ABC transporters and NR4A1 identify a quiescent subset of tissue-resident memory T cells

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Abstract

Immune surveillance in tissues is mediated by a long-lived subset of tissue-resident memory T cells (Trm cells). A putative subset of tissue-resident long-lived stem cells is characterized by the ability to efflux Hoechst dyes and is referred to as side population (SP) cells. Here, we have characterized a subset of SP T cells (Tsp cells) that exhibit a quiescent (G₀) phenotype in humans and mice. Human Trm cells in the gut and BM were enriched in Tsp cells that were predominantly in the G₀ stage of the cell cycle. Moreover, in histone 2B-GFP mice, the 2B-GFP label was retained in Tsp cells, indicative of a slow-cycling phenotype. Human Tsp cells displayed a distinct gene-expression profile that was enriched for genes overexpressed in Trm cells. In mice, proteins encoded by Tsp signature genes, including nuclear receptor subfamily 4 group A member 1 (NR4A1) and ATP-binding cassette (ABC) transporters, influenced the function and differentiation of Trm cells. Responses to adoptive transfer of human Tsp cells into immune-deficient mice and plerixafor therapy suggested that human Tsp cell mobilization could be manipulated as a potential cellular therapy. These data identify a distinct subset of human T cells with a quiescent/slow-cycling phenotype, propensity for tissue enrichment, and potential to mobilize into circulation, which may be harnessed for adoptive cellular therapy.

Authors

Chandra Sekhar Boddupalli, Shiny Nair, Simon M. Gray, Heba N. Nowyhed, Rakesh Verma, Joanna A. Gibson, Clara Abraham, Deepak Narayan, Juan Vasquez, Catherine C. Hedrick, Richard A. Flavell, Kavita M. Dhodapkar, Susan M. Kaech, Madhav V. Dhodapkar

TGF-β receptor maintains CD4 T helper cell identity during chronic viral infections

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Abstract

Suppression of CD8 and CD4 T cells is a hallmark in chronic viral infections, including hepatitis C and HIV. While multiple pathways are known to inhibit CD8 T cells, the host molecules that restrict CD4 T cell responses are less understood. Here, we used inducible and CD4 T cell–specific deletion of the gene encoding the TGF-β receptor during chronic lymphocytic choriomeningitis virus infection in mice, and determined that TGF-β signaling restricted proliferation and terminal differentiation of antiviral CD4 T cells. TGF-β signaling also inhibited a cytotoxic program that includes granzymes and perforin expression at both early and late stages of infection in vivo and repressed the transcription factor eomesodermin. Overexpression of eomesodermin was sufficient to recapitulate in great part the phenotype of TGF-β receptor–deficient CD4 T cells, while SMAD4 was necessary for CD4 T cell accumulation and differentiation. TGF-β signaling also restricted accumulation and differentiation of CD4 T cells and reduced the expression of cytotoxic molecules in mice and humans infected with other persistent viruses. These data uncovered an eomesodermin-driven CD4 T cell program that is continuously suppressed by TGF-β signaling. During chronic viral infection, this program limits CD4 T cell responses while maintaining CD4 T helper cell identity.

Authors

Gavin M. Lewis, Ellen J. Wehrens, Lara Labarta-Bajo, Hendrik Streeck, Elina I. Zuniga

A DOCK8-WIP-WASp complex links T cell receptors to the actin cytoskeleton

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Abstract

Wiskott-Aldrich syndrome (WAS) is associated with mutations in the WAS protein (WASp), which plays a critical role in the initiation of T cell receptor–driven (TCR-driven) actin polymerization. The clinical phenotype of WAS includes susceptibility to infection, allergy, autoimmunity, and malignancy and overlaps with the symptoms of dedicator of cytokinesis 8 (DOCK8) deficiency, suggesting that the 2 syndromes share common pathogenic mechanisms. Here, we demonstrated that the WASp-interacting protein (WIP) bridges DOCK8 to WASp and actin in T cells. We determined that the guanine nucleotide exchange factor activity of DOCK8 is essential for the integrity of the subcortical actin cytoskeleton as well as for TCR-driven WASp activation, F-actin assembly, immune synapse formation, actin foci formation, mechanotransduction, T cell transendothelial migration, and homing to lymph nodes, all of which also depend on WASp. These results indicate that DOCK8 and WASp are in the same signaling pathway that links TCRs to the actin cytoskeleton in TCR-driven actin assembly. Further, they provide an explanation for similarities in the clinical phenotypes of WAS and DOCK8 deficiency.

Authors

Erin Janssen, Mira Tohme, Mona Hedayat, Marion Leick, Sudha Kumari, Narayanaswamy Ramesh, Michel J. Massaad, Sumana Ullas, Veronica Azcutia, Christopher C. Goodnow, Katrina L. Randall, Qi Qiao, Hao Wu, Waleed Al-Herz, Dianne Cox, John Hartwig, Darrell J. Irvine, Francis W. Luscinskas, Raif S. Geha

Cognate HLA absence in trans diminishes human NK cell education

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Abstract

NK cells are innate lymphocytes with protective functions against viral infections and tumor formation. Human NK cells carry inhibitory killer cell Ig-like receptors (KIRs), which recognize distinct HLAs. NK cells with KIRs for self-HLA molecules acquire superior cytotoxicity against HLA^– tumor cells during education for improved missing-self recognition. Here, we reconstituted mice with human hematopoietic cells from donors with homozygous KIR ligands or with a mix of hematopoietic cells from these homozygous donors, allowing assessment of the resulting KIR repertoire and NK cell education. We found that co-reconstitution with 2 KIR ligand–mismatched compartments did not alter the frequency of KIR-expressing NK cells. However, NK cell education was diminished in mice reconstituted with parallel HLA compartments due to a lack of cognate HLA molecules on leukocytes for the corresponding KIRs. This change in NK cell education in mixed human donor–reconstituted mice improved NK cell–mediated immune control of EBV infection, indicating that mixed hematopoietic cell populations could be exploited to improve NK cell reactivity against leukotropic pathogens. Taken together, these findings indicate that leukocytes lacking cognate HLA ligands can disarm KIR⁺ NK cells in a manner that may decrease HLA^– tumor cell recognition but allows for improved NK cell–mediated immune control of a human γ-herpesvirus.

Authors

Vanessa Landtwing, Ana Raykova, Gaetana Pezzino, Vivien Béziat, Emanuela Marcenaro, Claudine Graf, Alessandro Moretta, Riccarda Capaul, Andrea Zbinden, Guido Ferlazzo, Karl-Johan Malmberg, Obinna Chijioke, Christian Münz

Lung-resident eosinophils represent a distinct regulatory eosinophil subset

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Abstract

Increases in eosinophil numbers are associated with infection and allergic diseases, including asthma, but there is also evidence that eosinophils contribute to homeostatic immune processes. In mice, the normal lung contains resident eosinophils (rEos), but their function has not been characterized. Here, we have reported that steady-state pulmonary rEos are IL-5–independent parenchymal Siglec-F^intCD62L⁺CD101^lo cells with a ring-shaped nucleus. During house dust mite–induced airway allergy, rEos features remained unchanged, and rEos were accompanied by recruited inflammatory eosinophils (iEos), which were defined as IL-5–dependent peribronchial Siglec-F^hiCD62L^–CD101^hi cells with a segmented nucleus. Gene expression analyses revealed a more regulatory profile for rEos than for iEos, and correspondingly, mice lacking lung rEos showed an increase in Th2 cell responses to inhaled allergens. Such elevation of Th2 responses was linked to the ability of rEos, but not iEos, to inhibit the maturation, and therefore the pro-Th2 function, of allergen-loaded DCs. Finally, we determined that the parenchymal rEos found in nonasthmatic human lungs (Siglec-8⁺CD62L⁺IL-3R^lo cells) were phenotypically distinct from the iEos isolated from the sputa of eosinophilic asthmatic patients (Siglec-8⁺CD62L^loIL-3R^hi cells), suggesting that our findings in mice are relevant to humans. In conclusion, our data define lung rEos as a distinct eosinophil subset with key homeostatic functions.

Authors

Claire Mesnil, Stéfanie Raulier, Geneviève Paulissen, Xue Xiao, Mark A. Birrell, Dimitri Pirottin, Thibaut Janss, Philipp Starkl, Eve Ramery, Monique Henket, Florence N. Schleich, Marc Radermecker, Kris Thielemans, Laurent Gillet, Marc Thiry, Maria G. Belvisi, Renaud Louis, Christophe Desmet, Thomas Marichal, Fabrice Bureau

BET bromodomain inhibition enhances T cell persistence and function in adoptive immunotherapy models

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Abstract

Adoptive immunotherapy is a potentially curative therapeutic approach for patients with advanced cancer. However, the in vitro expansion of antitumor T cells prior to infusion inevitably incurs differentiation towards effector T cells and impairs persistence following adoptive transfer. Epigenetic profiles regulate gene expression of key transcription factors over the course of immune cell differentiation, proliferation, and function. Using comprehensive screening of chemical probes with defined epigenetic targets, we found that JQ1, an inhibitor of bromodomain and extra-terminal motif (BET) proteins, maintained CD8⁺ T cells with functional properties of stem cell–like and central memory T cells. Mechanistically, the BET protein BRD4 directly regulated expression of the transcription factor BATF in CD8⁺ T cells, which was associated with differentiation of T cells into an effector memory phenotype. JQ1-treated T cells showed enhanced persistence and antitumor effects in murine T cell receptor and chimeric antigen receptor gene therapy models. Furthermore, we found that histone acetyltransferase p300 supported the recruitment of BRD4 to the BATF promoter region, and p300 inhibition similarly augmented antitumor effects of the adoptively transferred T cells. These results demonstrate that targeting the BRD4-p300 signaling cascade supports the generation of superior antitumor T cell grafts for adoptive immunotherapy.

Authors

Yuki Kagoya, Munehide Nakatsugawa, Yuki Yamashita, Toshiki Ochi, Tingxi Guo, Mark Anczurowski, Kayoko Saso, Marcus O. Butler, Cheryl H. Arrowsmith, Naoto Hirano

p21 mediates macrophage reprogramming through regulation of p50-p50 NF-κB and IFN-β

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Abstract

M1 and M2 macrophage phenotypes, which mediate proinflammatory and antiinflammatory functions, respectively, represent the extremes of immunoregulatory plasticity in the macrophage population. This plasticity can also result in intermediate macrophage states that support a balance between these opposing functions. In sepsis, M1 macrophages can compensate for hyperinflammation by acquiring an M2-like immunosuppressed status that increases the risk of secondary infection and death. The M1 to M2 macrophage reprogramming that develops during LPS tolerance resembles the pathological antiinflammatory response to sepsis. Here, we determined that p21 regulates macrophage reprogramming by shifting the balance between active p65-p50 and inhibitory p50-p50 NF-κB pathways. p21 deficiency reduced the DNA-binding affinity of the p50-p50 homodimer in LPS-primed and -rechallenged macrophages, impairing their ability to attenuate IFN-β production and acquire an M2-like hyporesponsive status. High p21 levels in sepsis patients correlated with low IFN-β expression, and p21 knockdown in human monocytes corroborated its role in IFN-β regulation. The data demonstrate that p21 adjusts the equilibrium between p65-p50 and p50-p50 NF-κB pathways to mediate macrophage plasticity in LPS tolerance. Identifying p21-related pathways involved in monocyte reprogramming may lead to potential targets for sepsis treatment.

Authors

Gorjana Rackov, Enrique Hernández-Jiménez, Rahman Shokri, Lorena Carmona-Rodríguez, Santos Mañes, Melchor Álvarez-Mon, Eduardo López-Collazo, Carlos Martínez-A, Dimitrios Balomenos

The composition of the microbiota modulates allograft rejection

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Abstract

Transplantation is the only cure for end-stage organ failure, but without immunosuppression, T cells rapidly reject allografts. While genetic disparities between donor and recipient are major determinants of the kinetics of transplant rejection, little is known about the contribution of environmental factors. Because colonized organs have worse transplant outcome than sterile organs, we tested the influence of host and donor microbiota on skin transplant rejection. Compared with untreated conventional mice, pretreatment of donors and recipients with broad-spectrum antibiotics (Abx) or use of germ-free (GF) donors and recipients resulted in prolonged survival of minor antigen–mismatched skin grafts. Increased graft survival correlated with reduced type I IFN signaling in antigen-presenting cells (APCs) and decreased priming of alloreactive T cells. Colonization of GF mice with fecal material from untreated conventional mice, but not from Abx-pretreated mice, enhanced the ability of APCs to prime alloreactive T cells and accelerated graft rejection, suggesting that alloimmunity is modulated by the composition of microbiota rather than the quantity of bacteria. Abx pretreatment of conventional mice also delayed rejection of major antigen–mismatched skin and MHC class II–mismatched cardiac allografts. This study demonstrates that Abx pretreatment prolongs graft survival, suggesting that targeting microbial constituents is a potential therapeutic strategy for enhancing graft acceptance.

Authors

Yuk Man Lei, Luqiu Chen, Ying Wang, Andrew T. Stefka, Luciana L. Molinero, Betty Theriault, Keston Aquino-Michaels, Ayelet S. Sivan, Cathryn R. Nagler, Thomas F. Gajewski, Anita S. Chong, Caroline Bartman, Maria-Luisa Alegre

Glycolysis determines dichotomous regulation of T cell subsets in hypoxia

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Abstract

Hypoxia occurs in many pathological conditions, including chronic inflammation and tumors, and is considered to be an inhibitor of T cell function. However, robust T cell responses occur at many hypoxic inflammatory sites, suggesting that functions of some subsets are stimulated under low oxygen conditions. Here, we investigated how hypoxic conditions influence human T cell functions and found that, in contrast to naive and central memory T cells (T_N and T_CM), hypoxia enhances the proliferation, viability, and cytotoxic action of effector memory T cells (T_EM). Enhanced TEM expansion in hypoxia corresponded to high hypoxia-inducible factor 1α (HIF1α) expression and glycolytic activity compared with that observed in T_N and T_CM. We determined that the glycolytic enzyme GAPDH negatively regulates HIF1A expression by binding to adenylate-uridylate–rich elements in the 3′-UTR region of HIF1A mRNA in glycolytically inactive T_N and T_CM. Conversely, active glycolysis with decreased GAPDH availability in T_EM resulted in elevated HIF1α expression. Furthermore, GAPDH overexpression reduced HIF1α expression and impaired proliferation and survival of T cells in hypoxia, indicating that high glycolytic metabolism drives increases in HIF1α to enhance T_EM function during hypoxia. This work demonstrates that glycolytic metabolism regulates the translation of HIF1A to determine T cell responses to hypoxia and implicates GAPDH as a potential mechanism for controlling T cell function in peripheral tissue.

Authors

Yang Xu, Arindam Chaudhury, Ming Zhang, Barbara Savoldo, Leonid S. Metelitsa, John Rodgers, Jason T. Yustein, Joel R. Neilson, Gianpietro Dotti

Uropathogenic Escherichia coli strain CFT073 disrupts NLRP3 inflammasome activation

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Abstract

Successful bacterial pathogens produce an array of virulence factors that allow subversion of the immune system and persistence within the host. For example, uropathogenic Escherichia coli strains, such as CFT073, express Toll/IL-1 receptor–containing (TIR-containing) protein C (TcpC), which impairs TLR signaling, thereby suppressing innate immunity in the urinary tract and enhancing persistence in the kidneys. Here, we have reported that TcpC also reduces secretion of IL-1β by directly interacting with the NACHT leucin-rich repeat PYD protein 3 (NLRP3) inflammasome, which is crucial for recognition of pathogens within the cytosol. At a low MOI, IL-1β secretion was minimal in CFT073-infected macrophages; however, IL-1β release was markedly increased in macrophages infected with CFT073 lacking tcpC. Induction of IL-1β secretion by CFT073 and tcpC–deficient CFT073 required the NLRP3 inflammasome. TcpC attenuated activation of the NLRP3 inflammasome by binding both NLRP3 and caspase-1 and thereby preventing processing and activation of caspase-1. Moreover, in a murine urinary tract infection model, CFT073 infection rapidly induced expression of the NLRP3 inflammasome in the bladder mucosa; however, the presence of TcpC in WT CFT073 reduced IL-1β levels in the urine of infected mice. Together, these findings illustrate how uropathogenic E. coli use the multifunctional virulence factor TcpC to attenuate innate immune responses in the urinary tract.

Authors

Anna Waldhuber, Manoj Puthia, Andreas Wieser, Christine Cirl, Susanne Dürr, Silke Neumann-Pfeifer, Simone Albrecht, Franziska Römmler, Tina Müller, Yunji Zheng, Sören Schubert, Olaf Groß, Catharina Svanborg, Thomas Miethke