Cryptic MHC-E epitope from influenza elicits a potent cytolytic T cell response.

The extent to which unconventional forms of antigen presentation drive T cell immunity is unknown. By convention, CD8 T cells recognize viral peptides, or epitopes, in association with classical major histocompatibility complex (MHC) class I, or MHC-Ia, but immune surveillance can, in some cases, be directed against peptides presented by nonclassical MHC-Ib, in particular the MHC-E proteins (Qa-1 in mice and HLA-E in humans); however, the overall importance of nonclassical responses in antiviral immunity remains unclear. Similarly uncertain is the importance of 'cryptic' viral epitopes, defined as those undetectable by conventional mapping techniques. Here we used an immunopeptidomic approach to search for unconventional epitopes that drive T cell responses in mice infected with influenza virus A/Puerto Rico/8/1934. We identified a nine amino acid epitope, termed M-SL9, that drives a co-immunodominant, cytolytic CD8 T cell response that is unconventional in two major ways: first, it is presented by Qa-1, and second, it has a cryptic origin, mapping to an unannotated alternative reading frame product of the influenza matrix gene segment. Presentation and immunogenicity of M-SL9 are dependent on the second AUG codon of the positive sense matrix RNA segment, suggesting translation initiation by leaky ribosomal scanning. During influenza virus A/Puerto Rico/8/1934 infection, M-SL9-specific T cells exhibit a low level of egress from the lungs and strong differentiation into tissue-resident memory cells. Importantly, we show that M-SL9/Qa-1-specific T cells can be strongly induced by messenger RNA vaccination and that they can mediate antigen-specific cytolysis in vivo. Our results demonstrate that noncanonical translation products can account for an important fraction of the T cell repertoire and add to a growing body of evidence that MHC-E-restricted T cells could have substantial therapeutic value.

PD-1 Mediated Regulation of Unique Activated CD8 + T Cells by NK Cells in the Submandibular Gland.

The increasing utilization of anti-PD-1 immune checkpoint blockade (ICB) has led to the emergence of immune-related adverse events (irAEs), including sicca syndrome. Interestingly, we found that the submandibular gland (SMG) of PD-1 deficient mice harbors a large population of CD8 + T cells, reminiscing ICB induced sicca. This phenotype was also observed in the SMG of both NK cell-depleted C57BL/6 animals and NK cell-deficient animals. Mechanistically, using mice conditionally deficient for PD-L1 in the NK cell lineage, we discovered that NK cells regulate CD8 + T cell homeostasis via the PD-1/PD-L1 axis in this organ. Importantly, single-cell RNA sequencing of PD-1 deficient SMG CD8 + T cells reveals a unique transcriptional profile consistent with TCR activation. These cells have limited TCR diversity and phenotypically overlap with GzmK + CD8 + T autoimmune cells identified in primary Sjögren's syndrome patients. These insights into NK cell immunoregulation in the SMG, and the consequences of disrupted CD8 + T cell homeostasis, provide opportunities for preventing the development of irAEs. Highlights: Elevated CD8 + T cells in the submandibular gland (SMG) of PD-1 deficient mice parallel sicca-like irAEs seen in ICB patients. In addition to their previously described hyporesponsive phenotype, NK cells in the SMG regulate CD8 + T cell homeostasis through the PD-L1/PD-1 axis. PD-1 deficient SMG CD8 + T cells display unique transcriptional profiles associated with proinflammatory functions, TCR activation, interferon stimulation, and exhaustion. Oligoclonal expansion and similarities in TCR sequences indicate T cell activation and a preference for recognizing specific antigens.

The Yin and Yang of Targeting KLRG1+ Tregs and Effector Cells.

The literature surrounding KLRG1 has primarily focused on NK and CD8+ T cells. However, there is evidence that the most suppressive Tregs express KLRG1. Until now, the role of KLRG1 on Tregs has been mostly overlooked and remains to be elucidated. Here we review the current literature on KLRG1 with an emphasis on the KLRG1+ Treg subset role during cancer development and autoimmunity. KLRG1 has been recently proposed as a new checkpoint inhibitor target, but these studies focused on the effects of KLRG1 blockade on effector cells. We propose that when designing anti-tumor therapies targeting KLRG1, the effects on both effector cells and Tregs will have to be considered.

SHP-2 deletion in CD4Cre expressing chondrocyte precursors leads to tumor development with wrist tropism.

Due to redundancy with other tyrosine phosphatases, the ubiquitously expressed tyrosine phosphatase SHP-2 (encoded by Ptpn11) is not required for T cell development. However, Ptpn11 gene deletion driven by CD4 Cre recombinase leads to cartilage tumors in the wrist. Using a fate mapping system, we demonstrate that wrist tumor development correlates with increased frequency and numbers of non-hematopoietic lineage negative CD45 negative cells with a bone chondrocyte stromal cell precursor cell (BCSP) phenotype. Importantly, the BCSP subset has a history of CD4 expression and a marked wrist location tropism, explaining why the wrist is the main site of tumor development. Mechanistically, we found that in SHP-2 absence, SOX-9 is no longer regulated, leading to an uncontrolled proliferation of the BCSP subset. Altogether, these results identify a unique subset of chondrocyte precursors tightly regulated by SHP-2. These findings underscore the need for the development of methods to therapeutically target this subset of cells, which could potentially have an impact on treatment of SHP-2 dysfunction linked debilitating diseases.

Sequential actions of EOMES and T-BET promote stepwise maturation of natural killer cells.

EOMES and T-BET are related T-box transcription factors that control natural killer (NK) cell development. Here we demonstrate that EOMES and T-BET regulate largely distinct gene sets during this process. EOMES is dominantly expressed in immature NK cells and drives early lineage specification by inducing hallmark receptors and functions. By contrast, T-BET is dominant in mature NK cells, where it induces responsiveness to IL-12 and represses the cell cycle, likely through transcriptional repressors. Regardless, many genes with distinct functions are co-regulated by the two transcription factors. By generating two gene-modified mice facilitating chromatin immunoprecipitation of endogenous EOMES and T-BET, we show a strong overlap in their DNA binding targets, as well as extensive epigenetic changes during NK cell differentiation. Our data thus suggest that EOMES and T-BET may distinctly govern, via differential expression and co-factors recruitment, NK cell maturation by inserting partially overlapping epigenetic regulations.

Combination blockade of KLRG1 and PD-1 promotes immune control of local and disseminated cancers.

Checkpoint blockade therapy is effective against many cancers; however, new targets need to be identified to treat patients who do not respond to current treatment or demonstrate immune escape. Here, we showed that blocking the inhibitory receptor Killer cell lectin-like receptor G1 (KLRG1) enhances anti-tumor immunity mediated by NK cells and CD8+ T cells. We found that loss of KLRG1 signaling alone significantly decreased melanoma and breast cancer tumor growth in the lungs of mice. In addition, we demonstrated that KLRG1 blockade can synergize with PD-1 checkpoint therapy to increase the therapeutic efficacy compared to either treatment alone. This effect was even observed with tumors that do not respond to PD-1 checkpoint therapy. Double blockade therapy led to significantly decreased tumor size, increased frequency and activation of CD8+ T cells, and increased NK cell frequency and maturation in the tumor microenvironment. These findings demonstrate that KLRG1 is a novel checkpoint inhibitor target that affects NK and T cell anti-tumor immunity, both alone and in conjunction with established immunotherapies.

The Invariant NKT Cell Response Has Differential Signaling Requirements during Antigen-Dependent and Antigen-Independent Activation.

Invariant NKT (iNKT) cells are an innate-like population characterized by their recognition of glycolipid Ags and rapid cytokine production upon activation. Unlike conventional T cells, which require TCR ligation, iNKT cells can also be stimulated independently of their TCR. This feature allows iNKT cells to respond even in the absence of glycolipid Ags, for example, during viral infections. Although the TCR-dependent and -independent activation of iNKT cells have been relatively well established, the exact contributions of IL-12, IL-18, and TLRs remain unclear for these two activation pathways. To definitively investigate how these components affect the direct and indirect stimulation of iNKT cells, we used mice deficient for either MyD88 or the IL-12Rß2 in the T cell lineage. Using these tools, we demonstrate that IL-12, IL-18, and TLRs are completely dispensable for the TCR activation pathway when a strong agonist is used. In contrast, during murine CMV infection, when the TCR is not engaged, IL-12 signaling is essential, and TLR signaling is expendable. Importantly, to our knowledge, we discovered an intrinsic requirement for IL-18 signaling by splenic iNKT cells but not liver iNKT cells, suggesting that there might be diversity, even within the NKT1 population.

Natural killers or ILC1s? That is the question.

Group 1 innate lymphoid cells (ILCs) comprise the natural killer (NK) cells and ILC1s. Both cells co-exist in peripheral tissues and despite effort to characterize the molecular identity and developmental pathways of ILC1s, their relationship with NK cells remains elusive. ILC1s and NK cells share many common features and analysis of ILC1s in tissues revealed a great heterogeneity and distinct transcriptional requirement of each ILC1 subsets complexifying the organization of this group. Here, we discuss whether ILC1 and NK cells can be considered as distinct lineages based on their origin, location, phenotype or transcriptional regulation. Discrimination of NK cells and ILC1s represent an important challenge to unravel the individual functions of these cells during infection and tumor immunosurveillance.

Inflammation-Induced Lactate Leads to Rapid Loss of Hepatic Tissue-Resident NK Cells.

The liver harbors two main innate lymphoid cell (ILC) populations: conventional NK (cNK) cells and tissue-resident NK (trNK) cells. Using the MCMV model of infection, we find that, in contrast to liver cNK cells, trNK cells initially undergo a contraction phase followed by a recovery phase to homeostatic levels. The contraction is MCMV independent because a similar phenotype is observed following poly(I:C)/CpG or α-GalCer injection. The rapid contraction phase is due to apoptosis, whereas the recovery phase occurs via proliferation in situ. Interestingly, trNK cell apoptosis is not mediated by fratricide and not induced by liver lymphocytes or inflammatory cytokines. Instead, we find that trNK cell apoptosis is the consequence of an increased sensitivity to lactic acid. Mechanistic analysis indicates that trNK cell sensitivity to lactate is linked to impaired mitochondrial function. These findings underscore the distinctive properties of the liver-resident NK cell compartment.

Qa-1-Restricted CD8+ T Cells Can Compensate for the Absence of Conventional T Cells during Viral Infection.

The role of non-classical T cells during viral infection remains poorly understood. Using the well-established murine model of CMV infection (MCMV) and mice deficient in MHC class Ia molecules, we found that non-classical CD8+ T cells robustly expand after MCMV challenge, become highly activated effectors, and are capable of forming durable memory. Interestingly, although these cells are restricted by MHC class Ib molecules, they respond similarly to conventional T cells. Remarkably, when acting as the sole component of the adaptive immune response, non-classical CD8+ T cells are sufficient to protect against MCMV-induced lethality. We also demonstrate that the MHC class Ib molecule Qa-1 (encoded by H2-T23) restricts a large, and critical, portion of this population. These findings reveal a potential adaptation of the host immune response to compensate for viral evasion of classical T cell immunity.

Shp-2 is critical for ERK and metabolic engagement downstream of IL-15 receptor in NK cells.

The phosphatase Shp-2 was implicated in NK cell development and functions due to its interaction with NK inhibitory receptors, but its exact role in NK cells is still unclear. Here we show, using mice conditionally deficient for Shp-2 in the NK lineage, that NK cell development and responsiveness are largely unaffected. Instead, we find that Shp-2 serves mainly to enforce NK cell responses to activation by IL-15 and IL-2. Shp-2-deficient NK cells have reduced proliferation and survival when treated with high dose IL-15 or IL-2. Mechanistically, Shp-2 deficiency hampers acute IL-15 stimulation-induced raise in glycolytic and respiration rates, and causes a dramatic defect in ERK activation. Moreover, inhibition of the ERK and mTOR cascades largely phenocopies the defect observed in the absence of Shp-2. Together, our data reveal a critical function of Shp-2 as a molecular nexus bridging acute IL-15 signaling with downstream metabolic burst and NK cell expansion.

Ptpn11 Deletion in CD4+ Cells Does Not Affect T Cell Development and Functions but Causes Cartilage Tumors in a T Cell-Independent Manner.

The ubiquitously expressed tyrosine phosphatase Src homology region 2 domain-containing phosphatase-2 (SHP-2, encoded by Ptpn11) is required for constitutive cellular processes including proliferation, differentiation, and the regulation of immune responses. During development and maturation, subsets of T cells express a variety of inhibitory receptors known to associate with phosphatases, which in turn, dephosphorylate key players of activating receptor signaling pathways. We hypothesized that SHP-2 deletion would have major effects on T cell development by altering the thresholds for activation, as well as positive and negative selection. Surprisingly, using mice conditionally deficient for SHP-2 in the T cell lineage, we show that the development of these lymphocytes is globally intact. In addition, our data demonstrate that SHP-2 absence does not compromise T cell effector functions, suggesting that SHP-2 is dispensable in these cells. Unexpectedly, in aging mice, Ptpn11 gene deletion driven by CD4 Cre recombinase leads to cartilage tumors in wrist bones in a T cell-independent manner. These tumors resemble miniature cartilaginous growth plates and contain CD4-lineage positive chondrocyte-like cells. Altogether these results indicate that SHP-2 is a cartilage tumor suppressor during aging.

Lacrimal Gland NK Cells Are Developmentally and Functionally Similar to Conventional NK Cells.

The murine lacrimal gland (LG), which produces crucial components of the ocular tear film, contains a population of natural killer (NK) cells. LG NK cells appear to belong to the conventional NK cell lineage, based on their cell surface receptor and transcription factor expression, absence in NFIL3-/- mice, and lack of RORγt expression during development. LG NK cells produce IFN-γ during the early stages of systemic murine cytomegalovirus (MCMV) infection. This effector response occurs in the absence of noticeable MCMV replication in the LG, indicating that LG NK cells are being activated by soluble factors. However, the magnitude of LG NK cell IFN-γ production during MCMV infection is significantly lower than spleen and liver NK cells. Adoptive transfer experiments in lymphopenic mice revealed that this hyporesponsive phenotype is tissue-specific, which indicates that that LG NK cells can produce a robust effector response.

NFIL3 Expression Distinguishes Tissue-Resident NK Cells and Conventional NK-like Cells in the Mouse Submandibular Glands.

The submandibular salivary gland (SMG), a major site of persistent infection for many viruses, contains a large NK cell population. Using NFIL3-deficient mice, PLZF reporter/fate mapping mice, and mixed bone marrow chimeras, we identified two distinct populations of NK cells in the SMG. Although phenotypically unique, the main population relies on NFIL3, but not PLZF, for development and, therefore, is developmentally similar to the conventional NK cell subset. In contrast, we found that approximately one quarter of the SMG NK cells develop independently of NFIL3. Interestingly, NFIL3-independent SMG tissue-resident NK (trNK) cells are developmentally distinct from liver trNK cells. We also demonstrated that the SMG NK cell hyporesponsive phenotype during murine CMV infection is tissue specific and not cell intrinsic. In contrast, NFIL3-independent SMG trNK cells are intrinsically hyporesponsive. Altogether, our data show that the SMG tissue environment shapes a unique repertoire of NK-like cells with distinct phenotypes.

The role of MHC class Ib-restricted T cells during infection.

Even though major histocompatibility complex (MHC) class Ia and many Ib molecules have similarities in structure, MHC class Ib molecules tend to have more specialized functions, which include the presentation of non-peptidic antigens to non-classical T cells. Likewise, non-classical T cells also have unique characteristics, including an innate-like phenotype in naïve animals and rapid effector functions. In this review, we discuss the role of MAIT and NKT cells during infection but also the contribution of less studied MHC class Ib-restricted T cells such as Qa-1-, Qa-2-, and M3-restricted T cells. We focus on describing the types of antigens presented to non-classical T cells, their response and cytokine profile following infection, as well as the overall impact of these T cells to the immune system.

Phenotype and functions of conventional and non-conventional NK cells.

Here we focus on the phenotypic and functional diversity of NK cells. We give an overview of the phenotype and developmental pathways of conventional and tissue-resident NK cells. We also discuss the potential complementary functions of conventional NK cells and tissue-resident NK cells in a variety of tissues.

Role of SHIP1 in Invariant NKT Cell Development and Functions.

SHIP1 is a 5'-inositol phosphatase known to negatively regulate the signaling product of the PI3K pathway, phosphatidylinositol (3-5)-trisphosphate. SHIP1 is recruited to a large number of inhibitory receptors expressed on invariant NK (iNKT) cells. We hypothesized that SHIP1 deletion would have major effects on iNKT cell development by altering the thresholds for positive and negative selection. Germline SHIP1 deletion has been shown to affect T cells as well as other immune cell populations. However, the role of SHIP1 on T cell function has been controversial, and its participation on iNKT cell development and function has not been examined. We evaluated the consequences of SHIP1 deletion on iNKT cells using germline-deficient mice, chimeric mice, and conditionally deficient mice. We found that T cell and iNKT cell development are impaired in germline-deficient animals. However, this phenotype can be rescued by extrinsic expression of SHIP1. In contrast, SHIP1 is required cell autonomously for optimal iNKT cell cytokine secretion. This suggests that SHIP1 calibrates the threshold of iNKT cell reactivity. These data further our understanding of how iNKT cell activation is regulated and provide insights into the biology of this unique cell lineage.

Role of type I interferon receptor signaling on NK cell development and functions.

Type I interferons (IFN) are unique cytokines transcribed from intronless genes. They have been extensively studied because of their anti-viral functions. The anti-viral effects of type I IFN are mediated in part by natural killer (NK) cells. However, the exact contribution of type I IFN on NK cell development, maturation and activation has been somewhat difficult to assess. In this study, we used a variety of approaches to define the consequences of the lack of type I interferon receptor (IFNAR) signaling on NK cells. Using IFNAR deficient mice, we found that type I IFN affect NK cell development at the pre-pro NK stage. We also found that systemic absence of IFNAR signaling impacts NK cell maturation with a significant increase in the CD27+CD11b+ double positive (DP) compartment in all organs. However, there is tissue specificity, and only in liver and bone marrow is the maturation defect strictly dependent on cell intrinsic IFNAR signaling. Finally, using adoptive transfer and mixed bone marrow approaches, we also show that cell intrinsic IFNAR signaling is not required for NK cell IFN-γ production in the context of MCMV infection. Taken together, our studies provide novel insights on how type I IFN receptor signaling regulates NK cell development and functions.

Mouse natural killer cell development and maturation are differentially regulated by SHIP-1.

The SH2-containing inositol phosphatase-1 (SHIP-1) is a 5' inositol phosphatase known to negatively regulate the product of phosphoinositide-3 kinase (PI3K), phosphatidylinositol-3.4,5-trisphosphate. SHIP-1 can be recruited to a large number of inhibitory receptors expressed on natural killer (NK) cells. However, its role in NK cell development, maturation, and functions is not well defined. In this study, we found that the absence of SHIP-1 results in a loss of peripheral NK cells. However, using chimeric mice we demonstrated that SHIP-1 expression is not required intrinsically for NK cell lineage development. In contrast, SHIP-1 is required cell autonomously for NK cell terminal differentiation. These findings reveal both a direct and indirect role for SHIP-1 at different NK cell development checkpoints. Notably, SHIP-1-deficient NK cells display an impaired ability to secrete IFN-γ during cytokine receptor-mediated responses, whereas immunoreceptor tyrosine-based activation motif containing receptor-mediated responses is not affected. Taken together, our results provide novel insights on how SHIP-1 participates in the development, maturation, and effector functions of NK cells.

Activated iNKT cells promote memory CD8+ T cell differentiation during viral infection.

α-Galactosylceramide (α-GalCer) is the prototypical lipid ligand for invariant NKT cells. Recent studies have proposed that α-GalCer is an effective adjuvant in vaccination against a range of immune challenges, however its mechanism of action has not been completely elucidated. A variety of delivery methods have been examined including pulsing dendritic cells with α-GalCer to optimize the potential of α-GalCer. These methods are currently being used in a variety of clinical trials in patients with advanced cancer but cannot be used in the context of vaccine development against pathogens due to their complexity. Using a simple delivery method, we evaluated α-GalCer adjuvant properties, using the mouse model for cytomegalovirus (MCMV). We measured several key parameters of the immune response to MCMV, including inflammation, effector, and central memory CD8(+) T cell responses. We found that α-GalCer injection at the time of the infection decreases viral titers, alters the kinetics of the inflammatory response, and promotes both increased frequencies and numbers of virus-specific memory CD8(+) T cells. Overall, our data suggest that iNKT cell activation by α-GalCer promotes the development of long-term protective immunity through increased fitness of central memory CD8(+) T cells, as a consequence of reduced inflammation.