Negative Immune Checkpoint Protein, VISTA, Regulates the CD4+ Treg Population During Sepsis Progression to Promote Acute Sepsis Recovery and Survival

Abstract

Sepsis is a systemic immune response to infection that is responsible for ~35% of in-hospital deaths and over 24 billion dollars in annual treatment costs. Strategic targeting of non-redundant negative immune checkpoint protein pathways can cater therapeutics to the individual septic patient and improve prognosis. B7-CD28 superfamily member V-domain Immunoglobulin Suppressor of T cell Activation (VISTA) is an ideal candidate for strategic targeting in sepsis. We hypothesized that immune checkpoint regulator, VISTA, controls T-regulatory cells (T_reg), in response to septic challenge, thus playing a protective role/reducing septic morbidity/mortality. Further, we investigated if changes in morbidity/mortality are due to a T_reg-mediated effect during the acute response to septic challenge. To test this, we used the cecal ligation and puncture model as a proxy for polymicrobial sepsis and assessed the phenotype of CD4⁺ T_regs in VISTA-gene deficient (VISTA^-/-) and wild-type mice. We also measured changes in survival, soluble indices of tissue injury, and circulating cytokines in the VISTA^-/- and wild-type mice. We found that in wild-type mice, CD4⁺ T_regs exhibit a significant upregulation of VISTA which correlates with higher T_reg abundance in the spleen and small intestine following septic insult. However, VISTA^-/- mice have reduced T_reg abundance in these compartments met with a higher expression of Foxp3, CTLA4, and CD25 compared to wild-type mice. VISTA^-/- mice also have a significant survival deficit, higher levels of soluble indicators of liver injury (i.e., ALT, AST, bilirubin), and increased circulating proinflammatory cytokines (i.e., IL-6, IL-10, TNFα, IL-17F, IL-23, and MCP-1) following septic challenge. To elucidate the role of T_regs in VISTA^-/- sepsis mortality, we adoptively transferred VISTA-expressing T_regs into VISTA^-/- mice. This adoptive transfer rescued VISTA^-/- survival to wild-type levels. Taken together, we propose a protective T_reg-mediated role for VISTA by which inflammation-induced tissue injury is suppressed and improves survival in early-stage murine sepsis. Thus, enhancing VISTA expression or adoptively transferring VISTA⁺ T_regs in early-stage sepsis may provide a novel therapeutic approach to ameliorate inflammation-induced death.

Keywords: CD25; CTLA4; Foxp3; Vista; cytokines; liver injury; regulatory T cells; sepsis.

Figure 1

Experimental timeline for study. (A) WT and VISTA^-/- mice underwent sham or CLP procedure, and tissues/blood were harvested for downstream analysis via flow cytometry or spectrophotometry. (B) WT and VISTA^-/- mice underwent a CLP procedure, and survival was tallied for 14 days. Surviving mice were euthanized on the 15th day. (C) WT mice were injected with Jurkat T_regs, and tissues were harvested 2 days postinjection for downstream analysis and validation of adoptive transfer via flow cytometry. (D) VISTA^-/- mice were injected with Jurkat T_regs then underwent CLP 2 days postinjection, and survival was tallied for 14 days. Surviving mice were euthanized on the 15th day.

Figure 2

VISTA⁺CD4⁺ T cells in mouse splenocytes and VISTA⁺CD3⁺ lymphocytes in patient blood increase following experimental or clinical sepsis. (A) Summary graph of VISTA⁺ CD4⁺ T cells in the wild-type mouse spleen. (B) Summary graph of CD4⁺ T cell frequency in the wild-type mouse spleen. (C) Summary graph of VISTA⁺ CD3⁺ T cell frequency in the peripheral blood lymphocytes. (D) Summary graph of CD3+ T cell frequency in the peripheral blood. (A–D) Summary graphs show mean ± SEM [WT-sham: n = 13, WT-CLP: n = 16]; significance **p < 0.01, ***p < 0.001. (E) Initial process (Strategy) for producing embryos deficient in the ~11.3-kb region containing exon (ex) 2 to exon 7 of the VISTA gene on mouse chromosome 10 with CRISPR/Cas9 followed by NHEJ-mediated repair. (F) Results of initial heterozygous cross of VISTA^-/+ founder mice resulting from CRISPR/Cas9 technology that produced homozygous VISTA^-/- mice for breeding (PCR genotyping strategy: 403/412: 11,745 bp from WT and ~0.4 kb from VISTA deletion alleles).

Figure 3

VISTA expression correlates with CD4⁺ T_reg population increase following CLP and VISTA^-/- mice fail to expand the CD4⁺T_reg population in the spleen. (A) Summary graph of VISTA⁺ CD4⁺ T_reg frequency in the spleen. (B) Summary graph of CD4⁺ T_reg frequency in the spleen and (C) representative flow cytometry plots comparing fluorescence minus one (FMO) control, sham (WT), CLP (WT), and CLP (VISTA^-/-) samples. Summary graphs show mean ± SEM [WT-sham: n = 8, WT-CLP: n = 8, VISTA^{-/ –} sham: n = 8, VISTA^-/- -CLP: n = 13]; significance *p < 0.05; **p < 0.01.

Figure 4

VISTA expression correlates with CD4⁺ T_reg population increase following CLP and VISTA^-/- mice fail to expand the CD4⁺ T_reg population in the intestinal intraepithelial compartment. (A) Summary graph of VISTA⁺ CD4⁺ T_reg frequency in the small intestine. (B) Summary graph of CD4⁺ T_reg frequency in the small intestine and (C) representative flow cytometry plots comparing fluorescence minus one (FMO) control, sham (WT), CLP (WT), and CLP (VISTA^-/-) samples. Summary graphs show mean ± SEM [WT-sham: n = 3, WT-CLP: n = 3, VISTA^-/-sham: n = 4, VISTA^-/- -CLP: n = 4]; significance *p < 0.05; **p < 0.01.

Figure 5

Expression of suppressive markers is upregulated on CD4⁺ T_regs in the spleen of VISTA^-/- mice. Median fluorescence intensity (MFI) of (A) Foxp3, (B) CTLA4, (C) CD25, and (D) CD69 on CD4⁺ T_regs in the spleen. Summary graphs show mean ± SEM [WT-sham: n = 3, WT-CLP: n = 3, VISTA^-/-sham: n = 4, VISTA^-/- -CLP: n = 4]; significance *p < 0.05; ***p < 0.001.

Figure 6

Expression of suppressive markers is upregulated on CD4⁺ T_regs and is downregulated on CD4⁺CD8⁺ T_regs in the thymus of VISTA^-/- mice. Median fluorescence intensity (MFI) of (A) Foxp3, (B) CTLA4, (C) CD25, and (D) CD69 on CD4⁺ T_regs in the thymus. Median fluorescence intensity (MFI) of (E) Foxp3, (F) CTLA4, (G) CD25, and (H) CD69 on CD4⁺CD8⁺ T_regs in the thymus. Summary graphs show mean ± SEM [WT-sham: n = 3, WT-CLP: n = 3, VISTA^-/-sham: n = 4, VISTA^-/- -CLP: n = 4]; significance *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 7

VISTA^-/- mice have significantly higher levels of several Th17 cytokines following septic challenge. (A–J) Plasma cytokine concentration of wild-type and VISTA^-/- mice. Summary graphs show mean ± SEM [WT-sham: n = 5, WT-CLP: n = 5, VISTA^-/-sham: n = 5, VISTA^-/- -CLP: n=5]; significance ^#p = 0.05; *p < 0.05; **p < 0.01; ****p < 0.0001.

Figure 8

VISTA^-/- mice have significantly worse survival and morbidities following septic challenge. (A) 14-day survival following CLP [WT: n = 28, VISTA^-/-: n = 26]. (B) Creatine kinase activity [WT-sham: n = 9, WT-CLP: n = 9, VISTA^-/-sham: n = 10, VISTA^-/- -CLP: n = 10]. (C) blood urea nitrogen [WT-sham: n = 4, WT-CLP: n = 9, VISTA^-/-sham: n = 5, VISTA^-/- -CLP: n = 8]. (D) α-Amylase activity [WT-sham: n = 5, WT-CLP: n = 6, VISTA^-/-sham: n = 5, VISTA^-/- -CLP: n = 6]. (E) Direct bilirubin concentration [WT-sham: n = 3, WT-CLP: n = 3, VISTA^-/-sham: n = 3, VISTA^-/- -CLP: n = 4]. (F) alanine aminotransferase activity [WT-sham: n = 4, WT-CLP: n = 8, VISTA^-/-sham: n = 4, VISTA^-/- -CLP: n = 8]. (G) Aspartate aminotransferase activity [WT-sham: n = 4, WT-CLP: n = 8, VISTA^-/-sham: n = 4, VISTA^-/- -CLP: n = 8] from plasma samples of wild-type and VISTA^-/- mice. (B–G) Summary graphs show mean ± SEM; significance *p < 0.05; **p < 0.01.

Figure 9

VISTA^-/- mice have significantly higher levels of several proinflammatory cytokines following septic challenge. (A–J) Plasma cytokine concentration of wild-type and VISTA^-/- mice. Summary graphs show mean ± SEM [WT-sham: n = 10, WT-CLP: n = 10, VISTA^-/-sham: n = 10, VISTA^-/- -CLP: n = 10]; significance ^#p = 0.05; *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 10

Adoptive transfer of Jurkat T_regs improves survival of VISTA^-/- mice post CLP. Blockade of VISTA in Jurkat T_regs in vitro reduces viability and cytokine production. (A) 14-day survival following adoptive transfer and CLP [VISTA^-/-(HBSS alone): n = 11, VISTA^-/-(HBSS+ Jurkat T_regs): n = 10]. (B) Alamar Blue viability assay of Jurkat T_regs following treatment with Ig control or 13F3. (C–I) Supernatant cytokine concentration of Jurkat T_regs following treatment with plasma from septic mouse (stimulated) or plasma and 13F3 (stim. + 13F3). Summary graphs show mean ± SEM; not significant ^ns; significance ^#p = 0.05; *p < 0.05; **p < 0.01.