Background: Checkpoint inhibitor pneumonitis (CIP) is a highly morbid complication of immune checkpoint immunotherapy (ICI), one which precludes the continuation of ICI. Yet, the mechanistic underpinnings of CIP are unknown. Methods: To better understand the mechanism of lung injury in CIP, we prospectively collected bronchoalveolar lavage (BAL) samples in ICI-treated patients with (n=12) and without CIP (n=6), prior to initiation of first-line therapy for CIP (high dose corticosteroids. We analyzed BAL immune cell populations using a combination of traditional multicolor flow cytometry gating, unsupervised clustering analysis and BAL supernatant cytokine measurements. Results: We found increased BAL lymphocytosis, predominantly CD4+ T cells, in CIP. Specifically, we observed increased numbers of BAL central memory T-cells (Tcm), evidence of Type I polarization, and decreased expression of CTLA-4 and PD-1 in BAL Tregs, suggesting both activation of pro-inflammatory subsets and an attenuated suppressive phenotype. CIP BAL myeloid immune populations displayed enhanced expression of IL-1β and decreased expression of counter-regulatory IL-1RA. We observed increased levels of T cell chemoattractants in the BAL supernatant, consistent with our pro-inflammatory, lymphocytic cellular landscape. Conclusion: We observe several immune cell subpopulations that are dysregulated in CIP, which may represent possible targets that could lead to therapeutics for this morbid immune related adverse event.
Karthik Suresh, Jarushka Naidoo, Qiong Zhong, Ye Xiong, Jennifer Mammen, Marcia Villegas de Flores, Laura Cappelli, Aanika Balaji, Tsvi Palmer, Patrick M. Forde, Valsamo Anagnostou, David S. Ettinger, Kristen A. Marrone, Ronan J. Kelly, Christine L. Hann, Benjamin Levy, Josephine L. Feliciano, Cheng-Ting Lin, David Feller-Kopman, Andrew D. Lerner, Hans Lee, Majid Shafiq, Lonny Yarmus, Evan J. Lipson, Mark Soloski, Julie R. Brahmer, Sonye K. Dannoff, Franco D'Alessio
BACKGROUND Persistence of HIV in sanctuary sites despite antiretroviral therapy (ART) presents a barrier to HIV remission and may affect neurocognitive function. We assessed HIV persistence in cerebrospinal fluid (CSF) and associations with inflammation and neurocognitive performance during long-term ART.METHODS Participants enrolled in the AIDS Clinical Trials Group (ACTG) HIV Reservoirs Cohort Study (A5321) underwent concurrent lumbar puncture, phlebotomy, and neurocognitive assessment. Cell-associated HIV DNA and HIV RNA (CA-DNA, CA-RNA) were measured by quantitative PCR (qPCR). in peripheral blood mononuclear cells (PBMCs) and in cell pellets from CSF. In CSF supernatant and blood plasma, cell-free HIV RNA was quantified by qPCR with single copy sensitivity, and inflammatory biomarkers were measured by enzyme immunoassay.RESULTS Sixty-nine participants (97% male, median age 50 years, CD4 696 cells/mm3, plasma HIV RNA <100 copies/mL) were assessed after a median 8.6 years of ART. In CSF, cell-free RNA was detected in 4%, CA-RNA in 9%, and CA-DNA in 48% of participants (median level 2.1 copies/103 cells). Detection of cell-free CSF HIV RNA was associated with higher plasma HIV RNA (P = 0.007). CSF inflammatory biomarkers did not correlate with HIV persistence measures. Detection of CSF CA-DNA HIV was associated with worse neurocognitive outcomes including global deficit score (P = 0.005), even after adjusting for age and nadir CD4 count.CONCLUSION HIV-infected cells persist in CSF in almost half of individuals on long-term ART, and their detection is associated with poorer neurocognitive performance.FUNDING This observational study, AIDS Clinical Trials Group (ACTG) HIV Reservoirs Cohort Study (A5321), was supported by the National Institutes of Health (NIAID and NIMH).
Serena Spudich, Kevin R. Robertson, Ronald J. Bosch, Rajesh T. Gandhi, Joshua C. Cyktor, Hanna Mar, Bernard J. Macatangay, Christina M. Lalama, Charles Rinaldo, Ann C. Collier, Catherine Godfrey, Joseph J. Eron, Deborah McMahon, Jana L. Jacobs, Dianna Koontz, Evelyn Hogg, Alyssa Vecchio, John W. Mellors
T follicular helper cells (Tfh), a subset of CD4+ T cells, provide requisite help to B cells in the germinal centers (GC) of lymphoid tissue. GC Tfh are identified by high expression of the chemokine receptor CXCR5 and the inhibitory molecule PD-1. Although more accessible, blood contains lower frequencies of CXCR5+ and PD-1+ cells that have been termed circulating Tfh (cTfh). However, it remains unclear whether GC Tfh exit lymphoid tissues and populate this cTfh pool. To examine exiting cells, we assessed the phenotype of Tfh present within the major conduit of efferent lymph from lymphoid tissues into blood, the human thoracic duct. Unlike what was found in blood, we consistently identified a CXCR5-bright PD-1–bright (CXCR5BrPD-1Br) Tfh population in thoracic duct lymph (TDL). These CXCR5BrPD-1Br TDL Tfh shared phenotypic and transcriptional similarities with GC Tfh. Moreover, components of the epigenetic profile of GC Tfh could be detected in CXCR5BrPD-1Br TDL Tfh and the transcriptional imprint of this epigenetic signature was enriched in an activated cTfh subset known to contain vaccine-responding cells. Together with data showing shared TCR sequences between the CXCR5BrPD-1Br TDL Tfh and cTfh, these studies identify a population in TDL as a circulatory intermediate connecting the biology of Tfh in blood to Tfh in lymphoid tissue.
Laura A. Vella, Marcus Buggert, Sasikanth Manne, Ramin S. Herati, Ismail Sayin, Leticia Kuri-Cervantes, Irene Bukh Brody, Kaitlin C. O’Boyle, Hagop Kaprielian, Josephine R. Giles, Son Nguyen, Alexander Muselman, Jack P. Antel, Amit Bar-Or, Matthew E. Johnson, David H. Canaday, Ali Naji, Vitaly V. Ganusov, Terri M. Laufer, Andrew D. Wells, Yoav Dori, Maxim G. Itkin, Michael R. Betts, E. John Wherry
Hedgehog (Hh) proteins regulate development and tissue homeostasis, but their role in atopic dermatitis (AD) remains unknown. We found that on induction of mouse AD, Sonic Hedgehog (Shh) expression in skin, and Hh pathway action in skin T cells were increased. Shh signaling reduced AD pathology and the levels of Shh expression determined disease severity. Hh-mediated transcription in skin T cells in AD-induced mice increased Treg populations and their suppressive function through increased active transforming growth factor–β (TGF-β) in Tregs signaling to skin T effector populations to reduce disease progression and pathology. RNA sequencing of skin CD4+ T cells from AD-induced mice demonstrated that Hh signaling increased expression of immunoregulatory genes and reduced expression of inflammatory and chemokine genes. Addition of recombinant Shh to cultures of naive human CD4+ T cells in iTreg culture conditions increased FOXP3 expression. Our findings establish an important role for Shh upregulation in preventing AD, by increased Gli-driven Treg cell–mediated immune suppression, paving the way for a potential new therapeutic strategy.
Eleftheria Papaioannou, Diana C. Yánez, Susan Ross, Ching-In Lau, Anisha Solanki, Mira Manilal Chawda, Alex Virasami, Ismael Ranz, Masahiro Ono, Ryan F. L. O’Shaughnessy, Tessa Crompton
Resident and inflammatory mononuclear phagocytes (MPh) with functional plasticity in the intestine are critically involved in the pathology of Inflammatory Bowel Diseases (IBD), in which the mechanism remains incompletely understood. In the present study, we found that increased expression of E3 ligase FBXW7 in the inflamed intestine was significantly correlated to IBD severity in both human diseases and mice model. Myeloid-Fbxw7 deﬁciency protected mice from dextran sodium sulfate (DSS) and 2,6,4-trinitrobenzene sulfonic acid (TNBS) induced colitis. Fbxw7 deficiency resulted in decreased production of chemokines CCL2 and CCL7 by colonic CX3CR1hi resident macrophages and reduced accumulation of CX3CR1int pro-inﬂammatory MPh in colitis colon tissue. Mice received AAV-shFbxw7 administration showed significantly improved survival rate and alleviated colitis. Mechanisms screening demonstrated that FBXW7 suppresses H3K27me3 modiﬁcation and promotes Ccl2 and Ccl7 expression via degradation of histone-lysine N-methyltransferase EZH2 in macrophages. Taken together, our results indicate that FBXW7 degrades EZH2 and increases Ccl2/Ccl7 in CX3CR1hi macrophages, which promotes the recruiting CX3CR1int pro-inﬂammatory MPh into local colon tissues with colitis. Targeting FBXW7 might represent a potential therapeutic approach for intestine inflammation intervention.
Jia He, Yinjing Song, Gaopeng Li, Peng Xiao, Yang Liu, Yue Xue, Qian Cao, Xintao Tu, Ting Pan, Zhinong Jiang, Xuetao Cao, Lihua Lai, Qingqing Wang
Lymph nodes (LNs) filter lymph to mount effective immune responses. Small soluble lymph-borne molecules from the periphery enter the draining LNs via a reticular conduit system. Intact antibodies and other larger molecules, in contrast, are physically unable to enter the conduits, and they are thought to be transported to the LNs only within migratory DCs after proteolytic degradation. Here, we discovered that lymph-borne antibodies and other large biomolecules enter within seconds into the parenchyma of the draining LN in an intact form. Mechanistically, we found that the uptake of large molecules is a receptor-independent, fluid-phase process that takes place by dynamin-dependent vesicular transcytosis through the lymphatic endothelial cells in the subcapsular sinus of the LN. Physiologically, this pathway mediates a very fast transfer of large protein antigens from the periphery to LN-resident DCs and macrophages. We show that exploitation of the transcytosis system allows enhanced whole-organ imaging and spatially controlled lymphocyte activation by s.c. administered antibodies in vivo. Transcytosis through the floor of the subcapsular sinus thus represents what we believe to be a new physiological and targetable mode of lymph filtering.
Laura Kähäri, Ruth Fair-Mäkelä, Kaisa Auvinen, Pia Rantakari, Sirpa Jalkanen, Johanna Ivaska, Marko Salmi
Induction of memory CD8 T cells is important for controlling infections such as malaria HIV/AIDS, and for cancer immunotherapy. Accurate assessment of antigen (Ag)-specific CD8 T-cells is critical for vaccine optimization and defining correlates of protection. However, conditions for determining Ag-specific CD8 T-cell responses ex-vivo using ICS may be variable, especially in humans with complex antigens. Here, we used an attenuated whole parasite malaria vaccine model in humans and various experimental infections in mice to show that the duration of antigenic stimulation and timing of brefeldin A (BFA) addition influences the magnitude of Ag-specific and bystander T cell responses. Indeed, following immunization with an attenuated whole sporozoite malaria vaccine in humans, significantly higher numbers of IFN-γ producing memory CD8 T-cells comprised of antigen specific and bystander responses were detected by increasing the duration of Ag-stimulation prior to addition of BFA. Mechanistic analyses of virus-specific CD8 T-cells in mice revealed that the increase in IFNg producing CD8 T-cells was due to bystander activation of Ag-experienced memory CD8 T-cells, and correlated with the proportion of Ag-experienced CD8 T-cells in the stimulated populations. Incubation with anti-cytokine antibodies (ex. IL-12) improved accuracy in detecting bona-fide memory CD8 T-cell responses suggesting this as the mechanism for the bystander activation. These data have important implications for accurate assessment of immune responses generated by vaccines intended to elicit protective memory CD8 T-cells.
Matthew D. Martin, Isaac J. Jensen, Andrew S. Ishizuka, Mitchell Lefebvre, Qiang Shan, Hai-Hui Xue, John T. Harty, Robert A. Seder, Vladimir P. Badovinac
A population of Natural Killer (NK) cells expressing the activating receptor NKG2C and the maturation marker CD57 expands in response to human cytomegalovirus (HCMV) infection. CD3–CD56dimCD57+NKG2C+ NK cells are similar to CD8+ memory T cells with rapid and robust effector function upon re-stimulation, persistence, and epigenetic remodeling of the IFNG locus. Chronic antigen stimulation drives CD8+ memory T cell proliferation while also inducing genome-wide epigenetic reprograming and dysfunction. We hypothesized that chronic stimulation could similarly induce epigenetic reprograming and dysfunction in NK cells. Here we show that chronic stimulation of adaptive NK cells through NKG2C using plate-bound agonistic antibodies in combination with IL-15 drove robust proliferation and activation of CD3–CD56dimCD57+NKG2C+ NK cells while simultaneously inducing high expression of the checkpoint inhibitory receptors LAG-3 and PD-1. Marked induction of checkpoint inhibitory receptors was also observed on the surface of adaptive NK cells co-cultured with HCMV-infected endothelial cells. Chronically stimulated adaptive NK cells were dysfunctional when challenged with tumor targets. These cells exhibited a pattern of epigenetic reprograming, with genome-wide alterations in DNA methylation. Our study has important implications for cancer immunotherapy and suggest that exhausted NK cells could be targeted with inhibitory checkpoint receptor blockade.
Aimee M. Merino, Bin Zhang, Phillip R. Dougherty, Xianghua Luo, Jinhua Wang, Bruce R. Blazar, Jeffrey S. Miller, Frank Cichocki
Resident microbiota activate regulatory cells that modulate intestinal inflammation and promote and maintain intestinal homeostasis. IL-10 is a key mediator of immune regulatory function. Our studies described the functional importance and mechanisms by which gut microbiota and specific microbial components influenced the development of intestinal IL-10-producing B cells. We used fecal transplant to germ-free (GF) Il10+/EGFP reporter and Il10-/- mice to demonstrate that microbiota from specific pathogen-free mice primarily stimulated IL-10-producing colon-specific B cells and T regulatory-1 cells in ex-GF mice. IL-10 in turn down-regulated microbiota-activated mucosal inflammatory cytokines. TLR2/9 ligands and enteric bacterial lysates preferentially induced IL-10 production and regulatory capacity of intestinal B cells. Analysis of Il10+/EGFP mice crossed with additional gene-deficient strains and B cell co-transfer studies demonstrated that microbiota-induced IL-10-producing intestinal B cells ameliorated chronic T cell-mediated colitis in a TLR2, MyD88 and PI3K-dependent fashion. In vitro studies implicated PI3Kp110δ and AKT downstream signaling. These studies demonstrated that resident enteric bacteria activated intestinal IL-10-producing B cells through TLR2, MyD88 and PI3K pathways. These B cells reduced colonic T cell activation and maintained mucosal homeostasis in response to intestinal microbiota.
Yoshiyuki Mishima, Akihiko Oka, Bo Liu, Jeremy W. Herzog, Chang Soo Eun, Ting-Jia Fan, Emily Bulik-Sullivan, Ian M. Carroll, Jonathan J. Hansen, Liang Chen, Justin E. Wilson, Nancy C. Fisher, Jenny P. Y. Ting, Tomonori Nochi, Angela Wahl, J. Victor Garcia, Christopher L. Karp, R. Balfour Sartor
Recent studies have demonstrated that CD4+ T cells can efficiently reject MHC-II–negative tumors. This requires indirect presentation of tumor-associated antigens on surrounding antigen-presenting cells. We hypothesized that intercellular transfer of proteins is not the sole consequence of cell death–mediated protein release, but depends on heat-shock cognate protein 70 (HSC70) and its KFERQ-like binding motif on substrate proteins. Using human Y chromosome antigen DBY, we showed that mutation of one of its 2 putative binding motifs markedly diminished T cell activation after indirect presentation and reduced protein-protein interaction with HSC70. Intercellular antigen transfer was shown to be independent of cell-cell contact, but relied on engulfment within secreted microvesicles. In vivo, alterations of the homologous KFERQ-like motif in murine DBY hampered tumor rejection, T cell activation, and migration into the tumor and substantially impaired survival. Collectively, we show that intercellular antigen transfer of DBY is tightly regulated via binding to HSC70 and that this mechanism influences recognition and rejection of MHC-II–negative tumors in vivo.
Sascha Kretschmann, Stefanie Herda, Heiko Bruns, Josefine Russ, Edith D. van der Meijden, Ursula Schlötzer-Schrehardt, Marieke Griffioen, Il-Kang Na, Andreas Mackensen, Anita N. Kremer