TNF receptor-associated factor 3 is required for T cell-mediated immunity and TCR/CD28 signaling

Abstract

We recently reported that TNFR-associated factor (TRAF)3, a ubiquitously expressed adaptor protein, promotes mature B cell apoptosis. However, the specific function of TRAF3 in T cells has remained unclear. In this article, we report the generation and characterization of T cell-specific TRAF3(-/-) mice, in which the traf3 gene was deleted from thymocytes and T cells. Ablation of TRAF3 in the T cell lineage did not affect CD4 or CD8 T cell populations in secondary lymphoid organs or the numbers or proportions of CD4(+),CD8(+) or double-positive or double-negative thymocytes, except that the T cell-specific TRAF3(-/-) mice had a 2-fold increase in FoxP3(+) T cells. In striking contrast to mice lacking TRAF3 in B cells, the T cell TRAF3-deficient mice exhibited defective IgG1 responses to a T-dependent Ag, as well as impaired T cell-mediated immunity to infection with Listeria monocytogenes. Surprisingly, we found that TRAF3 was recruited to the TCR/CD28 signaling complex upon costimulation and that TCR/CD28-mediated proximal and distal signaling events were compromised by TRAF3 deficiency. These findings provide insights into the roles played by TRAF3 in T cell activation and T cell-mediated immunity.

Figure 1. Normal thymocytes and peripheral B and T cell populations in T-TRAF3^−/− mice

(A) Detection of the excision of exons 1 and 2 of the TRAF3 gene (TRAF3⁻ allele) in thymocytes by genomic PCR analysis using primers U7 + BT6. A 2.6 kb PCR product was amplified from the TRAF3^flox allele, while a 645 bp PCR product was amplified from the TRAF3⁻ allele. (B) Verification of TRAF3 deletion in thymocytes and splenic T cells by Western blot analysis. Splenic pan T, CD8⁺, and CD4⁺ T cells were purified from LMC and T-TRAF3^−/− (T-T3^−/−) mice by negative selection using magnetic beads. Total cellular proteins were extracted from thymocytes, purified splenic non-T cells (N), T cells (T), CD8⁺, or CD4⁺ T cells. Each protein blot was first immunoblotted for TRAF3, then stripped and re-probed for TRAF2, TRAF1, TRAF6 and actin. NS, non-specific band. (C) Representative FACS profiles of thymocytes from LMC and T-TRAF3^−/− mice examined for CD4 and CD8 expression. (D) Percentages and numbers of CD4⁻CD8⁻ (DN), CD4⁺CD8⁺ (DP), CD4+, and CD8⁺ cells in the thymus of LMC and T-TRAF3^−/− mice. (E) Slight increase in spleen weight of T-TRAF3^−/− mice. (F) Percentages and numbers of B and T cells in spleens and LN of LMC and T-TRAF3^−/− mice. B cells and T cells were identified by FACS analysis using B220 and CD3, respectively. Data shown are results of five independent experiments (mean ± SEM). Mice analyzed were 8 to 12 weeks old.

Figure 2. Increased Treg cells, and altered CD44 and CD43 expression profiles on peripheral CD8 and CD4 T cells in T-TRAF3^−/− mice

(A) Representative FACS profiles of splenocytes from LMC and T-TRAF3^−/− mice stained for CD4, CD25 and Foxp3 expression. (B) Percentages and numbers of CD4 (CD4⁺CD8⁻CD25⁻), CD8 (CD4⁻ CD8⁺), and Treg (CD4⁺CD25⁺Foxp3⁺) cells in spleens and LN of LMC and T-TRAF3^−/− mice. Data shown are results of four independent experiments (mean ± SEM). (C) Representative FACS profiles of CD44 expression on CD8⁺ or CD4⁺ T cells in spleens, LN, and thymus of LMC (solid profile) and T-TRAF3^−/− (dashed profile) mice. (D) Representative FACS profiles of CD43 expression on CD8⁺ or CD4⁺ T cells in spleens, LN, and thymus of LMC (solid profile) and T-TRAF3^−/− (dashed profile) mice. Mice analyzed were 8 to 12 weeks old.

Figure 3. Defective T-dependent IgG1 response in T-TRAF3^−/− mice

LMC and T-TRAF3^−/− mice (8–10 weeks old, n=10 for each group) were immunized with the TD Ag TNP-KLH/Alum, and boosted on d21 after the first immunization. Sera were collected on d7, 14 and 28 after the first immunization. Serum levels of anti-TNP IgM and IgG1 were measured by ELISA. Multiple serial dilutions of each serum sample were tested to ensure the readout is within the linear range of the assay.

Figure 4. Impaired primary T cell responses to Listeria monocytogenes infections in T-TRAF3^−/− mice

(A) Survival rate of LMC and T-TRAF3^−/− mice after primary infection with 5×10³ CFU of virulent LM. (B) Bacterial numbers in livers on d7 post infection. (C) Representative FACS profiles of Ag-specific splenic CD8 and CD4 T cells on d7 post infection, determined in the absence or presence of peptide stimulation. (D) Percentages (among total splenocytes) and numbers of Ag-specific (IFNγ-producing or TNFα-producing) CD8 and CD4 T cells in spleens of LMC and T-TRAF3^−/− mice on d7 post primary infection. (E) Percentages and numbers of Ag-specific T cells in spleens of LMC and T-TRAF3^−/− mice on d10 post primary infection. The graph depicts the results of three independent experiments (mean ± SEM). Mice infected were 2 to 3 months old.

Figure 5. Diminished proliferation and enhanced apoptosis of splenic CD4 T cells following stimulation through CD3 and CD28

Splenic CD4⁺ T cells were purified from 2–3 month-old LMC and T-TRAF3^−/− mice, and Treg cells were depleted using anti-CD25 magnetic beads. Cells were cultured ex vivo in the absence or presence of stimulation with 0.5 μg/ml of plate-bound α-CD3 mAb, alone or in combination with 2 μg/ml of soluble α-CD28 mAb. (A) T cell survival ex vivo. The numbers of viable cells at each time point were determined by staining with Trypan blue. Data shown are results of three independent experiments (mean ± SEM). (B) Cell cycle analysis by PI staining and FACS. Representative histograms of PI staining are shown, and percentage of apoptotic cells (DNA content < 2n) and proliferating cells (2n < DNA content 4n) are indicated. (C) Western blot analysis of Bcl-xL, Bcl2 and Bim. Splenic pan T cells were purified from 2–3 month-old LMC and T-TRAF3^−/− mice by negative selection, and Treg cells were depleted using anti-CD25 Ab magnetic beads. Cells were stimulated with anti-CD3+anti-CD28 Abs at 37°C for indicated time periods. Total cellular lysates were immunoblotted for phosphorylated Bcl-xL, Bcl2, and Bim, followed by actin.

Figure 6. Decreased cytokine production by splenic CD4 and CD8 T cells following stimulation through CD3 and CD28

(A) Splenic CD4 T cells were purified from 2–3 month-old LMC and T-TRAF3^−/− mice, and Treg cells were depleted using anti-CD25 magnetic beads. (B) Splenic CD8 T cells were purified from 2–3 month-old LMC and T-TRAF3^−/− mice by negative selection. Cells were cultured ex vivo in the absence or presence of stimulation with 0.5 μg/ml of plate-bound α-CD3 mAb, alone or in combination with 2 μg/ml of soluble α-CD28 mAb. Levels of cytokines in the culture supernatants were measured by ELISA. Results shown are representative of at least 2 independent experiments.

Figure 7. TRAF3 is a proximal signaling component of TCR and CD28

(A) Splenic pan T cells were purified from 2–3 month-old LMC and T-TRAF3^−/− mice by negative selection, and Treg cells were depleted using anti-CD25 magnetic beads. Cells were stimulated with anti-CD3, or anti-CD3+anti-CD28, followed by a crosslinking Ab as described in the Methods at 37°C for indicated time periods. Total cellular lysates were immunoblotted for phosphorylated (P-) or total ERK, LAT, PLCγ1, ZAP70, and IκBα, followed by TRAF3. (B) Splenic naive Thy1⁺CD25⁻CD44^low T cells were sorted from 2–3 month-old LMC and T-TRAF3^−/− mice using a BD FACS Aria II. Cells were stimulated with anti-CD3+anti-CD28 Abs, followed by a crosslinking Ab as described in the Methods at 37°C for indicated time periods. Total cellular lysates were immunoblotted for phosphorylated (P-) or total ERK, LAT, PLCγ1, and ZAP70, followed by TRAF3. (C) Splenic pan T cells were purified from 2–3 month old LMC mice by negative selection. Cells were stimulated with anti-CD3, anti-CD28, or anti-CD3 + anti-CD28 mAbs, followed by Dynabeads coated with a crosslinking Ab as described in the Methods at 37°C for 3 minutes. The immunoprecipitates were analyzed by immunoblotting for TRAF3, Malt1, TRAF6 and ZAP70. (D) Splenic pan T cells were purified from 2–3 month-old LMC and T-TRAF3^−/− mice by negative selection. Cells were stimulated with anti-CD3 + anti-CD28 mAbs, followed by Dynabeads coated with a crosslinking Ab as described in the Methods at 37°C for indicated time periods. The immunoprecipitates and residual lysates after immunprecipitation were analyzed by immunoblotting for TRAF3 and ZAP70, followed by Actin.