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CD4+ T-helper 17 (Th17) T cells are important in the regulation of protective immunity during infection and in self-tolerance/autoimmunity. Th17 cells, through the secretion of IL-17, act in promotion of inflammation and are thus a major potential therapeutic target in autoimmune disorders. Recent reports show that the IL-17 family cytokines, IL-17A, IL-17F and IL-17AF, can directly act on CD4+ T-cells, inducing functional changes in these cells. Here we show that functional and transcriptomic changes were preferentially induced in naïve CD4+ T-cells, but not in memory CD4+ T-cells. As early as 48 hours post-IL-17 exposure, naïve cells showed transcriptome changes, whereas memory cells remained unaffected as late as 7 days. The functional differences occurred despite similar IL-17RC and IL-17RA receptor expression on these subsets. Also, the differential IL-17-mediated changes in naïve, but not memory, cells occurred even in transwell/co-culture systems, with each subset maintaining its respective functional phenotype. Notably, there were differences in downstream transcriptional signaling by the three IL-17 cytokines. The IL-17AF heterodimer conferred both the greatest transcriptional change and most altered functional consequences. Detailed analysis of the transcriptome provides important insights into the genes and pathways that are altered as a result of IL-17-induced signaling and may serve as targets of future therapies. This work was supported, in part, by grant awards from the NIH to N.J.K. (R01 AI121567) and N.B. (F30 CA29655).

Important immune regulatory mechanisms mediated by CD4+ and CD8+ T-cells keep destructive CD4+ T-cell responses in check. CD4+ 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. Here, we demonstrate that human CD4+ T-cells exposed to a Th17-differentiating milieu are highly 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 through the induction of IL-1b and IL-6/STAT3 pathways, indicating a novel feedback loop. These studies reveal a novel function for IL-17 cytokines in a CD4-intrinsic mechanism of immune resistance. The pathways induced in this process may serve as a critical target for intensive investigation and therapeutic intervention.

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 and is restored during quiescence, suggesting that lack of CD8+ T cell mediated regulatory function may contribute to relapses in MS. 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 as a treatment during ongoing disease. 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, LM-PLP175–194 infection during disease remission of PLP139–151-induced EAE could effectively prevent relapses. These studies support a potential immunotherapeutic strategy using LM vaccination to endogenously prime disease regulatory CD8+ T cells.

Pathogenesis of immune-mediated demyelinating diseases like multiple sclerosis (MS) is thought to be governed by a complex cellular interplay between immunopathological and immunoregulatory responses. We have previously shown that neuroantigen-specific CD8 T cells have an unexpected protective role in the mouse model of MS, experimental autoimmune encephalomyelitis (EAE). In this study, we interrogated the immunotherapeutic potential of PLP178–191-specific CD8 T cells. Here, we show that PLP178–191-specific CD8 T cells, when administered post-disease onset, swiftly ameliorated EAE progression and suppressed PLP178–191-specific CD4 T cell responses as measured by delayed-type hypersensitivity (DTH). To accomplish DTH suppression, PLP178–191-specific CD8 T cells required differential production of perforin and IFNγ. Perforin was ancillary for the rapid suppressive action of PLP178–191-specific CD8 T cells, but was necessary for maintenance of optimal longer term DTH suppression. Conversely, IFNγ production by PLP178–191-specific CD8 T cells was necessary for swift DTH suppression, but was less significant for maintenance of longer term suppression. These data indicate that neuroantigen-specific CD8 T cells employ an ordered regulatory mechanism program over a number of days in vivo during demyelinating disease and have mechanistic implications for this immunotherapeutic approach.

An association between intravascular large B-cell lymphoma (IVLBCL) and the mixed lineage leukemia (MLL) gene has never been demonstrated. Here, we report an IVLBCL in a 47-year-old Asian man. Morphologically, the atypical lymphoid infiltrate was entirely confined in the lumina of capillaries, small vessels, and sinusoidal space. Within the kidney, the neoplastic lymphoid cells exhibited both the glomerular and peritubular capillary distribution pattern. Conventional cytogenetic analysis from the bone marrow aspirates displayed a complex karyotype, with a notable triple tandem repeat at band segment q22–q25 of chromosome 11. Fluorescence in situ hybridization with an MLL probe set, performed on both interphase cells and metaphase spreads, confirmed the presence of 3 copies of the MLL gene on the derivative chromosome 11. From this finding and 3 other IVLBCL cases reported in the literature, we conclude that MLL may play an important role in the lymphomagenesis of IVLBCL at least in a subset of cases.

In this research, we investigate the problem of analyzing time-varying imagery using a feature-based approach. We assume a scenario where the imaged objects remain stationary while the camera moves. The goal is to compute the structure of the imaged objects and the motion of the camera from a sequence of video images. The proposed method exploits the principle of the invariance of rigid configurations during motion. Using the rigidity constraints, we specify equations based on distance and angular invariance to compute the structural parameters of the imaged 3D objects independently of the camera's motion. Once the structural parameters are recovered, the motion parameters can be computed. The strength of our approach is in the decomposition of the computations of structure and motion, and in the use of point and line correspondences simultaneously. The main advantage of using both points and lines is that our approach is not limited to objects whose images have only a particular type of feature in abundance. In this research, we consider the special case of four points and one line, which are the minimum feature sets required for computing the structure and motion parameters. We then consider additional features in an overdetermined system of equations to improve the reliability and accuracy of the computation. We present computer simulation results, as well as results from real image sequences, to demonstrate the algorithm's validity.

Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the central nervous system (CNS) that afflicts more than 400,000 people in the US. Although the etiology of the disease is unknown, pathogenic T cells are thought to underlie MS immune pathology. In contrast to the current paradigm, we recently showed that MS patients harbor CNS-specific CD8+ T regulatory cells (CD8 Tregs) that are deficient during disease relapse. In the current study, we demonstrate that the neuroantigen-specific CD8 Tregs were cytolytic and eliminated pathogenic CD4+ T cells. Sorting of CD8+ T cells using an array of surface cellular markers revealed that the CD8 Tregs were terminally differentiated (CD27-, CD45RO-). The CD8 Treg-mediated suppression was perforin, granzyme B, and interferon-γ-dependent. Interestingly, we found that MS patients with acute disease exacerbation displayed a significant loss (averaging 25%) in the terminally differentiated CD8+ T cells, with a concurrent loss in perforin and granzyme B expression. In order to restore the regulatory potential of impaired CD8 Tregs during exacerbation, we pre-treated exacerbation-derived bulk CD8+ T cells with the cytokine IL-12 and significantly increased the suppressive capability of the cells by ~48% through upregulation of granzyme B and perforin. Our studies uncover the immune suppressive mechanism of neuroantigen-specific CD8 Tregs, and may contribute to the design of clinically relevant immune therapies for MS patients.

Daily subcutaneous administration of FDA-approved glatiramer acetate (GA) has beneficial effects on clinical course of relapsing remitting multiple sclerosis (RRMS). Although mechanism of GA-action has been widely investigated and partially understood, immediate immune dynamics following GA-therapy and their significance are unknown. In the present study we characterized immediate effects of GA on phenotype, quantity and function of immune cell subsets in MS patients. PBMC from RRMS patients, either untreated or initiating GA, and healthy donors were obtained at time of admission and subsequently at 4h, 12h, 24h and 72h after first GA-injection. Prominent changes in immune cells were detected within 4-12h post first GA-injection. T-cell modulation included significantly decreased CD4/CD8 ratio, perturbation in homeostasis of predominantly CD8+ T-cells, significant enhancement in CD8+ T-cell mediated suppression and inhibitory potential of induced CD4-suppressors. Changes in APCs were restricted to monocytes and included reduced stimulatory capacity in MLR and significantly increased IL-10 and TNF-α. Our study provides first evidence that GA induced rapid immunologic changes even after first dose. Interestingly, these responses are not restricted to APCs but also include complex modulation of T-cell functionality. Long-term studies will evaluate sustenance of these effects over time and their significance in clinical efficacy of therapy.

Experimental autoimmune encephalomyelitis (EAE) studies have established that myelin reactive T cells contribute significantly to the pathology of multiple sclerosis (MS). While auto-reactive CD4+ T cells have long been considered to be pathogenic, the role of CD8+ T cells is relatively unknown. We previously showed that myelin oligodendrocyte glycoprotein-specific CD8+ T cells (MOG-CD8+) suppress EAE; however, the mechanism of disease suppression is not completely understood. Here we show that CD11c+ dendritic cells (DC) from MOG-CD8+ recipient mice are less efficient in priming naïve and in activating recall CD4+ T cell response as compared to control ovalbumin-CD8+ recipients. In response to lipopolysaccharide stimulation, DC from MOG-CD8+ recipients demonstrated an anti-inflammatory cytokine profile with significantly lower IL-12 but higher IL-10 secretion. This was also confirmed in CD8-/- mice where DC from MOG-immunized CD8-/- mice secreted higher levels of IL-12 as compared to wild-type mice. MOG-CD8+ transfer to naïve mice did not affect DC function indicating that DC modulation requires cognate antigen presentation. Kinetic studies revealed that DC were modulated prior to disease onset at day 7 and persisted until day 30 post CD8+ transfer. Overall, our data suggest that transfer of CNS-antigen specific CD8+ T cells modulates DC function and can be an important mechanism of maintaining peripheral tolerance.

Immune dysregulation is believed to be the causative factor in the debilitating neurological disease Multiple Sclerosis (MS). We have recently shown that neuroantigen-specific auto-reactive regulatory (autoregulatory) CD8+ T-cells are capable of suppressing the murine model of MS, experimental autoimmune encephalomyelitis (EAE), a disease mediated by neuroantigen-specific CD4+ T-cells. We now show that deficiency of CD8+ T-cells correlates with more severe disease and an increase in IL-17+ and TCRVβ8.2+ CD4+ T-cell responses with reduction in IL-10+ CD4+ T-cells. Disease suppression by autoregulatory CD8+ T-cells is dependent on recognition of cognate antigen in vivo in the context of MHC Class I and requires IFN-γ and perforin production by CD8+ T-cells, suggesting a cytotoxic/suppressor mechanism. Suppression mediated by direct CD8-CD4 interactions is suggested by the ability of these cells to inhibit adoptively transferred disease and direct in vitro killing of neuroantigen-loaded CD4+ T cells. Finally, neuroantigen-specific CD8+ T-cells were able to traffic to the central nervous system (CNS) during ongoing inflammation, suggesting both a peripheral and central mechanism of action. These studies define a novel and unexpected immune regulatory function for tissue-reactive “autoregulatory” CD8+ T-cells that can be harnessed for the development of immunotherapeutic intervention in MS and other autoimmune diseases.

Multiple Sclerosis (MS) is an immune-mediated, demyelinating disorder of the CNS. Glatiramer acetate (GA/Copaxone) is an FDA-approved treatment for relapsing-remitting MS that induces functional changes in antigen-presenting cells (APC) and CD4 and CD8 T-cells. We have previously shown that treatment-naïve MS patients have deficient CD8 T-cell responses to GA, which are restored after GA therapy. This is associated with enhanced cytotoxic/suppressive function in these cells. In this study, we assessed the immediate immunologic changes induced by GA therapy in treatment-naïve MS patients by multiparametric monitoring of T-cell and APC subsets prior to and at 4, 12, 24, 72 hrs of treatment. In untreated healthy control subjects quantitative and functional readouts showed stable dynamics over time. MS patients showed greater proportions of memory/effector CD4 and CD8 populations at baseline, which did not vary. Similarly, cytokine production by APC subsets was also stable. Interestingly, as early as 4 to 12 hrs after the initial GA injection, we observed significantly enhanced suppressive ability in CD8 T-cells. This rapid functional change consistently occurred in GA, as well as an IFN-b-treated control cohort. These studies show that the induction of T-cell suppressive function is an important early event during therapy. Future studies will delineate whether these changes are predictive of sustained longterm immunologic and clinical responsiveness.

CD4+CD25+FOXP3+ regulatory T-cells (Tregs) play a vital role in suppressing autoimmune responses. We have previously shown that, in humans, all activated T-cells attain a state of transient FOXP3 expression that correlates with transient suppressive ability. In fact, peripheral generation of induced Tregs (iTregs) is likely the dominant source of regulatory cells in healthy adults. Through the use of a sensitive flow-based suppression assay, we observed that both memory and naïve CD4+CD25-FOXP3- T-cells developed regulatory ability following a variety of activating stimuli. This suppressive ability was not due to competition for nutrients or antigen presenting cells. Interestingly, memory T-cells displayed significantly greater FOXP3 induction compared to naïve counterparts, and this phenotype correlated to significantly enhanced suppressive function. Furthermore, acquisition of suppressive ability in both the memory and naïve iTreg compartments was proportional to the strength of activating stimulus. While blockade of activation using anti-IL-2, CTLA-4 Ig, anti-TGF-B, indomethacin or cyclosporin A did not affect iTreg generation, methotrexate drastically preempted the induction of regulatory ability. Moreover, irradiation of iTregs also abrogated regulatory function. These studies suggest that iTreg development and function may vary dependent on precursor origin and emphasize the importance of characterizing the iTreg compartment prior to therapeutic application.

Intramembranous proteolysis by the γ-secretase complex is a key regulator of transmembrane protein turnover and intracellular signaling. Small molecule inhibitors of γ-secretase (γSI) have previously been shown to suppress Th1 differentiation and reduce the severity of EAE, presumably by affecting Notch1 signaling. In order to understand the mechanisms through which γSI affect T effector responses we examined helper T cell polarization and function in vitro and in EAE. In vitro γSI treatment reduced T cell production of the Th1 cytokine IFNγ. We found γSI do not change activation but have effects on proliferation. Additionally, expression of the Th1 transcription factor Tbet was also diminished in γSI treated T cells. Looking upstream of Tbet expression, we found that γSI modulated STAT1 phosphorylation induced by exposing the cells to IFNγ. These results suggest that γSI might work at preventing Th1 differentiation but do not address the affects γSI may have on fully differentiated Th1 cells. We found that treatment with a γSI reduced the severity of EAE induced by adoptive transfer of myelin-specific TCR-transgenic T cells. Antigen recall experiments demonstrated that there was a dramatic decrease in the production of IFNγ by T cells ex vivo. These results demonstrate that γSI act not just to diminish differentiation of myelin-specific T cells into autoimmune Th1 effectors but impair the ability of established Th1 cells to cause damage to the CNS.

Recent evidence suggests that B and T cell interactions may be paramount in relapsing remitting multiple sclerosis (RRMS) disease pathogenesis. We hypothesized that memory B cell pools from RRMS patients may specifically harbor a subset of potent neuro-antigen presenting cells that support neuro-antigen reactive T cell expansion and activation. To assess the potential influence of neuro-antigen specific memory B cells from RRMS patients on autologous CD4+ T cell activation, we compared CD80 and HLA-DR expression, IL-10 and LTα secretion, neuro-antigen binding capacity, and functional neuro-antigen presentation by memory B cells from RRMS patients and compared these parameters to naïve B cells from RRMS patients and to memory and naïve B cells from healthy donors (HDs). We identified memory B cells from a subset of RRMS patients that were functionally capable of presenting neuro-antigen to autologous T cells. Notwithstanding the fact that the phenotypic parameters that promote efficient antigen presentation were observed to be similar between RRMS and HDs memory B cells, a corresponding capability to present neuro-antigen was not observed in HD memory B cells. Our results demonstrate for the first time that the memory B cell pool in RRMS harbors neuro-antigen specific clones that can activate T cells.