Bone marrow is the preferred site of memory CD4+ T cell proliferation during recovery from sepsis

Abstract

Sepsis survivors suffer from increased vulnerability to infections, and lymphopenia presumably contributes to this problem. The mechanisms of the recovery of memory CD4+ T cells after sepsis remain elusive. We used the cecal ligation and puncture mouse model of sepsis to study the restoration of the memory CD4+ T cells during recovery from sepsis. Then, adoptive transfer of antigen-specific naive CD4+ T cells followed by immunization and BrdU labeling were performed to trace the proliferation and migration of memory CD4+ T cells. We revealed that the bone marrow (BM) is the primary site of CD4+ memory T cell homing and proliferation after sepsis-induced lymphopenia. Of interest, BM CD4+ T cells had a higher basal proliferation rate in comparison with splenic T cells. These cells also show features of resident memory T cells yet have the capacity to migrate outside the BM niche and engraft secondary lymphoid organs. The BM niche also sustains viability and functionality of CD4+ T cells. We also identified IL-7 as the major inducer of proliferation of the BM memory CD4+ T cells and showed that recombinant IL-7 improves the recovery of these cells. Taken together, we provide data on the mechanism and location of memory CD4+ T cell proliferation during recovery from septic lymphopenia, which are of relevance in studying immunostimulatory therapies in sepsis.

Keywords: Bone marrow; Immunology; Infectious disease; Memory; T cells.

Figure 1. Sepsis induces changes in CD4⁺ T cell subsets’ frequency.

(A) Representative flow cytometry plots showing CD4⁺ T cell gating strategy used for the analysis of naive (CD44^–CD62L⁺), central memory (CD44⁺CD62L⁺), effector memory (CD44⁺CD62L^–), and effector (CD44^–CD62L^–) CD4⁺ T cells. (B) Changes in the frequencies of CD4⁺ T cells in the lymph nodes after sepsis (left), with shifts in subset composition of the CD4⁺ T cells after sepsis (right graph). (C) Changes in the frequencies of CD4⁺ T cells in the spleen after sepsis (left), with shifts in subset composition of the CD4⁺ T cells after sepsis (right graph). (D) Changes in the frequencies of CD4⁺ T cells in the BM after sepsis (left), with shifts in subset composition of the CD4⁺ T cells after sepsis (right graph). Box-and-whiskers plots present 25th through 75th percentiles (p25-p75) (box), mean, and p10-p90 (whiskers). Data from 2 independent experiments (n = 6–8 in each group). *P < 0.05, and ***P < 0.001 using ANOVA with Tukey’s post hoc test. Superimposed graphs: sign on the left side of bar represents P < 0.05 between day 7 and controls; sign on the right side of bar represents P < 0.05 between days 14 and 7. “*” represents differences between effector; “&” effector memory; “#” central memory; and “§” naive CD4⁺ T cells at different time points using ANOVA with Tukey’s post hoc test.

Figure 2. BM contains actively proliferating CD4⁺ T cells after sepsis.

(A) Experimental design. Mice underwent CLP surgery and subsequent treatment with antibiotic and fluid resuscitation. On day 6 or 13 after surgery, mice were injected with a bolus of BrdU i.p. Twenty-four hours later the cells were isolated from organs and analyzed by flow cytometry. (B) Representative flow cytometry plots showing CD4⁺BrdU⁺ cells that were actively cycling after BrdU administration. Upper row shows plots from sham animals, and lower row shows plots from 7 days post-CLP mice. (C) Percentage of BrdU⁺ cells among CD4⁺ T cells from different organs at given time points after CLP (n = 6–8 in each group); box-and-whiskers plot presents p25-p75 (box), mean, and p10-p90 (whiskers). ****P < 0.0001 between BM and lymph nodes or spleen; §§§§P < 0.0001 between BM 7 days after CLP and control or 14 days post-CLP using ANOVA with Tukey’s multiple-comparisons test. (D) Changes in the subset composition of CD4⁺ T cells that incorporated BrdU. Data from 2 independent experiments are shown (n = 6–8). Superimposed graphs: sign on the left side of bar represents P < 0.05 between day 7 and control; sign on the right side of bar represents P < 0.05 between days 14 and 7. “*” represents differences between effector; “&” effector memory; “#” central memory; and “§” naive CD4⁺ T cells at different time points using ANOVA with Tukey’s post hoc test.

Figure 3. Sepsis leads to accumulation of CD69⁺ T cells in the BM.

(A) Representative flow cytometry plots showing expression of CD69 on CD4⁺ cells from lymph nodes, spleen, and BM of sham (upper row) and septic (lower row) mice. (B) Percentage of CD69⁺ cells among CD4⁺ T cells from different organs at given time points after CLP (n = 6–8 in each group). Box-and-whiskers plot presents p25-p75 (box), mean, and p10-p90 (whiskers). **P < 0.01, and ****P < 0.0001 between BM and lymph nodes or spleen; §§P < 0.01 between control BM and 7 days after CLP; and §§§§P < 0.0001 between BM 14 days after CLP and control or 7 days post-CLP using ANOVA with Tukey’s multiple-comparisons test. (C) Changes in the composition of CD4⁺ T cell subsets expressing CD69 after CLP sepsis. Data from 2 independent experiments are shown (n = 6–8 in each group). Superimposed graphs: sign on the left side of bar represents P < 0.05 between day 7 and control; sign on the right side of bar represents P < 0.05 between days 14 and 7. “*” represents differences between effector; “&” effector memory; “#” central memory; and “§” naive CD4⁺ T cells at different time points using ANOVA with Tukey’s post hoc test.

Figure 4. BM supports proliferation of specific Ag-experienced memory CD4⁺ T cells in sepsis.

(A) Experimental design. CD4⁺CD44^– naive T cells were isolated from young OT-II mice and transferred to C57BL/6 recipients. Then, mice were immunized with 100 μg of OVA i.p. and 100 μg s.c. Thirty days later mice were subjected to CLP and received a bolus of BrdU on day 6 after surgery. Cells were analyzed 24 hours or 30 days later by flow cytometry. (B) Plots showing flow cytometry gating strategy of the transplanted I-A^b OVA_329–337 tetramer-specific CD4⁺ T cells after nanobead enrichment. (C) Representative flow cytometry plots showing analysis of BrdU⁺ cells that are CD4⁺I-A^b OVA_329-337⁺CD44⁺ from mice that had CLP surgery 7 days (upper row) and 36 days (lower row) before. (D) Total number of the I-A^b OVA CD4⁺BrdU⁺ cells in lymph nodes, spleen, and BM of mice post-CLP and sham mice at different times after BrdU injection. (E) Percentage of BrdU⁺ cells among the I-A^b OVA CD4⁺ cells in lymph nodes, spleen, and BM of mice post-CLP and sham mice at different times after BrdU injection. (F) Normalized ratios of the number of I-A^b OVA CD4⁺BrdU⁺ cells in the lymph nodes and BM versus the number of I-A^b OVA⁺CD4⁺BrdU⁺ in the spleen. Box-and-whiskers plot presents p25-p75 (box), mean, and p10-p90 (whiskers). Results from the mice post-CLP and sham mice at different times after BrdU injection. Data from 2 independent experiments (n = 6–10 in each group). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 using ANOVA with Tukey’s post hoc test.

Figure 5. The capacity of T cell homing toward the BM niche is decreased in sepsis.

(A) Experimental design. CD3⁺ T cells were purified from healthy or post-CLP mice on day 6. Then cells were labeled with CTV and transplanted into healthy or post-CLP mice (on day 6). Twenty-four hours later fluorescently labeled cells were analyzed by flow cytometry. (B) Normalized ratio of the number of donor-origin splenic T cells in the lymph nodes or BM of recipient mice versus the number of donor-origin cells in the spleen (n = 5–8 in each group). (C) Normalized ratio of the number of donor-origin BM T cells in the lymph nodes or BM of recipient mice versus the number of donor-origin cells in the spleen. Box-and-whiskers plot presents p25-p75 (box), mean, and p10-p90 (whiskers). Data from 2 independent experiments (n = 5–8 in each group). *P < 0.05, ****P < 0.0001, ^§§§P < 0.001, and ^§§§§P < 0.0001 for differences between CD3 ratio in the lymph nodes of healthy-healthy and other combinations using ANOVA with Tukey’s post hoc test.

Figure 6. IL-7 drives proliferation of CD4⁺CD44⁺CD62L^– T cells in the BM after sepsis.

(A) Kinetics of IL-7 levels in the plasma and BM after sepsis. IL-7 concentration was measured in the sera and BM supernatants of healthy and postseptic mice at different time points (n = 6–8). For plasma: ^#P < 0.05 between 72 hours and 7 days. For BM: *P < 0.05, ***P < 0.001, and ****P < 0.0001 using ANOVA multiple-comparisons with Tukey’s post hoc test. (B) Confocal microscopy photographs of the BM sections show IL-7 (red) and CD4⁺ T cells (green) (representative of n = 4). Scale bar: 50 μm. (C) IL-7 is responsible for the proliferation of CD4⁺CD44⁺CD62L^– T cells after sepsis. Post-CLP mice received anti–IL-7R (anti-CD127) antibody on days 5 and 6 of sepsis and a bolus of BrdU on day 6. BrdU incorporation was evaluated by FACS 24 hours later. Representative plots are shown. Box-and-whiskers plot presents p25-p75 (box), mean, and p10-p90 (whiskers). Dashed line shows range of BrdU⁺CD4⁺CD44⁺CD62L^– cells in control mice. *P < 0.05 (n = 6 per group) using Student’s t test.

Figure 7. Exogenous IL-7 increases proliferation of CD4⁺ T cells including BM CD4⁺CD44⁺ T cells.

(A) Experimental design. On days 4 and 5 after CLP, mice were given 2 injections of recombinant murine IL-7 (rm-IL-7) s.c. and on day 6 a bolus of BrdU i.p. 24 hours later. Cell numbers and proliferation rates were analyzed by flow cytometry. (B) Representative dot plots and frequencies of BrdU-incorporating T cells from the lymph nodes are shown. Absolute counts of CD4⁺ and CD4⁺CD44⁺CD62L^– T cells from 2 axillary and 2 popliteal lymph nodes are shown. (C) Representative dot plots and frequencies of BrdU-incorporating cells from the BM are shown. Absolute counts of CD4⁺ and CD4⁺CD44⁺CD62L^– T cells from 2 femurs are shown. (D) Representative dot plots and frequencies of BrdU-incorporating cells from the spleen are shown. Box-and-whiskers plots present p25-p75 (box), mean, and p10-p90 (whiskers). Data from 2 independent experiments (n = 10 in each group). *P < 0.05, and ***P < 0.001 using Student’s t test.