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Purpose:Metastasis remains a major hurdle in treating aggressive malignancies such as pancreatic ductal adenocarcinoma (PDAC). Improving response to treatment, therefore, requires a more detailed characterization of the cellular populations involved in controlling metastatic burden.Experimental Design:PDAC patient tissue samples were subjected to RNA sequencing analysis to identify changes in immune infiltration following radiotherapy. Genetically engineered mouse strains in combination with orthotopic tumor models of PDAC were used to characterize disease progression. Flow cytometry was used to analyze tumor infiltrating, circulating, and nodal immune populations.Results:We demonstrate that although radiotherapy increases the infiltration and activation of dendritic cells (DC), it also increases the infiltration of regulatory T cells (Treg) while failing to recruit natural killer (NK) and CD8 T cells in PDAC patient tissue samples. In murine orthotopic tumor models, we show that genetic and pharmacologic depletion of Tregs and NK cells enhances and attenuates response to radiotherapy, respectively. We further demonstrate that targeted inhibition of STAT3 on Tregs results in improved control of local and distant disease progression and enhanced NK-mediated immunosurveillance of metastasis. Moreover, combination treatment of STAT3 antisense oligonucleotide (ASO) and radiotherapy invigorated systemic immune activation and conferred a survival advantage in orthotopic and metastatic tumor models. Finally, we show the response to STAT3 ASO + radiotherapy treatment is dependent on NK and DC subsets.Conclusions:Our results suggest targeting Treg-mediated immunosuppression is a critical step in mediating a response to treatment, and identifying NK cells as not only a prognostic marker of improved survival, but also as an effector population that functions to combat metastasis.

Supplementary Figure from Targeting Treg-Expressed STAT3 Enhances NK-Mediated Surveillance of Metastasis and Improves Therapeutic Response in Pancreatic Adenocarcinoma

In the peritoneal cavity, the omentum contains fat-associated lymphoid clusters (FALCs) whose role in response to infection is poorly understood. After intraperitoneal immunization with Toxoplasma gondii , conventional type 1 dendritic cells (cDC1s) were critical to induce innate sources of IFN-γ and cellular changes in the FALCs. Unexpectedly, infected peritoneal macrophages that migrated into the FALCs primed CD8 + T cells. Although T cell priming was cDC1 independent, these DCs were required for maximal CD8 + T cell expansion. An agent-based computational model and experimental data highlighted that cDC1s affected the magnitude of the proliferative burst and promoted CD8 + T cell expression of nutrient uptake receptors and cell survival. Thus, although FALCs lack the organization of secondary lymphoid organs, cDC1s resident in this tissue coordinate innate responses to microbial challenge and provide secondary signals required for T cell expansion and memory formation.

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 while clear cell renal cell carcinoma (ccRCC) was infiltrated by exhaustion-phenotype CD8(+) T cells which negatively correlated with patient prognosis, chromophobe RCC 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.

Despite the obvious knowledge that infectious particles can be shared through respiration, whether other constituents of the nasal/oral fluids can be passed between hosts has surprisingly never even been postulated, let alone investigated. The circumstances of the present pandemic facilitated a unique opportunity to fully examine this provocative idea. The data we show provides evidence for a new mechanism by which herd immunity may be manifested, the aerosol transfer of antibodies between immune and non-immune hosts.

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.

Purpose: Metastasis remains a major hurdle in treating aggressive malignancies such as pancreatic ductal adenocarcinoma (PDAC). Improving response to treatment, therefore, requires a more detailed characterization of the cellular populations involved in controlling metastatic burden. Experimental Design: PDAC patient tissue samples were subjected to RNA sequencing analysis to identify changes in immune infiltration following radiotherapy. Genetically engineered mouse strains in combination with orthotopic tumor models of PDAC were used to characterize disease progression. Flow cytometry was used to analyze tumor infiltrating, circulating, and nodal immune populations. Results: We demonstrate that although radiotherapy increases the infiltration and activation of dendritic cells (DC), it also increases the infiltration of regulatory T cells (Treg) while failing to recruit natural killer (NK) and CD8 T cells in PDAC patient tissue samples. In murine orthotopic tumor models, we show that genetic and pharmacologic depletion of Tregs and NK cells enhances and attenuates response to radiotherapy, respectively. We further demonstrate that targeted inhibition of STAT3 on Tregs results in improved control of local and distant disease progression and enhanced NK-mediated immunosurveillance of metastasis. Moreover, combination treatment of STAT3 antisense oligonucleotide (ASO) and radiotherapy invigorated systemic immune activation and conferred a survival advantage in orthotopic and metastatic tumor models. Finally, we show the response to STAT3 ASO + radiotherapy treatment is dependent on NK and DC subsets. Conclusions: Our results suggest targeting Treg-mediated immunosuppression is a critical step in mediating a response to treatment, and identifying NK cells as not only a prognostic marker of improved survival, but also as an effector population that functions to combat metastasis.

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.

The dried-tube specimen (DTS) procedure was used to develop the COVID-19 serology control panel (CSCP). The DTS offers the benefit of shipping materials without a cold chain, allowing for greater access without deterioration of material integrity. Samples in the panel were sourced from COVID-19 convalescent persons from March to May 2020. The immunoglobulin subtypes (total Ig, IgM, and IgG) and their respective reactivity to severe acute respiratory syndrome coronavirus 2 nucleocapsid, spike, and receptor-binding domain antigens of the samples were delineated and compared with the WHO International Standard to elucidate the exact binding antibody units of each CSCP sample and ensure the CSCP provides adequate reactivity for different types of serological test platforms. We distribute the CSCP as a kit with five coded tubes to laboratories around the world to be used to compare test kits for external quality assurance, for harmonizing laboratory testing, and for use as training materials for laboratory workers.

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). Chemokine receptor 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 dendrtitic cells (DCs) and monocyte-derived DCs internalized and processed vaccine antigen in lungs. We also found that Basic Leucine Zipper ATF-Like Transcription Factor 3 (BATF-3)-dependent DCs were essential for eliciting T cell responses, but CCR2 deficiency enhanced the differentiation of CD127HI/KLRG-1LO, OX40+veCD62L+ve and mucosally imprinted CD69+veCD103+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 SARS-CoV-2.ImportanceWhile antibody-based immunity to influenza A virus (IAV) is type and sub-type 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 anti-viral lung-resident memory T cells, following intranasal vaccination. These findings suggested that strategies that limit monocyte infiltration can potentiate vaccine-induced frontline T-cell immunity to respiratory viruses such as IAV and SARS-CoV-2.

We used the dried tube specimen (DTS) procedure to develop the COVID-19 Serology Control Panel (CSCP). The CSCP contains five well-characterized SARS-CoV-2 pooled plasma samples made available for labs around the world to compare test kits, use for external quality assurance, harmonize laboratory testing, and train laboratory workers.Article Summary LineThe dried tube specimen system is a highly effective and resilient way to provide laboratories with well-characterized serology materials. The CSCP can help clinical laboratories inform their choice of diagnostic test to supplement clinical diagnoses of SARS-CoV-2 infection.

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

Virus-specific immune responses persist over time in subjects recovering from asymptomatic SARS-CoV-2 infection., The dynamics of immune responses in asymptomatic SARS-CoV-2–infected subjects remain to be fully characterized. The work presented in this issue of JEM by Le Bert et al. (2021. J. Exp. Med. https://doi.org/10.1084/jem.20202617) sheds some light on these issues and ultimately provides some degree of confidence in the magnitude and persistence of immunity over time after asymptomatic infection with SARS-CoV-2.

SUMMARY Using non-human primates (NHPs), mice, and human primary cells, we found a role for interleukin-10 (IL-10) in the upregulation of the tissue-resident memory T cell (TRM) marker CD103. In NHPs, intravenous, but not subcutaneous, immunization with peptide antigen and an adjuvant combining an agonistic anti-CD40 antibody plus poly(IC:LC) induced high levels of CD103+ TRMs in the lung, which correlated with early plasma IL-10 levels. Blocking IL-10 reduced CD103 expression on human T cells stimulated in vitro with the adjuvant combination as well as diminished CD103 on lung-resident T cells in vivo in mice. Monocyte-produced IL-10 induced the release of surface-bound transforming growth factor β (TGF-β), which in turn upregulated CD103 on T cells. Early TGF-β imprinted increased sensitivity to TGF-β restimulation, indicating an early commitment of the T cell lineage toward TRMs during the priming stage of activation. IL-10-mediated TGF-β signaling may therefore have a critical role in the generation of TRM following vaccination., Graphical Abstract, In Brief Thompson et al. outline a role for early IL-10 following immunization in the release of TGF-β and subsequent differentiation of CD103+ TRMs. Using a combination of non-human primate, human, and murine systems, the authors demonstrate that blocking IL-10 reduces CD103 expression, whereas delivery of IL-10 can augment a CD103+ phenotype.

The pleiotropic actions of interleukin-2 (IL-2) are essential for regulation of immune responses and maintenance of immune tolerance. The IL-2 receptor (IL-2R) is composed of IL-2Rα, IL-2Rβ, and IL-2Rγ subunits, with defects in IL-2Rα and IL-2Rγ and their downstream signaling effectors resulting in known primary immunodeficiency disorders. Here, we report the first human defect in IL-2Rβ, occurring in two infant siblings with a homozygous IL2RB mutation in the WSXWS motif, manifesting as multisystem autoimmunity and susceptibility to CMV infection. The hypomorphic mutation results in diminished IL-2Rβ surface expression and dysregulated IL-2/15 signaling, with an anticipated reduction in regulatory T cells. However, in contrast to the IL-2Rβ−/− animal model, which lacks NK cells, these siblings demonstrate an expansion of NK cells, particularly the CD56bright subset, and a lack of terminally differentiated NK cells. Thus, the early-onset autoimmunity and immunodeficiency are linked to functional deficits arising from altered IL-2Rβ expression and signaling in T and NK cells.

Inhibitory receptors (IR) are a diverse group of cell surface molecules that modulate T cell activation, but there are gaps in our knowledge of the cell-extrinsic factors that regulate their expression. The present study found that in vivo overexpression of IL-27 in mice led to increased T cell expression of PD-L1, LAG-3, TIGIT, and TIM-3. In vitro, TCR stimulation alone promoted expression of multiple IRs, whereas IL-27 alone induced expression of PD-L1. However, the combination of intermediate TCR stimulation and IL-27 resulted in synergistic induction of LAG-3, CTLA-4, and TIGIT. In vivo, infection with Toxoplasma gondii resulted in parasite-specific effector T cells that expressed high levels of IR, and at local sites of infection where IL-27 production was highest, IL-27 was required for maximal effector cell expression of PD-L1, LAG-3, CTLA-4, and TIGIT. Together, these results affirm the critical role of TCR signals in the induction of IR expression but find that during infection, IL-27 promotes T cell expression of IR.

Naive T cell activation in secondary lymphoid organs such as lymph nodes (LNs) occurs upon recognition of cognate antigens presented by antigen presenting cells (APCs). T cell activation requires cytoskeleton rearrangement and sustained interactions with APCs. Ena/VASP proteins are a family of cytoskeletal effector proteins responsible for actin polymerization and are frequently found at the leading edge of motile cells. Ena/VASP proteins have been implicated in motility and adhesion in various cell types, but their role in primary T cell activation has not been explored. Our goal was to determine the contribution of Ena/VASP proteins to T cell activation and expansion in vivo. Our results showed that naïve T cells from Ena/VASP-deficient mice have a significant reduction in antigen-specific T cell accumulation following Listeria monocytogenes infection. The kinetics of antigen-specific T cell impairment were further confirmed in Ena/VASP-deficient T cells stimulated via dendritic cell immunization. To investigate the cause of this T cell expansion defect, we analyzed T cell-APC interactions in vivo by 2-photon microscopy and observed fewer Ena/VASP-deficient naïve T cells interacting with APCs in LNs during priming. We also found that Ena/VASP-deficient T cells formed conjugates with significantly less actin polymerization at the T cell-APC synapse, and that these conjugates were less stable than their WT counterparts. Thus, we conclude that Ena/VASP proteins contribute to T cell actin remodeling downstream of T-APC interactions required for the initiation of stable T cell conjugates during APC scanning and for efficient activation and expansion of T cells in vivo. Supported by NIH (R01AI125553; T32AI007405)

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.

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.

The close relationship between B cells and CD4 T cells has been carefully studied, revealing a collaborative effort, where B cells promote the activation, differentiation, and expansion of CD4 T cells, while the so-called “helper” cells provide signals to B cells, influencing their class-switching and fate. Interactions between B cells and CD8 T cells are not as well studied, although CD8 T cells do exhibit an accelerated contraction after certain infections in B cell-deficient mice. Here, we found that B cells significantly enhance primary CD8 T cell responses following vaccination. Moreover, memory CD8 numbers and function are impaired in B cell-deficient animals, leading to increased susceptibility to bacterial challenge. We also found that IL-27 production by B cells contributes to their impact on primary, but not memory CD8 responses. Better understanding of the interactions between CD8 T cells and B cells may aid in the design of more effective future vaccine strategies.

Supplemental Digital Content is available in the text, Objectives: Define the seroprevalence and risk factors for SARS-CoV-2 antibodies in Arapahoe County, Colorado first responders (eg, law enforcement, human services, fire departments). Methods: Two hundred sixty four first responders were enrolled June to July 2020. SARS-CoV-2 seropositivity was defined as detection of immunoglobulin G (IgG) antibodies to both spike receptor binding domain and nucleocapsid in venous blood by validated enzyme-linked immunosorbent assay. We compared risk factors for being seropositive versus seronegative. Results: 4% (11/264) were SARS-CoV-2 seropositive. Seropositive participants were significantly more likely to have lung disease (% seropositive, % seronegative; P-value) (36%, 8%; P = 0.01), prior SARS-CoV-2/COVID-19 testing (36%, 8%; P ≤ 0.01), a prior positive result (18%, less than 1%), and to believe they previously had COVID-19 (64%, 15%; P

The omentum in the peritoneal cavity contains fat associated lymphoid clusters (FALCs) whose role in the response to microbial challenge are poorly understood. After intraperitoneal immunization with Toxoplasma gondii, type I dendritic cells (cDC1) were critical to induce innate sources of IFN-γ required to recruit monocytes to the FALCs. The migration of infected peritoneal macrophages into T and B cell rich areas of the FALCs allowed the TCR-induced activation of parasite-specific T cells. Unexpectedly, cDC1 were not required for T cell priming but rather supported the expansion of parasite-specific CD8+ T cells. An agent-based mathematical model predicted that the lack of cDC1 would impact the early proliferative burst, and we confirmed that cDC1 were required for optimal T cell expression of nutrient uptake receptors and cell survival. These studies highlight that cDC1 in the FALCs have distinct roles in the co-ordination of the innate and adaptive responses to microbial challenge.

Induction of protective mucosal T cell memory remains a formidable challenge to vaccinologists. Using a combination adjuvant strategy that elicits potent CD8 and CD4 T cell responses, we define the tenets of vaccine-induced pulmonary T cell immunity. An acrylic-acid-based adjuvant (ADJ), in combination with Toll-like receptor (TLR) agonists glucopyranosyl lipid adjuvant (GLA) or CpG, promotes mucosal imprinting but engages distinct transcription programs to drive different degrees of terminal differentiation and disparate polarization of TH1/TC1/TH17/TC17 effector/memory T cells. Combination of ADJ with GLA, but not CpG, dampens T cell receptor (TCR) signaling, mitigates terminal differentiation of effectors, and enhances the development of CD4 and CD8 TRM cells that protect against H1N1 and H5N1 influenza viruses. Mechanistically, vaccine-elicited CD4 T cells play a vital role in optimal programming of CD8 TRM and viral control. Taken together, these findings provide further insights into vaccine-induced multifaceted mucosal T cell immunity with implications in the development of vaccines against respiratorypathogens, including influenza virus and SARS-CoV-2., Graphical Abstract, Highlights Combination adjuvants stimulate potent TRM cell immunity in the respiratory tract Differentiation and functional programming depend on adjuvant and TCR signaling Vaccine-induced T cell immunity to influenza requires CD4 and CD8 T cells CD4 T cells regulate optimal positioning and programming of CD8 TRM in lungs, Marinaik et al. report that carbomer and TLR-based combination adjuvants elicit airway- and lung-resident memory and confer heterosubtypic immunity to influenza virus. Further, they show that combination adjuvants stimulate varying levels of TCR signaling and cytokines in lungs and drive disparate patterns of effector/memory differentiation, mucosal imprinting, and functional polarization.

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.

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.

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 varied 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. Here, using different in vivo 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 role of T cell-produced chemokines in infectious and other diseases.

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.

While 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, in order to set a universally applicable benchmark for adjuvant evaluation. Using an IL-27 reporter transgenic mouse model, we show here that cDC1 IL-27 production in the draining lymph node 12 hours after subcutaneous 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.

Eya proteins are critical developmental regulators that are highly expressed in embryogenesis but downregulated after development. Amplification and/or re-expression of Eyas occurs in many tumor types. In breast cancer, Eyas regulate tumor progression by acting as transcriptional cofactors and tyrosine phosphatases. Intriguingly, Eyas harbor a separate threonine (Thr) phosphatase activity, which was previously implicated in innate immunity. Here we describe what we believe to be a novel role for Eya3 in mediating triple-negative breast cancer-associated immune suppression. Eya3 loss decreases tumor growth in immune-competent mice and is associated with increased numbers of infiltrated CD8+ T cells, which, when depleted, reverse the effects of Eya3 knockdown. Mechanistically, Eya3 utilizes its Thr phosphatase activity to dephosphorylate Myc at pT58, resulting in a stabilized form. We show that Myc is required for Eya3-mediated increases in PD-L1, and that rescue of PD-L1 in Eya3-knockdown cells restores tumor progression. Finally, we demonstrate that Eya3 significantly correlates with PD-L1 in human breast tumors, and that tumors expressing high levels of Eya3 have a decreased CD8+ T cell signature. Our data uncover a role for Eya3 in mediating tumor-associated immune suppression, and suggest that its inhibition may enhance checkpoint therapies.

It is well accepted that the innate response is a necessary prerequisite to the formation of the adaptive response. This is true for T cell responses against infections or adjuvanted subunit vaccination. However, specific innate parameters with predictive value for the magnitude of an adjuvant-elicited T cell response have yet to be identified. We previously reported how T cell responses induced by subunit vaccination were dependent on the cytokine IL-27. These findings were unexpected, given that T cell responses to an infection typically increase in the absence of IL-27. Using a novel IL-27p28–eGFP reporter mouse, we now show that the degree to which an adjuvant induces IL-27p28 production from dendritic cells and monocytes directly predicts the magnitude of the T cell response elicited. To our knowledge, these data are the first to identify a concrete innate correlate of vaccine-elicited cellular immunity, and they have significant practical and mechanistic implications for subunit vaccine biology.

Antigens derived from viral infection or vaccination can persist within a host for many weeks after resolution of the infection or vaccine responses. We previously identified lymphatic endothelial cells (LEC) as the repository for this antigen archival, yet LECs are unable to present their archived antigens to CD8+ T cells, and instead transfer their antigens to CD11c+ antigen-presenting cells (APC). Here we show that the exchange of archived antigens between LECs and APCs is mediated by migratory dendritic cells (DC). After vaccination, both migratory basic leucine zipper ATF-like transcription factor 3 (BatF3)-dependent and BatF3-independent DCs are responsible for antigen exchange and cross-presentation. However, exchange of archived viral antigens is mediated only by BatF3-dependent migratory DCs potentially acquiring apoptotic LECs. In conclusion, LEC-archived antigens are exchanged with migratory DCs, both directly and through LEC apoptosis, to cross-present archived antigens to circulating T cells., Viral infection and vaccination both induce lasting persistence of antigens for protective responses. Here the authors show that migratory dendritic cells, independent of the transcription factor BatF3 for their development, contribute to “archived antigen” exchange with lymphatic endothelial cells.

SUMMARY The relationship between B cells and CD4 T cells has been carefully studied, revealing a collaborative effort in which B cells promote the activation, differentiation, and expansion of CD4 T cells while the so-called “helper” cells provide signals to B cells, influencing their class switching and fate. Interactions between B cells and CD8 T cells are not as well studied, although CD8 T cells exhibit an accelerated contraction after certain infections in B-cell-deficient mice. Here, we find that B cells significantly enhance primary CD8 T cell responses after vaccination. Moreover, memory CD8 numbers and function are impaired in B-cell-deficient animals, leading to increased susceptibility to bacterial challenge. We also show that interleukin-27 production by B cells contributes to their impact on primary, but not memory, CD8 responses. Better understanding of the interactions between CD8 T cells and B cells may aid in the design of more effective future vaccine strategies., In brief Generating cytotoxic CD8 T cell responses with vaccines can greatly improve their efficacy, but inducing adequate numbers of these cells can be challenging. Klarquist et al. reveal that the magnitude, persistence, and function of CD8 T cell vaccine responses depend on B cells., Graphical abstract