Signaling through the adaptor molecule MyD88 in CD4+ T cells is required to overcome suppression by regulatory T cells

Abstract

Innate immune recognition controls adaptive immune responses through multiple mechanisms. The MyD88 signaling adaptor operates in many cell types downstream of Toll-like receptors (TLRs) and interleukin-1 (IL-1) receptor family members. Cell-type-specific functions of MyD88 signaling remain poorly characterized. Here, we have shown that the T cell-specific ablation of MyD88 in mice impairs not only T helper 17 (Th17) cell responses, but also Th1 cell responses. MyD88 relayed signals of TLR-induced IL-1, which became dispensable for Th1 cell responses in the absence of T regulatory (Treg) cells. Treg cell-specific ablation of MyD88 had no effect, suggesting that IL-1 acts on naive CD4(+) T cells instead of Treg cells themselves. Together, these findings demonstrate that IL-1 renders naive CD4(+) T cells refractory to Treg cell-mediated suppression in order to allow their differentiation into Th1 cells. In addition, IL-1 was also important for the generation of functional CD4(+) memory T cells.

Figure 1. Impaired CD4⁺ T cells response in MyD88^T-KO mice

(A) Proliferation of CD4⁺ T cells from MyD88^T-KO mice and wild-type controls. CD4⁺ T cells were isolated from the draining lymph nodes 7 days after immunization in the feet with OVA + LPS in IFA and re-stimulated with OVA in the presence of irradiated splenocytes. Proliferation was measured 3 days later by ³H-thymidine incorporation. A representative of 3 experiments is shown. (B). Cytokine secretion of CD4⁺ T cells following restimulation with OVA. (C). T_H1 response in MyD88^T-KO mice and wild-type controls after immunization with 2W peptide + LPS in IFA and restimulation with 2W peptide. (D). Frequency of 2W:I-A^b+ CD4⁺ T cells in MyD88^T-KO and wild-type control mice T cells 7 days after footpad immunization with 2W peptide + LPS in IFA. (E). Cytokine production of 2W:I-A^b+ CD4⁺ T cells in MyD88^T-KO and wild-type control mice after restimulation with an αCD3e antibody. (F). Absolute numbers of all cells, CD4⁺ T cells, and 2W:I-A^b+ CD4⁺ T cells in the draining lymph nodes of MyD88^T-KO and wild-type controls immunized with 2W peptide + LPS in IFA 7 days earlier.

Figure 2. Proliferation, viability, and differentation of 2W:IA(b)⁺ CD4⁺ T cells from MyD88^T-KO and wild-type control mice

(A) Proliferation of antigen-specific 2W:I-A(b)⁺ CD4⁺ T cells in the draining lymph nodes of MyD88^T-KO and wild-type control mice between day 4–7 after immunization with 2W peptide + LPS in IFA as measured by incorporation of BrdU. Left panel: A representative experiment is shown. Right panel: Statistical representation of all experiments normalized to the level of BrdU incorporation of 2W:I-A(b)⁺ CD4⁺ T cells of MyD88^WT mice. Each dot represents one mouse. (B) Frequency of apoptotic antigen-specific 2W:I-A(b)⁺ CD4⁺ T cells in the draining lymph nodes of MyD88^T-KO and wild-type control mice on day 7 after immunization with 2W peptide + LPS in IFA, measured by staining for active caspase-3. Shown is the mean ± standard deviation. (C) T-bet expression by qPCR in immunized CD4+ T cells in the draining lymph nodes of MyD88T-KO and wild-type control mice. A representative experiment using the pooled samples from 5–10 mice per genotype is shown.

Figure 3. Impaired CD4⁺ T cells response in MyD88^T-KO mice is caused by defective IL-1 signaling

(A, B) Expression of the receptors for IL-1 (A) and IL-18 (B) in CD4⁺ T cells in the draining lymph nodes of immunized MyD88^T-KO and wild-type control mice as measured by qPCR. The expression levels were normalized to the level of naïve CD4⁺ T cells. A representative experiment using the pooled samples from 5–10 mice per genotype is shown. (C, D) CD4⁺ T cell response of Rag2-deficient mice reconstituted with a 3:1 mix of bone marrow from TCRβ-deficient mice and IL1R^KO, IL18^KO, or TLR2^KO; TLR4^KO mice, respectively. CD4⁺ T cells were restimulated with OVA in the presence of irradiated splenocytes 7 days after the immunzation of the bone marrow chimeras in the feet with OVA + LPS in IFA. The proliferation was measured 3 days later by ³H-thymidine incorporation (C) and the secretion of IFNγ and IL-17 of the restimulated CD4⁺ T cells was measured by ELISA (D). A epresentative experiment is shown. (E, F). CD4⁺ T cell response of mixed bone marrow chimeras harboring bone marrow of IL1R^KO, IL18^KO, or wild-type mice mixed with bone marrow of TCRβ^KO mice in a ratio of 1:3 after footpad immunization with OVA + LPS in IFA. A representative experiment is shown.

Figure 4. Induction of a T_H1 response requires T cell-specific MyD88 signaling in naïve or effector CD4⁺ T cells in order to overcome Treg-mediated suppression

(A, B) Restoration of the T_H1 response after transient depletion of CD25⁺ Tregs in MyD88^T-KO mice. MyD88^T-KO and control mice were immunized with OVA + LPS in IFA in the footpads following the injection of an αCD25 antibody 3 days earlier in order to deplete CD25⁺ Tregs. CD4⁺ T cells were isolated 7 days after immunization and restimulated with OVA in vitro in the presence of irradiated splenocytes. Proliferation (A) and cytokine secretion (B) of the CD4⁺ T cells were measured 3 days later. A representative experiment is shown. (C, D) CD4⁺ T cell response in MyD88^TREG-KO mice and wild-type controls. The mice were immunized with OVA + LPS in IFA, the proliferation (C) and cytokine secretion (D) was measured as described before. A representative experiment is shown.

Figure 5. Frequency of Tregs in MyD88^T-KO mice

(A) Frequency of FoxP3⁺ Tregs as percentage of CD4⁺ T cells in unimmunized wild-type and MyD88T-KO mice. A representative of 3 independent experiments is shown. (B) Frequency of FoxP3⁺ Tregs after immunization with OVA + LPS in IFA and subsequent restimulation of isolated CD4+ T cells with OVA in the presence of irradiated splenocytes. An experiment representing 3 independent experiments is shown. (C) Frequency of FoxP3⁺ Tregs in wild-type and MyD88^T-KO mice in the draining lymph nodes 7 days after immunization with OVA + LPS in IFA. A representative of three experiments is shown. (D, E) Frequency of antigen-specific FoxP3⁺ 2W:I-A(b)⁺ Tregs in the draining lymph nodes 7 days after immunization with 2W peptide + LPS in IFA, shown as percentage of all CD4⁺ T cells (D) or percentage of all 2W:I-A(b)⁺ CD4+ T cells (E). A representative of 3 experiments is shown. (F) Absolute numbers of FoxP3⁺ 2W:I-A(b)⁺ Tregs in the draining lymph nodes 7 days after immunization with 2W peptide + LPS in IFA.

Figure 6. Memory CD4⁺ T cell response in MyD88^T-KO mice

(A, B) Frequency of 2W:I-A(b)⁺ memory CD4⁺ T cells in MyD88^T-KO mice and wild-type controls. The frequency of 2W:I-A(b)⁺ CD4⁺ T cells and 2W:I-A(b)⁺ memory CD4⁺ T cells was determined 40–60 days after immunization with 2W peptide + LPS in IFA. The mice were immunized either in the presence (− αCD25) or transient absence (+ αCD25) of Tregs during the primary immunization. (A). Frequency of 2W:I-A(b)⁺ CD4⁺ T cells as a percentage of total CD4⁺ T cells (upper panels) and frequency of 2W:I-A(b)⁺ CD44⁺ CD62L⁺ memory CD4⁺ T cells as a percentage of total 2W:I-A(b)⁺ CD4⁺ T cells (lower panels). Values represent mean ± standard error. (B). Frequency of 2W:I-A(b)⁺ CXCR5⁻ PD-1⁻ effector memory CD4⁺ T, 2W:I-A(b)⁺ CXCR5⁺ PD-1⁻ central memory CD4⁺ T cells, and 2W:I-A(b)⁺ CXCR5⁺ PD-1⁺ T follicular helper (T_FH) cells as a percentage of total 2W:I-A(b)⁺ CD4⁺ T cells. A representative experiment is shown. (C, D). CD4⁺ memory T cell response after transient depletion of CD25⁺ Tregs prior to both the primary and secondary immunization. MyD88^T-KO mice and control mice were injected with an αCD25 antibody 3 days prior to the primary immunization with OVA + LPS in IFA. After 60 days, Tregs were transiently depleted again and the mice were challenged 3 days later with OVA + LPS in IFA. CD4⁺ T cells were isolated 7 days later and restimulated with OVA in vitro in the presence of irradiated splenocytes. The proliferation measured by ³H-thymidine incorporation (C) and cytokine secretion measured by ELISA are shown (D). The data reflect representative results from 2 independent experiments involving a total of 4 mice per genotype.

Figure 7. Gene expression analysis of MyD88^T-KO and IL6Rα^T-KO mice compared to wild-type controls

(A) RNASeq of antigen-specific 2W:I-A(b)⁺ CD4⁺ T cells from the draining lymph nodes of MyD88^T-KO, IL6Rα^T-KO, and wild-type control mice that were isolated by flow cytometry 7 days after immunization with 2W peptide + LPS in IFA. (B) Principal component analysis MyD88^T-KO, IL6Rα^T-KO, and wild-type control mice.