IL-7 treatment augments and prolongs sepsis-induced expansion of IL-10-producing B lymphocytes and myeloid-derived suppressor cells

<div><p>Immunological dysregulation in sepsis is associated with often lethal secondary infections. Loss of effector cells and an expansion of immunoregulatory cell populations both contribute to sepsis-induced immunosuppression. The extent and duration of this immunosuppression are unknown. Interleukin 7 (IL-7) is important for the maintenance of lymphocytes and can accelerate the reconstitution of effector lymphocytes in sepsis. How IL-7 influences immunosuppressive cell populations is unknown. We have used the mouse model of peritoneal contamination and infection (PCI) to investigate the expansion of immunoregulatory cells as long-term sequelae of sepsis with or without IL-7 treatment. We analysed the frequencies and numbers of regulatory T cells (Tregs), double negative T cells, IL-10 producing B cells and myeloid-derived suppressor cells (MDSCs) for 3.5 months after sepsis induction. Sepsis induced an increase in IL-10⁺ B cells, which was enhanced and prolonged by IL-7 treatment. An increased frequency of MDSCs in the spleen was still detectable 3.5 months after sepsis induction and this was more pronounced in IL-7-treated mice. MDSCs from septic mice were more potent at suppressing T cell proliferation than MDSCs from control mice. Our data reveal that sepsis induces a long lasting increase in IL-10⁺ B cells and MDSCs. Late-onset IL-7 treatment augments this increase, which should be relevant for clinical interventions.</p></div

MDSCs from septic mice efficiently suppress T cell proliferation.

<p>Mice were injected with PBS i.p. (Sham) or subjected to sepsis induction. IL-7 (Sepsis + IL-7) or PBS (Sepsis + PBS) was injected daily for 5 days from day 5–9 post sepsis induction. The proliferation of CD4⁺ T cells in the presence of Gr1⁺ cells from spleen was analysed 1 week, 1 month and 3.5 months after sepsis induction. <b>(A)</b> Representative flow cytometry images from analysis after 1 month showing proliferation of T cells (determined by dilution of cell proliferation dye, CPD) from septic mice treated with IL-7 when cultured with MDSCs from sham mice (left), sepsis + PBS mice (middle) and sepsis + IL-7 mice (right). <b>(B)</b> Graph representing frequency of proliferating CD4⁺ T cells from spleen when cultured with MDSCs from spleen from different groups of mice for 3 days. This graph is representative of the experiment performed 1 month post-sepsis induction. n = 3–4 (for all time points). *<i>P</i>< 0.05, **<i>P</i>< 0.01, ***<i>P</i>< 0.001 (ANOVA). Data are expressed as mean ± SEM. Data are representative of one of three experiments.</p

Sepsis results in a sustained expansion of MDSCs.

<p>Mice were injected with PBS i.p. (Sham) or subjected to sepsis induction. IL-7 (Sepsis + IL-7) or PBS (Sepsis + PBS) was injected daily for 5 days from day 5–9 post sepsis induction. Gr1⁺CD11b⁺ cells from the spleen and bone marrow were analysed 1 week, 1 month and 3.5 months after sepsis induction. <b>(A, B)</b> Representative flow cytometry images from spleen (A) and bone marrow (B) from analysis after 3.5 months. <b>(C)</b> Frequency of Gr1⁺CD11b⁺ cells among total spleen cells (top) and among total bone marrow cells (bottom). <b>(D)</b> Number of Gr1⁺CD11b⁺ cells in spleen (top) and bone marrow (bottom). n = 6–9 (1 week), 5–12 (1 month), 8–20 (3.5 months). *<i>P</i>< 0.05, **<i>P</i>< 0.01, ***<i>P</i>< 0.001 (ANOVA). Data are expressed as mean ± SEM. Data are representative of three experiments.</p

Representative gating strategy.

Following singlet gating, lymphocytes were selected with forward/side scatter parameters. Live CD3+ cells were identified by excluding 7AAD stained cells, and CD8+ T cells were selected by CD8a expression. Next, CD8+ T cells (blue gate and arrow) were gated into CD45.2+ single and CD45.1+CD45.2+ double positive populations in mixed bone chimeric animals. Population arising from each bone marrow was progressively gated to define naive and primed CD8+ T cells based on CD44 and CD11a expression. Central memory cells were distinguished from effector cells by CD62L and CD27 expression within primed population. Similarly, short lived effector and memory cell populations (SLEC and Mem) were gated according to KLRG1 and CD27 expression within primed CD8+ T cells (green gate and arrow). Tetramer+ (M38+ and m139+) and CX3CR1+ populations were defined by gating directly on CD45.2+ CD8+ T cells (red gate and arrow) or control CD45.1+CD45.2+ CD8+ T cell populations. (TIF)</p

Sepsis induces a sustained increase of IL-10⁺ B cells.

<p>Mice were injected with PBS i.p. (Sham) or subjected to sepsis induction. IL-7 (Sepsis + IL-7) or PBS (Sepsis + PBS) was injected daily for 5 days from day 5–9 post sepsis induction. IL-10⁺ and CD1d^hi B cells from the spleen were analysed 1 week, 1 month and 3.5 months after sepsis induction. <b>(A)</b> Representative flow cytometry images from analyses 1 week after sepsis induction showing IL-10⁺ cells among CD19⁺ B cells (left panel) and distribution of IL-10⁺ cells (blue) with respect to CD1d and CD5 markers (right panel). <b>(B)</b> Frequency of IL-10⁺ cells among CD19⁺ B cells. (C) Number of IL-10⁺ CD19⁺ cells in the spleen. n (number of mice per group) = 6–10 (1 week), 9–16 (1 month), 7–16 (3.5 months). *<i>P</i>< 0.05, **<i>P</i>< 0.01 (ANOVA). Data are expressed as mean ± SEM. Data are representative of three experiments.</p

NFAT signaling regulates effector CD8⁺ T cell differentiation during chronic infection.

Mixed BMC animals were infected with 106 PFU of MCMV and analyzed at 90 dpi. (A) Representative flow-cytometric plots showing KLRG1+CD27- and KLRG1-CD27+populations from BMC mice with WT and NFATc1c2 DKO BM. The plots are pre-gated on primed (CD44+CD11a+) CD45.2+ CD8+ T cells (live CD3+CD8+). (B) Pairwise comparison of KLRG1+CD27- and KLRG1-CD27+CD8+ T cell frequencies in blood, spleen and lungs of individual mice during chronic MCMV infection. Lines connect data from individual animals (C) Flow-cytometric plots showing representative central memory (CM) populations among primed blood CD8+ T cells of chronically infected mice (left). Kinetics of these CM populations in blood are shown on the right Lines connect group means at indicated time points, error bars are SEM. (D) Percentage of CM CD8+ T cells in spleen and lungs at 90 dpi. Bar plots represent the group average, error bars are SEM and each dot represents mouse. (E) Percentage of CM cells among M45 and M38 tetramer specific CD8+ T cells from spleen at 90 dpi. Bar plots represent mean ± SEM and each dot is a mouse. (F) Representative flow-cytometric plots of blood CD8+ T cells showing CXCR3+ population among primed (CD44+CD11a+) cells (left). Mean CXCR3+ CD8+ T cells population from blood, spleen and mesenteric LN at 90 dpi are shown as mean ± SEM, each dot is a mouse. (G) Representative flow-cytometric plots of blood CD8+ T cells showing CX3CR1+ population among primed (CD44+CD11a+) cells (left). Mean CX3CR1+ CD8+ T cells population from blood, spleen and lungs at 90 dpi are shown as mean ± SEM, each dot is a mouse. Data are pooled from two experiments and each dot represents one mouse, n≥7. Statistically significant differences are highlighted; *, p < 0.05; **, p < 0.01; ***, p < 0.001; (Mann-Whitney U Test); mean ± SEM values are plotted.</p

CD8⁺ T cell differentiation status during chronic infection.

Mixed bone marrow chimeric animals were infected with 106 PFU of MCMV and sacrificed at 90 dpi. (A) Absolute count of KLRG1+CD27- CD8+ T cells in spleen and lungs of BMC animals at 90 dpi. (B) Frequency of CM and KLRG1-CD27+ T cells among primed CD45.2+/+ CD8+ T cells (CD44+CD11a+) from mesenteric LN of chronically infected mice. (C) Quantification of CM CD8+ T cells from CD45.2+/+ compartment in blood, spleen, lungs and mesenteric LN. (D) Absolute count of CX3CR1+ CD8+ T cells in spleen and lungs of BMC animals at 90 dpi. Data are pooled from two independent experiments and each dot represent one mouse. Statistically significant differences are highlighted; *, p (TIF)</p

Sepsis results in sustained activation of DN T cells.

<p>Mice were injected with PBS i.p. (Sham) or subjected to sepsis induction. IL-7 (Sepsis + IL-7) or PBS (Sepsis + PBS) was injected daily for 5 days from day 5–9 post sepsis induction. The cytokine expression in CD3⁺NK1.1^-γδTCR^-CD4^-CD8^- (double negative, DN) T cells from the spleen was analysed 1 week, 1 month and 3.5 months after sepsis induction. <b>(A)</b> Representative flow cytometry images from analysis after 1 week showing DN T cells and IFN-γ and IL-10 expression. <b>(B)</b> Frequency of DN T cells among CD3⁺ cells (left) and their absolute numbers (right). <b>(C)</b> Frequency of IFN-γ⁺ cells among DN T cells (left) and their absolute numbers (right). <b>(D)</b> Frequency of IL-10⁺ cells among DN T cells (left) and their absolute numbers (right). n = 7–13 (1 week), 5–10 (1 month), 3–6 (3.5 months) for IL-10 staining and 3–12 (3.5 months) for IFN-γ staining. *<i>P</i>< 0.05, **<i>P</i>< 0.01, ***<i>P</i>< 0.001 (ANOVA). Data are expressed as mean ± SEM. Data are representative of three experiments for the sham and sepsis + PBS groups. Data are representative of two experiments for the sepsis + IL-7 group.</p

Sepsis induces a transient expansion of Tregs.

<p>Mice were injected with PBS i.p. (Sham) or subjected to sepsis induction. IL-7 (Sepsis + IL-7) or PBS (Sepsis + PBS) was injected daily for 5 days from day 5–9 post sepsis induction. CD4⁺ T cells from the spleen were analysed for expression of Foxp3 and CD25, 1 week, 1 month and 3.5 months after sepsis induction. <b>(A)</b> Representative flow cytometry images from analysis after 1 month showing CD25⁺ and Foxp3⁺ cells among CD4⁺ T cells. <b>(B)</b> Frequency of Foxp3⁺CD25⁺ (top) and Foxp3⁺CD25^- (bottom) cells among CD4⁺ T cells. <b>(C)</b> Number of Foxp3⁺CD25⁺CD4⁺ cells (top) and Foxp3⁺CD25^-CD4⁺ T cells (bottom). n = 7–13 (1 week), 9–16 (1 month), 5–18 (3.5 months). *<i>P</i>< 0.05, **<i>P</i>< 0.01 (ANOVA). Data are expressed as mean ± SEM. Data are representative of three experiments.</p

Phenotype of inflationary CD8⁺ T cells during chronic infection.

Lymphocytes from spleen (A) or lungs (B) of mice infected for 3 months were stained for CD45.1, CD45.2 and CD8 expression as well as the indicated cell-surface molecules. The plots shown are gated on CD45.1+/-CD45.2+/- CD8+ T cells (shaded histogram) or CD45.1-/-CD45.2+/+ CD8+ T cells (black line) from the same sample. Data are representative of at least six individual mice per stain and two independent experiments. (TIF)</p