TNF-α antagonists provide benefit to patients with inflammatory autoimmune disorders such as Crohn’s disease, rheumatoid arthritis, and ankylosing spondylitis. However, TNF antagonism unexplainably exacerbates CNS autoimmunity, including multiple sclerosis and neuromyelitis optica. The underlying mechanisms remain enigmatic. We demonstrate that TNFR2 deficiency results in female-biased spontaneous autoimmune CNS demyelination in myelin oligodendrocyte glycoprotein–specific 2D2 TCR transgenic mice. Disease in TNFR2−/− 2D2 mice was associated with CNS infiltration of T and B cells as well as increased production of myelin oligodendrocyte glycoprotein–specific IL-17, IFN-γ, and IgG2b. Attenuated disease in TNF−/− 2D2 mice relative to TNFR2−/− 2D2 mice identified distinctive roles for TNFR1 and TNFR2. Oral antibiotic treatment eliminated spontaneous autoimmunity in TNFR2−/− 2D2 mice to suggest role for gut microbiota. Illumina sequencing of fecal 16S rRNA identified a distinct microbiota profile in male TNFR2−/− 2D2 that was associated with disease protection. Akkermansia muciniphila, Sutterella sp., Oscillospira sp., Bacteroides acidifaciens, and Anaeroplasma sp. were selectively more abundant in male TNFR2−/− 2D2 mice. In contrast, Bacteroides sp., Bacteroides uniformis, and Parabacteroides sp. were more abundant in affected female TNFR2−/− 2D2 mice, suggesting a role in disease causation. Overall, TNFR2 blockade appears to disrupt commensal bacteria–host immune symbiosis to reveal autoimmune demyelination in genetically susceptible mice. Under this paradigm, microbes likely contribute to an individual’s response to anti-TNF therapy. This model provides a foundation for host immune–microbiota-directed measures for the prevention and treatment of CNS-demyelinating autoimmune disorders.

Summary The double-edged sword nature by which IL-2 regulates autoimmunity and the unpredictable outcomes of anti-TNF therapy in autoimmunity highlight the importance for understanding how TNF regulates IL-2. Transmembrane (tm) TNF preferentially binds TNFR2 while soluble (s) TNF binds TNFR1. We have previously shown reduced IL-2 production in TNFR1−/− TNFR2−/− CD4+ T cells. Here, we generated TNFR1−/−, TNFR2−/−, or TNFR1−/− TNFR2−/− 5C.C7 TCR Il2-GFP mice and report that CD4+ T cell-intrinsic tmTNF/TNFR2 stimulates Il2 promoter activity and Il2 mRNA stability. We further utilize tmTNF Foxp3 GFP reporter mice and pharmacological TNF blockade in wild-type mice to report a tmTNF/TNFR2 interaction for Il2 expression. IL-17 is critical for host defense but its overabundance promotes autoimmunity. IL-2 represses Th17 differentiation but the role for TNFR2 in this process is not well understood. Here, we report elevated expression of TNFR2 under Th17 polarization conditions. Genetic loss-of-function experimental models as well as selective TNF blockade by etanercept and XPro™1595 in wild-type mice demonstrate that impaired tmTNF/TNFR2, but not sTNF/TNFR1, promotes Th17 differentiation in vivo and in vitro. Under Th17 polarizing conditions, elevated IL-17 production by TNFR2 KO CD4+ T cells was associated with increased STAT3 activity and decreased STAT5 activity. Increased IL-17 production in TNFR2 KO T cells was prevented by adding exogenous IL-2. We conclude that CD4+ T cell-intrinsic tmTNF/TNFR2 promotes IL-2 production that inhibits the generation of Th17 cells in a FoxP3-independent manner. Moreover, under Th17 polarizing conditions, selective blockade of CD4+ T cell-intrinsic TNFR2 appears to be sufficient to promote Th17 differentiation.

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Tumor necrosis factor (TNF) plays dualistic pro-inflammatory and immune suppressive roles that lead to unpredictable outcomes of TNF blockade in autoimmune disease. Recent evidence has identified contrasting co-stimulatory effects of TNF on effector T cells and regulatory T cells in controlling autoimmunity. The objective of this study was to determine whether CD4+ T cell autonomous TNFR1 and TNFR2 differentially regulate IL-2 production. We utilize B10.A 5C.C7 TCR Tg Rag2-/- IL-2-GFP reporter mice that are deficient for TNFR1 and/or TNFR2 and memTNFΔ1-12 FoxP3-GFP reporter mice that encode an uncleavable tmTNF to demonstrate that TNFR2, but not TNFR1, and membrane, but not soluble, TNF promote Il2 promoter activity and decrease Il2 mRNA decay to increase IL-2 production in response to CD4+ T cell stimulation. We further demonstrate differential DNA binding of NF-κB in TNFR1- and TNFR2-deficient CD4+ T cells following activation. Adoptive transfer of naive CD4+ T cells from TNFR2-deficient IL-2-GFP heterozygous (IL-2+/-), but not IL-2-GFP homozygous (IL-2-/-), 5C.C7 Rag2-/- TCR Tg mice into B10.A Rag2-/- hosts and subsequent immunization with MCC88-103 + LPS yielded greater IL-17 production upon CD4+ T cell re-challenge in comparison to transfer of TNFR2-sufficient 5C.C7 TCR CD4+ T cells. Taken together, these results suggest that selective blockade of TNFR2 on CD4+ T cells is sufficient to inhibit IL-2 production and promote TH17 differentiation.

Host-commensal interactions influence the development and plasticity of gut-associated T helper (Th) and T regulatory (Treg) cells. In mice, the gut commensal segmented filamentous bacteria (SFB) modulate TH17 and FoxP3+ Treg differentiation to enhance host protection against external pathogens. However, in the context of a susceptible genetic background, SFB has the potential to augment the development of autoimmunity, including multiple sclerosis (MS). We have previously demonstrated that greater than 90% of female, but few male, tumor necrosis alpha receptor type 2 (TNFR2)-deficient mice crossed onto the myelin oligodendrocyte glycoprotein peptide fragment 35-55 (MOG35-55)-specific T cell receptor (TCR) transgenic background rapidly develop spontaneous experimental autoimmune encephalomyelitis (EAE). We now report that oral administration of trimethoprim/sulfamethoxazole, an antibiotic which has broad spectrum of activity against a variety of bacteria prevents the development of spontaneous EAE in TNFR2-deficient 2D2 TCR Tg mice. We have further combined Illumina MiSeq sequencing and RT-PCR to demonstrate a sex-biased microbiome profile in TNFR2-deficient MOG35-55-specific TCR mice, including increased SFB expression in the females relative to their male cohorts. Collectively, these findings suggest a role for TNFR2 in regulating commensal microbiota in the context of myelin autoantigen to control autoreactive T cells and autoimmune demyelination.

Tumor necrosis factor-α (TNF) antagonism has improved the treatment of autoimmune disorders including rheumatoid arthritis and inflammatory bowel disease but is contraindicated in multiple sclerosis (MS) due to disease exacerbation. The mechanisms underlying these contrasting responses remain elusive. The objective of this study was to investigate the effect of selective TNFR2 ablation on the autoreactivity of myelin oligodendrocyte glycoprotein (MOG)-specific T cells. We report that 92% of female C57BL/6 TNFR2-/- MOG-specific (2D2) TCR transgenic mice spontaneously develop experimental autoimmune encephalomyelitis (EAE); whereas, only 8% of male TNFR2-/- 2D2 littermates developed an autoimmune phenotype. Demyelination accompanied B cell and CD4 T cell infiltration in the spinal cords and optic nerves of TNFR2-/- 2D2 mice with EAE. CNS inflammation in these mice correlated with increased production of IFN-γ and IL-17 and elevated sera titers of MOG35-55-specific IgG2b. Ex vivo co-culture studies demonstrated autonomous TNFR2-/- 2D2 CD4 FoxP3+ regulatory T cell (Treg) dysfunction as well as increased resilience by FoxP3-/- 2D2 CD4 T cells to respond to Treg suppression. These results suggest that TNFR2 plays a key role in the functional tolerance of MOG-specific CD4 T and B cells in a sex-related manner. This novel spontaneous autoimmune model provides a unique paradigm to study T and B cell interactions as well as sex-related factors in multiple sclerosis susceptibility.

While IL-17 is critical for host defense, its overabundance promotes autoimmunity. IL-2 represses TH17 differentiation, but a role for tumor necrosis factor (TNF) in this process is not well defined. TNF binds TNF receptor 1 (TNFR1) and TNFR2 to stimulate opposing signaling cascades. Whereas sTNF signals through TNFR1, tmTNF preferentially activates TNFR2. We have previously demonstrated reduced IL-2 production in TNFR1−/− TNFR2−/− double knockout CD4+ T cells. To further explore the mechanism by which TNF regulates IL-2 production, we generated TNFR1−/−, TNFR2−/−, and TNFR1−/− TNFR2−/− 5C.C7 TCR Il2-GFP mice to study Il2 transcription at the single cell level. Our findings indicate that CD4+ T cell–intrinsic tmTNF/TNFR2 stimulates Il2 promoter activity and Il2 mRNA stability. Pharmacological blockade of TNF in wild-type C57BL/6 mice and tmTNF Foxp3-GFP mice also demonstrated tmTNF/TNFR2-mediated augmentation of Il2 expression. We further report elevated expression of TNFR2 on the surface of TH17-polarized CD4+ T cells and show that tmTNF/TNFR2, but not sTNF/TNFR1, inhibits TH17 differentiation. Under TH17-polarizing conditions, elevated IL-17 production by TNFR2-knockout CD4+ T cells correlated with increased STAT3 activity and was prevented by exogenous IL-2. We conclude that increased IL-2 production in response to CD4+ T cell–intrinsic tmTNF/TNFR2 signaling is sufficient to inhibit TH17 differentiation in a Foxp3-independent manner.