Critical Role for SLAM/SAP Signaling in the Thymic Developmental Programming of IL-17- and IFN-γ-Producing γδ T Cells.

During thymic development, mouse γδ T cells commit to either an IFN-γ- or an IL-17-producing phenotype through mechanisms that remain unclear. In this study, we investigated the extent to which the SLAM/SAP signaling pathway regulates the functional programming of γδ T cells. Characterization of SLAM family receptor expression revealed that thymic γδ T cell subsets were each marked by distinct coexpression profiles of SLAMF1, SLAMF4, and SLAMF6. In the thymus, Vγ1 and Vγ4 T cells that exhibited an SLAMF1+SLAMF6+ double positive phenotype were largely contained within immature CD24+CD73- and CD24+CD73+ subsets, whereas SLAMF1 single positive, SLAMF6 single positive, or SLAMF1SLAMF6 double negative cells were found within mature CD24-CD73+ and CD24-CD73- subsets. In the periphery, SLAMF1 and SLAMF6 expression distinguished IL-17- and IFN-γ-producing γδ T cells, respectively. Disruption of SLAM family receptor signaling through deletion of SAP resulted in impaired thymic Vγ1 and Vγ4 T cell maturation at the CD24+CD73-SLAMF1+SLAMF6+ double positive stage that was associated with a decreased frequency of CD44+RORγt+ γδ T cells. Impaired development was in turn associated with decreased γδ T cell IL-17 and IFN-γ production in the thymus as well as in peripheral tissues. The role for SAP was subset-specific, as Vγ1Vδ6.3, Vγ4, Vγ5, but not Vγ6 subsets were SAP-dependent. Together, these data suggest that the SLAM/SAP signaling pathway plays a larger role in γδ T cell development than previously appreciated and represents a critical checkpoint in the functional programming of both IL-17- and IFN-γ-producing γδ T cell subsets.

Regulation of invariant NKT cell development and function by a 0.14 Mbp locus on chromosome 1: a possible role for Fcgr3.

Invariant NKT (iNKT) cells are tissue-resident innate-like T cells critical to the host immune response. We previously identified a 6.6 Mbp region on chromosome 1 as a major regulator of iNKT cell number and function in C57BL/6 and 129X1/SvJ mice. Here, we fine-mapped this locus by assessing the iNKT cell response to alpha-galactosylceramide (αGalCer) in a series of B6.129 congenic lines. This analysis revealed the presence of at least two genetic elements that regulate iNKT cell cytokine production in response to αGalCer. While one of these genetic elements mapped to the B6.129c6 interval containing Slam genes, the dominant regulator in this region mapped to the 0.14 Mbp B6.129c3 interval. In addition, we found that numbers of thymic iNKT cells and DP thymocytes were significantly lower in B6.129c3 mice, indicating that this interval also regulates iNKT cell development. Candidate gene analysis revealed a fivefold increase in Fcgr3 expression in B6.129c3 iNKT cells, and we observed increased expression of FcγR3 protein on B6.129c3 iNKT cells, NK cells, and neutrophils. These data identify the B6.129c3 interval as a novel locus regulating the response of iNKT cells to glycosphingolipid, revealing a link between this phenotype and a polymorphism that regulates Fcgr3 expression.

The role of iNKT cells on the phenotypes of allergic airways in a mouse model.

iNKT cells and mast cells have both been implicated in the syndrome of allergic asthma through their activation-induced release of Th2 type cytokines and secretion of histamine and other mediators, respectively, which can promote airways hyperresponsiveness (AHR) to agents such as methacholine. However, a mechanistic link between iNKT cells and mast cell recruitment or activation has never been explored. Our objective was to determine whether iNKT cells are necessary for the recruitment of mast cells and if iNKT cells can influence the acute allergen induced bronchoconstriction (AIB) caused by mast cell mediator release. To do so, we pharmacologically eliminated iNKT cells using a specific antibody (NKT-14) and examined its impact on airway inflammation and physiological phenotype. In mice treated with NKT-14, the elimination of iNKT cells was sufficient to prevent AHR and pulmonary eosinophilic inflammation elicited by administration of the iNKT cell agonist αGalCer. In mice treated with NKT-14 and then sensitized and challenged with house dust mite extract (HDM), eliminating the iNKT cells significantly reduced both AHR and AIB but did not affect pulmonary inflammation, the mast cell population, nor the release of the mast cell mediators mast cell protease-1 and prostaglandin D2. We conclude that while iNKT cells contribute to the phenotype of allergic airways disease through the manifestation of AIB and AHR, their presence is not required for mast cell recruitment and activation, or to generate the characteristic inflammatory response subsequent to allergen challenge.

Polymorphisms in the CD1d promoter that regulate CD1d gene expression are associated with impaired NKT cell development.

CD1d-restricted NKT cells comprise an innate-like T cell population that exerts significant influence over early events in the developing immune response. The frequency of NKT cells is highly variable in humans and in mice, but the basis for this variability remains unclear. In this study, we report a striking deficiency of type I NKT cells in the wild-derived inbred strains PWD/PhJ, SPRET/EiJ, and CAST/EiJ. Investigation of the underlying basis for the lack of type I NKT cells revealed that one strain, PWD/PhJ, exhibited a significant impairment in thymocyte and splenocyte CD1d gene and protein expression. Accordingly, both thymocytes and bone marrow-derived dendritic cells from PWD mice exhibited a significant impairment in the ability to present α-galactosylceramide to NKT cells. The impaired PWD CD1d gene expression was due to impaired CD1d promoter activity. Fine-mapping of the promoter activity revealed that two single nucleotide substitutions at positions -331 and -164 in the proximal promoter were each sufficient to account for the diminished PWD CD1d promoter activity. Examination of the strain distribution pattern of these polymorphisms revealed that, of 19 strains analyzed, only PWD and PWK mice possessed both CD1d promoter polymorphisms. A subsequent examination of the PWK strain revealed that it also exhibited impaired thymocyte CD1d expression and very low numbers of NKT cells. Taken together, these results provide new insight into the control of CD1d gene expression, and they have implications for the evolution of CD1d and type I NKT cells.

An Empirical Antigen Selection Method Identifies Neoantigens That Either Elicit Broad Antitumor T-cell Responses or Drive Tumor Growth.

Neoantigens are critical targets of antitumor T-cell responses. The ATLAS bioassay was developed to identify neoantigens empirically by expressing each unique patient-specific tumor mutation individually in Escherichia coli, pulsing autologous dendritic cells in an ordered array, and testing the patient's T cells for recognition in an overnight assay. Profiling of T cells from patients with lung cancer revealed both stimulatory and inhibitory responses to individual neoantigens. In the murine B16F10 melanoma model, therapeutic immunization with ATLAS-identified stimulatory neoantigens protected animals, whereas immunization with peptides associated with inhibitory ATLAS responses resulted in accelerated tumor growth and abolished efficacy of an otherwise protective vaccine. A planned interim analysis of a clinical study testing a poly-ICLC adjuvanted personalized vaccine containing ATLAS-identified stimulatory neoantigens showed that it is well tolerated. In an adjuvant setting, immunized patients generated both CD4+ and CD8+ T-cell responses, with immune responses to 99% of the vaccinated peptide antigens. SIGNIFICANCE: Predicting neoantigens in silico has progressed, but empirical testing shows that T-cell responses are more nuanced than straightforward MHC antigen recognition. The ATLAS bioassay screens tumor mutations to uncover preexisting, patient-relevant neoantigen T-cell responses and reveals a new class of putatively deleterious responses that could affect cancer immunotherapy design.This article is highlighted in the In This Issue feature, p. 521.

Resolution of Chlamydia trachomatis Infection Is Associated with a Distinct T Cell Response Profile.

Chlamydia trachomatis is the causative agent of the most frequently reported bacterial sexually transmitted infection, the total burden of which is underestimated due to the asymptomatic nature of the infection. Untreated C. trachomatis infections can cause significant morbidities, including pelvic inflammatory disease and tubal factor infertility (TFI). The human immune response against C. trachomatis, an obligate intracellular bacterium, is poorly characterized but is thought to rely on cell-mediated immunity, with CD4(+) and CD8(+) T cells implicated in protection. In this report, we present immune profiling data of subjects enrolled in a multicenter study of C. trachomatis genital infection. CD4(+) and CD8(+) T cells from subjects grouped into disease-specific cohorts were screened using a C. trachomatis proteomic library to identify the antigen specificities of recall T cell responses after natural exposure by measuring interferon gamma (IFN-γ) levels. We identified specific T cell responses associated with the resolution of infection, including unique antigens identified in subjects who spontaneously cleared infection and different antigens associated with C. trachomatis-related sequelae, such as TFI. These data suggest that novel and unique C. trachomatis T cell antigens identified in individuals with effective immune responses can be considered as targets for vaccine development, and by excluding antigens associated with deleterious sequelae, immune-mediated pathologies may be circumvented.