Immunology
Abstract
In prior studies, we delineated the landscape of neoantigens arising from nonsynonymous point mutations in a murine pancreatic cancer model, Panc02. We developed a peptide vaccine by targeting neoantigens predicted using a pipeline that incorporates the MHC binding algorithm NetMHC. The vaccine, when combined with immune checkpoint modulators, elicited a robust neoepitope-specific antitumor immune response and led to tumor clearance. However, only a small fraction of the predicted neoepitopes induced T cell immunity, similarly to that reported for neoantigen vaccines tested in clinical studies. While these studies have used binding affinities to MHC I as surrogates for T cell immunity, this approach does not include spatial information on the mutated residue that is crucial for TCR activation. Here, we investigate conformational alterations in and around the MHC binding groove induced by selected minimal neoepitopes, and we examine the influence of a given mutated residue as a function of its spatial position. We found that structural parameters, including the solvent-accessible surface area (SASA) of the neoepitope and the position and spatial configuration of the mutated residue within the sequence, can be used to improve the prediction of immunogenic neoepitopes for inclusion in cancer vaccines.
Authors
Neeha Zaidi, Mariya Soban, Fangluo Chen, Heather Kinkead, Jocelyn Mathew, Mark Yarchoan, Todd D. Armstrong, Shozeb Haider, Elizabeth M. Jaffee
Abstract
Despite advances in identifying the key immunoregulatory roles of many of the human leukocyte immunoglobulin (Ig)-like receptor (LILR) family members, the function of the inhibitory molecule LILRB3 (ILT5, CD85a, LIR3) remains unclear. Studies indicate a predominant myeloid expression; however, high homology within the LILR family and a relative paucity of reagents have hindered progress for this receptor. To investigate its function and potential immunomodulatory capacity, a panel of LILRB3-specific monoclonal antibodies (mAb) was generated. LILBR3-specific mAb bound to discrete epitopes in either Ig-like domain two or four. LILRB3 ligation on primary human monocytes by an agonistic mAb resulted in phenotypic and functional changes, leading to potent inhibition of immune responses in vitro, including significant reduction in T cell proliferation. Importantly, agonizing LILRB3 in humanized mice induced tolerance and permitted efficient engraftment of allogeneic cells. Our findings reveal powerful immunosuppressive functions of LILRB3 and identify it as an important myeloid checkpoint receptor.
Authors
Muchaala J. Yeboah, Charys Papagregoriou, Des C. Jones, H. T. Claude Chan, Guangan Hu, Justine S. McPartlan, Torbjörn Schiött, Ulrika T. Mattson, C. Ian Mockridge, Ulla-Carin Tornberg, Björn Hambe, Anne Ljungars, Mikael Mattsson, Ivo Tews, Martin J. Glennie, Stephen M. Thirdborough, John Trowsdale, Björn Frendéus, Jianzhu Chen, Mark S. Cragg, Ali Roghanian
Abstract
Hidradenitis Suppurativa (HS) is a debilitating chronic inflammatory skin disease characterized by chronic abscess formation and development of multiple draining sinus tracts in the groin, axillae, and perineum. Utilizing proteomic and transcriptomic approaches, we characterized the inflammatory responses in HS in depth, revealing immune responses centered around IFN-γ, IL-36, and TNF, with lesser contribution from IL-17A. We further identified B cells and plasma cells, with associated increases in immunoglobulin production and complement activation, as pivotal players in HS pathogenesis, with BTK and SYK pathway activation as a central signal transduction networks in HS. These data provide preclinical evidence to accelerate the path towards clinical trials targeting BTK and SYK signaling in moderate to severe HS.
Authors
Johann E. Gudjonsson, Lam C. Tsoi, Feiyang Ma, Allison C. Billi, Kelsey R. van Straalen, Allard R.J.V. Vossen, H.H. Zee, Paul W. Harms, Rachael Wasikowski, Christine M. Yee, Syed Monem Rizvi, Xianying Xing, Enze Xing, Olesya Plazyo, Chang Zeng, Matthew T. Patrick, Margaret M. Lowe, Richard E. Burney, Jeffrey H. Kozlow, Jill R. Cherry-Bukowiec, Yanyun Jiang, Joseph Kirma, Stephan Weidinger, Kelly C. Cushing, Michael D. Rosenblum, Celine C. Berthier, Amanda S. MacLeod, John J. Voorhees, Fei Wen, J. Michelle Kahlenberg, Emanual Maverakis, Robert L. Modlin, Errol P. Prens
Abstract
African green monkeys (AGMs) are natural hosts of Simian immunodeficiency virus (SIV) that post-thymically down-regulate CD4 to maintain a large population of CD4-CD8aa+ virus-resistant cells with T-helper functionality, which can result in AGMs becoming apparently cured of SIVagm infection. To understand the mechanisms of this process we performed genome-wide transcriptional analysis on T cells induced to down-regulate CD4 in vitro from AGMs and closely-related Patas monkeys, and T cells that maintain CD4 expression from rhesus macaques. In T cells that down-regulated CD4, pathway analysis revealed an atypical regulation ofthe DNA methylation machinery, which was reversible when pharmacologically targeted with 5-aza-2deoxycytidine. This signature was driven largely by the dioxygenase TET3 that became down-regulated with loss of CD4 expression. CpG motifs within the AGM CD4 promoter region became methylated during CD4 downregulation in vitro and were stably imprinted in AGM CD4-CD8aa+ T cells sorted directly ex vivo. These results suggest AGMs employ epigenetic mechanisms to durably silence the CD4 gene. Manipulation of these mechanisms could provide avenues for modulating SIV and human immunodeficiency virus (HIV)-1 entry receptor expression in hosts that become progressively SIV-infected, which could lead to novel therapeutic interventions aimed to reduce HIV viremia in vivo.
Authors
Joseph C. Mudd, Stephen Lai, Sanjana Shah, Andrew R. Rahmberg, Jacob K. Flynn, Carly E. Starke, Molly R. Perkins, Amy Ransier, Samuel Darko, Daniel Douek, Vanessa Hirsch, Mark J. Cameron, Jason M. Brenchley
Abstract
Myeloid cells orchestrate the anti-tumor immune response and influence the efficacy of immune checkpoint blockade (ICB) therapies. We and others have previously shown that interleukin 32 (IL-32) mediates dendritic cell (DC) differentiation and macrophage activation. Here, we demonstrate that IL-32 expression in human melanoma positively correlates with overall survival, response to ICB, and an immune inflamed tumor microenvironment (TME) enriched in mature DC, M1 macrophages and CD8+ T cells. Treatment of B16F10 murine melanomas with IL-32 increased the frequencies of activated, tumor-specific CD8+ T cells, leading to the induction of systemic tumor immunity. Our mechanistic in vivo studies revealed a novel role of IL-32 in activating intra-tumoral DC and macrophages to act in concert to prime CD8+ T cells and recruit them into the TME through CCL5. Thereby, IL-32 treatment reduced tumor growth and rendered ICB resistant B16F10 tumors responsive to anti-PD-1 therapy without toxicity. Furthermore, increased baseline IL-32 gene expression was associated with response to nivolumab and pembrolizumab in two independent human melanoma patient cohorts, implying IL-32 as a predictive biomarker for anti-PD-1 therapy. Collectively, this study suggests IL-32 as a potent adjuvant in immunotherapy to enhance the efficacy of ICB to patients with non-T cell inflamed TME.
Authors
Thomas Gruber, Mirela Kremenovic, Hassan Sadozai, Nives Rombini, Lukas Baeriswyl, Fabienne Maibach, Robert L. Modlin, Michel Gilliet, Diego Von Werdt, Robert E. Hunger, Giulia Parisi, Gabriel Abril-Rodriguez, Antoni Ribas, Mirjam Schenk
Abstract
Hidradenitis suppurativa (HS) is a highly prevalent and morbid inflammatory skin disease with limited treatment options. The major cell types and inflammatory pathways in skin of HS patients are poorly understood. In addition, it is currently unknown which patients will respond to TNFα blockade. Herein, we comprehensively elucidate and functionally define the immune cell infiltrate and major inflammatory pathways in HS skin, before and after anti-TNFα therapy. We discovered that clinically and histologically healthy appearing skin (i.e., nonlesional skin) is dysfunctional in HS patients with a relative loss of immune regulatory pathways. At the cellular level, HS skin lesions were characterized by quantitative and qualitative dysfunction of type 2 dendritic cells (cDC2s), relatively reduced regulatory T cells (Tregs), an influx of memory B cells and a plasma cell/plasmablast infiltrate predominantly in end-stage fibrotic skin. At the molecular level, there was a relative bias towards the IL-1 pathway and type 1 T cell responses when compared to both healthy skin and skin from psoriasis patients. Anti-TNFα therapy significantly attenuated B cell activation with minimal effect on other inflammatory pathways. Finally, we identified an immune activation signature in skin prior to anti-TNFα treatment that correlated with subsequent lack of response to this modality. Taken together, our results reveal the fundamental immunopathogenesis of HS and provide a molecular foundation for future studies focused on stratifying patients based on likelihood of clinical response to TNFα blockade.
Authors
Margaret M. Lowe, Haley B. Naik, Sean Clancy, Mariela Pauli, Kathleen M. Smith, Yingtao Bi, Robert Dunstan, Johann Gudjonsson, Maia Paul, Hobart W. Harris, Esther A. Kim, Uk Sok Shin, Richard Ahn, Wilson Liao, Scott L. Hansen, Michael Rosenblum
Abstract
Metabolic reprogramming dictates the fate and function of stimulated T cells, yet these pathways can be suppressed in T cells in tumor microenvironments. We previously showed that glycolytic and mitochondrial adaptations directly contribute to reducing the effector function of renal cell carcinoma (RCC) CD8+ tumor-infiltrating lymphocytes (TILs). Here we define the role of these metabolic pathways in the activation and effector functions of CD8+ RCC TILs. CD28 costimulation plays a key role in augmenting T cell activation and metabolism, and is antagonized by the inhibitory and checkpoint immunotherapy receptors CTLA4 and PD-1. While RCC CD8+ TILs were activated at a low level when stimulated through the T cell receptor alone, addition of CD28 costimulation greatly enhanced activation, function, and proliferation. CD28 costimulation reprogrammed RCC CD8+ TIL metabolism with increased glycolysis and mitochondrial oxidative metabolism, possibly through upregulation of GLUT3. Mitochondria also fused to a greater degree, with higher membrane potential and overall mass. These phenotypes were dependent on glucose metabolism, as the glycolytic inhibitor 2-deoxyglucose both prevented changes to mitochondria and suppressed RCC CD8+ TIL activation and function. These data show that CD28 costimulation can restore RCC CD8+ TIL metabolism and function through rescue of T cell glycolysis that supports mitochondrial mass and activity.
Authors
Kathryn E. Beckermann, Rachel Hongo, Xiang Ye, Kirsten Young, Katie Carbonell, Diana C. Contreras Healey, Peter J. Siska, Sierra Barone, Caroline E. Roe, Christof C. Smith, Benjamin G. Vincent, Frank M. Mason, Jonathan M. Irish, W. Kimryn Rathmell, Jeffrey C. Rathmell
Abstract
Granulosa cell tumors (GCT) are rare ovarian malignancies. Due to the lack of effective treatment in late relapse, there is a clear unmet need for novel therapies. Forkhead Box L2 (FOXL2) is a protein mainly expressed in granulosa cells (GC) and therefore is a rational therapeutic target. Since we identified tumor infiltrating lymphocytes (TILs) as the main immune population within GCT, TILs from 11 GCT patients were expanded, and their phenotypes were interrogated to determine that T cells acquired late antigen-experienced phenotypes and lower levels of PD1 expression. Importantly, TILs maintained their functionality after ex vivo expansion as they vigorously reacted against autologous tumors (100% of patients) and against FOXL2 peptides (57.1% of patients). To validate the relevance of FOXL2 as a target for immune therapy, we developed a plasmid DNA vaccine (FoxL2–tetanus toxin; FoxL2-TT) by fusing Foxl2 cDNA with the immune-enhancing domain of TT. Mice immunization with FoxL2-TT controlled growth of FOXL2-expressing ovarian (BR5) and breast (4T1) cancers in a T cell–mediated manner. Combination of anti–PD-L1 with FoxL2-TT vaccination further reduced tumor progression and improved mouse survival without affecting the female reproductive system and pregnancy. Together, our results suggest that FOXL2 immune targeting can produce substantial long-term clinical benefits. Our study can serve as a foundation for trials testing immunotherapeutic approaches in patients with ovarian GCT.
Authors
Stefano Pierini, Janos L. Tanyi, Fiona Simpkins, Erin George, Mireia Uribe-Herranz, Ronny Drapkin, Robert Burger, Mark A. Morgan, Andrea Facciabene
Human Tregs at the maternal-fetal interface show site-specific adaptation reminiscent of tumor Tregs
Abstract
Regulatory T cells (Tregs) are crucial for maintaining maternal immune-tolerance against the semi-allogeneic fetus. We investigated the elusive transcriptional profile and functional adaptation of human uterine Tregs (uTregs) during pregnancy. Uterine biopsies, from placental bed (=maternal-fetal interface) and incision site (=control), and blood were obtained from women with uneventful pregnancies undergoing Caesarean section. Tregs and CD4+ non-Tregs were isolated for transcriptomic profiling by Cel-Seq2. Results were validated on protein and single cell level by flow cytometry. Placental bed uterine Tregs (uTregs) showed elevated expression of Treg signature markers, including FOXP3, CTLA-4 and TIGIT. Their transcriptional profile was indicative of late-stage effector Treg differentiation and chronic activation, with increased expression of immune checkpoints GITR, TNFR2, OX-40, 4-1BB, genes associated with suppressive capacity (HAVCR2, IL10, LAYN, PDCD1), and transcription factors MAF, PRDM1, BATF, and VDR. uTregs mirrored non-Treg Th1 polarization and tissue-residency. The particular transcriptional signature of placental bed uTregs overlapped strongly with that of tumor-infiltrating Tregs, and was remarkably pronounced at the placental bed compared to uterine control site. Concluding, human uTregs acquire a differentiated effector Treg profile similar to tumor-infiltrating Tregs, specifically at the maternal-fetal interface. This introduces the novel concept of site-specific transcriptional adaptation of Tregs within one organ.
Authors
Judith Wienke, Laura Brouwers, Leone M. van der Burg, Michal Mokry, Rianne C. Scholman, Peter G. J. Nikkels, Bas B. van Rijn, Femke van Wijk
Abstract
Acute graft-versus-host disease (aGVHD) can occur after hematopoietic cell transplant in patients undergoing treatment for hematological malignancies or inborn errors. Although CD4 T helper (Th) cells play a major role in aGVHD, the mechanisms by which they contribute, particularly within the intestines, have remained elusive. We have identified a novel subset of Th cells that accumulated in the intestines and produced the serine protease granzyme A (GrA). GrA+ Th cells were distinct from other Th lineages and exhibited a non-cytolytic phenotype. In vitro, GrA+ Th cells differentiated in the presence of IL-4, IL-6, and IL-21 and were transcriptionally unique from cells cultured with either IL-4 or the IL-6/IL-21 combination alone. In vivo, both STAT3 and STAT6 were required for GrA+ Th cell differentiation and played roles in maintenance of the lineage identity. Importantly, GrA+ Th cells promoted aGVHD-associated morbidity and mortality and contributed to crypt destruction within intestines but were not required for the beneficial graft-versus-leukemia effect. Our data indicate that GrA+ Th cells represent a distinct Th subset and are critical mediators of aGVHD.
Authors
Sungtae Park, Brad Griesenauer, Hua Jiang, Djamilatou Adom, Pegah Mehrpouya-Bahrami, Srishti Chakravorty, Majid Kazemian, Tanbeena Imam, Rajneesh Srivastava, Tristan A. Hayes, Julian Pardo, Sarath Chandra Janga, Sophie Paczesny, Mark H. Kaplan, Matthew R. Olson
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