Alterations in antigen-specific naive CD4 T cell precursors after sepsis impairs their responsiveness to pathogen challenge

Abstract

Patients surviving the acute stages of sepsis develop compromised T cell immunity and increased susceptibility to infection. Little is known about the decreased CD4 T cell function after sepsis. We tracked the loss and recovery of endogenous Ag-specific CD4 T cell populations after cecal ligation and puncture-induced sepsis and analyzed the CD4 T cell response to heterologous infection during or after recovery. We observed that the sepsis-induced early loss of CD4 T cells was followed by thymic-independent numerical recovery in the total CD4 T cell compartment. Despite this numerical recovery, we detected alterations in the composition of naive CD4 T cell precursor pools, with sustained quantitative reductions in some populations. Mice that had experienced sepsis and were then challenged with epitope-bearing, heterologous pathogens demonstrated significantly reduced priming of recovery-impaired Ag-specific CD4 T cell responses, with regard to both magnitude of expansion and functional capacity on a per-cell basis, which also correlated with intrinsic changes in Vβ clonotype heterogeneity. Our results demonstrate that the recovery of CD4 T cells from sepsis-induced lymphopenia is accompanied by alterations to the composition and function of the Ag-specific CD4 T cell repertoire.

FIGURE 1. Numerical recovery of CD4 T cells after cecal ligation and puncture (CLP)-induced sepsis occurs by a thymus-independent mechanism

A. Kaplan-Meier survival curve of experimental cohorts after undergoing sham or CLP surgery. B. Number of CD4 T cells in spleen and inguinal lymph nodes (pLN) on d 2 and 30 after sham or CLP surgery. C. Thymectomized and euthymic mice underwent CLP surgery, and the number of CD4 T cells in the peripheral blood was measured over time. D. Thymectomized and euthymic mice underwent sham or CLP surgery. BrdU was injected i.p. 6 d later, and the frequency of peripheral blood CD4 T cells incorporating BrdU was determined 24 h later. Statistical significance was determined using Mann-Whitney U test (A) or one-way ANOVA (B-D) with multiple-testing correction using the Holm-Sidak method, and α = 0.05, when deemed appropriate. **** p < 0.001; *** p < 0.005; * p < 0.05; and n.s. – not significant. Data shown are representative of at least 2 independent experiments of 4-5 mice/group in each experiment.

FIGURE 2. Numerical recovery of CD4 T cells after sepsis is accompanied by the acquisition of an “Ag-experienced” phenotype in an Ag-independent manner

A. The phenotype of CD4 T cells in the spleen was assessed 30 d after sham or CLP surgery. Representative flow cytometry plots depicting CD11a/CD49d and CD44/CD127 expression in CD4 T cells 30 d after sham or CLP surgery. B. Frequency of CD11a^hi CD49d^hi and CD44^h CD127^hi CD4 T cells 2 and 30 d after sham or CLP surgery. C. Salmonella FliC_447-458-specific Sm1 (CD90.1/CD90.1; 5 × 10⁵/mouse) and LCMV gp_61-77-specific SMARTA (CD90.1/CD90.2; 10⁶/mouse) CD4 T cells were adoptively transferred together into naïve CD90.2 B6 mice 1 d before sham or CLP surgery. After 30 d, the frequency of CD11a^hi CD49d^hi endogenous CD90.2/CD90.2 CD4 T cells and adoptively transferred TCR-tg CD4 T cells was determined. Statistical significance was determined using one-way ANOVA with multiple-testing correction using the Holm-Sidak method, and α= 0.05, when deemed appropriate. **** p < 0.001; ** p < 0.01; * p < 0.05; and n.s. – not significant. Data shown are representative of at least 2 independent experiments of 4-5 mice/group in each experiment.

FIGURE 3. Numerical recovery of CD4 T cells after sepsis is accompanied by asymmetric changes in Ag-specific repertoire heterogeneity

Mice underwent sham or CLP surgery, and the number of Ag-specific CD4 T cells specific for 2W1S, L. monocytogenes LLO_190-201, influenza A virus NP_311-325, LCMV gp_66-77, HSV gD_290-302, and OVA_323-339 was determined 2 and 30 d later using p:I-A^b tetramer enrichment. A. Representative flow plots showing gating strategy used in tetramer-enriched cell fractions to detect the frequency of Ag-specific CD4 T cell populations. Shown is an example used to detect 2W1S:I-A^b-specific CD4⁺ T cells. Gating for p:I-A^b-specific cells was determined using CD8⁺ T cells as an internal negative control for tetramer binding. B. Number of Ag-specific, naïve CD4 T cell precursors across the 6 epitopes in sham- and CLP-treated mice 2 or 30 d after surgery. Statistical significance was determined using group-wise, one-way ANOVA with multiple-testing correction using the Holm-Sidak method, and α = 0.05. **** p < 0.001; and n.s. – not significant. Data shown are the combined results from 2-4 independent experiments per population analyzed, with 3-5 mice/group in each experiment.

FIGURE 4. Expansion of epitope-specific populations correlates with precursor pool recovery after septic injury

A. Experimental design. Mice were infected with 2W1S-expressing C. albicans (C. albicans-2W; 5×10⁴ yeasts in 0.1 ml i.v.), attenuated 2W-expressing L. monocytogenes (Lm-2W or Lm-OVA; 10⁷ CFU in 0.1 ml i.v.), HSV-1 (2.5×10⁴ PFU in 0.1 ml i.v.), or influenza A virus (×31; 3000 EID50 in 0.02 ml i.n.) 30 d after sham or CLP surgery. After another 7-12 d, the frequency and number of Ag-specific CD4 T cells was determined in the spleen. B-C. Representative flow plots showing the frequency (B) and number (C) of 2W1S-specific CD4 T cells in the spleens from sham- and CLP-treated mice 7 d after i.v. infection with C. albicans-2W. D-E. Representative flow plots showing the frequency (D) and number (E) of LLO₁₉₀- and 2W1S-specific CD4 T cells in the spleens from sham- and CLP-treated mice 7 d after i.v. infection with Lm-2W. F-G. Representative flow plots showing the frequency (F) and number (G) of OVA₃₂₃-specific CD4 T cells in the spleens from sham- and CLP-treated mice 7 d after i.v. infection with Lm-OVA. H-I. Representative flow plots showing the frequency (H) and number (I) of gD₂₉₀-specific CD4 T cells in the spleens from sham- and CLP-treated mice 9 d after i.v. infection with HSV-1. J-K. Representative flow plots showing the frequency (J) and number (K) of NP₃₁₁-specific CD4 T cells in the lungs and spleens from sham- and CLP-treated mice 12 d after i.n. infection with ×31. Statistical significance was determined using group-wise, one-way ANOVA analyses followed by multiple-testing correction using the Holm-Sidak method, with α = 0.05. ** p < 0.01; * p < 0.05; and n.s. – not significant. Data shown are the combined results from 2-4 independent experiments per pathogen tested, with 3-5 mice/group in each experiment.

FIGURE 5. Inadequate recovery of precursor population correlates with inadequate CD4 T cell function after pathogen challenge

A. Experimental design. Mice were infected with attenuated 2W- or OVA-expressing L. monocytogenes (Lm-2W or Lm-OVA; 10⁷ CFU in 100 μl i.v.) 2 or 30 d after sham or CLP surgery. After another 7d, the mice were injected i.v. with 100 μg 2W1S_56-68 or OVA_323-339 peptide. Spleens were harvested 2 h later, and the frequency and number of IFNγ⁺ or TNFα⁺IFNγ⁺ CD44⁺2W:I-A^b+ or OVA₃₂₃:I-A^b+ CD4 T cells was determined. B. Representative flow plots of intracellular IFNγ and TNFα detection in the CD44⁺2W:I-A^b+ CD4 T cells after in vivo peptide restimulation. Plots show cells gated from internal control populations (CD44^lo2W:I-A^b- CD4 T cells) denoted as background (“BKG”) or 2W:I-A^b-enriched CD4 T cells from sham- or CLP-treated mice. C-D. Frequency and number of CD44⁺2W:I-A^b+-specific CD4 T cells in the spleen producing IFNγ (C) or TNFα and IFNγ (D). E. Representative flow plots of intracellular IFNγ and TNFα detection in the CD44⁺OVA₃₂₃:I-A^b+ CD4 T cells after in vivo peptide restimulation. Plots show cells gated from internal control populations (CD44^loOVA₃₂₃:I-A^b− CD4 T cells) denoted as background (“BKG”) or OVA₃₂₃:I-A^b-enriched CD4 T cells from sham- or CLP-treated mice. F. Frequency and number of CD44⁺ OVA₃₂₃:I-A^b+-specific CD4 T cells in the spleen producing TNFα and IFNγ. Statistical significance was determined using group-wise, one-way ANOVA analyses followed by Holm-Sidak correction with α = 0.05. **** p < 0.001; *** p < 0.005; and ** p < 0.01. Data shown are the combined results of 2 independent experiments, with 3-5 mice/group in each experiment.

FIGURE 6. Ag-specific CD4 T cells have functional deficits in Th17-polarized responses

A. Experimental design. On d 30 after sham or CLP surgery, mice were infected epicutaneously with 2W1S-expressing C. albicans (C. albicans-2W; 10⁸ yeasts in 0.05 ml). After 7 d, lymphocytes obtained from the skin-draining (inguinal, brachial, axillary and cervical) LN of infected mice were stimulated for 4 h with PMA/ionomycin. The stimulated samples were enriched for 2W1S-specific CD4 T cells and production of IFNγ and IL-17A was assayed by flow cytometry. B. Representative flow plots showing the gating strategy to identify IL-17A⁺IFNγ⁻ cells within bulk and 2W:I-A^b-specific CD4 T cells. C-D. Frequency and number of IL-17A⁺ in bulk (C) and 2W1S-specific CD4 T cells (D) in infected sham- or CLP-treated mice. Statistical significance was determined using Welch”s t-test. ** p < 0.01; * p < 0.05; and n.s. – not significant. Data shown are representative results from 2 independent experiments, with 5 mice/group in each experiment.

FIGURE 7. Sepsis alters the T cell receptor clonotype composition of Ag-specific CD4 T cell population

A. Experimental design. Mice were injected i.v. with 50 μg 2W_56-68 peptide (along with LPS) 30 d after sham or CLP surgery. Splenocytes were harvested 4 d later and tetramer-enriched as previously described. The resultant sample was then used to determine the clonotype composition using two multiplexed flow cytometry panels consisting of the indicated murine TCR Vβ mAb. B. Representative flow plots showing the gating strategy to identify Vβ usage on 2W:I-A^b-specific CD4 T cells using TCR panel #1. C. Usage profile for TCR Vβ gene segments in total 2W:I-A^b-specific CD4 T cells from 3 representative individual sham- or CLP-treated mice. D. Averaged frequency of TCR Vβ of 2W:I-A^b-specific CD4 T cells in sham- or CLP-treated mice. Statistical significance was determined using group-wise, one-way ANOVA analyses followed by Holm-Sidak correction with α = 0.05. ** p < 0.01; and * p < 0.05. Data are combined from 2 independent experiments, each having 5 mice/group.