Nitin J. Karandikar

This study investigates the role of T‐cell‐intrinsic IL‐6 in Th17 differentiation and effector resistance. Using CRISPR‐Cas9‐mediated Il6 knockdown in primary human CD4 ⁺ T‐cells, we demonstrate that T‐cell‐produced IL‐6 is crucial for the optimal development of Th17 cells. Lack of T‐cell‐endogenous Il6 resulted in impaired IL‐17A production as well as significantly increased responsiveness to immune suppression. Importantly, these effects could not be reversed by the addition of exogenous IL‐6, likely due to interference with Il6R expression in the absence of endogenous Il6 , with consequent reduction in STAT3 phosphorylation. Blockade of IL‐6 receptor replicated the functional effects of Il6 knockdown and revealed a feedback loop between Il6 and Il6R expression. Of note, IL‐21 was able to bypass the need for IL‐6 in both Th17 differentiation and the development of resistance. Our findings emphasize T‐cell‐endogenous IL‐6 as a critical cytokine in Th17 cell biology and highlight IL‐6 and IL‐21 as potential immunotherapeutic targets.

Extracorporeal photopheresis (ECP) is a widely utilized immunomodulatory procedure with an incompletely defined mechanism. In graft‐versus‐host disease (GvHD) and transplant rejection, ECP is thought to induce immune tolerance by increasing regulatory CD4+ T‐cells, whereas in cutaneous T cell lymphoa it may enhance dendritic cell–mediated antigen presentation and cytotoxic T cell activity. We investigated the role of CD8+ T cells in ECP using a murine model of multiple sclerosis (MS). ECP protected mice from disease, mitigated CNS pathology, and was dependent on CD8+ T cells. Translation to patients revealed increased numbers of suppressive CD8+ T‐cells. Functional assays identified enhanced suppressive capacity of CD8+ T‐cells in ECP patients and longitudinal studies found this occurred within 1 month of starting ECP. Using both a murine model and clinical samples, our findings reveal a mechanistic role for suppressive CD8+ T‐cells in mediating the effects of ECP, potentially providing a unifying mechanism for ECP's apparently dichotomous effects.

This study investigates the role of T-cell-intrinsic IL-6 in Th17 differentiation and effector resistance. Using CRISPR-Cas9-mediated Il6 knockdown in primary human CD4⁺ T-cells, we demonstrate that T-cell-produced IL-6 is crucial for the optimal development of Th17 cells. Lack of T-cell-endogenous Il6 resulted in impaired IL-17A production as well as significantly increased responsiveness to immune suppression. Importantly, these effects could not be reversed by the addition of exogenous IL-6, likely due to interference with Il6R expression in the absence of endogenous Il6, with consequent reduction in STAT3 phosphorylation. Blockade of IL-6 receptor replicated the functional effects of Il6 knockdown and revealed a feedback loop between Il6 and Il6R expression. Of note, IL-21 was able to bypass the need for IL-6 in both Th17 differentiation and the development of resistance. Our findings emphasize T-cell-endogenous IL-6 as a critical cytokine in Th17 cell biology and highlight IL-6 and IL-21 as potential immunotherapeutic targets.

Multiple sclerosis (MS), an autoimmune demyelinating disease of the central nervous system, is modeled in mice as experimental autoimmune encephalomyelitis (EAE). While CD4⁺ T cells, primarily Th1 and Th17 subsets, drive disease pathogenesis, the exact function of CD8⁺ T cells remains unclear. We previously demonstrated that adoptively transferred myelin-reactive CD8⁺ T cells (PLP-CD8) prevent EAE induction and suppress ongoing disease through the engagement of MHC Class-I in recipient mice. Here, we show that PLP-CD8 induce regulatory changes in both subsets of conventional dendritic cells (cDC1 and CD11b⁺ cDC) in vivo and in vitro. Adoptively transferred PLP-CD8 promoted both cDC subsets to adopt a mature and regulatory phenotype with an anti-inflammatory cytokine profile and a reduced capacity to support CD4⁺ T cell proliferation. In vitro, PLP-CD8 induced similar phenotypic changes in both cDC subsets in an antigen-specific, dose-dependent manner. PLP-CD8 directly interacted with cDC1 and indirectly influenced CD11b⁺ cDC through paracrine signaling. Notably, direct interaction with PLP-CD8 had detrimental effects on CD11b⁺ cDC. Single-cell RNA sequencing revealed upregulation of key immunoregulatory genes, such as Foxo3, in both cDC subsets with enrichment of pathways involved in immune regulation and T cell differentiation. Our study highlights a novel mechanism in which myelin-reactive CD8⁺ T cells directly interact with cDC1 and modulate CD11b⁺ cDC through paracrine mechanisms to induce mature, regulatory dendritic cells, which leads to inhibited CD4⁺ T cell responses and reduced EAE pathogenesis.

An imbalance between proinflammatory and regulatory processes underlies autoimmune disease pathogenesis. We have shown that acute relapses of multiple sclerosis are characterized by a deficit in the immune suppressive ability of CD8+ T cells. These cells play an important immune regulatory role, mediated in part through cytotoxicity (perforin [PRF]/granzyme [GZM]) and IFNγ secretion. In this study, we further investigated the importance of IFNγ–, GZMB-, PRF1-, and LYST-associated pathways in CD8+ T cell–mediated suppression. Using the CRISPR-Cas9 ribonucleoprotein transfection system, we first optimized efficient gene knockout while maintaining high viability in primary bulk human CD8+ T cells. Knockout was confirmed through quantitative real-time PCR assays in all cases, combined with flow cytometry where appropriate, as well as confirmation of insertions and/or deletions at genomic target sites. We observed that the knockout of IFNγ, GZMB, PRF1, or LYST, but not the knockout of IL4 or IL5, resulted in significantly diminished in vitro suppressive ability in these cells. Collectively, these results reveal a pivotal role for these pathways in CD8+ T cell–mediated immune suppression and provide important insights into the biology of human CD8+ T cell–mediated suppression that could be targeted for immunotherapeutic intervention.

The pathogenic role B cells play in multiple sclerosis is underscored by the success of B cell depletion therapies. Yet, it remains unclear how B cells contribute to disease, although it is increasingly accepted that mechanisms beyond Ab production are involved. Better understanding of pathogenic interactions between B cells and autoreactive CD4 T cells will be critical for novel therapeutics. To focus the investigation on B cell:CD4 T cell interactions in vivo and in vitro, we previously developed a B cell–dependent, Ab-independent experimental autoimmune encephalomyelitis (EAE) mouse model driven by a peptide encompassing the extracellular domains of myelin proteolipid protein (PLPECD). In this study, we demonstrate that B cell depletion significantly inhibited PLPECD-induced EAE disease, blunted PLPECD-elicited delayed-type hypersensitivity reactions in vivo, and reduced CD4 T cell activation, proliferation, and proinflammatory cytokine production. Further, PLPECD-reactive CD4 T cells sourced from B cell–depleted donor mice failed to transfer EAE to naive recipients. Importantly, we identified B cell–mediated Ag presentation as the critical mechanism explaining B cell dependence in PLPECD-induced EAE, where bone marrow chimeric mice harboring a B cell–restricted MHC class II deficiency failed to develop EAE. B cells were ultimately observed to restimulate significantly higher Ag-specific proliferation from PLP178–191–reactive CD4 T cells compared with dendritic cells when provided PLPECD peptide in head-to-head cultures. We therefore conclude that PLPECD-induced EAE features a required pathogenic B cell–mediated Ag presentation function, providing for investigable B cell:CD4 T cell interactions in the context of autoimmune demyelinating disease.

CD4⁺ T-helper 17 (Th17) T cells are a key population in protective immunity during infection and in self-tolerance/autoimmunity. Through the secretion of IL-17, Th17 cells act in promotion of inflammation and are thus a major potential therapeutic target in autoimmune disorders. Recent reports have brought to light that the IL-17 family cytokines, IL-17A, IL-17F and IL-17AF, can directly act on CD4⁺ T-cells, both in murine and human systems, inducing functional changes in these cells. Here we show that this action is preferentially targeted toward naïve, but not memory, CD4⁺ T-cells. Naïve cells showed transcriptome changes as early as 48 hours post-IL-17 exposure, whereas memory cells remained unaffected as late as 7 days. These functional differences occurred despite similar IL-17 receptor expression on these subsets and were maintained in co-culture/transwell systems, with each subset maintaining its functional response to IL-17. Importantly, there were differences in downstream transcriptional signaling by the three IL-17 cytokines, with the IL-17AF heterodimer conferring both the greatest transcriptional change and most altered functional consequences. Detailed transcriptome analysis provides important insights into the genes and pathways that are modulated as a result of IL-17-mediated signaling and may serve as targets of future therapies.

Multiple sclerosis (MS) is a chronic immune-mediated disease of the central nervous system (CNS); the brain and spinal cord. The symptomology of MS includes pain, numbness, walking difficulty, and/or cognitive dysfunction. As of 2020, 2.8 million individuals worldwide are diagnosed with MS. While the exact cause of MS is unknown, the disease is thought to arise from a complex interaction of genetic and environmental factors. Interestingly, genetic variations account for 30% of disease risk while 70% of risk stems from environmental factors. Recent evidence suggests that the gut microbiome – the trillions of microorganisms that reside in the intestinal tract – is an environmental factor that contributes to disease pathogenesis. Several studies have demonstrated that the composition of the gut microbiome within MS patients is distinct from that of healthy individuals, including a loss of phytoestrogen metabolizing bacteria in MS patients. The pathogenic relevance of these bacterial differences remains elusive. My thesis work focuses on the influence of phytoestrogen-metabolizing bacteria on the immune system and its subsequent impact on a mouse model of MS.Utilizing mouse diets either containing or lacking dietary isoflavones (a type of phytoestrogen), I demonstrate that the gut microbiomes of mice on either diet is distinct. Interestingly, the gut microbiomes of mice on an isoflavone and isoflavone-free diet exhibit some parallels to the gut microbiomes of healthy individuals and MS patients, respectively. Dietary isoflavones do not alter the levels of broad immune cell populations in the spleen, blood, inguinal lymph nodes, and mesenteric lymph nodes. However, dietary isoflavones influence the levels of certain immune cell populations in the intestinal lamina propria, specifically CD8+ T cells. Moreover, isoflavones influence intestinal barrier permeability and cytokine production in mice and human cell lines, respectively. Utilizing the experimental autoimmune encephalomyelitis (EAE) mouse model of MS, I demonstrate that mice consuming an isoflavone diet exhibit an amelioration of EAE compared to mice on a diet lacking dietary isoflavones. Mice on an isoflavone diet exhibit less spinal cord pathology and pro-inflammatory cellular infiltration into the CNS compared to mice on an isoflavone-free diet. Importantly, the ability of an isoflavone diet to protect was dependent on isoflavone-metabolizing bacteria in the intestines. Strikingly, bacteria that do not metabolize isoflavones do not confer protection against disease in mice on an isoflavone diet. This suggests that isoflavone-metabolizing bacteria provide unique anti-inflammatory benefits to hosts consuming dietary isoflavones. However, treatment with exogenous equol, the end product of isoflavone metabolism, provides protection against EAE in mice on either diet. These results demonstrate that metabolism of isoflavones into equol, by intestinal gut bacteria, provides protection against EAE and possibly MS. Cumulatively, my thesis demonstrates that dietary isoflavones influence the gut microbiome and intestinal immunity. An isoflavone diet enhances the abundance of bacteria that are depleted in MS patients compared to healthy individuals. Furthermore, the same bacteria that are enhanced as a result of dietary isoflavones can uniquely protect against EAE in hosts on an isoflavone diet. Overall, these results suggest that MS patients are lacking gut bacteria that provide anti-inflammatory benefits.

Peripheral blood abnormalities like variant lymphocytes, Pelger-Huët anomaly, and granulocytic left shift have been reported in patients with active severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. However, the specificity of these findings for SARS-CoV-2 infection is unknown as there is a lack of comparative data between SARS-CoV-2 and other viral respiratory illnesses. In this study, we examined the peripheral blood findings in patients admitted with acute SARS-CoV-2 and other viral respiratory infections. We reviewed the complete blood differential counts and peripheral blood leukocyte morphology on peripheral smears received from patients admitted with a positive viral respiratory panel (VRP) or SARS-CoV-2 test from March 20^th – May 31^st, 2020. Cases where the specimen was collected 10 days after the symptom onset were excluded. During the study period, we reviewed 122 blood specimens from 59 patients (mean±SD age: 46.7±21.7 years, female: 49.2%). Of these, 53 (43.4%) specimens were from patients with SARS-CoV-2 infection and 69 (56.6%) specimens from patients with other viral respiratory infections. There were no significant differences in the demographic characteristics of the two groups. For the blood cell counts, absolute leukopenia (11.3% versus 40.6%, P), low red blood cell count (45.3% versus 78.3%, P), low hemoglobin (56.6% versus 76.8%, P0.018), low hematocrit (43.4% versus 78.3%, P), high mean corpuscular volume (3.8% versus 31.9%, P), high mean corpuscular hemoglobin concentration (1.9% versus 14.5%, P0.016), thrombocytopenia (11.3% versus 58.0%, P), low mean platelet volume (3.9% versus 15.0%, P0.048), and high red cell distribution width (13.2% versus 53.6%, P) were all less common in patients with SARS-CoV-2 infection as compared to those with other viral respiratory infections. There were no significant differences in the differential leukocyte counts between the two groups, except neutropenia was less common in SARS-CoV-2 infection (17.3% versus 44.1%, P0.002). Among the peripheral blood smear findings, toxic granulation in neutrophils (1.9% versus 42.01%, P) and the presence of myelocytes (13.2% versus 29.0%, P0.042) were less commonly seen in SARS-CoV-2 infection. Other findings like elevated band neutrophils (8.3% versus 27.5%, P0.081), presence of metamyelocytes (13.2% versus 27.5%, P0.060), presence of promyelocytes (5.7% versus 13.0%, P0.186), plasmacytoid lymphocytes (66.7% versus 91.7%, P0.177), and hypogranular neutrophils (7.6% versus 8.7%, P0.819) showed a trend towards being less common in patients with SARS-CoV-2 infection but the differences were not statistically significant. On the contrary, reactive lymphocytes (43.4% versus 33.3%, P0.257) and uni- or bi-lobate neutrophils showed a trend towards being more common in SARS-CoV-2 infection (18.9% versus 17.4%, P0.833), however, the differences were not statistically significant. Abnormalities in peripheral blood count and blood smear findings seem to be less frequent in hospitalized patients with SARS-CoV-2 infection as compared to those with other viral respiratory infections.

Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the CNS. We have previously demonstrated that CNS-specific CD8 T cells possess a disease-suppressive function in MS and variations of its animal model, experimental autoimmune encephalomyelitis (EAE), including the highly clinically relevant relapsing-remitting EAE disease course. Regulatory CD8 T cell subsets have been identified in EAE and other autoimmune diseases, but studies vary in defining phenotypic properties of these cells. In relapsing-remitting EAE, PLP178-191 CD8 T cells suppress disease, whereas PLP139-151 CD8 T cells lack this function. In this study, we used this model to delineate the unique phenotypic properties of CNS-specific regulatory PLP178-191 CD8 T cells versus nonregulatory PLP139-151 or OVA323-339 CD8 T cells. Using multiparametric flow cytometric analyses of phenotypic marker expression, we identified a CXCR3+ subpopulation among activated regulatory CD8 T cells, relative to nonregulatory counterparts. This subset exhibited increased degranulation and IFN-γ and IL-10 coproduction. A similar subset was also identified in C57BL/6 mice within autoregulatory PLP178-191 CD8 T cells but not within nonregulatory OVA323-339 CD8 T cells. This disease-suppressing CD8 T cell subpopulation provides better insights into functional regulatory mechanisms, and targeted enhancement of this subset could represent a novel immunotherapeutic approach for MS.