A longstanding goal has been to find an antigen-specific preventive therapy, i.e., a vaccine, for autoimmune diseases. It has been difficult to find safe ways to steer the targeting of natural regulatory antigen. Here, we show that the administration of exogenous mouse major histocompatibility complex class II protein bounding a unique galactosylated collagen type II (COL2) peptide (A ^q –galCOL2) directly interacts with the antigen-specific TCR through a positively charged tag. This leads to expanding a VISTA-positive nonconventional regulatory T cells, resulting in a potent dominant suppressive effect and protection against arthritis in mice. The therapeutic effect is dominant and tissue specific as the suppression can be transferred with regulatory T cells, which downregulate various autoimmune arthritis models including antibody-induced arthritis. Thus, the tolerogenic approach described here may be a promising dominant antigen-specific therapy for rheumatoid arthritis, and in principle, for autoimmune diseases in general.

B cells undergo several rounds of selection to eliminate potentially pathogenic autoreactive clones, but in contrast to T cells, evidence of positive selection of autoreactive B cells remains moot. Using unique tetramers, we traced natural autoreactive B cells (C1-B) specific for a defined triple-helical epitope on collagen type-II (COL2), constituting a sizeable fraction of the physiological B cell repertoire in mice, rats, and humans. Adoptive transfer of C1-B suppressed arthritis independently of IL10, separating them from IL10-secreting regulatory B cells. Single-cell sequencing revealed an antigen processing and presentation signature, including induced expression of CD72 and CCR7 as surface markers. C1-B presented COL2 to T cells and induced the expansion of regulatory T cells in a contact-dependent manner. CD72 blockade impeded this effect suggesting a new downstream suppressor mechanism that regulates antigen-specific T cell tolerization. Thus, our results indicate that autoreactive antigen-specific naïve B cells tolerize infiltrating T cells against self-antigens to impede the development of tissue-specific autoimmune inflammation.

Studying how single nucleotide polymorphisms (SNPs) crosstalk with non-autologous factors to cause complex autoimmune diseases is challenging. An amino acid replacement in the neutrophil cytosolic factor 1 (NCF1-339/NCF1 ^R90H ) leading to lower reactive oxygen species induction has been reported as the major SNP for systemic lupus erythematosus (SLE). Here we show that infection with the murine norovirus (MNV) contributes to the induction of lupus in Ncf1 ^90H mice. Mutant NCF1^90H upregulates the IFN-α/JAK1/STAT1 pathway in macrophages and anti-MNV-antibody production. In parallel, the MNV infection of NCF1^90H mice upregulates Toll-like receptor 7 in macrophages, plasmacytoid dendritic cells and B220⁺ splenocytes, thereby promoting germinal center formation and lupus-associated autoantibodies production. These compounded effects lead to protection against MNV infection but also glomeruloneph ritis with proteinuria and lupus arthritis in the absence of chemical inducers such as pristane. Our data thus suggest that this SLE-associated SNP, NCF1^90H, synergizes with MNV infection to induce the development of mouse lupus.

Auranofin, an FDA-approved antirheumatic drug and thioredoxin reductase 1 (TXNRD1) inhibitor, has demonstrated anti-tumoral properties, but its immunological effects are not well characterized. Here, we report that auranofin unexpectedly promotes regulatory T cell (Treg) expansion. In a B16F10 melanoma model, auranofin treatment increased lung tumor coverage, IL-10 serum levels, and FOXP3⁺CD44⁺CD4⁺ T cell frequencies. It also altered the proportion of antigen-presenting cells (APCs), increasing B cells and reducing dendritic cells. To test whether Treg expansion occurs independently of tumor antigens, we stimulated T cells ex vivo in lymph node cultures from naïve mice using anti-CD3/CD28, with or without auranofin. Auranofin increased Treg frequency in these cultures, as well as in treated human PBMCs. Similar effects were observed with the TXNRD1 inhibitor TRi-1, suggesting a ROS-dependent mechanism. Using mice with conditional expression of neutrophil cytosolic factor 1 (NCF1), we found that both TXNRD1 inhibition and APC-specific NCF1-NOX2-ROS expression enhanced tumor burden and Treg expansion. Alternatively, sorted T cells from mice harboring conditional TXNRD1 knockouts showed reduced FOXP3 and GITR expression in the naïve state and reduced tumor burden when challenged with B16F10. These data suggest TXNRD1 inhibitors likely drive Treg expansion by elevating ROS levels in APCs during T cell priming and less by intrinsic Treg TXNRD1 blockade. Our findings reveal a paradoxical immunosuppressive effect of TXNRD1 inhibitors that may contribute to their limited efficacy in immunocompetent cancer models. This work provides mechanistic insight and underscores the need to consider Treg-mediated immune suppression when designing TXNRD1-targeted therapies.

The positional cloning of single nucleotide polymorphisms (SNPs) of the neutrophil cytosolic factor 1 (Ncf1) gene, advocating that a low oxidative burst drives autoimmune disease, demands an understanding of the underlying molecular causes. A cellular target could be T cells, which have been shown to be regulated by reactive oxygen species (ROS). However, the pathways by which ROS mediate T cell signaling remain unclear. The adaptor molecule linker for activation of T cells (LAT) is essential for coupling T cell receptor-mediated antigen recognition to downstream responses, and it contains several cysteine residues that have previously been suggested to be involved in redox regulation. To address the possibility that ROS regulate T cell-dependent inflammation through LAT, we established a mouse strain with cysteine-to-serine mutations at positions 120 and 172 (LAT^SS). We found that redox regulation of LAT through C120 and C172 mediate its localization and phosphorylation. LAT^SS mice had reduced numbers of double-positive thymocytes and naïve peripheral T cells. Importantly, redox insensitivity of LAT enhanced T cell-dependent autoimmune inflammation in collagen-induced arthritis (CIA), a mouse model of rheumatoid arthritis (RA). This effect was reversed on an NCF1-mutated (NCF1^m1j), ROS-deficient, background. Overall, our data show that LAT is redox-regulated, acts to repress T cell activation, and is targeted by ROS induced by NCF1 in antigen-presenting cells (APCs).

The positional cloning of single nucleotide polymorphisms (SNPs) of the neutrophil cytosolic factor 1 (Ncf1) gene, advocating that a low oxidative burst drives autoimmune disease, demands an understanding of the underlying molecular causes. A cellular target could be T cells, which have been shown to be regulated by reactive oxygen species (ROS). However, the pathways by which ROS mediate T cell signaling remain unclear. The adaptor molecule linker for activation of T cells (LAT) is essential for coupling T cell receptor-mediated antigen recognition to downstream responses, and it contains several cysteine residues that have previously been suggested to be involved in redox regulation. To address the possibility that ROS regulate T cell-dependent inflammation through LAT, we established a mouse strain with cysteine-to-serine mutations at positions 120 and 172 (LATSS). We found that redox regulation of LAT through C120 and C172 mediate its localization and phosphorylation. LATSS mice had reduced numbers of double-positive thymocytes and naïve peripheral T cells. Importantly, redox insensitivity of LAT enhanced T cell-dependent autoimmune inflammation in collagen-induced arthritis (CIA), a mouse model of rheumatoid arthritis (RA). This effect was reversed on an NCF1-mutated (NCF1m1j), ROS-deficient, background. Overall, our data shows that LAT is redox-regulated, acts to repress T cell activation, and is targeted by ROS induced by NCF1 in antigen-presenting cells (APCs).