Chikungunya virus (CHIKV) causes acute and chronic rheumatologic disease. Pathogenic CHIKV strains persist in joints of immunocompetent mice, while the attenuated CHIKV strain 181/25 is cleared by adaptive immunity. We analyzed the draining lymph node (dLN) to define events in lymphoid tissue that may contribute to CHIKV persistence or clearance. Acute 181/25 infection resulted in dLN enlargement and germinal center (GC) formation, while the dLN of mice infected with pathogenic CHIKV became highly disorganized and depleted of lymphocytes. Using CHIKV strains encoding ovalbumin-specific TCR epitopes, we found that lymphocyte depletion was not due to impaired lymphocyte proliferation. Instead, the accumulation of naive lymphocytes transferred from the vasculature to the dLN was reduced, which was associated with fewer high endothelial venule cells and decreased CCL21 production. Following NP-OVA immunization, NP-specific GC B cells in the dLN were decreased during pathogenic, but not attenuated, CHIKV infection. Our data suggest that pathogenic, persistent strains of CHIKV disable the development of adaptive immune responses within the dLN.
Mary K. McCarthy, Bennett J. Davenport, Glennys V. Reynoso, Erin D. Lucas, Nicholas A. May, Susan A. Elmore, Beth A. Tamburini, Heather D. Hickman, Thomas E. Morrison
BACKGROUND. Immune checkpoint inhibitors provide significant clinical benefit to a subset of patients, but novel prognostic markers are needed to predict which patients will respond. This study was initiated to determine if features of patient T cell repertoires could provide insights into the mechanisms of immunotherapy, while also predicting outcomes. METHODS. We examined T cell receptor (TCR) repertoires in peripheral blood of 25 metastatic pancreatic cancer patients treated with ipilimumab with or without GVAX (a pancreatic cancer vaccine), as well as peripheral blood and tumor biopsies from 32 patients treated with GVAX and mesothelin-expressing Listeria monocytogenes with or without nivolumab. Statistics from these repertoires were then tested for their association with clinical response and treatment group. RESULTS. We demonstrate that, first, the majority of patients receiving these treatments experience a net diversification of their peripheral TCR repertoires. Second, patients receiving ipilimumab experienced larger changes in their repertoires, especially in combination with GVAX. Finally, both a low baseline clonality and a high number of expanded clones following treatment were associated with significantly longer survival in patients who received ipilimumab but not in patients receiving nivolumab. CONCLUSIONS. We show that these therapies have measurably different effects on the peripheral repertoire, consistent with their mechanisms of action, and demonstrate the potential for TCR repertoire profiling to serve as a biomarker of clinical response in pancreatic cancer patients receiving immunotherapy. In addition, our results suggest testing sequential administration of anti–CTLA-4 and anti–PD-1 antibodies to achieve optimal therapeutic benefit. TRIAL REGISTRATION. Samples used in this study were collected from the NCT00836407 and NCT02243371 clinical trials. FUNDING. Research supported by a Stand Up To Cancer Lustgarten Foundation Pancreatic Cancer Convergence Dream Team Translational Research grant (SU2C-AACR-DT14-14). Stand Up To Cancer is a program of the Entertainment Industry Foundation administered by the American Association for Cancer Research (AACR). Additional clinical trial funding was provided by AACR-Pancreatic Cancer Action Network Research Acceleration Network grant (14-90-25-LE), NCI SPORE in GI Cancer (CA062924), Quick-Trials for Novel Cancer Therapies: Exploratory Grants (R21CA126058-01A2), and the US Food and Drug Administration (R01FD004819). Research collaboration and financial support were provided by Adaptive Biotechnologies.
Alexander C. Hopkins, Mark Yarchoan, Jennifer N. Durham, Erik C. Yusko, Julie A. Rytlewski, Harlan S. Robins, Daniel A. Laheru, Dung T. Le, Eric R. Lutz, Elizabeth M. Jaffee
Recent years have witnessed the groundbreaking success of immune checkpoint blockage (ICB) in metastasized malignant melanoma. However, biomarkers predicting the response to ICB are still urgently needed. In the present study, we investigated CTLA4 promoter methylation (mCTLA4) in 470 malignant melanoma patients from The Cancer Genome Atlas (non-ICB cohort) and in 50 individuals with metastasized malignant melanomas under PD-1/CTLA-4–targeted immunotherapy (ICB cohort). mCTLA4 levels were quantified using the Infinium HumanMethylation450 BeadChip (non-ICB cohort) and methylation-specific quantitative real-time PCR in DNA formalin-fixed and paraffin-embedded tissues (ICB cohort). Methylation levels were associated with molecular and clinicopathological variables and analyzed with respect to response (irRECIST) and overall survival. CTLA-4 mRNA and mCTLA4 showed a significant inverse correlation (non-ICB cohort: Spearman’s ρ = –0.416, P < 0.001). In ICB-treated melanoma patients, low mCTLA4 was further strongly correlated with response to therapy (P = 0.009, ANOVA) and overall survival (hazard ratio = 2.06 [95% CI: 1.29–3.29], P = 0.003). Our data strongly support the assumption that mCTLA4 predicts response to both anti–PD-1 and anti–CTLA-4 targeted ICB in melanoma and provides paramount information for the selection of patients likely to respond to ICB.
Diane Goltz, Heidrun Gevensleben, Timo J. Vogt, Joern Dietrich, Carsten Golletz, Friedrich Bootz, Glen Kristiansen, Jennifer Landsberg, Dimo Dietrich
Success of immune checkpoint inhibitors in advanced non-small-cell lung cancer (NSCLC) has invigorated their use in the neoadjuvant setting for early-stage disease. However, the cellular and molecular mechanisms of the early immune responses to therapy remain poorly understood. Through an integrated analysis of early-stage NSCLC patients and a Kras mutant mouse model, we show a prevalent programmed cell death 1/programmed cell death 1 ligand 1 (PD-1/PD-L1) axis exemplified by increased intratumoral PD-1+ T cells and PD-L1 expression. Notably, tumor progression was associated with spatiotemporal modulation of the immune microenvironment with dominant immunosuppressive phenotypes at later phases of tumor growth. Importantly, PD-1 inhibition controlled tumor growth, improved overall survival, and reprogrammed tumor-associated lymphoid and myeloid cells. Depletion of T lymphocyte subsets demonstrated synergistic effects of those populations on PD-1 inhibition of tumor growth. Transcriptome analyses revealed T cell subset–specific alterations corresponding to degree of response to the treatment. These results provide insights into temporal evolution of the phenotypic effects of PD-1/PD-L1 activation and inhibition and motivate targeting of this axis early in lung cancer progression.
Geoffrey J. Markowitz, Lauren S. Havel, Michael J.P. Crowley, Yi Ban, Sharrell B. Lee, Jennifer S. Thalappillil, Navneet Narula, Bhavneet Bhinder, Olivier Elemento, Stephen T.C. Wong, Dingcheng Gao, Nasser K. Altorki, Vivek Mittal
The peripheral blood represents only a small fraction of the total number of lymphocytes in the body. To develop a more thorough understanding of T cell dynamics, including the effects of SIV/SHIV/HIV infection on immune cell depletion and immune reconstitution following combination antiretroviral therapy (cART), one needs to utilize approaches that allow direct visualization of lymphoid tissues. In the present study, noninvasive in vivo imaging of the CD4+ T cell pool has revealed that the timing of the CD4+ T cell pool reconstitution following initiation of ART in SIV-infected nonhuman primates (NHPs) appears seemingly stochastic among clusters of lymph nodes within the same host. At 4 weeks following initiation or interruption of cART, the changes observed in peripheral blood (PB) are primarily related to changes in the whole-body CD4 pool rather than changes in lymphocyte trafficking. Lymph node CD4 pools in long-term antiretroviral-treated and plasma viral load–suppressed hosts appear suboptimally reconstituted compared with healthy controls, while splenic CD4 pools appear similar between the 2 groups.
Michele Di Mascio, Sharat Srinivasula, Insook Kim, Gorka Duralde, Alexis St. Claire, Paula DeGrange, Marisa St. Claire, Keith A. Reimann, Erin E. Gabriel, Jorge Carrasquillo, Richard C. Reba, Chang Paik, Henry C. Lane
T cells engineered to express chimeric antigen receptors (CARs) against B cell antigens are being investigated as cellular immunotherapies. Similar approaches designed to target T cell malignancies have been hampered by the critical issue of T-on-T cytotoxicity, whereby fratricide or self-destruction of healthy T cells prohibits cell product manufacture. To date, there have been no reports of T cells engineered to target the definitive T cell marker, CD3 (3CAR). Recent improvements in gene editing now provide access to efficient disruption of such molecules on T cells, and this has provided a route to generation of 3CAR, CD3-specific CAR T cells. T cells were transduced with a lentiviral vector incorporating an anti-CD3ε CAR derived from OKT3, either before or after TALEN-mediated disruption of the endogenous TCRαβ/CD3 complex. Only transduction after disrupting assembly of TCRαβ/CD3 yielded viable 3CAR T cells, and these cultures were found to undergo self-enrichment for 3CAR+TCR–CD3– T cells without any further processing. Specific cytotoxicity against CD3ε was demonstrated against primary T cells and against childhood T cell acute lymphoblastic leukemia (T-ALL). 3CAR T cells mediated potent antileukemic effects in a human/murine chimeric model, supporting the application of cellular immunotherapy strategies against T cell malignancies. 3CAR provides a bridging strategy to achieve T cell eradication and leukemic remission ahead of conditioned allogeneic stem cell transplantation.
Jane Rasaiyaah, Christos Georgiadis, Roland Preece, Ulrike Mock, Waseem Qasim
Coinhibitory receptors play an important role in the prevention of autoimmune diseases, such as systemic lupus erythematosus (SLE), by limiting T cell activation. B and T lymphocyte attenuator (BTLA) is an inhibitory receptor, similar to cytotoxic T lymphocyte–associated protein 4 (CTLA-4) and programmed death 1 (PD1), that negatively regulates the immune response. The role of BTLA in the pathogenesis of autoimmune diseases in humans and, more specifically, in SLE is largely unknown. We investigated BTLA expression on various T cell subsets, and we did not observe significant variations of BTLA expression between lupus patients and healthy controls. However, the enhancement of BTLA expression after activation was significantly lower in SLE patients compared with that in healthy controls. Furthermore, we found an impaired capacity of BTLA to inhibit T cell activation in SLE due to a poor BTLA recruitment to the immunological synapse following T cell stimulation. Finally, we demonstrated that defective BTLA function can be corrected by restoring intracellular trafficking and by normalizing the lipid metabolism in lupus CD4+ T cells. Collectively, our results evidence that the BTLA signaling pathway is altered in SLE T cells and highlight the potential of targeting this pathway for the development of new therapeutic strategies in lupus.
Matthieu Sawaf, Jean-Daniel Fauny, Renaud Felten, Flora Sagez, Jacques-Eric Gottenberg, Hélène Dumortier, Fanny Monneaux
Data from clinical trials for hemophilia B using adeno-associated virus (AAV) vectors have demonstrated decreased transgenic coagulation factor IX (hFIX) expression 6–10 weeks after administration of a high vector dose. While it is likely that capsid-specific cytotoxic T lymphocytes eliminate vector-transduced hepatocytes, thereby resulting in decreased hFIX, this observation is not intuitively consistent with restored hFIX levels following prednisone application. Although the innate immune response is immediately activated following AAV vector infection via TLR pathways, no studies exist regarding the role of the innate immune response at later time points after AAV vector transduction. Herein, activation of the innate immune response in cell lines, primary human hepatocytes, and hepatocytes in a human chimeric mouse model was observed at later time points following AAV vector transduction. Mechanistic analysis demonstrated that the double-stranded RNA (dsRNA) sensor MDA5 was necessary for innate immune response activation and that transient knockdown of MDA5, or MAVS, decreased IFN-β expression while increasing transgene production in AAV-transduced cells. These results both highlight the role of the dsRNA-triggered innate immune response in therapeutic transgene expression at later time points following AAV transduction and facilitate the execution of effective strategies to block the dsRNA innate immune response in future clinical trials.
Wenwei Shao, Lauriel F. Earley, Zheng Chai, Xiaojing Chen, Junjiang Sun, Ting He, Meng Deng, Matthew L. Hirsch, Jenny Ting, R. Jude Samulski, Chengwen Li
Despite the discovery of key pattern recognition receptors and CD4+ T cell subsets in laboratory mice, there is ongoing discussion of the value of murine models to reflect human disease. Pneumocystis is an AIDS-defining illness, in which risk of infection is inversely correlated with peripheral CD4+ T cell counts. Due to medical advances in the control of HIV, the current epidemiology of Pneumocystis infection is predominantly due to primary human immunodeficiencies and immunosuppressive therapies. To this end, we found that every human genetic immunodeficiency associated with Pneumocystis infection that has been tested in mice recapitulated susceptibility. For example, humans with a loss-of-function IL21R mutation are severely immunocompromised. We found that IL-21R, in addition to CD4+ T cell intrinsic STAT3 signaling, were required for generating protective antifungal class-switched antibody responses, as well as effector T cell–mediated protection. Furthermore, CD4+ T cell intrinsic IL-21R/STAT3 signaling was required for CD4+ T cell effector responses, including IL-22 production. Recombinant IL-22 administration to Il21r–/– mice induced the expression of a fungicidal peptide, cathelicidin antimicrobial peptide, which showed in vitro fungicidal activity. In conclusion, SPF laboratory mice faithfully replicate many aspects of human primary immunodeficiency and provide useful tools to understand the generation and nature of effector CD4+ T cell immunity.
Waleed Elsegeiny, Mingquan Zheng, Taylor Eddens, Richard L. Gallo, Guixiang Dai, Giraldina Trevejo-Nunez, Patricia Castillo, Kara Kracinovsky, Hillary Cleveland, William Horne, Jonathan Franks, Derek Pociask, Mark Pilarski, John F. Alcorn, Kong Chen, Jay K. Kolls
The gut-liver axis is of clinical importance as a potential therapeutic target in a wide range of liver diseases; however, the mechanisms underlying interactions between microbial products and immune responses in the liver remain unknown. In this study, we demonstrated that IL-10–producing macrophages contribute to immune tolerance in the inflamed liver under intestinal barrier disruption in a murine tandem model of dextran sulfate sodium (DSS) colitis and concanavalin A (Con A) hepatitis. Intestinal barrier disruption protected mice from subsequent liver injury, and the severity of colitis directly affected susceptibility to such injury. The protective effect of DSS–Con A was canceled in gut-sterilized mice, suggesting that gut microbiota play a substantial role in this process. Altered gut microbiota and their metabolites, along with a disrupted intestinal barrier, directly gave rise to immunological permissiveness in the inflamed liver. We identified 1-methylnicotinamide (1-MNA) as a candidate metabolite capable of suppressing liver injury with the potential to induce IL-10–producing macrophages. Consistently, expression of nicotinamide N-methyltransferase, which converts nicotinamide to 1-MNA, was upregulated in the liver of DSS–Con A mice, and this effect was abrogated by gut sterilization. Collectively, our results provide a mechanistic insight into the regulation of immunological balance in the liver via the gut-liver axis.
Nobuhito Taniki, Nobuhiro Nakamoto, Po-Sung Chu, Yohei Mikami, Takeru Amiya, Toshiaki Teratani, Takahiro Suzuki, Tomoya Tsukimi, Shinji Fukuda, Akihiro Yamaguchi, Shunsuke Shiba, Rei Miyake, Tadashi Katayama, Hirotoshi Ebinuma, Takanori Kanai
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