The surface receptor CD8α is present on 20-80% of human (but not mouse) NK cells, yet its function on NK cells remains poorly understood. CD8α expression on donor NK cells was associated with a lack of therapeutic responses for leukemia patients in prior studies, thus we hypothesized that CD8α may impact critical NK cell functions. Here, we discovered that CD8α- NK cells had improved control of leukemia in xenograft models, compared to CD8α+ NK cells, likely due to an enhanced capacity for proliferation. Unexpectedly, CD8α expression was induced on approximately 30% of previously CD8α- NK cells following IL-15 stimulation. These ‘induced’ CD8α+ (‘iCD8α+’) NK cells had the greatest proliferation, responses to IL-15 signaling, and metabolic activity, compared to those that sustained existing CD8α expression (‘sustained CD8α+) or those that remained CD8α- (‘persistent CD8α-‘). These iCD8α+ cells originated from an IL-15Rβ high NK cell population, with CD8α expression dependent on the transcription factor RUNX3. Moreover, CD8A CRISPR/Cas9 deletion resulted in enhanced responses through the activating receptor NKp30, possibly by modulating KIR inhibitory function. Thus, CD8α status identifies human NK cell capacity for IL-15-induced proliferation and metabolism in a time-dependent fashion and exhibits a suppressive effect on NK cell activating receptors.
Celia C. Cubitt, Pamela Wong, Hannah K. Dorando, Jennifer A. Foltz, Jennifer Tran, Lynne Marsala, Nancy D. Marin, Mark Foster, Timothy Schappe, Hijab Fatima, Michelle Becker-Hapak, Alice Y. Zhou, Kimberly Hwang, Miriam T. Jacobs, David A. Russler-Germain, Emily M. Mace, Melissa M. Berrien-Elliott, Jacqueline E. Payton, Todd A. Fehniger
Intratumoral regulatory T cells (Tregs) are key mediators of cancer immunotherapy resistance, including anti-PD-(L)1 immune checkpoint blockade (ICB). The mechanisms driving Treg infiltration into the tumor microenvironment (TME) and the consequence on CD8+ T cell exhaustion remains elusive. Herein, we report that heat shock protein gp96 (GRP94) is indispensable for Treg tumor infiltration, primarily through gp96’s roles in chaperoning integrins. Among various gp96-dependent integrins, we found that only LFA-1 (αL integrin) but not αV, CD103 (αE) or β7 integrin was required for Treg tumor homing. Loss of Treg infiltration into the TME by genetically deleting gp96/LFA-1 potently induces rejection of multiple ICB-resistant murine cancer models in a CD8+ T cell-dependent manner without loss of self-tolerance. Moreover, gp96 deletion impeded Treg activation primarily by suppressing IL-2/STAT5 signaling, which also contributes to tumor regression. By competing for intratumoral IL-2, Tregs prevent activation of CD8+ tumor-infiltrating lymphocytes (TILs), drive TOX induction and induce bona fide CD8+ T cell exhaustion. By contrast, Treg ablation leads to striking CD8+ T cell activation without TOX induction, demonstrating clear uncoupling of the two processes. Our study reveals that the gp96/LFA-1 axis plays a fundamental role in Treg biology and suggests that Treg-specific gp96/LFA-1 targeting represents a valuable strategy for cancer immunotherapy without inflicting autoinflammatory conditions.
Lei Zhou, Maria Velegraki, Yi Wang, J K Mandula, Yuzhou Chang, Weiwei Liu, No-Joon Song, Hyunwoo Kwon, Tong Xiao, Chelsea Bolyard, Feng Hong, Gang Xin, Qin Ma, Mark P. Rubinstein, Haitao Wen, Zihai Li
Jarmila Stremenova Spegarova, Praisoody Sinnappurajar, Dalila Al Julandani, Rokas Navickas, Helen Griffin, Manisha Ahuja, Angela Grainger, Katie Livingstone, Gillian I. Rice, Fraser Sutherland, Corinne Hayes, Simon Parke, Lewis Pang, Marion R. Roderick, Mary Slatter, Yanick Crow, Athimalaipet V. Ramanan, Sophie Hambleton
Group 3 innate lymphoid cells (ILC3s) are key players in intestinal homeostasis. Endoplasmic reticulum (ER) stress is linked to inflammatory bowel disease (IBD). Herein, we used cell culture, mouse models, and human specimens to examine if ER stress in ILC3s impacts IBD pathophysiology. We show that mouse intestinal ILC3s exhibited a 24h-rhythmic expression pattern of the master ER stress response regulator, IRE1α/XBP1. Proinflammatory cytokine IL-23 selectively stimulated IRE1α/XBP1 in mouse ILC3s through mitochondrial reactive oxygen species (mtROS). IRE1α/XBP1 was activated in ILC3s of mice exposed to experimental colitis and in inflamed human IBD specimens. Mice with Ire1α deletion in ILC3s (Ire1αΔRorc) showed reduced expression of ER stress response and cytokine genes including Il22 in ILC3s and were highly vulnerable to infections and colitis. Administration of IL-22 counteracted their colitis susceptibility. In human ILC3s, IRE1 inhibitors suppressed cytokine production, which was upregulated by an IRE1 activator. Moreover, the frequencies of intestinal XBP1s+ ILC3s in Crohn’s disease patients before administration of ustekinumab, an anti-IL-12/IL-23 antibody, positively correlated with response to treatment. We demonstrate that a non-canonical mtROS-IRE1α/XBP1 pathway augments cytokine production by ILC3s and identify XBP1s+ ILC3s as a potential biomarker for predicting response to anti-IL-23 therapies in IBD.
Siyan Cao, Jose Luis Fachi, Kaiming Ma, Alina Ulezko Antonova, Qianli Wang, Zhangying Cai, Randal J. Kaufman, Matthew A Ciorba, Parakkal Deepak, Marco Colonna
Alyssa Duffy, Maryam I. Azeem, Smriti Kanangat, Melinda Yushak, David Lawson, Madhav V. Dhodapkar, Kavita M. Dhodapkar
We describe a previously-unappreciated role for Bruton's tyrosine kinase (BTK) in fungal immune surveillance against aspergillosis, an unforeseen complication of BTK inhibitors (BTKi) used for treating B-cell lymphoid malignancies. We studied BTK-dependent fungal responses in neutrophils from diverse populations, including healthy donors, BTKi-treated patients, and X-linked agammaglobulinemia patients. Upon fungal exposure, BTK was activated in human neutrophils in a TLR2-, Dectin-1-, and FcγR-dependent manner, triggering the oxidative burst. BTK inhibition selectively impeded neutrophil-mediated damage to Aspergillus hyphae, primary granule release, and the fungus-induced oxidative burst by abrogating NADPH oxidase subunit p40phox and GTPase RAC2 activation. Moreover, neutrophil-specific Btk deletion in mice enhanced aspergillosis susceptibility by impairing neutrophil function, not recruitment or lifespan. Conversely, GM-CSF partially mitigated these deficits by enhancing p47phox activation. Our findings underline the crucial role of BTK signaling in neutrophils for antifungal immunity and provide a rationale for GM-CSF use to offset these deficits in susceptible patients.
Jigar V. Desai, Marissa A. Zarakas, Andrew L. Wishart, Mark Roschewski, Mariano A. Aufiero, Ágnes Donkó, Gustaf Wigerblad, Neta Shlezinger, Markus Plate, Matthew R. James, Jean K. Lim, Gulbu Uzel, Jenna R.E. Bergerson, Ivan Fuss, Robert A. Cramer, Luis M. Franco, Emily S. Clark, Wasif N. Khan, Daisuke Yamanaka, Georgios Chamilos, Jamel El-Benna, Mariana J. Kaplan, Louis M. Staudt, Thomas L. Leto, Steven M. Holland, Wyndham H. Wilson, Tobias M. Hohl, Michail S. Lionakis
BACKGROUND Patients hospitalized for COVID-19 exhibit diverse clinical outcomes, with outcomes for some individuals diverging over time even though their initial disease severity appears similar to that of other patients. A systematic evaluation of molecular and cellular profiles over the full disease course can link immune programs and their coordination with progression heterogeneity.METHODS We performed deep immunophenotyping and conducted longitudinal multiomics modeling, integrating 10 assays for 1,152 Immunophenotyping Assessment in a COVID-19 Cohort (IMPACC) study participants and identifying several immune cascades that were significant drivers of differential clinical outcomes.RESULTS Increasing disease severity was driven by a temporal pattern that began with the early upregulation of immunosuppressive metabolites and then elevated levels of inflammatory cytokines, signatures of coagulation, formation of neutrophil extracellular traps, and T cell functional dysregulation. A second immune cascade, predictive of 28-day mortality among critically ill patients, was characterized by reduced total plasma Igs and B cells and dysregulated IFN responsiveness. We demonstrated that the balance disruption between IFN-stimulated genes and IFN inhibitors is a crucial biomarker of COVID-19 mortality, potentially contributing to failure of viral clearance in patients with fatal illness.CONCLUSION Our longitudinal multiomics profiling study revealed temporal coordination across diverse omics that potentially explain the disease progression, providing insights that can inform the targeted development of therapies for patients hospitalized with COVID-19, especially those who are critically ill.TRIAL REGISTRATION ClinicalTrials.gov NCT04378777.FUNDING NIH (5R01AI135803-03, 5U19AI118608-04, 5U19AI128910-04, 4U19AI090023-11, 4U19AI118610-06, R01AI145835-01A1S1, 5U19AI062629-17, 5U19AI057229-17, 5U19AI125357-05, 5U19AI128913-03, 3U19AI077439-13, 5U54AI142766-03, 5R01AI104870-07, 3U19AI089992-09, 3U19AI128913-03, and 5T32DA018926-18); NIAID, NIH (3U19AI1289130, U19AI128913-04S1, and R01AI122220); and National Science Foundation (DMS2310836).
Jeremy P. Gygi, Cole Maguire, Ravi K. Patel, Pramod Shinde, Anna Konstorum, Casey P. Shannon, Leqi Xu, Annmarie Hoch, Naresh Doni Jayavelu, Elias K. Haddad, IMPACC Network, Elaine F. Reed, Monica Kraft, Grace A. McComsey, Jordan P. Metcalf, Al Ozonoff, Denise Esserman, Charles B. Cairns, Nadine Rouphael, Steven E. Bosinger, Seunghee Kim-Schulze, Florian Krammer, Lindsey B. Rosen, Harm van Bakel, Michael Wilson, Walter L. Eckalbar, Holden T. Maecker, Charles R. Langelier, Hanno Steen, Matthew C. Altman, Ruth R. Montgomery, Ofer Levy, Esther Melamed, Bali Pulendran, Joann Diray-Arce, Kinga K. Smolen, Gabriela K. Fragiadakis, Patrice M. Becker, Rafick P. Sekaly, Lauren I.R. Ehrlich, Slim Fourati, Bjoern Peters, Steven H. Kleinstein, Leying Guan
The immune system can control cancer progression. However, even though some innate immune sensors of cellular stress are expressed intrinsically in epithelial cells, their potential role in cancer aggressiveness and subsequent overall survival in humans is mainly unknown. Here, we show that NLR family CARD Domain Containing 4 (NLRC4) is downregulated in epithelial tumor cells of colorectal cancer (CRC) patients by using spatial tissue imaging. Strikingly, only the loss of tumor NLRC4 but not stromal is associated with poor immune infiltration (mainly dendritic and CD4+/CD8+ T cells) and accurately predicts progression to metastatic Stage IV and decrease of overall survival. By combining multi-omics approaches, we show that restoring NLRC4 expression in human colorectal cancer cells triggers a broad inflammasome-independent immune reprogramming consisting of Type-I IFN signaling genes and the release of chemokines and myeloid growth factors involved in the tumor infiltration and activation of dendritic cells (DCs) and T cells. Consistently, such reprogramming in cancer cells is sufficient to directly mature human DCs towards a Th1 antitumor immune response through IL-12 production in vitro. In multiple human carcinomas (colorectal, lung, and skin), we confirmed that NLRC4 expression in patient tumors is strongly associated with Type-I IFN genes, immune infiltrates and high microsatellite instability. Thus, we shed light on the epithelial innate immune sensor NLRC4 as a novel therapeutic target to promote an efficient antitumor immune response against the aggressiveness of various carcinomas.
Charlotte Domblides, Steven Crampton, Hong Liu, Juliet M. Bartleson, Annie Nguyen, Claudia Champagne, Emily E. Landy, Lindsey Spiker, Christopher Proffitt, Sunil Bhattarai, Anissa P. Grawe, Matias Fuentealba Valenzuela, Lydia Lartigue, Isabelle Mahouche, Jeremy Dupaul-Chicoine, Kazuho Nishimura, Félix Lefort, Marie Decraecker, Valérie Velasco, Sonia Netzer, Vincent Pitard, Christian Roy, Isabelle Soubeyran, Victor Racine, Patrick Blanco, Julie Déchanet-Merville, Maya Saleh, Scott W. Canna, David Furman, Benjamin Faustin
Administration of anti-RhD immunoglobulin (Ig) to decrease maternal alloimmunization (antibody-mediated immune suppression [AMIS]) was a landmark clinical development. However, IgG has potent immune-stimulatory effects in other settings (antibody-mediated immune enhancement [AMIE]). The dominant thinking has been that IgG causes AMIS for antigens on RBCs but AMIE for soluble antigens. However, we have recently reported that IgG against RBC antigens can cause either AMIS or AMIE as a function of an IgG subclass. Recent advances in mechanistic understanding have demonstrated that RBC alloimmunization requires the IFN-α/-β receptor (IFNAR) and is inhibited by the complement C3 protein. Here, we demonstrate the opposite for AMIE of an RBC alloantigen (IFNAR is not required and C3 enhances). RBC clearance, C3 deposition, and antigen modulation all preceded AMIE, and both CD4+ T cells and marginal zone B cells were required. We detected no significant increase in antigen-specific germinal center B cells, consistent with other studies of RBC alloimmunization that show extrafollicular-like responses. To the best of our knowledge, these findings provide the first evidence of an RBC alloimmunization pathway which is IFNAR independent and C3 dependent, thus further advancing our understanding of RBCs as an immunogen and AMIE as a phenomenon.
Arijita Jash, Thomas Pridmore, James B. Collins, Ariel M. Hay, Krystalyn E. Hudson, Chance John Luckey, James C. Zimring
Individuals with clonal hematopoiesis of indeterminate potential (CHIP) are at increased risk of aging related health conditions and all-cause mortality, but whether CHIP impacts risk of infection is much less clear. Using UK Biobank data, we revealed a positive association between CHIP and incident pneumonia in 438,421 individuals. We show that inflammation enhanced pneumonia risk, as CHIP carriers with a hypomorphic IL6 receptor polymorphism were protected. To better characterize the pathways of susceptibility, we challenged hematopoietic Tet Methylcytosine Dioxygenase 2 knockout (Tet2–/–) and floxed control mice (Tet2f/f) with Streptococcus pneumoniae. As with human CHIP carriers, Tet2–/– mice had hematopoietic abnormalities resulting in the expansion of inflammatory monocytes and neutrophils in peripheral blood. Yet, these cells were insufficient in defending against S. pneumoniae and resulted in increased pathology, impaired bacterial clearance, and higher mortality in Tet2–/– mice. We delineated the transcriptional landscape of Tet2–/– neutrophils and found that while inflammation-related pathways were upregulated in Tet2–/– neutrophils, migration and motility pathways were compromised. Using live-imaging techniques, we demonstrated impairments in motility, pathogen uptake and neutrophil extracellular trap (NET) formation by Tet2–/– neutrophils. Collectively, we show that CHIP is a risk factor for bacterial pneumonia related to innate immune impairments.
Candice Quin, Erica N. DeJong, Elina K. Cook, Yi Zhen Luo, Caitlyn Vlasschaert, Sanathan Sadh, Amy J.M. McNaughton, Marco M. Buttigieg, Jessica A Breznik, Allison E. Kennedy, Kevin Zhao, Jeffrey Mewburn, Kimberly J. Dunham-Snary, Charles C.T. Hindmarch, Alexander G. Bick, Stephen L. Archer, Michael J. Rauh, Dawn M.E. Bowdish