyze populations of CD4+ cells most different between the two groups. Results indicated that relative to non-cancer controls, cancer mice contained more resting memory CD4+ T cells, more activated CD4+ effectors, and fewer naïve CD4+ T cells during sepsis, suggesting that the CD4+ T cell compartment in cancer septic hosts is one of increased activation and differentiation. Moreover, cancer septic animals exhibited expansion of two distinct subsets of CD4+ T cells relative to previously healthy septic controls. Specifically, we identified increases in both a PD-1hi population and a distinct 2B4hi BTLAhi LAG-3hi population in cancer septic animals. By combining phenotypic analysis of exhaustion markers with functional analysis of cytokine production, we found that PD-1+ CD4+ cells in cancer hosts failed to make any cytokines following CLP, while the 2B4+ PD-1lo cells in cancer mice secreted increased TNF during sepsis. In sum, the immunophenotypic landscape of cancer septic animals is characterized by both increased CD4+ T cell activation and exhaustion, findings that may underlie the observed increased mortality in mice with pre-existing malignancy following sepsis.