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Despite successful endovascular therapy, a proportion of stroke patients exhibit long-term functional decline, regardless of the cortical reperfusion. Our objective was to evaluate the early activation of the adaptive immune response and its impact on neurological recovery in patients with large vessel occlusion (LVO).Nineteen (13 females, 6 males) patients with acute LVO were enrolled in a single-arm prospective cohort study. During endovascular therapy (EVT), blood samples were collected from pre and post-occlusion, distal femoral artery, and median cubital vein (controls). Cytokines, chemokines, cellular and functional profiles were evaluated with immediate and follow-up clinical and radiographic parameters, including cognitive performance and functional recovery.In the hyperacute phase (within hours), adaptive immune activation was observed in the post-occlusion intra-arterial environment (post). Ischemic vascular tissue had a significant increase in T-cell-related cytokines, including IFN-γ and MMP-9, while GM-CSF, IL-17, TNF-α, IL-6, MIP-1a, and MIP-1b were decreased. Cellularity analysis revealed an increase in inflammatory IL-17+ and GM-CSF+ helper T-cells, while natural killer (NK), monocytes and B-cells were decreased. A correlation was observed between hypoperfused tissue, infarct volume, inflammatory helper, and cytotoxic T-cells. Moreover, helper and cytotoxic T-cells were also significantly increased in patients with improved motor function at 3 months.We provide evidence of the activation of the inflammatory adaptive immune response during the hyperacute phase and the association of pro-inflammatory cytokines with greater ischemic tissue and worsening recovery after successful reperfusion. Further characterization of these immune pathways is warranted to test selective immunomodulators during the early stages of stroke rehabilitation.

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.

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.

Objectives We sought to investigate the clinical utility of flow cytometry (FC) and fluorescence in situ hybridization (FISH) in the workup of myeloma. Methods We retrospectively reviewed the reports of bone marrow biopsies received for myeloma evaluation between October 2015 and January 2019. Results A total of 1,708 biopsy specimens from 469 myeloma patients (mean age, 64.5 years [SD, 9.3]; female, 41.4%) were reviewed. Both FC and FISH had comparable detection rates at the time of initial diagnosis (97.6% vs 98.8%) and for follow-up cases (28.6% vs 28.2%). FC and FISH results were concordant in 98.8% of the initial diagnosis cases and 89.6% of the follow-up cases. The FISH-positive (FISH+)/FC-negative (FC−) discordance and FISH−/FC+ discordance occurred among 81 (5.0%) and 87 (5.4%) follow-up cases. In comparison with all concordant cases, FISH+/FC− discordant cases were more likely to have received treatment with daratumumab (P < .05). Conclusions Plasma cell–enriched FISH and FC have comparable abnormal plasma cell detection rates, and approximately 10% of the follow-up cases have discordant FISH and FC results in which residual disease is detected by only one of these modalities. FISH testing should be considered for cases with negative FC, especially in patients who have received treatment with daratumumab or in cases in which there is concern about specimen adequacy.

A disrupted equilibrium between IL-17A-producing CD4 T-cells (Th17) and CD4+CD25+FoxP3+ regulatory T cells (Tregs) play an important role in the pathobiology of Multiple sclerosis (MS). Gut bacteria help in maintaining immune homeostasis by regulating the balance between anti-inflammatory Tregs and pro-inflammatory Th17 cells. Although, both gut bacteria and Tregs can regulate Th17 cells, the impact of IL-17A on gut microbiota and Tregs is unclear. Utilizing HLA-DR3 transgenic mouse model of MS, we show that IL-17A deficiency (HLA-DR3.IL17A-/- mice) expands Treg-inducing gut bacteria such as Prevotella, Parabacteroides, and Bacteroides and consequently Tregs, resulting in a milder disease in an animal model of MS. Notably, IL-17A sufficient DR3 mice develop milder disease on cohousing with IL-17A-deficient mice, highlighting a dominant role for gut microbiota in inducing Treg and reducing disease severity. Further, we observed an enrichment of bacterial-specific Treg promoting short-chain-fatty-acid metabolic pathways and induction of tolerogenic dendritic cells in HLA-DR3.IL17A-/- mice. Thus, our study shows a novel role of IL-17A in immune homeostasis and inflammation through regulation of the gut microbiota-Treg axis which can be used for the development of gut bacteria as therapeutics for MS.Significance of our workIL-17A a pro-inflammatory cytokine, is linked with pathobiology of multiple inflammatory diseases including multiple sclerosis (MS) and regulated by both gut microbiota and regulatory CD4 T cells (Tregs). However, the importance of IL-17A in the regulation of gut microbiota and Treg is unknown. Here we show that IL-17A can regulate Treg and disease phenotype by modulating gut microbiota and provide a novel mechanism by which immune-mediators such as IL-17A impact the gut microbiota to alter immune cell function and ultimately disease outcomes. Transfer of milder disease phenotype from IL-17A deficient mice to IL-17A sufficient mice on cohousing indicate a dominant role of gut microbiota in disease suppression. Thus, our study lays the foundation for future studies to unravel the interplay between immunological responses and the gut microbiota which will result in the development of microbiota-based therapeutics to treat autoimmune diseases.

Intravascular (IV) perfusion of tissue fixative is commonly used in the field of neuroscience as the central nervous system tissues are exquisitely sensitive to handling and fixation artifacts which can affect downstream microscopic analysis. Both 10% neutral-buffered formalin (NBF) and 4% paraformaldehyde (PFA) are used, although IV perfusion with PFA is most commonly referenced. The study objective was to compare the severity of handling and fixation artifacts, semiquantitative scores of inflammatory and neurodegenerative changes, and quantitative immunohistochemistry following terminal IV perfusion of mice with either 10% NBF or 4% PFA in a model of experimental autoimmune encephalitis (EAE). The study included 24 mice; 12 were control animals not immunized and an additional 12 were immunized with PLP139–151subcutaneously, harvested at day 20, and fixed in the same fashion. Equal numbers (4 per group) were perfused with 10% NBF or 4% PFA, and 4 were immersion-fixed in 10% NBF. NBF-perfused mice had less severe dark neuron artifact than PFA-perfused mice ( P < .001). Immersion-fixed animals had significantly higher scores for oligodendrocyte halos, dark neuron artifact, and perivascular clefts than perfusion-fixed animals. Histopathology scores in EAE mice for inflammation, demyelination, and necrosis did not differ among fixation methods. Also, no significant differences in quantitative immunohistochemistry for CD3 and Iba-1 were observed in immunized animals regardless of the method of fixation. These findings indicate that IV perfusion of mice with 10% NBF and 4% PFA are similar and adequate fixation techniques in this model.

BACKGROUND Flow cytometry is widely used for minimal residual disease (MRD) detection in plasma cell myeloma (PCM). Recently, an increasing number of studies have demonstrated that polytypic plasma cells (PPCs) display greater immunophenotypic variation than previously appreciated. Our aim was to further characterize the immunophenotype of PPC in this setting. METHODS PPC in 102 bone marrow specimens (93 MRD-negative post-treatment PCM and 9 negative initial evaluations for plasma cell neoplasm) were evaluated by 10-color flow cytometry. Frequency of CD19, CD27, CD28, CD56, CD117, and CD138 expression was assessed. RESULTS All cases showed CD27 and CD19 positivity. CD117 was uniformly negative. Percentage of CD138 expression was variable with one negative case (

Multiple genome-wide association studies have shown that the single-nucleotide polymorphism (SNP) rs2281808 TT variant, present within the signal regulatory protein gamma (SIRPG) gene, is associated with autoimmune diseases, such as type 1 diabetes. SIRPγ is the only SIRP expressed on T cells. The role of SIRPγ in human T-cells or the effect of the TT variant are poorly understood. In this short report, we demonstrate the rather unusual finding that this intronic SNP is associated with a reduction of SIRPγ expression on T cells, both in healthy subjects as well as patients with type 1 diabetes. Using this information, we propose that a simple flow cytometric detection of SIRPγ could be a potential diagnostic testing approach for the presence of SNP in the appropriate clinical context.

Introduction: Acute Ischemic Stroke (AIS) is one of the leading causes of disability and death in US. Although Endovascular Therapy (EVT) remains the mainstay therapy during acute phase for large vessel occlusions (LVOs), functional outcome varies among the treated patients. This ischemic injury results in an inflammatory response which plays an important role in the functional and neurological outcomes. We hypothesize that the early changes in the inflammatory response near the site of occlusion can be used as predictor of long-term neurofunctional decline Methods: AIS-LVO patients presenting to an academic comprehensive stroke center (CSC) within 24 hours from their last known well and undergoing EVT were included. Blood was collected proximal and distal to the thrombus during thrombectomy. Control samples were collected from the femoral artery and median cubital vein. Cytokine analysis and deep immune profiling was performed using a 20-parameter bead array and 13-parameter flow cytometer. Least Absolute Shrinkage and Selection Operator (LASSO) models were used for cell selection and correlation was evaluated for outcomes including mRS, NIHSS, MOCA and mortality, using R-software. Results: With 19 patients meeting the inclusion criteria, cytokine analysis revealed a significant increase in MMP and IFN-g, and decrease in GM-CSF, IL17, TNF-α, IL6, MIP-1a, and MIP-1b distal to clot. Flow cytometry analysis revealed a significant decrease in NK-T-cells, and CD8 T-cells counts and a relative increase in GM-CSF+ and IL17+ CD4 T-cells distal to clot. Immunological and neurological analysis revealed a correlation with CD4 + IFN-γ - IL10 + (r=0.7) & CD8 + IFN-γ - GMCSF + (0.6) with mRS, and CD4 + IFN-γ - IL10 + (r=0.7), CD4+ IFN-γ - IL17 + (r= -0.6), & CD8 + IFN-γ + IL17 + (r=0.7) cells with mortality. Conclusion: Our results indicate that local ischemia results in a hyperacute adaptive immune response at the site of occlusion. This immune response is predictive of functional outcome among AIS patients and is impactful in multiple ways, including the use of supportive therapy for patients with a poor functional trajectory and the use of immune-modulators at the site of ischemic injury.

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 20th – May 31st, 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

Human CD8+ T-cells play an important immune regulatory role during autoimmune diseases, characterized by suppressing the activation and proliferation of pathogenic effector cells, mediated in part through cytotoxicity (PRF/GZM), IFNγ secretion or by Fas/FasL interactions. Recent studies from our group demonstrated that IL-12 exposure during in vitro differentiation of naïve CD8+ T-cells induced an immune-suppressive deficit, with Tc1 cells showing significant loss of suppressive ability against CD4+ T-cells. RNAseq analysis revealed that various cytokines, transcription factors, and other regulatory molecules were differentially regulated in these cells. In this study, we further investigated the importance of IFNγ-, PRF1-, GZMB- and LYST-associated pathways in the suppressive ability of CD8+ T-cells. Using the CRISPR-Cas9 RNP optimized transfection system in primary bulk human CD8+ T-cells. Knockout was confirmed through quantitative real-time PCR assays in all cases, combined with flow cytometry when possible, as well confirmation of indels at genomic target sites. We observed that knockout of each of the four molecules, but not the knockout of IL-4 or IL-5, resulted in significantly diminished suppressive ability, as measured through in vitro immune suppression assays. Interestingly, in some of the knockouts, we observed differential expression of non-target molecules indicating potential functional links between these pathways. 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. This work was supported, in part, by grant awards to NJK from the NIH (R01 AI121567) and VA.

Given the success of B cell depletion therapy in multiple sclerosis (MS), deeper understanding of pathogenic B cells will be key in developing novel treatments. Notably, B cell depletion therapy leaves plasma cells and immunoglobulin levels largely intact, indicating that B cells provide pathogenic functions beyond antibodies. To better understand this, we recently developed a novel B cell-dependent, antibody-independent experimental autoimmune encephalomyelitis (EAE) mouse model of MS featuring immunoreactivity to the extracellular domains of myelin proteolipid protein (PLPECD). In contrast to a B cell-independent EAE model, we demonstrate here that PLPECD induced EAE in WT but not B cell-depleted mice, and that established disease was ameliorated by B cell depletion. We tested antigen presentation by B cells through mixed bone marrow chimeras and demonstrate that mice in which MHC II deficiency is restricted to B cells alone failed to exhibit EAE compared to MHC II sufficient mice. Given that HLA class II is the largest molecular link to MS incidence and CD4 T cells are causative in EAE, we tested B cells’ influence on CD4 T cells. In the absence of B cells, we observed not only lower activation and proliferation of CD4 T cells, but also noted a lower proinflammatory functional profile amongst the activated cells. Further, adoptively transferred CD4 T cells derived from B cell-depleted donors induced significantly less EAE compared to those from B cell-sufficient donors. These results demonstrate that antigen presentation by B cells plays a critical role in the activation, proliferation, function and pathogenicity of autoreactive CD4 T cells in demyelinating disease. Supported by grants from NIAID/NIH AI156123 and US Veterans Affairs I01BX003677.

Autoimmune diseases are characterized by regulatory deficit in both the CD4+ and CD8+ T-cell compartments. We have shown that CD8+ T-cells associated with acute relapse of multiple sclerosis are significantly deficient in their immune suppressive ability. We hypothesized that distinct CD8+ cytotoxic T-cell (Tc) lineages, determined by cytokine milieu during naïve T-cell differentiation, may harbor differential ability to suppress effector CD4+ T-cells. We differentiated purified human naïve CD8+ T-cells in vitro toward Tc0 (media control), Tc1 and Tc17 lineages. Using in vitro flow cytometric suppression assays, we observed that Tc0 and Tc17 cells had similar suppressive ability. In contrast, Tc1 cells showed significant loss of suppressive ability against ex vivo CD4+ T-cells and in vitro-differentiated Th0, Th1 and Th17 cells. Of note, Tc1 cells were also suboptimal in suppressing CD4-induced acute xenogeneic graft versus host disease (xGVHD) in vivo. Tc subtypes derived under various cytokine combinations revealed that IL-12-containing conditions resulted in less suppressive cells exhibiting dysregulated cytotoxic degranulation. RNA sequencing transcriptome analyses indicated differential regulation of inflammatory genes and enrichment in GM-CSF-associated pathways. These studies provide insights into the role of T-cell differentiation in CD8 suppressive biology and may reveal therapeutically targetable pathways to reverse suppressive deficit during immune-mediated disease.

Multiple Sclerosis (MS) is an immune-mediated demyelinating disease of the central nervous system (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 disease course (RR-EAE). 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 RR-EAE, PLP(178–191) CD8 T cells suppress disease, while PLP(139–151) CD8 T cells lack this function. In this study, we utilized this model to delineate the unique phenotypic properties of CNS-specific regulatory PLP(178–191) CD8 T cells vs. non-regulatory PLP(139–151) or OVA(323–339) CD8 T cells. Using multiparametric flow cytometric analyses of phenotypic marker expression, we identified a CXCR3+ subpopulation amongst activated regulatory CD8 T cells, relative to non-regulatory counterparts. This subset exhibited increased degranulation and IFNγ and IL-10 co-production. A similar subset was also identified in C57BL/6 mice within autoregulatory PLP(178–191) CD8 T cells, but not within non-regulatory OVA(323–339) 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.

Untoward effector CD4+ T cell responses are kept in check by immune regulatory mechanisms mediated by CD4+ and CD8+ T cells. CD4+ T helper 17 (Th17) cells, characterized by IL-17 production, play important roles in the pathogenesis of autoimmune diseases (such as arthritis, multiple sclerosis, psoriasis, inflammatory bowel disease, among others) and in the host response to infection and cancer. Here, we demonstrate that human CD4+ T cells cells exposed to a Th17-differentiating milieu are significantly more resistant to immune suppression by CD8+ T cells compared to control Th0 cells. This resistance is mediated, in part, through the action of IL-17A, IL-17F, and IL-17AF heterodimer through their receptors (IL-17RA and IL-17RC) on CD4+ T cells themselves, but not through their action on CD8+ T cells or APC. We further show that IL-17 can directly act on non-Th17 effector CD4+ T cells to induce suppressive resistance, and this resistance can be reversed by blockade of IL-1β, IL-6, or STAT3. These studies reveal a role for IL-17 cytokines in mediating CD4-intrinsic immune resistance. The pathways induced in this process may serve as a critical target for future investigation and immunotherapeutic intervention.

Cerebral vasospasm (VSP) is a common phenomenon after aneurysmal subarachnoid hemorrhage (aSAH) and contributes to neurocognitive decline. The natural history of the pro-inflammatory immune response after aSAH has not been prospectively studied in human cerebrospinal fluid (CSF). In this pilot study, we aimed to identify specific immune mediators of VSP after aSAH. Peripheral blood (PB) and CSF samples from patients with aSAH were prospectively collected at different time-points after hemorrhage: days 0–1 (acute); days 2–4 (pre-VSP); days 5–9 (VSP) and days 10 + (post-VSP peak). Presence and severity of VSP was assessed with computed tomography angiography/perfusion imaging and clinical examination. Cytokine and immune mediators’ levels were quantified using ELISA. Innate and adaptive immune cells were characterized by flow cytometry, and cell counts at different time-points were compared with ANOVA. Confocal immunostaining was used to determine the presence of specific immune cell populations detected in flow cytometry. Thirteen patients/aneurysms were included. Five (38.5%) patients developed VSP after a mean of 6.8 days from hemorrhage. Flow cytometry demonstrated decreased numbers of CD45+ cells during the acute phase in PB of aSAH patients compared with healthy controls. In CSF of VSP patients, NK cells (CD3-CD161 +) were increased during the acute phase and progressively declined, whereas CD8+CD161+ lymphocytes significantly increased at days 5–9. Microglia cells (CD45dimCD11b +) increased over time after SAH. This increase was particularly significant in patients with VSP. Levels of VEGF and MMP-9 were consistently higher in VSP patients, with the highest difference occurring at the acute phase. Confocal immunostaining demonstrated the presence of CD8+CD161+ lymphocytes in the arterial wall of two unruptured intracranial aneurysms. In this preliminary study, human CSF showed active presence of innate and adaptive immune cells after aSAH. CD8+CD161+ lymphocytes may have an important role in the inflammatory response after aneurysmal rupture and were identified in the aneurysmal wall of unruptured brain aneurysms. Microglia activation occurs 6 + days after aSAH.

Therapeutic success of B cell-targeting approaches in multiple sclerosis (MS) has intensified research into the role B cells play in pathogenesis and regulation of disease. Historical dissection of B cell function in the MS mouse model experimental autoimmune encephalomyelitis (EAE) has largely been confined to disease induced with either the immunodominant epitope of myelin oligodendrocyte glycoprotein (MOG35–55) or full length recombinant human MOG protein (hrMOG), the latter representing the only available B cell-dependent EAE model. We have demonstrated that myelin proteolipid protein (PLP178–191)-specific CD8 T cells possess striking EAE-suppression capacity. However, little is known regarding the role of B cells in these contexts. Interestingly, unlike MOG35–55, where genetic absence or depletion of B cells yields a more severe disease course, PLP178–191 yields identical EAE progression in both WT and μMT mice, suggesting suboptimal B cell engagement. Given the reports that hrMOG drives a B cell-dependent demyelinating disease versus its MOG35–55 counterpart, we hypothesized that a longer PLP antigen consisting of more epitopes may better engage B cells and provide an additional model for the field. To this end, we designed a novel peptide encompassing the extracellular domains (ECD) of PLP and compared its immunogenicity and disease-inducing capacity with PLP178–191 in μMT and WT mice. We demonstrate here that PLPECD-immunized B cell-deficient mice failed to exhibit EAE disease compared to WT counterparts. PLPECD also induced EAE in B cell-sufficient mice incapable of secreting antibodies, suggesting a predominant antigen presentation role. These results establish a novel, efficient B cell-dependent EAE model.

Investigating the complex cellular interplay controlling immunopathogenic and immunoregulatory responses is critical for understanding multiple sclerosis (MS) and for developing successful immunotherapies. Our group has demonstrated that CNS myelin-specific CD8 T cells unexpectedly harbor immune regulatory capacity in both mouse and human. In particular, PLP178-191–specific CD8 T cells (PLP-CD8) robustly suppress the MS mouse model experimental autoimmune encephalomyelitis. We have recently shown that this depends on PLP-CD8 elaborating IFN-γ and perforin in a coordinated suppression program over time. However, the cellular target and downstream effects of CD8 T cell–derived IFN-γ remains poorly understood. In this study, we show that although wild-type (WT) PLP-CD8 were robustly suppressive in IFN-γR–deficient mice, IFN-γR–deficient PLP-CD8 exhibited suboptimal suppression in WT mice. Compared with WT counterparts, IFN-γR–deficient PLP-CD8 were defective in suppressing disease in IFN-γ–deficient recipients, a scenario in which the only IFN-γ available to WT PLP-CD8 is that which they produce themselves. Further, we found that IFN-γR–deficient PLP-CD8 exhibited altered granzyme/IFN-γ profiles, altered migration in recipients, and deficits in killing capacity in vivo. Collectively, this work suggests that IFN-γ responsiveness allows myelin-specific CD8 T cells to optimally perform autoregulatory function in vivo. These insights may help elucidate future adoptive immunotherapeutic approaches for MS patients.

Factors regulating self-antigen directed immune-responses in autoimmunity are poorly understood. Signal regulatory protein gamma (SIRPγ) is a human T-cell specific protein with genetic variants associated with type 1 diabetes (T1D). SIRPγ's function in the immune system remains unclear. We show that T1D and relapsing remitting multiple sclerosis (RRMS) subjects have significantly greater frequency of rs2281808 T genetic variant, that correlates with reduced SIRPγ-expression in T-cells. Importantly, reduced SIRPγ-expression in RRMS and T1D subjects was not restricted to T variant, suggesting SIRPγ-expression is also regulated by disease specific factors in autoimmunity. Interestingly, increased frequencies of SIRPγlow T-cells in RRMS and T1D positively correlated with proinflammatory molecules from T-cells. Finally, we show that SIRPγlow T-cells have enhanced pathogenecity in vivo in a GVHD model. These findings suggest that decreased-SIRPγ expression, either determined by genetic variants or through peripherally acquired processes, may have a mechanistic link to autoimmunity through induction of hyperactive T-cells.

Multiple sclerosis (MS) is a chronic demyelinating disorder of the central nervous system (CNS). Its corresponding animal model, experimental autoimmune encephalomyelitis (EAE), is widely used to understand disease pathogenesis and test novel therapeutic agents. However, existing methods to score EAE disease severity are subjective and often vary between individual researchers, making it difficult to translate findings across different studies. An enhanced automated method of disease scoring would eliminate subjectivity and reduce operator-dependent errors. Here, we used an Infra-Red Activity Monitoring System (IRAMS) to measure murine locomotor activity as a surrogate measure of disease severity and compared it to standard EAE scoring methods. In mice immunized with CNS-specific myelin antigens, we observed an inverse correlation between disease severity and mouse activity, with the IRAMS showing enhanced disease scoring compared to standard EAE scoring methods. Relative to standard EAE scoring methods, IRAMS showed comparable measurement of disease relapses and remissions in the SJL/J-relapsing-remitting model of EAE, and could comparably assess the therapeutic efficiency of the MS drug, Copaxone (Glatiramer acetate-GA). Thus, the IRAMS is a method to measure disease severity in EAE without subjective bias and is a tool to consistently assess the efficacy of novel therapeutic agents for MS.

BACKGROUND Multiparametric flow cytometry is a useful tool for diagnosis of plasma cell (PC) dyscrasias and assessment of minimal residual disease in plasma cell myeloma (PCM). However, the immunophenotypic differences between the clonal PCs of PCM and those of monoclonal gammopathy of undetermined significance (MGUS) as well as the correlation of these flow cytometric markers with pertinent laboratory parameters have not been evaluated. METHODS We retrospectively identified all newly diagnosed treatment-naive PCM and MGUS patients between 09/2014 and 06/2015 who underwent 10-color flow-cytometric evaluation: CD45, CD38, CD138, cKappa, cLambda, CD19, CD27, CD28, CD56, CD117. FACSDiva analysis was used to identify antigenic aberrancies and associations with pertinent laboratory parameters were evaluated. RESULTS All cases demonstrated at least two aberrancies. There was a trend toward a greater number of aberrancies in PCM, with 68% showing >/= 4 aberrancies compared with 44% in MGUS (P = 0.11). The only marker more frequently aberrant in one disease class was CD19, aberrant in 68% of PCM and 25% of MGUS (P

Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the central nervous system, and among all the genes associated with MS, HLA class II genes have the highest relative risk. Although IL-17A is thought to be a major cytokine in the pathogenesis of MS, the exact role of IL-17A in the MS is still poorly understood. Specifically, it is unclear whether IL-17A is required for disease initiation or disease severity or in both. Therefore, in the present study, utilizing HLA-DR3 transgenic mice lacking IL-17A (HLA-DR3.IL-17A−/−), we investigated the importance of IL-17A in disease development and progression in experimental autoimmune encephalomnyelitis (EAE) an animal model of MS. We observed that HLA-DR3.IL-17A−/− mice had a higher frequency of CD4+CD25+FoxP3+ regulatory T cells (Treg)s and developed milder EAE compared to IL-17 sufficient HLA-DR3 mice. Further characterization of mechanism identified a novel role of IL-17A in regulating the disease progression through modulation of immunoregulatory responses. We also identified an important role of gut microbiota in IL-17A dependent disease regulation specifically depletion of gut bacteria with ability to promote Treg population in IL-17A sufficient HLA-DR3 mice. Fecal transplantation of HLA.DR3 mice with HLA-DR3. IL17A−/− showed milder EAE and an increase in Treg cells in HLA-DR3 mice confirming a role of gut microbiota in shaping Treg repertoire and function. Additionally, we observed higher frequency of metabolic pathway involved with Treg promoting short chain fatty acid (SCFA) metabolism in HLA-DR3.IL17A−/− mice. Thus our study shows a novel role of IL-17A in immune homeostasis and inflammation by regulating levels of Tregs through modulation of gut microbiota.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system. Despite IL-17A being linked to the immunopathogenesis of MS, the mechanism of IL-17A in disease development and maintenance is unclear. In the present study, we investigated the association of IL-17A and gut microbiota in disease development and severity, utilizing HLA-DR3 transgenic mice lacking IL-17A (IL-17A−/−). We found that HLA-DR3.IL-17A−/− mice had a higher frequency of CD4+CD25+FoxP3+ Treg cells and develop milder EAE compared to IL-17A suffcient HLA-DR3 mice. Utilizing 16S metagenocmic sequencing and analysis of gut microbiota we observed that HLA-DR3.IL-17A−/− mice had an increased abundance of regulatory T cells promoting bacteria in the gut such as Lactobacillus and Clostridia. Fecal transplantation followed by co-housing of HLA.DR3 mice with HLA-DR3.IL17A−/− resulted in development of milder EAE and an increase in CD4+CD25+FoxP3+ Treg cells in HLA-DR3 mice. Further whole-genome shotgun metagenomic analysis showed modulation of pathogen-associated molecular patterns (PAMPs) and O-antigen pathways linked with the host innate immune responses in HLA-DR3.IL-17A−/− mice. Thus our study suggests that gut microbiota changes due to the absence of IL17A induce regulatory T cells and suppresses disease in an animal model of MS by modulating the host-bacterial dynamics via PRR-PAMPS interactions.

Investigating the complex cellular interplay controlling immunopathogenic and immunoregulatory responses is critical for understanding multiple sclerosis (MS) and for developing successful immunotherapies. Our group has demonstrated that central nervous system (CNS) myelin-specific CD8 T cells unexpectedly harbor striking regulatory capacity in both mouse and human. In particular, PLP178–191-specific CD8 T cells (PLP-CD8) robustly suppress the MS mouse model experimental autoimmune encephalomyelitis (EAE). We have recently shown this depends on PLP-CD8 elaborating IFNγ and perforin in a coordinated suppression program over time. However, the cellular target and downstream effects of CD8 T cell-elaborated IFNγ remains poorly understood. Here, we show that while WT PLP-CD8 were robustly suppressive in IFNγR-deficient mice, IFNγR-deficient PLP-CD8 exhibited defective suppression in WT mice. Further, compared to WT counterparts, IFNγR-deficient PLP-CD8 were defective in suppressing disease in IFNγ-deficient recipients, a scenario in which the only IFNγ WT PLP-CD8 “see” is their own. These data indicate that myelin-specific CD8 T cells must sense their own IFNγ in an autocrine/paracrine manner to optimally perform autoregulatory function in vivo. Further mechanistic dissection showed that IFNγR-deficient PLP-CD8 exhibited altered degranulation/granzyme profiles, altered migration in recipients, and potential deficits in killing capacity. Collectively, this work suggests that myelin-specific CD8 T cells require IFNγ-responsiveness to optimally regulate CNS autoimmunity and may help elucidate future adoptive immunotherapeutic approaches in MS.

The incidence of stroke has risen over the past decade and will continue to be one of the leading causes of adult disability and death worldwide. Reperfusion therapy or endovascular thrombectomy (EVT) for large vessel occlusion (LVO) remains the only proven stroke therapy during the acute phase. But a select set of patients still exhibit poor recovery of neurological function, despite active cortical reorganization. The mechanisms that underlie this neurological dysfunction is still poorly understood. Subsequent to ischemic injury, a massive inflammatory response is seen for days, which exacerbates neuropathology in stroke models. Within the draining lymph nodes of ischemic patients, innate immune cells are actively phagocytizing brain antigen and presenting them to adaptive immune cells. The focus of this project is to determine if early changes in immune cells near the site of occlusion can be used as predictors of long-term neurofunctional decline. Acute anterior circulation LVO patients treated with EVT will be enrolled in this study. At the time of thrombectomy, blood was collected proximal and distal to the thrombus prior to recanalization. As a control, blood was also collected from the femoral artery. Deep immune profiling of innate and adaptive immune cells using 13-parameter flow cytometry was performed revealing a specific increase in post-thrombus CD4 T-cells, as compared to pre-thrombus. Cytokine analysis revealed a decrease in pre- and post-thrombus IFN-γ and IL1α. Induction of in vitro hypoxia in healthy peripheral blood mononuclear cells revealed a similar reduction in IFN-γ. We hypothesize that ischemia precipitates a shift in immune cells at the site of cerebrovascular blockage that modulates stroke recovery.

Multiple GWAS studies have shown that the SNP rs2281808 TT variant, present within the SIRPG gene, is associated with autoimmune diseases, such as type 1 diabetes. However, the role of SIRPγ in human T-cells is not known, neither is the functional significance of TT variant. Here we investigated SIRPG genotypes and their effects on the fate and function of human T-cells. We found that the presence of T variant resulted in reduction of SIRPγ expression on T-cells. Functionally, SIRPγlow CD8 T-cells in CT and TT individuals existed in a heightened effector state with lower activation threshold and had greater expression of genes and molecules associated with migratory and cytotoxic potential. Further, SIRPγlow CD8 T-cells were deficient in transcription factors associated with long-term functional memory formation. Our study reveals biological consequences of the SNP rs2281808 and provides novel insights into the potential mechanisms by which SIRPγ might regulate human immune responses.

We report an instructive case of acute myeloid leukemia with histiocytic differentiation (acute histiocytic leukemia) arising in a patient, a 52-year-old woman with a history of follicular lymphoma. The results of genetic studies proved a clonal relationship between the lymphoma and the leukemic cells. To our knowledge, this is the first report of leukemic transdifferentiation of follicular lymphoma into modified base 5-methylcytosine (M5c)–like acute histiocytic leukemia and the first reported karyotype on a transdifferentiated neoplasm.

Nature Communications 5: Article number: 4802 (2014); Published 5 September 2014; Updated 29 November 2017 The HTML version of this Article previously published had an incorrect volume number of 2; it should have been 5. This has now been corrected in the HTML; the PDF version of the paper was correct from the time of publication.

BACKGROUND: Multiparametric flow cytometry (MFC) is a useful tool for diagnosis of plasma cell dyscrasias and assessment of minimal residual disease (MRD) in plasma cell myeloma (PCM). However, the immunophenotypic differences between the clonal plasma cells (PCs) of plasma cell myeloma (PCM) and those of monoclonal gammopathy of undetermined significance (MGUS) as well as the correlation of these flow cytometric markers with pertinent laboratory parameters have not been evaluated. METHODS: We retrospectively identified all newly diagnosed treatment-naive PCM and MGUS patients between 09/2014 and 06/2015 who underwent 10-color flow-cytometric evaluation: CD45, CD38, CD138, cKappa, cLambda, CD19, CD27, CD28, CD56, CD117. FACSDiva analysis was utilized to identify antigenic aberrancies and associations with pertinent laboratory parameters were evaluated. RESULTS: All cases demonstrated at least 2 aberrancies. There was a trend toward a greater number of aberrancies in PCM, with 68% showing >/= 4 aberrancies compared to 44% in MGUS (p=0.11). The only marker more frequently aberrant in one disease class was CD19, aberrant in 68% of PCM and 25% of MGUS (p

Training in patient safety, quality, and management is widely recognized as an important element of graduate medical education. These concepts have been intertwined in pathology graduate medical education for many years, although training programs face challenges in creating explicit learning opportunities in these fields. Tangibly involving pathology residents in management and quality improvement projects has the potential to teach and reinforce key concepts and further fulfill Accreditation Council for Graduate Medical Education goals for pursuing projects related to patient safety and quality improvement. In this report, we present our experience at a pathology residency program (University of Iowa) in engaging pathology residents in projects related to practical issues of laboratory management, process improvement, and informatics. In this program, at least 1 management/quality improvement project, typically performed during a clinical chemistry/management rotation, was required and ideally resulted in a journal publication. The residency program also initiated a monthly management/informatics series for pathology externs, residents, and fellows that covers a wide range of topics. Since 2010, all pathology residents at the University of Iowa have completed at least 1 management/quality improvement project. Many of the projects involved aspects of laboratory test utilization, with some projects focused on other areas such as human resources, informatics, or process improvement. Since 2012, 31 peer-reviewed journal articles involving effort from 26 residents have been published. Multiple projects resulted in changes in ongoing practice, particularly within the hospital electronic health record. Focused management/quality improvement projects involving pathology residents can result in both meaningful quality improvement and scholarly output.

CD8 T-cells predominate in CNS lesions of MS patients and display oligoclonal expansion. However, the role of myelin-specific CD8 T-cells in disease remains unclear, with studies showing protective and pathogenic roles in EAE. We demonstrated a disease-suppressive function for CNS-specific CD8 T-cells in a model where the antigen is exogenously administered in vivo and used for in vitro activation. To probe the nature of the CD8 response elicited by endogenously presented myelin antigens in vivo, we developed a novel approach utilizing infection with Listeria monocytogenes (LM) encoding proteolipid protein peptide (PLP) amino acids 178-191 (LM-PLP). LM-PLP infection preferentially induced PLP-specific CD8 T-cell responses. Despite the induction of PLP-specific CD8 T-cells, LM-PLP infection did not result in disease. In fact, LM-PLP infection resulted in significant amelioration of PLP178-191-induced EAE. Disease suppression was not observed in mice deficient in CD8 T-cells, IFN-γ or perforin. DTH responses and CNS infiltration were reduced in protected mice, and their CD4 T-cells had reduced capacity to induce tissue inflammation. Importantly, infection with LM-PLP ameliorated established disease. Our studies indicate that CD8 T-cells induced by endogenous presentation of PLP178-191 attenuate CNS autoimmunity in models of EAE, implicating the potential of this approach as a novel immunotherapeutic strategy.

Background: Acute megakaryoblastic leukemia (AMKL) is a rare form of acute myeloid leukemia that encompasses three distinct subgroups, children with Down syndrome (DS-AMKL), children without DS (non-DS-AMKL) and adults (adult-AMKL). We hypothesize that the biological differences in the subtypes of AMKL may be reflected in distinct immunophenotypic (IP) features. Our aims were to employ mutiparameter flow cytometry to establish a comprehensive IP spectrum of AMKL in multiple demographic groups and to determine whether the subtypes of AMKL demonstrate distinct immunophenotypes. Methods: Thirty-seven AMKL cases were retrieved and subdivided into these three groups. Multiparameter flow cytometry was used to determine and compare the immunophenotypes. Available clinical, cytogenetic, and morphologic data were reviewed to confirm diagnoses. Results: While there is commonality in the expression pattern for a wide range of myelomonocytic markers in these three subtypes of AMKL, blasts in DS-AMKL were more likely to express CD7 and CD11b than those in non-DS-AMKL and adult-AMKL. Furthermore, blasts in DS-AMKL were more likely to express CD13, CD33 and CD36 than non-DS pediatric AMKL, and showed greater CD56 expression compared to adult AMKL. Conclusions: These results indicate that DS-AMKL is related to but immunophenotypically distinct from non-DS-AMKL and adult-AMKL. This distinct immunophenotypic pattern, co-expression of CD7 and CD11b, has practical implications for characterization of AMKL. © 2014 Clinical Cytometry Society.

The role of CD8+ T cells in the process of autoimmune pathology has been both understudied and controversial. Multiple sclerosis (MS) is an inflammatory, demyelinating disorder of the central nervous system (CNS) with underlying T cell-mediated immunopathology. CD8+ T cells are the predominant T cells in human MS lesions, showing oligoclonal expansion at the site of pathology. It is still unclear whether these cells represent pathogenic immune responses or disease-regulating elements. Through studies in human MS and its animal model, experimental autoimmune encephalomyelitis (EAE), we have discovered two novel CD8+ T cell populations that play an essential immunoregulatory role in disease: (1) MHC class Ia-restricted neuroantigen-specific "autoregulatory" CD8+ T cells and (2) glatiramer acetate (GA/Copaxone(®)) therapy-induced Qa-1/HLA-E-restricted GA-specific CD8+ T cells. These CD8+ Tregs suppress proliferation of pathogenic CD4+ CD25- T cells when stimulated by their cognate antigens. Similarly, CD8+ Tregs significantly suppress EAE when transferred either pre-disease induction or during peak disease. The mechanism of disease inhibition depends, at least in part, on an antigen-specific, contact-dependent process and works through modulation of CD4+ T cell responses as well as antigen-presenting cells through a combination of cytotoxicity and cytokine-mediated modulation. This review provides an overview of our understanding of CD8+ T cells in immune-mediated disease, focusing particularly on our findings regarding regulatory CD8+ T cells both in MS and in EAE. Clinical relevance of these novel CD8-regulatory populations is discussed, providing insights into a potentially intriguing, novel therapeutic strategy for these diseases.

Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the central nervous system (CNS). Although its etiology remains unknown, pathogenic T cells are thought to underlie MS immune pathology. We recently showed that MS patients harbor CNS-specific CD8+ Tregs that are deficient during disease relapse. We now demonstrate that CNS-specific CD8+ Tregs were cytolytic and could eliminate pathogenic CD4+ T cells. These CD8+ Tregs were present primarily in terminally differentiated (CD27-, CD45RO-) subset and their suppression was IFNγ, perforin and granzyme B-dependent. Interestingly, MS patients with acute relapse displayed a significant loss in terminally differentiated CD8+ T cells, with a concurrent loss in expression of perforin and granzyme B. Pre-treatment of exacerbation-derived CD8+ T cells with IL-12 significantly restored suppressive capability of these cells through upregulation of granzyme B. Our studies uncover immune-suppressive mechanisms of CNS-specific CD8+ Tregs, and may contribute to design of novel immune therapies for MS.

Investigating the complex cellular interplay controlling immunopathogenic and immunoregulatory responses is critical for understanding multiple sclerosis (MS) pathogenesis and for developing successful immunotherapies for patients. We have shown previously that CNS-specific CD8 T cells (CNS-CD8) have an unexpected regulatory role in the MS mouse model experimental autoimmune encephalomyelitis (EAE). CNS-CD8 fail to transfer or exacerbate disease but rather protect mice from EAE, and use IFNγ and perforin production to exert suppression. However, the cellular target and downstream effects of IFNγ are less well understood. Here, we evaluated IFNγ dynamics, its receptor and perforin during inhibition of CNS-specific DTH and EAE and show that both IFNγ and perforin production by CNS-CD8 were required at different times to suppress CD4 responses and inhibit EAE disease. Interestingly, while WT CNS-CD8 suppressed CD4 responses in IFNγR-deficient mice, IFNγR-deficient CD8 T cells showed suboptimal inhibition of CD4 responses in WT mice. Likewise, IFNγR-deficient CD8 T cells also exhibited delayed and suboptimal EAE suppression. These data suggest that autoregulatory CD8 T cells’ ability to suppress pathogenic responses during demyelinating disease is influenced by different mechanisms during different phases of disease. Moreover, the results suggest that IFNγ may have an effect on the CD8 T cells themselves, revealing a potential previously unappreciated autocrine/paracrine IFNγ-mediated pathway. Results from these studies have mechanistic implications for the interaction between IFNγ-mediated and perforin-mediated suppression pathways, which may help educate future adoptive immunotherapeutic approaches for MS.

Multiple Sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system (CNS). We have shown that CNS-specific CD8+ T cells possess a disease suppressive function in MS and its animal model, experimental autoimmune encephalomyelitis (EAE). In humans, this suppressive function of CD8+ T cells is deficient during MS relapses. We thus evaluated the role of CD8+ T cells in the context of the relapsing-remitting model (RR-EAE), using SJL mice. We demonstrate here that PLP178–191- and MBP84–104-specific CD8+ T cells ameliorated disease severity in an antigen-specific manner. Moreover, PLP178–191 CD8+ T cells reduced the number of relapses in PLP178–191-induced disease, even when transferred during ongoing disease. We were further able to ascertain that the suppressor population of CD8+ T cells express CD25, and show that a very low number of CD25+ CD8+ T cells are sufficient to suppress RR-EAE. We also probed the role of endogenously generated CNS-specific CD8+ T cell responses by using Listeria monocytogenes (LM) encoding CNS antigens to preferentially prime suppressive CD8+ T cells in vivo. Using this system, we show infection with LM expressing PLP175–194 endogenously induced disease suppressive CD8+ T cells that protected and treated PLP178–191 disease. Importantly, a combination of CD8+ T cell transfer boosted by LM infection also successfully treated ongoing disease induced by a non-cognate peptide (PLP139–151), indicating that this approach could be effective even in the context of epitope spreading. These studies support a potential immunotherapeutic strategy using LM vaccination to prime disease regulatory CD8+ T cells.

Regulatory T-cells keep untoward immune responses under control. In several autoimmune diseases, there is a substantial regulatory deficit in both the CD4+ and CD8+ T-cell compartments. We have shown that acute relapses of Multiple Sclerosis are characterized by a deficit of CD8+ T-cell mediated immune-suppression. Similar to CD4+ T-cells, lineages for CD8+ cytotoxic T-cells (Tc) are predominantly determined by the cytokine milieu during differentiation. We hypothesized that distinct Tc lineages may harbor differential ability to suppress effector CD4+ T-cell responses. To test this hypothesis, we obtained purified human naïve CD8+ T-cells and activated them in different in-vitro conditions to differentiate them toward Tc0 (control), Tc1, Tc2 and Tc17 lineages. We then tested their ability to suppress various types of CD4+ T-cells in flow cytometric suppression assays. We observed that Tc0 controls and Tc2 cells had comparable suppression. However, Tc1 cells showed significant loss of suppressive ability. Interestingly, Tc17 cells exhibited significantly greater suppression of effector cells of various lineages (including bulk ex-vivo CD4+ T-cells as well as in-vitro differentiated Th1 and Th17 cells). Tc17 cells also showed the greatest proportion of CD8+ T-cells with a terminally differentiated phenotype (CD27−/CD45RO−), which we have previously shown to harbor the highest suppressive ability in a neuroantigen-specific setting. These studies illustrate that Tc17 cells, known to play a vital role in infection, tumor and autoimmunity, may have an important immune-regulatory function against effector CD4+ T-cells that could be harnessed in the design of CD8+ T-cell based immune-modulatory therapy.

CD4+ T-helper (Th) cells are an important component of the immune system that come in several flavors, largely defined by the cytokines they produce. Important immune regulatory mechanisms mediated by CD4+ and CD8+ T-cells keep untoward effector T-cell responses in check. T-helper 17 (Th17) cells, characterized by IL-17 production, play critical roles in the body’s response to infections and cancer and in the pathogenesis of autoimmune diseases such as multiple sclerosis, psoriasis, arthritis, IBD, among others. We hypothesized that different lineages of effector Th cells would exert differential resistance to suppression. To test this, we differentiated purified naïve human CD4+ T-cells along different lineages (Th0, Th1, Th2, Th17) and tested the ability of CD8+ T-cells to suppress them. We observed that Th17 cells were highly resistant to immune suppression by CD8+ T-cells, compared to control Th0 cells. In contrast, Th1 cells were significantly more sensitive to suppression. Th17 resistance was mediated through the action of IL-17A, IL-17F and IL-17AF heterodimer through their receptors (IL-17RA and IL-17RC) on CD4+ T-cells themselves, but not through their action on CD8+ T-cells or APC. Interestingly, Th17 cells derived in different cytokine milieus showed distinct suppressive resistance, with the typical “non-pathogenic” conditions resulting in Th17 cells that were as sensitive to suppression as control Th0 cells. These studies reveal a novel function for IL-17 cytokines in a CD4-intrinsic mechanism of immune resistance that may serve as a critical target for intensive investigation and therapeutic intervention.

The autoimmune disease systemic lupus erythematosus is characterized by loss of tolerance to nuclear Ags and a heightened inflammatory environment, which together result in end organ damage. Lyn-deficient mice, a model of systemic lupus erythematosus, lack an inhibitor of B-cell and myeloid cell activation. This results in B-cell hyper-responsiveness, plasma cell accumulation, autoantibodies, and glomerulonephritis (GN). IL-21 is associated with autoimmunity in mice and humans and promotes B-cell differentiation and class switching. Here, we explore the role of IL-21 in the autoimmune phenotypes of lyn–/– mice. We find that IL-21 mRNA is reduced in the spleens of lyn–/–IL-6–/– and lyn–/–Btklo mice, neither of which produce pathogenic autoantibodies or develop significant GN. While IL-21 is dispensable for plasma cell accumulation and IgM autoantibodies in lyn–/– mice, it is required for anti-DNA IgG antibodies and some aspects of T-cell activation. Surprisingly, GN still develops in lyn–/–IL-21–/– mice. This likely results from the presence of IgG autoantibodies against a limited set of non-DNA Ags. These studies identify a specific role for IL-21 in the class switching of anti-DNA B cells and demonstrate that neither IL-21 nor anti-DNA IgG is required for kidney damage in lyn–/– mice.