Background: Spinal cord injury (SCI) elicits robust neuroinflammation that eventually exacerbates the initial damage to the spinal cord. L-arginine is critical for the responsiveness of T cells, which are important contributors to neuroinflammation after SCI. Furthermore, L-arginine is the substrate for nitric oxide (NO) production, which is a known inducer of secondary damage. Methods: To accomplish systemic L-arginine depletion, repetitive injections of recombinant arginase-1 (rArg-I) were performed. Functional recovery and histopathological parameters were analyzed. Splenic immune responses were evaluated by flow cytometry. Pro-inflammatory gene expression and nitrite concentrations were measured. Results: We show for the first time that systemic L-arginine depletion improves locomotor recovery. Flow cytometry and immunohistological analysis showed that intraspinal T-cell infiltration was reduced by 65%, and peripheral numbers of Th1 and Th17 cells were suppressed. Moreover, rArg-I treatment reduced the intraspinal NO production by 40%. Histopathological analyses revealed a 37% and 36% decrease in the number of apoptotic neurons and neuron-macrophage/microglia contacts in the spinal cord, respectively. Conclusions: Targeting detrimental T-cell responses and NO-production via rArg-I led to a reduced neuronal cell death and an improved functional recovery. These findings indicate that L-arginine depletion holds promise as a therapeutic strategy after SCI.

Multiple sclerosis (MS) is a common and devastating chronic inflammatory disease of the CNS. CD4⁺ T cells are assumed to be the first to cross the blood–central nervous system (CNS) barrier and trigger local inflammation. Here, we explored how pathogenicity‐associated effector programs define CD4⁺ T cell subsets with brain‐homing ability in MS. Runx3‐ and Eomes‐, but not T‐bet‐expressing CD4⁺ memory cells were diminished in the blood of MS patients. This decline reversed following natalizumab treatment and was supported by a Runx3⁺Eomes⁺T‐bet⁻ enrichment in cerebrospinal fluid samples of treatment‐naïve MS patients. This transcription factor profile was associated with high granzyme K (GZMK) and CCR5 levels and was most prominent in Th17.1 cells (CCR6⁺CXCR3⁺CCR4^−/dim). Previously published CD28⁻ CD4 T cells were characterized by a Runx3⁺Eomes⁻T‐bet⁺ phenotype that coincided with intermediate CCR5 and a higher granzyme B (GZMB) and perforin expression, indicating the presence of two separate subsets. Under steady‐state conditions, granzyme K^high Th17.1 cells spontaneously passed the blood–brain barrier in vitro. This was only found for other subsets including CD28⁻ cells when using inflamed barriers. Altogether, CD4⁺ T cells contain small fractions with separate pathogenic features, of which Th17.1 seems to breach the blood–brain barrier as a possible early event in MS.

Cytotoxic CD4⁺ T cells (CD4 CTL) are terminally differentiated T helper cells that contribute to autoimmune diseases, such as multiple sclerosis. We developed a novel triple co-culture transwell assay to study mutual interactions between CD4 CTL, conventional TH cells, and regulatory T cells (Tregs) simultaneously. We show that, while CD4 CTL are resistant to suppression by Tregs in vitro, the conditioned medium of CD4 CTL accentuates the suppressive phenotype of Tregs by upregulating IL-10, Granzyme B, CTLA-4, and PD-1. We demonstrate that CD4 CTL conditioned medium skews memory TH cells to a TH17 phenotype, suggesting that the CD4 CTL induce bystander polarization. In our triple co-culture assay, the CD4 CTL secretome promotes the proliferation of TH cells, even in the presence of Tregs. However, when cell−cell contact is established between CD4 CTL and TH cells, the proliferation of TH cells is no longer increased and Treg-mediated suppression is restored. Taken together, our results suggest that when TH cells acquire cytotoxic properties, these Treg-resistant CD4 CTL affect the proliferation and phenotype of conventional TH cells in their vicinity. By creating such a pro-inflammatory microenvironment, CD4 CTL may favor their own persistence and expansion, and that of other potentially pathogenic TH cells, thereby contributing to pathogenic responses in autoimmune disorders.

The effector programs of CD8⁺ memory T cells are influenced by the transcription factors RUNX3, EOMES and T-bet. How these factors define brain-homing CD8⁺ memory T cells in multiple sclerosis (MS) remains unknown. To address this, we analyzed blood, CSF and brain tissues from MS patients for the impact of differential RUNX3, EOMES and T-bet expression on CD8⁺ T cell effector phenotypes. The frequencies of RUNX3- and EOMES-, but not T-bet-expressing CD8⁺ memory T cells were reduced in the blood of treatment-naïve MS patients as compared to healthy controls. Such reductions were not seen in MS patients treated with natalizumab (anti-VLA-4 Ab). We found an additional loss of T-bet in RUNX3-expressing cells, which was associated with the presence of MS risk SNP rs6672420 (RUNX3). RUNX3⁺EOMES⁺T-bet⁻ CD8⁺ memory T cells were enriched for the brain residency-associated markers CCR5, granzyme K, CD20 and CD69 and selectively dominated the MS CSF. In MS brain tissues, T-bet coexpression was recovered in CD20^dim and CD69⁺ CD8⁺ T cells, and was accompanied by increased coproduction of granzyme K and B. These results indicate that coexpression of RUNX3 and EOMES, but not T-bet, defines CD8⁺ memory T cells with a pre-existing brain residency-associated phenotype such that they are prone to enter the CNS in MS.

In autoimmunity, FOXP3 ⁺ Tregs skew toward a proinflammatory, nonsuppressive phenotype and are, therefore, unable to control the exaggerated autoimmune response. This largely affects the success of autologous Treg therapy, which is currently under investigation for autoimmune diseases, including multiple sclerosis (MS). There is a need to ensure in vivo Treg stability before successful application of Treg therapy. Using genetic fate-mapping mice, we demonstrate that inflammatory, cytokine-expressing exFOXP3 T cells accumulate in the CNS during experimental autoimmune encephalomyelitis. In a human in vitro model, we discovered that interaction with inflamed blood-brain barrier endothelial cells (BBB-ECs) induces loss of function by Tregs. Transcriptome and cytokine analysis revealed that in vitro migrated Tregs have disrupted regenerative potential and a proinflammatory Th1/17 signature, and they upregulate the mTORC1 signaling pathway. In vitro treatment of migrated human Tregs with the clinically approved mTORC1 inhibitor rapamycin restored suppression. Finally, flow cytometric analysis indicated an enrichment of inflammatory, less-suppressive CD49d ⁺ Tregs in the cerebrospinal fluid of people with MS. In summary, interaction with BBB-ECs is sufficient to affect Treg function, and transmigration triggers an additive proinflammatory phenotype switch. These insights help improve the efficacy of autologous Treg therapy of MS.

Chronic immune activation is a hallmark of latent viral infections and autoimmune disorders, profoundly shaping immune cell phenotypes, including CD4⁺ cytotoxic T lymphocytes (CD4 CTL). The mechanisms underlying CD4 CTL development remain elusive, although antigenic triggers and the local microenvironment are thought to influence their phenotype. In this study, it was investigated if CD4 CTL induced under different circumstances exhibit phenotypic differences. Using single cell multiomics, we analyzed CD4 CTL from healthy cytomegalovirus (CMV)-seropositive donors, patients with CMV-seronegative relapsing-remitting multiple sclerosis (RR-MS) (autoimmune trigger), and patients with CMV-seropositive RR-MS (combination of viral and autoimmune trigger). Our findings reveal that the heterogeneous pool of CD4 CTL encompasses distinct subsets with divergent expression of proinflammatory, cytotoxic, and migratory markers. Moreover, we identified a pathogenic CD4 CTL subset coexpressing the MS-associated transcription factor eomesodermin (EOMES) and the migratory receptor class I-restricted T-cell-associated molecule, which accumulates in MS lesions and demonstrates resistance to natalizumab treatment. CMV was implicated as a dominant driver of development of highly cytotoxic CD4⁺ T cells, as these cells were markedly enriched in CMV-seropositive individuals. This comprehensive phenotypic atlas of CD4 CTL advances our understanding of their development and highlights potential targets for diagnosing, treating, and preventing MS progression.