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Background - Helper T cell activity is dysregulated in a number of diseases including those associated with rheumatic autoimmunity. Treatment options are limited and usually consist of systemic immune suppression, resulting in undesirable consequences from compromised immunity. Hedgehog (Hh) signaling has been implicated in the activation of T cells and the formation of the immune synapse, but remains understudied in the context of autoimmunity. Modulation of Hh signaling has the potential to enable controlled immunosuppression but a potential therapy has not yet been developed to leverage this opportunity.
Methods - In this work, we developed biodegradable nanoparticles to enable targeted delivery of eggmanone (Egm), a specific Hh inhibitor, to CD4 T cell subsets. We utilized two FDA-approved polymers, poly(lactic-co-glycolic acid) and polyethylene glycol, to generate hydrolytically degradable nanoparticles. Furthermore, we employed maleimide-thiol mediated conjugation chemistry to decorate nanoparticles with anti-CD4 F(ab') antibody fragments to enable targeted delivery of Egm.
Results - Our novel delivery system achieved a highly specific association with the majority of CD4 T cells present among a complex cell population. Additionally, we have demonstrated antigen-specific inhibition of CD4 T cell responses mediated by nanoparticle-formulated Egm.
Conclusion - This work is the first characterization of Egm's immunomodulatory potential. Importantly, this study also suggests the potential benefit of a biodegradable delivery vehicle that is rationally designed for preferential interaction with a specific immune cell subtype for targeted modulation of Hh signaling.
© 2020 Haycook et al.
Intestinal intraepithelial lymphocytes (IEL) comprise a diverse population of cells residing in the epithelium at the interface between the intestinal lumen and the sterile environment of the lamina propria. Because of this anatomical location, IEL are considered critical components of intestinal immune responses. Indeed, IEL are involved in many different immunological processes, ranging from pathogen control to tissue stability. However, despite their critical importance in mucosal immune responses, very little is known about the homeostasis of different IEL subpopulations. The phosphoprotein osteopontin is important for critical physiological processes, including cellular immune responses, such as survival of Th17 cells and homeostasis of NK cells among others. Because of its impact in the immune system, we investigated the role of osteopontin in the homeostasis of IEL. In this study, we report that mice deficient in the expression of osteopontin exhibit reduced numbers of the IEL subpopulations TCRγδ, TCRβCD4, TCRβCD4CD8α, and TCRβCD8αα cells in comparison with wild-type mice. For some IEL subpopulations, the decrease in cell numbers could be attributed to apoptosis and reduced cell division. Moreover, we show in vitro that exogenous osteopontin stimulates the survival of murine IEL subpopulations and unfractionated IEL derived from human intestines, an effect mediated by CD44, a known osteopontin receptor. We also show that iCD8α IEL but not TCRγδ IEL, TCRβ IEL, or intestinal epithelial cells, can promote survival of different IEL populations via osteopontin, indicating an important role for iCD8α cells in the homeostasis of IEL.
Copyright © 2020 by The American Association of Immunologists, Inc.
Our previous studies of protective immunity and pathology against blood stage malaria parasites have shown that not only CD4 T cells, but also CD8 T cells and macrophages, are important for host defense against blood stage malaria infection. Furthermore, we found that 17XNL (PyNL) parasitizes erythroblasts, the red blood cell (RBC) precursor cells, which then express MHC class I molecules. In the present study, we analyzed spleen cytokine production. In CD8 T cell-depleted mice, IL-10 production in early stage infection was increased over two-fold relative to infected control animals and IL-10 CD3 cells were increased, whereas IFN-γ production in the late stage of infection was decreased. At day 16 after PyNL infection, CD8 T cells produced more IFN-γ than CD4 T cells. We evaluated the involvement of the immunoproteasome in induction of immune CD8 T cells, and the role of Fas in protection against PyNL both of which are downstream of IFN-γ. In cell transfer experiments, at least the single molecules LMP7, LMP2, and PA28 are not essential for CD8 T cell induction. The Fas mutant LPR mouse was weaker in resistance to PyNL infection than WT mice, and 20% of the animals died. LPR-derived parasitized erythroid cells exhibited less externalization of phosphatidylserine (PS), and phagocytosis by macrophages was impaired. Furthermore, we tried to identify the cause of death in malaria infection. Blood lactate concentration was increased in the CD8 T cell-depleted PyNL-infected group at day 19 (around peak parasitemia) to similar levels as day 7 after infection with a lethal strain of Py. When we injected mice with lactate at day 4 and 6 of PyNL infection, all mice died at day 8 despite demonstrating low parasitemia, suggesting that hyperlactatemia is one of the causes of death in CD8 T cell-depleted PyNL-infected mice. We conclude that CD8 T cells might control cytokine production to some extent and regulate hyperparasitemia and hyperlactatemia in protection against blood stage malaria parasites.
Copyright © 2019 Imai, Suzue, Ngo-Thanh, Ono, Orita, Suzuki, Shimokawa, Olia, Obi, Taniguchi, Ishida, Van Kaer, Murata, Tanaka and Hisaeda.
Dengue Virus (DENV) associated disease is a major public health problem. Assessment of HLA class II restricted DENV-specific responses is relevant for immunopathology and definition of correlates of protection. While previous studies characterized responses restricted by the HLA-DRB1 locus, the responses associated with other class II loci have not been characterized to date. Accordingly, we mapped HLA-DP, DQ, and DRB3/4/5 restricted DENV-specific CD4 T cell epitopes in PBMCs derived from the DENV endemic region Sri Lanka. We studied 12 DP, DQ, and DRB3/4/5 alleles that are commonly expressed and provide worldwide coverage >82% for each of the loci analyzed and >99% when combined. CD4+ T cells purified by negative selection were stimulated with pools of HLA-predicted binders for 2 weeks with autologous APC. Epitope reactive T cells were enumerated using IFNγ ELISPOT assay. This strategy was previously applied to identify DRB1 restricted epitopes. In parallel, membrane expression levels of HLA-DR, DP, and DQ proteins was assessed using flow cytometry. Epitopes were identified for all DP, DQ, and DRB3/4/5 allelic variants albeit with magnitudes significantly lower than the ones previously observed for the DRB1 locus. This was in line with lower membrane expression of HLA-DP and DQ molecules on the PBMCs tested, as compared to HLA-DR. Significant differences between loci were observed in antigen immunodominance. Capsid responses were dominant for DRB1/3/4/5 and DP alleles but negligible for the DQ alleles. NS3 responses were dominant in the case of DRB1/3/4/5 and DQ but absent in the case of DP. NS1 responses were prominent in the case of the DP alleles, but negligible in the case of DR and DQ. In terms of epitope specificity, repertoire was largely overlapping between DRB1 and DRB3/4/5, while DP and DQ loci recognized largely distinct epitope sets. The HLA-DP, DQ, and DRB3/4/5 loci mediate DENV-CD4 specific immune responses of lower magnitude as compared to HLA-DRB1, consistent with their lower levels of expression. The responses are associated with distinct and characteristic patterns of immunodominance, and variable epitope overlap across loci.
Checkpoint inhibitors produce durable responses in numerous metastatic cancers, but immune-related adverse events (irAEs) complicate and limit their benefit. IrAEs can affect organ systems idiosyncratically; presentations range from mild and self-limited to fulminant and fatal. The molecular mechanisms underlying irAEs are poorly understood. Here, we report a fatal case of encephalitis arising during anti-programmed cell death receptor 1 therapy in a patient with metastatic melanoma. Histologic analyses revealed robust T cell infiltration and prominent programmed death ligand 1 expression. We identified 209 reported cases in global pharmacovigilance databases (across multiple cancer types) of encephalitis associated with checkpoint inhibitor regimens, with a 19% fatality rate. We performed further analyses from the index case and two additional cases to shed light on this recurrent and fulminant irAE. Spatial and multi-omic analyses pinpointed activated memory CD4 T cells as highly enriched in the inflamed, affected region. We identified a highly oligoclonal T cell receptor repertoire, which we localized to activated memory cytotoxic (CD45ROGZMBKi67) CD4 cells. We also identified Epstein-Barr virus-specific T cell receptors and EBV lymphocytes in the affected region, which we speculate contributed to neural inflammation in the index case. Collectively, the three cases studied here identify CD4 and CD8 T cells as culprits of checkpoint inhibitor-associated immune encephalitis.
Tau protein is found to be aggregated and hyperphosphorylated (p-tau) in many neurologic disorders, including Parkinson disease (PD) and related parkinsonisms, Alzheimer disease, traumatic brain injury, and even in normal aging. Although not known to produce autoimmune responses, we hypothesized that the appearance of aggregated tau and p-tau with disease could activate the immune system. We thus compared T cell responses to tau and p-tau-derived peptides between PD patients, age-matched healthy controls, and young healthy controls (<35 y old; who are less likely to have high levels of tau aggregates). All groups exhibited CD4 T cell responses to tau-derived peptides, which were associated with secretion of IFN-γ, IL-5, and/or IL-4. The PD and control participants exhibited a similar magnitude and breadth of responses. Some tau-derived epitopes, consisting of both unmodified and p-tau residues, were more highly represented in PD participants. These results were verified in an independent set of PD and control donors (either age-matched or young controls). Thus, T cells recognizing tau epitopes escape central and peripheral tolerance in relatively high numbers, and the magnitude and nature of these responses are not modulated by age or PD disease.
Copyright © 2019 by The American Association of Immunologists, Inc.
BACKGROUND - Family screening of a 48-year-old male with recently diagnosed IgG4-related disease (IgG4-RD) revealed unanticipated elevations in plasma IgG4 in his two healthy teenaged sons.
METHODS - We performed gene sequencing, immune cell studies, HLA typing, and analyses of circulating cytotoxic CD4+ T lymphocytes and plasmablasts to seek clues to pathogenesis. DNA from a separate cohort of 99 patients with known IgG4-RD was also sequenced for the presence of genetic variants in a specific gene, FGFBP2.
RESULTS - The three share a previously unreported heterozygous single base deletion in fibroblast growth factor binding protein type 2 (FGFBP2), which causes a frameshift in the coding sequence. The FGFBP2 protein is secreted by cytotoxic T-lymphocytes and binds fibroblast growth factor. The variant sequence in the FGFBP2 protein is predicted to form a disordered random coil rather than a helical-turn-helix structure, unable to adopt a stable conformation. The proband and the two sons had 5-10-fold higher numbers of circulating cytotoxic CD4 + T cells and plasmablasts compared to matched controls. The three members also share a homozygous missense common variant in FGFBP2 found in heterozygous form in ~40% of the population. This common variant was found in 73% of an independent, well characterized IgG4-RD cohort, showing enrichment in idiopathic IgG4-RD.
CONCLUSIONS - The presence of a shared deleterious variant and homozygous common variant in FGFBP2 in the proband and sons strongly implicates this cytotoxic T cell product in the pathophysiology of IgG4-RD. The high prevalence of a common FGFBP2 variant in sporadic IgG4-RD supports the likelihood of participation in disease.
© 2019 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.
T cell help in humoral immunity includes interactions of B cells with activated extrafollicular CD4 and follicular T helper (Tfh) cells. Each can promote antibody responses but Tfh cells play critical roles during germinal center (GC) reactions. After restimulation of their antigen receptor (TCR) by B cells, helper T cells act on B cells via CD40 ligand and secreted cytokines that guide Ig class switching. Hypoxia is a normal feature of GC, raising questions about molecular mechanisms governing the relationship between hypoxia response mechanisms and T cell help to antibody responses. Hypoxia-inducible factors (HIF) are prominent among mechanisms that mediate cellular responses to limited oxygen but also are induced by lymphocyte activation. We now show that loss of HIF-1α or of both HIF-1α and HIF-2α in CD4 T cells compromised essential functions in help during antibody responses. HIF-1α depletion from CD4 T cells reduced frequencies of antigen-specific GC B cells, Tfh cells, and overall antigen-specific Ab after immunization with sheep red blood cells. Compound deficiency of HIF-1α and HIF-2α led to humoral defects after hapten-carrier immunization. Further, HIF promoted CD40L expression while restraining the FoxP3-positive CD4 cells in the CXCR5 follicular regulatory population. Glycolysis increases T helper cytokine expression, and HIF promoted glycolysis in T helper cells via TCR or cytokine stimulation, as well as their production of cytokines that direct antibody class switching. Indeed, IFN-γ elaboration by HIF-deficient in vivo-generated Tfh cells was impaired. Collectively, the results indicate that HIF transcription factors are vital components of the mechanisms of help during humoral responses.
Cannabis use is prevalent among HIV-positive persons, but evidence regarding the impact of cannabis in HIV-positive persons is limited. We conducted a retrospective cohort study of HIV-positive adults initiating their first antiretroviral therapy (ART) regimen. A dedicated intake form assessed self-reported cannabis use in the preceding 7 days at each visit. The relationships between time-varying cannabis use and body mass index (BMI), CD4+ T-cell count, and HIV-1 RNA levels were assessed using random effects models adjusted for age, sex, race, and other reported substance use. 4290 patient-visits from 2008 to 2011 were available from 1010 patients. Overall, there were no statistically significant differences in CD4+ T-cell count and BMI across multiple adjusted models using different measures of cannabis use (ever use during the study period, any use, and number of times used in the preceding 7 days). Cannabis use by all three measures was associated with greater odds of having a detectable viral load at a given visit than no reported use (OR 2.02, 1.72, and 1.08, respectively; all adjusted p < 0.05). Self-reported cannabis use was not associated with changes in BMI or CD4+ T-cell count in ART-naïve HIV-positive persons starting treatment. However, reported cannabis use by multiple categories was associated with having a detectable HIV-1 RNA during the study period. Associations between cannabis use, adherence, and HIV-related outcomes merit further study.
infection (CDI) is a major public health threat worldwide. The use of nonsteroidal anti-inflammatory drugs (NSAIDs) is associated with enhanced susceptibility to and severity of CDI; however, the mechanisms driving this phenomenon have not been elucidated. NSAIDs alter prostaglandin (PG) metabolism by inhibiting cyclooxygenase (COX) enzymes. Here, we found that treatment with the NSAID indomethacin prior to infection altered the microbiota and dramatically increased mortality and the intestinal pathology associated with CDI in mice. We demonstrated that in -infected animals, indomethacin treatment led to PG deregulation, an altered proinflammatory transcriptional and protein profile, and perturbed epithelial cell junctions. These effects were paralleled by increased recruitment of intestinal neutrophils and CD4 cells and also by a perturbation of the gut microbiota. Together, these data implicate NSAIDs in the disruption of protective COX-mediated PG production during CDI, resulting in altered epithelial integrity and associated immune responses. infection (CDI) is a spore-forming anaerobic bacterium and leading cause of antibiotic-associated colitis. Epidemiological data suggest that use of nonsteroidal anti-inflammatory drugs (NSAIDs) increases the risk for CDI in humans, a potentially important observation given the widespread use of NSAIDs. Prior studies in rodent models of CDI found that NSAID exposure following infection increases the severity of CDI, but mechanisms to explain this are lacking. Here we present new data from a mouse model of antibiotic-associated CDI suggesting that brief NSAID exposure prior to CDI increases the severity of the infectious colitis. These data shed new light on potential mechanisms linking NSAID use to worsened CDI, including drug-induced disturbances to the gut microbiome and colonic epithelial integrity. Studies were limited to a single NSAID (indomethacin), so future studies are needed to assess the generalizability of our findings and to establish a direct link to the human condition.
Copyright © 2019 Maseda et al.