IFN-γR/STAT1 signaling in recipient hematopoietic antigen-presenting cells suppresses graft-versus-host disease

The absence of IFN-γ receptor (IFN-γR) or STAT1 signaling in donor cells has been shown to result in reduced induction of acute graft-versus-host disease (GVHD). In this study, we unexpectedly observed increased activation and expansion of donor lymphocytes in both lymphohematopoietic organs and GVHD target tissues of IFN-γR/STAT1–deficient recipient mice, leading to rapid mortality following the induction of GVHD. LPS-matured, BM-derived Ifngr1^–/– Stat1^–/– DCs (BMDCs) were more potent allogeneic stimulators and expressed increased levels of MHC II and costimulatory molecules. Similar effects were observed in human antigen-presenting cells (APCs) with knockdown of Stat1 by CRISPR/Cas9 and treatment with a JAK1/2 inhibitor. Furthermore, we demonstrated that the absence of IFN-γR/STAT1 signaling in hematopoietic APCs impaired the presentation of exogenous antigens, while promoting the presentation of endogenous antigens. Thus, the indirect presentation of host antigens to donor lymphocytes was defective in IFN-γR/STAT1–deficient, donor-derived APCs in fully donor chimeric mice. The differential effects of IFN-γR/STAT1 signaling on endogenous and exogenous antigen presentation could provide further insight into the roles of the IFN-γ/STAT1 signaling pathway in the pathogenesis of GVHD, organ rejection, and autoimmune diseases.     

Durable SARS-CoV-2 B cell immunity after mild or severe disease

Multiple studies have shown loss of severe acute respiratory syndrome coronavirus 2–specific (SARS-CoV-2–specific) antibodies over time after infection, raising concern that humoral immunity against the virus is not durable. If immunity wanes quickly, millions of people may be at risk for reinfection after recovery from coronavirus disease 2019 (COVID-19). However, memory B cells (MBCs) could provide durable humoral immunity even if serum neutralizing antibody titers decline. We performed multidimensional flow cytometric analysis of S protein receptor binding domain–specific (S-RBD–specific) MBCs in cohorts of ambulatory patients with COVID-19 with mild disease (n = 7), and hospitalized patients with moderate to severe disease (n = 7), at a median of 54 days (range, 39–104 days) after symptom onset. We detected S-RBD–specific class-switched MBCs in 13 of 14 participants, failing only in the individual with the lowest plasma levels of anti–S-RBD IgG and neutralizing antibodies. Resting MBCs (rMBCs) made up the largest proportion of S-RBD–specific MBCs in both cohorts. FCRL5, a marker of functional memory on rMBCs, was more dramatically upregulated on S-RBD–specific rMBCs after mild infection than after severe infection. These data indicate that most SARS-CoV-2-infected individuals develop S-RBD–specific, class-switched rMBCs that resemble germinal center–derived B cells induced by effective vaccination against other pathogens, providing evidence for durable B cell–mediated immunity against SARS-CoV-2 after mild or severe disease.     

IFN-α with dasatinib broadens the immune repertoire in patients with chronic-phase chronic myeloid leukemia

In chronic myeloid leukemia (CML), combination therapies with tyrosine kinase inhibitors (TKIs) aim to improve the achievement of deep molecular remission that would allow therapy discontinuation. IFN-α is one promising candidate, as it has long-lasting effects on both malignant and immune cells. In connection with a multicenter clinical trial combining dasatinib with IFN-α in 40 patients with chronic-phase CML (NordCML007, {"type":"clinical-trial","attrs":{"text":"NCT01725204","term_id":"NCT01725204"}}NCT01725204), we performed immune monitoring with single-cell RNA and T cell receptor (TCR) sequencing (n = 4, 12 samples), bulk TCRβ sequencing (n = 13, 26 samples), flow cytometry (n = 40, 106 samples), cytokine analyses (n = 17, 80 samples), and ex vivo functional studies (n = 39, 80 samples). Dasatinib drove the immune repertoire toward terminally differentiated NK and CD8⁺ T cells with dampened functional capabilities. Patients with dasatinib-associated pleural effusions had increased numbers of CD8⁺ recently activated effector memory T (Temra) cells. In vitro, dasatinib prevented CD3-induced cell death by blocking TCR signaling. The addition of IFN-α reversed the terminally differentiated phenotypes and increased the number of costimulatory intercellular interactions and the number of unique putative epitope-specific TCR clusters. In vitro IFN-α had costimulatory effects on TCR signaling. Our work supports the combination of IFN-α with TKI therapy, as IFN-α broadens the immune repertoire and restores immunological function.     

Longitudinal liver sampling in patients with chronic hepatitis B starting antiviral therapy reveals hepatotoxic CD8+ T cells

Accumulation of activated immune cells results in nonspecific hepatocyte killing in chronic hepatitis B (CHB), leading to fibrosis and cirrhosis. This study aims to understand the underlying mechanisms in humans and to define whether these are driven by widespread activation or a subpopulation of immune cells. We enrolled CHB patients with active liver damage to receive antiviral therapy and performed longitudinal liver sampling using fine-needle aspiration to investigate mechanisms of CHB pathogenesis in the human liver. Single-cell sequencing of total liver cells revealed a distinct liver-resident, polyclonal CD8⁺ T cell population that was enriched at baseline and displayed a highly activated immune signature during liver damage. Cytokine combinations, identified by in silico prediction of ligand-receptor interaction, induced the activated phenotype in healthy liver CD8⁺ T cells, resulting in nonspecific Fas ligand–mediated killing of target cells. These results define a CD8⁺ T cell population in the human liver that can drive pathogenesis and a key pathway involved in their function in CHB patients.     

Enhanced neoepitope-specific immunity following neoadjuvant PD-L1 and TGF-β blockade in HPV-unrelated head and neck cancer

BACKGROUNDHead and neck squamous cell carcinoma not associated with HPV (HPV-unrelated HNSCC) is associated with a high rate of recurrence and poor survival.METHODSWe conducted a clinical trial in 14 patients with newly diagnosed HPV-unrelated HNSCC to evaluate the safety and efficacy of neoadjuvant bintrafusp alfa, a bifunctional fusion protein that blocks programmed death ligand 1 (PD-L1) and neutralizes TGF-β.RESULTSBintrafusp alfa was well tolerated, and no treatment-associated surgical delays or complications occurred. Objective pathologic responses (PRs) were observed, and 12 of the 14 (86%) patients were alive and disease free at 1 year. Alterations in Treg infiltration and spatial distribution relative to proliferating CD8⁺ T cells indicated a reversal of Treg immunosuppression in the primary tumor. Detection of neoepitope-specific tumor T cell responses, but not virus-specific responses, correlated with the development of a PR. Detection of neoepitope-specific responses and PRs in tumors was not correlated with genomic features or tumor antigenicity but was associated with reduced pretreatment myeloid cell tumor infiltration. These results indicate that dual PD-L1 and TGF-β blockade can safely enhance tumor antigen–specific immunity and highlight the feasibility of multimechanism neoadjuvant immunotherapy for patients with HPV-unrelated HNSCC.CONCLUSIONOur studies provide insight into the ability of neoadjuvant immunotherapy to induce polyclonal neoadjuvant–specific T cell responses in tumors and suggest that features of the tumor microenvironment, such as myeloid cell infiltration, may be a major determinant of enhanced antitumor immunity following such treatment.TRIAL REGISTRATIONClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT04247282","term_id":"NCT04247282"}}NCT04247282.FUNDINGThis work was funded by the Center for Cancer Research, the NCI, and the Intramural Research Program of the NIDCD, NIH. Bintrafusp alfa was provided by the health care business of Merck KGaA (Darmstadt, Germany), through a Cooperative Research and Development Agreement with the NCI. Additional funding was provided by ImmunityBio through a Cooperative Research and Development Agreement with the NIDCD.     

Blocking Borrelia burgdorferi transmission from infected ticks to nonhuman primates with a human monoclonal antibody

Disrupting transmission of Borrelia burgdorferi sensu lato complex (B. burgdorferi) from infected ticks to humans is one strategy to prevent the significant morbidity from Lyme disease. We have previously shown that an anti-OspA human mAb, 2217, prevents transmission of B. burgdorferi from infected ticks in animal models. Maintenance of a protective plasma concentration of a human mAb for tick season presents a significant challenge for a preexposure prophylaxis strategy. Here, we describe the optimization of mAb 2217 by amino acid substitutions (2217LS: M428L and N434S) in the Fc domain. The LS mutation led to a 2-fold increase in half-life in cynomolgus monkeys. In a rhesus macaque model, 2217LS protected animals from tick transmission of spirochetes at a dose of 3 mg/kg. Crystallographic analysis of Fab in complex with OspA revealed that 2217 bound an epitope that was highly conserved among the B. burgdorferi, B. garinii, and B. afzelii species. Unlike most vaccines that may require boosters to achieve protection, our work supports the development of 2217LS as an effective preexposure prophylaxis in Lyme-endemic regions, with a single dose at the beginning of tick season offering immediate protection that remains for the duration of exposure risk.     

Tissue-resident-like CD4+ T cells secreting IL-17 control Mycobacterium tuberculosis in the human lung

T cell immunity is essential for the control of tuberculosis (TB), an important disease of the lung, and is generally studied in humans using peripheral blood cells. Mounting evidence, however, indicates that tissue-resident memory T cells (Trms) are superior at controlling many pathogens, including Mycobacterium tuberculosis (M. tuberculosis), and can be quite different from those in circulation. Using freshly resected lung tissue, from individuals with active or previous TB, we identified distinct CD4⁺ and CD8⁺ Trm-like clusters within TB-diseased lung tissue that were functional and enriched for IL-17–producing cells. M. tuberculosis–specific CD4⁺ T cells producing TNF-α, IL-2, and IL-17 were highly expanded in the lung compared with matched blood samples, in which IL-17⁺ cells were largely absent. Strikingly, the frequency of M. tuberculosis–specific lung T cells making IL-17, but not other cytokines, inversely correlated with the plasma IL-1β levels, suggesting a potential link with disease severity. Using a human granuloma model, we showed the addition of either exogenous IL-17 or IL-2 enhanced immune control of M. tuberculosis and was associated with increased NO production. Taken together, these data support an important role for M. tuberculosis–specific Trm-like, IL-17–producing cells in the immune control of M. tuberculosis in the human lung.     

B cells,antibody-secreting cells,and virus-specific antibodies respond to herpes simplex virus 2 reactivation in skin

Tissue-based T cells are important effectors in the prevention and control of mucosal viral infections; less is known about tissue-based B cells. We demonstrate that B cells and antibody-secreting cells (ASCs) are present in inflammatory infiltrates in skin biopsy specimens from study participants during symptomatic herpes simplex virus 2 (HSV-2) reactivation and early healing. Both CD20⁺ B cells, most of which are antigen inexperienced based on their coexpression of IgD, and ASCs — characterized by dense IgG RNA expression in combination with CD138, IRF4, and Blimp-1 RNA — were found to colocalize with T cells. ASCs clustered with CD4⁺ T cells, suggesting the potential for crosstalk. HSV-2–specific antibodies to virus surface antigens were also present in tissue and increased in concentration during HSV-2 reactivation and healing, unlike in serum, where concentrations remained static over time. B cells, ASCs, and HSV-specific antibody were rarely detected in biopsies of unaffected skin. Evaluation of samples from serial biopsies demonstrated that B cells and ASCs followed a more migratory than resident pattern of infiltration in HSV-affected genital skin, in contrast to T cells. Together, these observations suggest the presence of distinct phenotypes of B cells in HSV-affected tissue; dissecting their role in reactivation may reveal new therapeutic avenues to control these infections.     

mTOR regulates T cell exhaustion and PD-1–targeted immunotherapy response during chronic viral infection

T cell exhaustion is a state of T cell dysfunction associated with expression of programmed death 1 (PD-1). Exhausted CD8⁺ T cells are maintained by self-renewing stem-like T cells that provide differentiated TIM3⁺ cells, a part of which possesses effector-like properties. PD-1–targeted therapies enhance T cell response by promoting differentiation of stem-like T cells toward TIM3⁺ cells, but the role of mTOR during T cell exhaustion remains elusive. Here, we showed that mTOR inhibition has distinct outcomes during the beginning of and after the establishment of chronic viral infection. Blocking mTOR during the T cell expansion phase enhanced the T cell response by causing accumulation of stem-like T cells, leading to improved efficacy of PD-1 immunotherapy; whereas, after exhaustion progressed, mTOR inhibition caused immunosuppression, characterized by decreased TIM3⁺ cells and increased viral load with minimal changes in stem-like T cells. Mechanistically, a cell-intrinsic mTOR signal was vital for differentiation of stem-like T cells into the TIM3⁺ state in the early and late phases of chronic infection as well as during PD-1 immunotherapy. Thus, PD-1 blockade worked after cessation of mTOR inhibition, but simultaneous treatment failed to induce functional TIM3⁺ cells, reducing efficacy of PD-1 immunotherapy. Our data demonstrate that mTOR regulates T cell exhaustion and have important implications for combination cancer therapies with PD-1 blockade.     

Lac water extract inhibits IFN-γ signaling through JAK2-STAT1-IRF1 axis in human melanoma

Interferon-γ (IFN-γ) is a cytokine that plays an important role in the host defense of infectious diseases and in immune surveillance during tumor development; however, it has adverse effects in the pathogenesis of autoimmune diseases and in immunosuppressive microenvironments, promoting the immunoevasion of cancer cells. In this study, we identified lac water extract (Lac) as a candidate that can suppress IFN-γ signaling amongst 112 types of natural products, using PD-L1 promoter as a readout for the IFN-γ signaling. Moreover, we determined that Lac inhibits IFN-γ-induced PD-L1 and MHC class I expression on the cell surface in melanoma cells, both of which have been identified as the downstream molecules of IFN-γ signaling. We also determined that Lac inhibited the JAK2-STAT1-IRF1 pathway. Finally, we identified laccaic acids, encompassing laccaic acid A, B, C, and E, as the active components in Lac that inhibit IFN-γ signaling. Collectively, the laccaic acids are lead compounds for a novel inhibitor that targets the JAK2-STAT1-IRF1 pathway for diseases caused by the aberrant activation of IFN-γ signaling.

Interferon-γ (IFN-γ) is a cytokine that plays an important role in the host defense of infectious diseases and in immune surveillance during tumor development.     

FcγRIIB regulates autoantibody responses by limiting marginal zone B cell activation

FcγRIIB is an inhibitory receptor expressed throughout B cell development. Diminished expression or function is associated with lupus in mice and humans, in particular through an effect on autoantibody production and plasma cell (PC) differentiation. Here, we analyzed the effect of B cell–intrinsic FcγRIIB expression on B cell activation and PC differentiation. Loss of FcγRIIB on B cells in Fcgr2b–conditional KO (Fcgr2b-cKO) mice led to a spontaneous increase in autoantibody titers. This increase was most striking for IgG3, suggestive of increased extrafollicular responses. Marginal zone (MZ) B cells had the highest expression of FcγRIIB in both mice and humans. This high expression of FcγRIIB was linked to increased MZ B cell activation, Erk phosphorylation, and calcium flux in the absence of FcγRIIB triggering. We observed a marked increase in IgG3⁺ PCs and B cells during extrafollicular PC responses in Fcgr2b-cKO mice. The increased IgG3 response following immunization of Fcgr2b-cKO mice was lost in MZ-deficient Notch2 Fcgr2b–double KO mice. Importantly, patients with systemic lupus erythematosus (SLE) had a decrease in FcγRIIB expression that was strongest in MZ B cells. Thus, we present a model in which high FcγRIIB expression in MZ B cells prevented their hyperactivation and ensuing autoimmunity.     

Combined immunodeficiency due to a mutation in the γ1 subunit of the coat protein I complex

The coat protein I (COPI) complex mediates retrograde trafficking from the Golgi to the endoplasmic reticulum (ER). Five siblings with persistent bacterial and viral infections and defective humoral and cellular immunity had a homozygous p.K652E mutation in the γ1 subunit of COPI (γ1-COP). The mutation disrupts COPI binding to the KDEL receptor and impairs the retrieval of KDEL-bearing chaperones from the Golgi to the ER. Homozygous Copg1^K652E mice had increased ER stress in activated T and B cells, poor antibody responses, and normal numbers of T cells that proliferated normally, but underwent increased apoptosis upon activation. Exposure of the mutants to pet store mice caused weight loss, lymphopenia, and defective T cell proliferation that recapitulated the findings in the patients. The ER stress-relieving agent tauroursodeoxycholic acid corrected the immune defects of the mutants and reversed the phenotype they acquired following exposure to pet store mice. This study establishes the role of γ1-COP in the ER retrieval of KDEL-bearing chaperones and thereby the importance of ER homeostasis in adaptive immunity.