Cytotoxic innate lymphoid cells sense cancer cell-expressed interleukin-15 to suppress human and murine malignancies.

Malignancy can be suppressed by the immune system. However, the classes of immunosurveillance responses and their mode of tumor sensing remain incompletely understood. Here, we show that although clear cell renal cell carcinoma (ccRCC) was infiltrated by exhaustion-phenotype CD8+ T cells that negatively correlated with patient prognosis, chromophobe RCC (chRCC) had abundant infiltration of granzyme A-expressing intraepithelial type 1 innate lymphoid cells (ILC1s) that positively associated with patient survival. Interleukin-15 (IL-15) promoted ILC1 granzyme A expression and cytotoxicity, and IL-15 expression in chRCC tumor tissue positively tracked with the ILC1 response. An ILC1 gene signature also predicted survival of a subset of breast cancer patients in association with IL-15 expression. Notably, ILC1s directly interacted with cancer cells, and IL-15 produced by cancer cells supported the expansion and anti-tumor function of ILC1s in a murine breast cancer model. Thus, ILC1 sensing of cancer cell IL-15 defines an immunosurveillance mechanism of epithelial malignancies.

Conserved and Novel Mouse CD8 T Cell Epitopes within SARS-CoV-2 Spike Receptor Binding Domain Protein Identified following Subunit Vaccination.

The prior existence of human ACE2 protein-expressing mice used to study SARS-CoV and the rapid development of mouse-adapted virus strains have allowed the study of SARS-CoV-2 in mice, even as we are still learning about its natural pathology in humans. With myriad genetically altered strains on the C57BL/6 background and the abundance of immunological reagents available to interrogate its immune responses, the C57BL/6 mice may provide useful insight into the immunology of SARS-CoV-2 infection and vaccination. To conduct more detailed studies on their T cell responses to vaccines and infection, the epitopes eliciting those responses must be characterized in further detail. In this study, we mapped CD8 T cell epitopes within the receptor binding domain of the SARS-CoV-2 spike protein in C57BL/6 mice. Our study identified five major CD8 T cell epitopes in immunized C57BL/6 mice, including one, VVLSFELL, presented by H-2Kb and common between SARS-CoV and SARS-CoV-2.

CD40 Agonist Overcomes T Cell Exhaustion Induced by Chronic Myeloid Cell IL-27 Production in a Pancreatic Cancer Preclinical Model.

Pancreatic cancer is a particularly lethal malignancy that resists immunotherapy. In this study, using a preclinical pancreatic cancer murine model, we demonstrate a progressive decrease in IFN-γ and granzyme B and a concomitant increase in Tox and IL-10 in intratumoral tumor-specific T cells. Intratumoral myeloid cells produced elevated IL-27, a cytokine that correlates with poor patient outcome. Abrogating IL-27 signaling significantly decreased intratumoral Tox+ T cells and delayed tumor growth yet was not curative. Agonistic αCD40 decreased intratumoral IL-27-producing myeloid cells, decreased IL-10-producing intratumoral T cells, and promoted intratumoral Klrg1+Gzmb+ short-lived effector T cells. Combination agonistic αCD40+αPD-L1 cured 63% of tumor-bearing animals, promoted rejection following tumor rechallenge, and correlated with a 2-log increase in pancreas-residing tumor-specific T cells. Interfering with Ifngr1 expression in nontumor/host cells abrogated agonistic αCD40+αPD-L1 efficacy. In contrast, interfering with nontumor/host cell Tnfrsf1a led to cure in 100% of animals following agonistic αCD40+αPD-L1 and promoted the formation of circulating central memory T cells rather than long-lived effector T cells. In summary, we identify a mechanistic basis for T cell exhaustion in pancreatic cancer and a feasible clinical strategy to overcome it.

CCR2 Regulates Vaccine-Induced Mucosal T-Cell Memory to Influenza A Virus.

Elicitation of lung tissue-resident memory CD8 T cells (TRMs) is a goal of T cell-based vaccines against respiratory viral pathogens, such as influenza A virus (IAV). C-C chemokine receptor type 2 (CCR2)-dependent monocyte trafficking plays an essential role in the establishment of CD8 TRMs in lungs of IAV-infected mice. Here, we used a combination adjuvant-based subunit vaccine strategy that evokes multifaceted (TC1/TC17/TH1/TH17) IAV nucleoprotein-specific lung TRMs to determine whether CCR2 and monocyte infiltration are essential for vaccine-induced TRM development and protective immunity to IAV in lungs. Following intranasal vaccination, neutrophils, monocytes, conventional dendritic cells (DCs), and monocyte-derived dendritic cells internalized and processed vaccine antigen in lungs. We found that basic leucine zipper ATF-like transcription factor 3 (BATF3)-dependent DCs were essential for eliciting T cell responses, but CCR2 deficiency enhanced the differentiation of CD127hi, KLRG-1lo, OX40+ve CD62L+ve, and mucosally imprinted CD69+ve CD103+ve effector and memory CD8 T cells in lungs and airways of vaccinated mice. Mechanistically, increased development of lung TRMs induced by CCR2 deficiency was linked to dampened expression of T-bet but not altered TCF-1 levels or T cell receptor signaling in CD8 T cells. T1/T17 functional programming, parenchymal localization of CD8/CD4 effector and memory T cells, recall T cell responses, and protective immunity to a lethal IAV infection were unaffected in CCR2-deficient mice. Taken together, we identified a negative regulatory role for CCR2 and monocyte trafficking in mucosal imprinting and differentiation of vaccine-induced TRMs. Mechanistic insights from this study may aid the development of T-cell-based vaccines against respiratory viral pathogens, including IAV and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). IMPORTANCE While antibody-based immunity to influenza A virus (IAV) is type and subtype specific, lung- and airway-resident memory T cells that recognize conserved epitopes in the internal viral proteins are known to provide heterosubtypic immunity. Hence, broadly protective IAV vaccines need to elicit robust T cell memory in the respiratory tract. We have developed a combination adjuvant-based IAV nucleoprotein vaccine that elicits strong CD4 and CD8 T cell memory in lungs and protects against H1N1 and H5N1 strains of IAV. In this study, we examined the mechanisms that control vaccine-induced protective memory T cells in the respiratory tract. We found that trafficking of monocytes into lungs might limit the development of antiviral lung-resident memory T cells following intranasal vaccination. These findings suggest that strategies that limit monocyte infiltration can potentiate vaccine-induced frontline T-cell immunity to respiratory viruses, such as IAV and SARS-CoV-2.

cDC1 IL-27p28 Production Predicts Vaccine-Elicited CD8+ T Cell Memory and Protective Immunity.

Although adjuvants and formulations are often either empirically derived, or at best judged by their ability to elicit broad inflammation, it would be ideal if specific innate correlates of adaptive immunity could be identified to set a universally applicable benchmark for adjuvant evaluation. Using an IL-27 reporter transgenic mouse model, we show in this study that conventional type 1 dendritic cell IL-27 production in the draining lymph node 12 h after s.c. vaccination directly correlates with downstream CD8+ T cell memory and protective immunity against infectious challenge. This correlation is robust, reproducible, predictive, entirely unique to vaccine biology, and is the only innate correlate of CD8+ T cell immune memory yet to be identified. Our results provide new insights into the basic biology of adjuvant-elicited cellular immunity and have clear implications for the screening and evaluation of novel adjuvants.

Chemokine Signatures of Pathogen-Specific T Cells II: Memory T Cells in Acute and Chronic Infection.

Pathogen-specific memory T cells (TM) contribute to enhanced immune protection under conditions of reinfection, and their effective recruitment into a recall response relies, in part, on cues imparted by chemokines that coordinate their spatiotemporal positioning. An integrated perspective, however, needs to consider TM as a potentially relevant chemokine source themselves. In this study, we employed a comprehensive transcriptional/translational profiling strategy to delineate the identities, expression patterns, and dynamic regulation of chemokines produced by murine pathogen-specific TM CD8+TM, and to a lesser extent CD4+TM, are a prodigious source for six select chemokines (CCL1/3/4/5, CCL9/10, and XCL1) that collectively constitute a prominent and largely invariant signature across acute and chronic infections. Notably, constitutive CCL5 expression by CD8+TM serves as a unique functional imprint of prior antigenic experience; induced CCL1 production identifies highly polyfunctional CD8+ and CD4+TM subsets; long-term CD8+TM maintenance is associated with a pronounced increase of XCL1 production capacity; chemokines dominate the earliest stages of the CD8+TM recall response because of expeditious synthesis/secretion kinetics (CCL3/4/5) and low activation thresholds (CCL1/3/4/5/XCL1); and TM chemokine profiles modulated by persisting viral Ags exhibit both discrete functional deficits and a notable surplus. Nevertheless, recall responses and partial virus control in chronic infection appear little affected by the absence of major TM chemokines. Although specific contributions of TM-derived chemokines to enhanced immune protection therefore remain to be elucidated in other experimental scenarios, the ready visualization of TM chemokine-expression patterns permits a detailed stratification of TM functionalities that may be correlated with differentiation status, protective capacities, and potential fates.

Chemokine Signatures of Pathogen-Specific T Cells I: Effector T Cells.

The choreography of complex immune responses, including the priming, differentiation, and modulation of specific effector T cell populations generated in the immediate wake of an acute pathogen challenge, is in part controlled by chemokines, a large family of mostly secreted molecules involved in chemotaxis and other patho/physiological processes. T cells are both responsive to various chemokine cues and a relevant source for certain chemokines themselves; yet, the actual range, regulation, and role of effector T cell-derived chemokines remains incompletely understood. In this study, using different in vivo mouse models of viral and bacterial infection as well as protective vaccination, we have defined the entire spectrum of chemokines produced by pathogen-specific CD8+ and CD4+T effector cells and delineated several unique properties pertaining to the temporospatial organization of chemokine expression patterns, synthesis and secretion kinetics, and cooperative regulation. Collectively, our results position the "T cell chemokine response" as a notably prominent, largely invariant, yet distinctive force at the forefront of pathogen-specific effector T cell activities and establish novel practical and conceptual approaches that may serve as a foundation for future investigations into the role of T cell-produced chemokines in infectious and other diseases.

Development and Validation of a Multiplex Microsphere Immunoassay Using Dried Blood Spots for SARS-CoV-2 Seroprevalence: Application in First Responders in Colorado, USA.

Serological testing of large representative populations for antibodies to SARS-CoV-2 is needed to estimate seroprevalence, transmission dynamics, and the duration of antibody responses from natural infection and vaccination. In this study, a high-throughput SARS-CoV-2 multiplex microsphere immunoassay (MMIA) was developed for the receptor binding domain (RBD) and nucleocapsid (N) that was more sensitive than enzyme-linked immunosorbent assay (ELISA) (98% versus 87%). The MMIA was then applied and validated in 264 first responders in Colorado using serum and dried blood spot (DBS) eluates, compared to ELISA, and evaluated for neutralizing antibodies. Four percent (11/264) of first responders were seropositive in July to August 2020. Serum and DBS were highly correlated for anti-RBD and anti-N antibodies (R = 0.83, P < 0.0001 and R = 0.87, P < 0.0001, respectively) by MMIA. The MMIA accurately predicted SARS-CoV-2 neutralizing antibodies using DBS (R = 0.76, P = 0.037). On repeat antibody testing 3 months later, anti-RBD IgG decreased less rapidly than anti-N IgG measured by MMIA, with a median change in geometric median fluorescence intensity of 62% versus 79% (P < 0.01) for anti-RBD and anti-N IgG, respectively. This novel MMIA using DBS could be scalable for rapid and affordable SARS-CoV-2 serosurveillance in the United States and globally.

Chikungunya Virus Evades Antiviral CD8+ T Cell Responses To Establish Persistent Infection in Joint-Associated Tissues.

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes explosive epidemics of a febrile illness characterized by debilitating arthralgia and arthritis that can endure for months to years following infection. In mouse models, CHIKV persists in joint tissues for weeks to months and is associated with chronic synovitis. Using a recombinant CHIKV strain encoding a CD8+ T cell receptor epitope from ovalbumin, as well as a viral peptide-specific major histocompatibility complex class I tetramer, we interrogated CD8+ T cell responses during CHIKV infection. Epitope-specific CD8+ T cells, which were reduced in Batf3-/- and Wdfy4-/- mice with known defects in antigen cross-presentation, accumulated in joint tissue and the spleen. Antigen-specific ex vivo restimulation assays and in vivo killing assays demonstrated that CD8+ T cells produce cytokine and have cytolytic activity. Despite the induction of a virus-specific CD8+ T cell response, the CHIKV burden in joint-associated tissues and the spleen were equivalent in wild-type (WT) and CD8α-/- mice during both the acute and the chronic phases of infection. In comparison, CD8+ T cells were essential for the control of acute and chronic lymphocytic choriomeningitis virus infection in the joint and spleen. Moreover, adoptive transfer of virus-specific effector CD8+ T cells or immunization with a vaccine that induces virus-specific effector CD8+ T cells prior to infection enhanced the clearance of CHIKV infection in the spleen but had a minimal impact on CHIKV infection in the joint. Collectively, these data suggest that CHIKV establishes and maintains a persistent infection in joint-associated tissue in part by evading CD8+ T cell immunity.IMPORTANCE CHIKV is a reemerging mosquito-transmitted virus that in the last decade has spread into Europe, Asia, the Pacific Region, and the Americas. Joint pain, swelling, and stiffness can endure for months to years after CHIKV infection, and epidemics have a severe economic impact. Elucidating the mechanisms by which CHIKV subverts antiviral immunity to establish and maintain a persistent infection may lead to the development of new therapeutic strategies against chronic CHIKV disease. In this study, we found that CHIKV establishes and maintains a persistent infection in joint-associated tissue in part by evading antiviral CD8+ T cell immunity. Thus, immunomodulatory therapies that improve CD8+ T cell immune surveillance and clearance of CHIKV infection could be a strategy for mitigating chronic CHIKV disease.

T Cell Vaccinology: Beyond the Reflection of Infectious Responses.

Inducing sustained, robust CD8(+) T cell responses is necessary for therapeutic intervention in chronic infectious diseases and cancer. Unfortunately, most adjuvant formulations fail to induce substantial cellular immunity in humans. Attenuated acute infectious agents induce strong CD8(+) T cell immunity, and are thought to therefore represent a good road map for guiding the development of subunit vaccines capable of inducing the same. However, recent evidence suggests that this assumption may need reconsideration. Here we provide an overview of subunit vaccine history as it pertains to instigating T cell responses. We argue that in light of evidence demonstrating that T cell responses to vaccination differ from those induced by infectious challenge, research in pursuit of cellular immunity-inducing vaccine adjuvants should no longer follow only the infection paradigm.

Immune Surveillance by Natural IgM Is Required for Early Neoantigen Recognition and Initiation of Adaptive Immunity.

Early recognition of neoantigen-expressing cells is complex, involving multiple immune cell types. In this study, in vivo, we examined how antigen-presenting cell subtypes coordinate and induce an immunological response against neoantigen-expressing cells, particularly in the absence of a pathogen-associated molecular pattern, which is normally required to license antigen-presenting cells to present foreign or self-antigens as immunogens. Using two reductionist models of neoantigen-expressing cells and two cancer models, we demonstrated that natural IgM is essential for the recognition and initiation of adaptive immunity against neoantigen-expressing cells. Natural IgM antibodies form a cellular immune complex with the neoantigen-expressing cells. This immune complex licenses surveying monocytes to present neoantigens as immunogens to CD4+ T cells. CD4+ T helper cells, in turn, use CD40L to license cross-presenting CD40+ Batf3+ dendritic cells to elicit a cytotoxic T cell response against neoantigen-expressing cells. Any break along this immunological chain reaction results in the escape of neoantigen-expressing cells. This study demonstrates the surprising, essential role of natural IgM as the initiator of a sequential signaling cascade involving multiple immune cell subtypes. This sequence is required to coordinate an adaptive immune response against neoantigen-expressing cells.

Type I interferon signaling is required for the APOBEC3/Rfv3-dependent neutralizing antibody response but not innate retrovirus restriction.

BACKGROUND: APOBEC3/Rfv3 restricts acute Friend retrovirus (FV) infection and promotes virus-specific neutralizing antibody (NAb) responses. Classical Rfv3 studies utilized FV stocks containing lactate-dehydrogenase elevating virus (LDV), a potent type I interferon inducer. Previously, we showed that APOBEC3 is required for the anti-FV activity of exogenous IFN-alpha treatment. Thus, type I interferon receptor (IFNAR) signaling may be required for the APOBEC3/Rfv3 response. RESULTS: To test if the APOBEC3/Rfv3 response is dependent on type I IFN signaling, we infected IFNAR knockout versus IFNAR/APOBEC3 double-knockout mice with FV/LDV or LDV-free FV, and evaluated acute FV infection and subsequent NAb titers. We show that LDV co-infection and type I IFN signaling are not required for innate APOBEC3-mediated restriction. By contrast, removal of LDV and/or type I IFN signaling abrogated the APOBEC3-dependent NAb response. CONCLUSIONS: APOBEC3 can restrict retroviruses in a type I IFN-independent manner in vivo. By contrast, the ability of APOBEC3 to promote NAb responses is type I IFN-dependent. These findings reveal novel insights on the interplay between type I IFNs and APOBEC3 in vivo that may have implications for augmenting antiretroviral NAb responses.

Human Anti-CD40 Antibody and Poly IC:LC Adjuvant Combination Induces Potent T Cell Responses in the Lung of Nonhuman Primates.

Nonlive vaccine platforms that induce potent cellular immune responses in mucosal tissue would have broad application for vaccines against infectious diseases and tumors. Induction of cellular immunity could be optimized by targeted activation of multiple innate and costimulatory signaling pathways, such as CD40 or TLRs. In this study, we evaluated immune activation and elicitation of T cell responses in nonhuman primates after immunization with peptide Ags adjuvanted with an agonistic anti-CD40Ab, with or without the TLR3 ligand poly IC:LC. We found that i.v. administration of the anti-CD40Ab induced rapid and transient innate activation characterized by IL-12 production and upregulated costimulatory and lymph node homing molecules on dendritic cells. Using fluorescently labeled Abs for in vivo tracking, we found that the anti-CD40Ab bound to all leukocytes, except T cells, and disseminated to multiple organs. CD4(+) and CD8(+) T cell responses were significantly enhanced when the anti-CD40Ab was coadministered with poly IC:LC compared with either adjuvant given alone and were almost exclusively compartmentalized to the lung. Notably, Ag-specific T cells in the bronchoalveolar lavage were sustained at ∼5-10%. These data indicate that systemic administration of anti-CD40Ab may be particularly advantageous for vaccines and/or therapies that require T cell immunity in the lung.

Cutting Edge: Roles for Batf3-Dependent APCs in the Rejection of Minor Histocompatibility Antigen-Mismatched Grafts.

In transplantation, a major obstacle for graft acceptance in MHC-matched individuals is the mismatch of minor histocompatibility Ags. Minor histocompatibility Ags are peptides derived from polymorphic proteins that can be presented by APCs on MHC molecules. The APC subtype uniquely responsible for the rejection of minor Ag-mismatched grafts has not yet been identified. In this study, we examined graft rejection in three mouse models: 1) mismatch of male-specific minor Ags, 2) mismatch of minor Ags distinct from male-specific minor Ags, and 3) skin transplant. This study demonstrates that in the absence of pathogen-associated molecular patterns, Batf3-dependent dendritic cells elicit the rejection of cells and grafts expressing mismatched minor Ags. The implication of our findings in clinical transplantation may be significant, as minor Ag reactivity has been implicated in the pathogenesis of multiple allograft tissues.

IL-27 is required for shaping the magnitude, affinity distribution, and memory of T cells responding to subunit immunization.

An elusive goal of cellular immune vaccines is the generation of large numbers of antigen-specific T cells in response to subunit immunization. A broad spectrum of cytokines and cell-surface costimulatory molecules are known to shape the programming, magnitude, and repertoire of T cells responding to vaccination. We show here that the majority of innate immune receptor agonist-based vaccine adjuvants unexpectedly depend on IL-27 for eliciting CD4(+) and CD8(+) T-cell responses. This is in sharp contrast to infectious challenge, which generates T-cell responses that are IL-27-independent. Mixed bone marrow chimera experiments demonstrate that IL-27 dependency is T cell-intrinsic, requiring T-cell expression of IL-27Rα. Further, we show that IL-27 dependency not only dictates the magnitude of vaccine-elicited T-cell responses but also is critical for the programming and persistence of high-affinity T cells to subunit immunization. Collectively, our data highlight the unexpected central importance of IL-27 in the generation of robust, high-affinity cellular immune responses to subunit immunization.