IL-6 and TNF-α synergized to impair the suppressive function of Tregs. (A) CD4⁺CD25⁻ T cells were stimulated with soluble anti-CD3 with or without the co-culture of CD4⁺CD25⁺ Tregs. Recombinant IL-6, TNF-α, or both were added to the cultures. Results are presented as counts per minute or percentage suppression of T effector proliferation mediated by Tregs. (B) C57BL/6 CD4⁺CD25⁻ T cells were stimulated with BALB/c APCs with or without co-culture of CD4⁺CD25⁺ Tregs. Recombinant IL-6, TNF-α, or both were added to the cultures. Results are presented as counts per minute or percentage suppression of T effector proliferation mediated by Tregs. (C) CD4⁺CD25⁻ T cells were stimulated with soluble anti-CD3 and cultured alone or co-cultured with increasing numbers of CD4⁺CD25⁺ Tregs from C57BL/6 WT and IL-6^−/−TNF-α^−/− mice, and proliferation was measured. IL-6^−/−TNF-α^−/− Tregs exhibited a similar ability to suppress T effector proliferation compared with WT Tregs.

Inhibition of IL-6 and TNF-α impairs the ability of TLR-dependent cytokines to augment T cell alloimmunity. Conditioned media (CM) were harvested from LPS-activated BMDCs and used to perform an MLR. Inhibition of either IL-6 or TNF-α reduced T cell proliferation (A) or T cell IFN-γ (B) production. Similar results were noted for CD4⁺ and CD8⁺ T cells. Isotype control antibodies did not inhibit T cell responses in this assay (data not shown). *P < 0.05.

T cell intrinsic IL-6 and TNF-α are dispensable for T cell alloimmunity. (A) Polyclonal T cells were purified from C57BL/6 WT, IL-6^−/−, TNF-α^−/−, and IL-6^−/−TNF-α^−/− mice and stimulated by allogeneic APCs in standard media (lacking cytokines). No differences in T cell proliferation were noted between the groups. (B) Polyclonal T cells (1 × 10⁶) were purified from either C57BL/6 WT or IL-6^−/−TNF-α^−/− mice and adoptively transferred via intravenous tail vein to syngeneic RAG^−/−OTI TCR transgenic mice, which received a BALB/c skin allograft. The rate of graft rejection between the groups was similar. Nonadoptively transferred mice did not reject their allografts.

Deletion of IL-6 and TNF-α reduces the ability of TLR-dependent cytokines to augment T cell antigen–specific, alloimmune responses. Addition of IL-6 and TNF-α together augments T cell alloimmunity versus either cytokine alone. (A) CM were harvested from WT, IL-6^−/−, TNF-α^−/−, and IL-6^−/−TNF-α^−/− TLR-activated BMDCs and used to culture T cells in an MLR. The absence of IL-6 but not TNF-α reduced T cell responses in this assay (*P < 0.05). Absence of both cytokines further reduced T cell responses to a small degree (*P < 0.05). (B) TLR4^−/− T cells were cultured in standard media or in CM harvested from WT, IL-6^−/−, TNF-α^−/−, and IL-6^−/−TNF-α^−/− TLR-activated BMDCs without the addition of allogeneic APCs, and T cell proliferation was measured. The use of TLR4^−/− T cells (i.e., hyporesponsive to LPS) was to assess the ability of the CM (which contained LPS) to promote T cell proliferation independent of the effect of LPS on T cells. (C) Addition of both IL-6 and TNF-α to the MLR augmented T cell alloimmune responses as compared with T cells stimulated in standard media, devoid of cytokines (*P < 0.05). Addition of either cytokine augmented T cell alloimmune proliferation to a small degree, which was less than that induced by both cytokines (*P < 0.05).

Inflammatory cytokines augment T cell proliferation in an IRAK-M–dependent manner. IRAK-M^−/− transplant recipients resist the graft-prolonging effects of anti-CD45RB and anti-CD154 to extend allograft survival. (A) WT and IRAK-M^−/− macrophages were stimulated with LPS, and CM were harvested. These media were used to culture T cells that were stimulated with allogeneic APCs. IRAK-M^−/− CM augmented T cell alloimmune proliferation versus WT CM (*P < 0.05). This difference was blocked by either IL-6 or TNF-α inhibition. (B) C57BL/6 IRAK-M^−/− recipients of BALB/c skin allografts that were treated perioperatively with anti-CD45RB and anti-CD154 rejected their allografts at a faster rate than similarly treated WT recipients (P = 0.03, log rank). Both groups rejected their allografts at a similar rate when co-stimulatory blockade was not administered (data not shown). (C) Bulk purified T cells from recipient mice in B were harvested 21 d after transplantation and were re-stimulated with irradiated BALB/c or third-party CBA (H2^K) APCs. IFN-γ response was then measured via ELISPOT. T cells from IRAK-M^−/− recipients exhibited an enhanced donor-specific IFN-γ versus WT recipients (P = 0.03; n = 3 per group, two independent experiments).

Absence of both recipient IL-6 and TNF-α synergizes with the administration of co-stimulatory blockade to induce permanent allograft acceptance. (A and B) C57BL/6 WT, IL-6^−/−, TNF-α^−/−, and IL-6^−/−TNF-α^−/− recipients received a BALB/c skin allograft and were untreated (A) or administered co-stimulatory blockade (CB; CTLA4-Ig and anti-CD154) (B). Without co-stimulatory blockade, all experimental groups rejected their allografts at a similar rate. When co-stimulatory blockade was administered, most IL-6^−/−TNF-α^−/− recipients accepted their allograft for >100 d (P < 0.0001, log rank versus WT; P = 0.004 versus IL-6^−/−). Only a minority of IL-6^−/− recipients exhibited extended allograft survival compared with the WT group, although this reached statistical significance (P = 0.01). TNF-α^−/− rejected their allografts at a similar rate to WT. (C) Full-thickness C57BL/6 WT and IL-6^−/−TNF-α^−/− skin was transplanted to BALB/c recipients, which were administered CTLA4-Ig and anti-CD154. There was no difference in allograft survival times between the groups. (D and E) C57BL/6 WT, IL-6^−/−, TNF-α^−/−, and IL-6^−/− TNF-α^−/− recipients (n > 3 per group) received a BALB/c skin allograft and were treated with co-stimulatory blockade (CTLA4-Ig and anti-CD154). At day 21 after transplantation, bulk T cells, CD4⁺ and CD8⁺ T cells, were purified from spleens and stimulated ex vivo with irradiated donor spleen cells for <24 h. T cell proliferation (D) and IFN-γ responses were then measured (E). T cells purified from and IL-6^−/−TNF-α^−/− recipients manifest significantly reduced responses versus the other groups in all of the assays (P < 0.05).