Human cytolytic T lymphocytes and NK cells can limit tumor growth and are being increasingly harnessed for tumor immunotherapy. One way cytolytic lymphocytes recognize tumor cells is by engagement of their activating receptor, NKG2D, by stress-antigens of the MICA/B and ULBP families. This study shows that surface upregulation of NKG2D ligands by human epithelial cells in response to ultraviolet irradiation, osmotic shock, oxidative stress, and growth factor provision, is attributable to activation of the EGF-receptor (EGFR). EGFR activation causes intracellular re-localisation of AUF1 proteins that ordinarily destabilise NKG2D ligand mRNAs by targeting an AU-rich element conserved within the 3′ ends of most human but not murine NKG2D ligand genes. Consistent with these findings, NKG2D ligand expression by primary human carcinomas positively correlated with EGFR expression that is commonly hyper-activated in such tumours, and was reduced by clinical EGFR inhibitors. Thus, stress-induced activation of EGFR not only regulates cell growth but concomitantly regulates the cells’ immunological visibility. Thus, therapeutics designed to limit cancer cell growth should also be considered in terms of their impact on immunosurveillance.
B cells, αβ T cells and γδ T cells are conserved lymphocyte subtypes encoding their antigen receptors from somatically rearranged genes. αβ T cells undergo positive selection in the thymus by engagement of their T cell receptors (TCRs) with self-peptides presented by major histocompatibility complex molecules1. The molecules that select γδ T cells are unknown2–4. Vγ5+Vδ1+ cells comprise 90% of mouse epidermal cd T cells4. By mapping and genetic complementation using a strain showing loss of Vγ5+Vδ1+ cells due to a failure of thymic selection, we show that this defect is caused by mutation in Skint1, a newly identified gene expressed in thymus and skin that encodes a protein with immunoglobulin-like and transmembrane domains. Skint1 is the prototypic member of a rapidly evolving family of at least 11 genes in mouse, with greatest similarity to the butyrophilin genes. These findings define a new family of proteins mediating key epithelial-immune interactions.
The production of cytokines such as interferon-γ and interleukin 17 by αβ and γδ T cells influences the outcome of immune responses. Here we show that most γδ T lymphocytes expressed the tumor necrosis factor receptor family member CD27 and secreted interferon-γ, whereas interleukin 17 production was restricted to CD27- γδ T cells. In contrast to the apparent plasticity of αβ T cells, the cytokine profiles of these distinct γδ T cell subsets were essentially stable, even during infection. These phenotypes were established during thymic development, when CD27 functions as a regulator of the differentiation of γδ T cells at least in part by inducing expression of the lymphotoxin-β receptor and genes associated with trans-conditioning and interferon-γ production. Thus, the cytokine profiles of peripheral γδ T cells are predetermined mainly by a mechanism involving CD27.
In this study we demonstrate a new form of immunoregulation: engagement on CD4+ T cells of the complement regulator CD46 promoted the effector potential of T helper type 1 cells (TH1 cells), but as interleukin 2 (IL-2) accumulated, it switched cells toward a regulatory phenotype, attenuating IL-2 production via the transcriptional regulator ICER/CREM and upregulating IL-10 after interaction of the CD46 tail with the serine-threonine kinase SPAK. Activated CD4+ T cells produced CD46 ligands, and blocking CD46 inhibited IL-10 production. Furthermore, CD4+ T cells in rheumatoid arthritis failed to switch, consequently producing excessive interferon-γ (IFN-γ). Finally, γδ T cells, which rarely produce IL-10, expressed an alternative CD46 isoform and were unable to switch. Nonetheless, coengagement of T cell antigen receptor (TCR) γδ and CD46 suppressed effector cytokine production, establishing that CD46 uses distinct mechanisms to regulate different T cell subsets during an immune response.
Microbes and viruses provoke immune responses because certain of their molecular determinants engage and activate dendritic cells (DC). However, evidence is growing for lymphocyte activation by tissue dysregulation. Thus, murine γδ T cells and NK cells can respond rapidly in vivo to Major Histocompatibility Complex (MHC) class I–related “stress-antigens” displayed by cells experiencing DNA damage and/or other physico-chemical stress. Such “lymphoid stress-surveillance” (LSS) can limit tumor formation, but may also promote immunopathology. MICA is a highly polymorphic human stress-antigen implicated in tumor-surveillance, inflammation, and transplant rejection. However, neither the generality of LSS in humans, nor a functional context for MICA polymorphism has been established. Here we show that MICA coding-sequence polymorphisms substantially affect RNA and protein expression. All donors tested showed LSS responses of γδ T and NK cells, but unexpectedly each was individually “tuned”. Hence, some responded optimally to high MICA expression, while others responded better to poorly-expressed MICA alleles, challenging the orthodoxy that higher stress-antigen levels promote greater responsiveness. The routine clinical monitoring of individual tuning should provide practical insight into individual variation in tumor immune-surveillance, transplant rejection and inflammation, and introduce new perspectives on immuno-evasion and immune-suppression in these scenarios.
Psoriasis is a common, chronic, inflammatory skin disorder. A number of genetic loci have been shown to confer risk for psoriasis. Collectively, these offer an integrated model for the inherited basis for susceptibility to psoriasis that combines altered skin barrier function together with the dysregulation of innate immune pathogen sensing and adaptive immunity. The major histocompatibility complex (MHC) harbours the psoriasis susceptibility region which exhibits the largest effect size, driven in part by variation contained on the HLA-Cw*0602 allele. However, the resolution of the number and genomic location of potential independent risk loci are hampered by extensive linkage disequilibrium across the region. We leveraged the power of large psoriasis case and control data sets and the statistical approach of conditional analysis to identify potential further association signals distributed across the MHC. In addition to the major loci at HLA-C (P = 2.20 × 10−236), we observed and replicated four additional independent signals for disease association, three of which are novel. We detected evidence for association at SNPs rs2507971 (P = 6.73 × 10−14), rs9260313 (P = 7.93 × 10−09), rs66609536 (P = 3.54 × 10−07) and rs380924 (P = 6.24 × 10−06), located within the class I region of the MHC, with each observation replicated in an independent sample (P ≤ 0.01). The previously identified locus is close to MICA, the other three lie near MICB, HLA-A and HCG9 (a non-coding RNA gene). The identification of disease associations with both MICA and MICB is particularly intriguing, since each encodes an MHC class I-related protein with potent immunological function.
γδ T cells, a lineage of innate-like lymphocytes, are distinguished from conventional αβ T cells in their antigen-recognition, cell activation requirements and effector functions. γδ T cells have been implicated in the pathology of several human autoimmune and inflammatory diseases and their corresponding mouse models, but their specific roles in these diseases have not been elucidated. We report that γδTCR+ cells including both the CD27−CD44hi and CD27+CD44lo subsets infiltrate islets of pre-diabetic non-obese diabetic (NOD) mice. Moreover, NOD CD27−CD44hi and CD27+CD44lo γδ T cells were pre-programmed to secrete IL-17, or IFN-γ upon activation. Adoptive transfer of T1D to T and B lymphocyte-deficient NOD recipients was greatly potentiated when γδ T cells, and specifically the CD27− γδ T cell subset, were included compared to transfer of αβ T cells alone. Antibody-mediated blockade of IL-17 prevented T1D transfer in this setting. Moreover, introgression of genetic Tcrd deficiency onto the NOD background provided robust T1D protection, supporting a non-redundant, pathogenic role of γδ T cells in this model. The potent contributions of CD27− γδ T cells and IL-17 to islet inflammation and diabetes reported here suggest that these mechanisms may also underlie human T1D.
Epithelial cells respond to physico-chemical damage with up-regulation of major histocompatbility complex–like ligands that can activate the cytolytic potential of neighboring intraepithelial T cells by binding the activating receptor, NKG2D. The systemic implications of this lymphoid stress-surveillance response, however, are unknown. We found that antigens encountered at the same time as cutaneous epithelial stress induced strong primary and secondary systemic, T helper 2 (TH2)–associated atopic responses in mice. These responses required NKG2D-dependent communication between dysregulated epithelial cells and tissue-associated lymphoid cells. These data are germane to uncertainty over the afferent induction of TH2 responses and provide a molecular framework for considering atopy as an important component of the response to tissue damage and carcinogenesis.
Polyaromatic hydrocarbons (PAHs) are prevalent, potent carcinogens, and 7,12-dimethylbenz[a]anthracene (DMBA) is a model PAH widely used to study tumorigenesis. Mice lacking Langerhans cells (LCs), a signatory epidermal dendritic cell (DC), are protected from cutaneous chemical carcinogenesis, independent of T cell immunity. Investigation of the underlying mechanism revealed that LC-deficient skin was relatively resistant to DMBA-induced DNA damage. LCs efficiently metabolized DMBA to DMBA-trans-3,4-diol, an intermediate proximal to oncogenic Hras mutation, and DMBA-treated LC-deficient skin contained significantly fewer Hras mutations. Moreover, DMBA-trans-3,4-diol application bypassed tumor resistance in LC-deficient mice. Additionally, the genotoxic impact of DMBA on human keratinocytes was significantly increased by prior incubation with human-derived LC. Thus, tissue-associated DC can enhance chemical carcinogenesis via PAH metabolism, highlighting the complex relation between immune cells and carcinogenesis.
Psoriasis is an immune-mediated skin disorder that is inherited as a complex genetic trait. Although genome-wide association scans (GWAS) have identified 36 disease susceptibility regions, more than 50% of the genetic variance can be attributed to a single Major Histocompatibility Complex (MHC) locus, known as PSORS1. Genetic studies indicate that HLA-C is the strongest PSORS1 candidate gene, since markers tagging HLA-Cw*0602 consistently generate the most significant association signals in GWAS. However, it is unclear whether HLA-Cw*0602 is itself the causal PSORS1 allele, especially as the role of SNPs that may affect its expression has not been investigated. Here, we have undertaken an in-depth molecular characterization of the PSORS1 interval, with a view to identifying regulatory variants that may contribute to disease susceptibility. By analysing high-density SNP data, we refined PSORS1 to a 179 kb region encompassing HLA-C and the neighbouring HCG27 pseudogene. We compared multiple MHC sequences spanning this refined locus and identified 144 candidate susceptibility variants, which are unique to chromosomes bearing HLA-Cw*0602. In parallel, we investigated the epigenetic profile of the critical PSORS1 interval and uncovered three enhancer elements likely to be active in T lymphocytes. Finally we showed that nine candidate susceptibility SNPs map within a HLA-C enhancer and that three of these variants co-localise with binding sites for immune-related transcription factors. These data indicate that SNPs affecting HLA-Cw*0602 expression are likely to contribute to psoriasis susceptibility and highlight the importance of integrating multiple experimental approaches in the investigation of complex genomic regions such as the MHC.
To gain further insight into the genetic architecture of psoriasis, we conducted a meta-analysis of three genome-wide association studies (GWAS) and two independent datasets genotyped on the Immunochip, involving 10,588 cases and 22,806 controls in total. We identified 15 new disease susceptibility regions, increasing the number of psoriasis-associated loci to 36 for Caucasians. Conditional analyses identified five independent signals within previously known loci. The newly identified shared disease regions encompassed a number of genes whose products regulate T-cell function (e.g. RUNX3, TAGAP and STAT3). The new psoriasis-specific regions were notable for candidate genes whose products are involved in innate host defense, encoding proteins with roles in interferon-mediated antiviral responses (DDX58), macrophage activation (ZC3H12C), and NF-κB signaling (CARD14 and CARM1). These results portend a better understanding of shared and distinctive genetic determinants of immune-mediated inflammatory disorders and emphasize the importance of the skin in innate and acquired host defense.
Psoriasis is an inflammatory skin disorder that is inherited as a complex trait. Genetic studies have repeatedly highlighted HLA-C as the major determinant for psoriasis susceptibility, with the Cw*0602 allele conferring significant disease risk in a wide-range of populations. Despite the potential importance of HLA-C variation in psoriasis, either via an effect on peptide presentation or immuno-inhibitory activity, allele-specific expression patterns have not been investigated. Here, we used reporter assays to characterize two regulatory variants, which virtually abolished the response to TNF-α (rs2524094) and IFN-γ (rs10657191) in HLA-Cw*0602 and a cluster of related alleles. We validated these findings through the analysis of HLA-Cw*0602 expression in primary keratinocytes treated with TNF-α and IFN-γ. Finally, we showed that HLA-Cw*0602 transcripts are not increased in psoriatic skin lesions, despite highly elevated TNF-α levels. Thus, our findings demonstrate the presence of allele-specific differences in HLA-C expression and indicate that HLA-Cw*0602 is unresponsive to up-regulation by key pro-inflammatory cytokines in psoriasis. These data pave the way for functional studies into the pathogenic role of the major psoriasis susceptibility allele.
The Regulator of G Protein Signaling 1 [RGS1] gene is associated with celiac disease, multiple sclerosis (MS) and Type I diabetes (T1D), which are all T cell-mediated pathologies. And yet there is no reported analysis of RGS1 biology in human T cells. This study shows that RGS1 expression is substantially higher in T cells from human gut versus peripheral blood, and that this can be exaggerated in intestinal inflammation. Elevated RGS1 levels profoundly reduce T cell migration to lymphoid-homing chemokines, whereas RGS-1 depletion selectively enhances such chemotaxis in gut T cells, and impairs their colitogenic potential. These findings provide a revised framework in which to view the linkage of RGS1 to inflammatory disease.
The Infectious Diseases BioBank (IDB) has consistently archived peripheral blood mononuclear cell (PBMNC) RNA for transcriptome analyses. RNA is particularly labile, and hence, these samples provide a sensitive indicator for assessing the IDB's quality-assurance measures. Independent analyses of 104 PBMNC RNA specimens from 26 volunteers revealed that the mean RNA integrity number (RIN) was high (9.02), although RIN ranged between scores of 7 and 10. This variation of RIN values was not associated with ischemic time, PBMNC quality, number of samples processed per day, self-medication after immunization, freezer location, donor characteristics, differential white blood cell counts, or daily variation in RNA extractions (all P>0.05). RIN values were related to the date of collection, with those processed during mid-summer having highest RIN scores (P=0.0001). Amongst specimens with the lowest RIN scores, no common feature could be identified. Thus, no technical explanation for the variation in RNA quality could be ascertained and these may represent normal physiological variations. These data provide strong evidence that current IDB protocols for the isolation and preservation PBMNC RNA are robust.
Infection or stimulation of the innate immune system by nonspecific microbial antigens is thought to educate the immune system to respond appropriately to allergens, preventing allergy.
To determine the immunologic pathways that might explain how infection/microbial exposure inhibits allergic sensitization.
Immunologic studies of non-antigen-specific functions of CD8 memory cells, their maturation in vivo, and their effects in a mouse asthma model, to test the hypothesis that CD8 memory is shaped by innate immunity in a way that can inhibit allergic disease.
We found that CD8 memory T-cell (CD8 Tm) populations bridge innate and adaptive immunity by responding to either antigen or cytokines alone. CD8 Tm populations partially subvert the clonal selection process by activating their neighbors through induction of dendritic cell IL-12. Stimulation of innate or acquired immunity in the lung or gut causes expansion/maturation of CD8 Tm populations, which provide an early source of cytokines, enhance TH1 immunity, and inhibit allergic sensitization and airway inflammation/hyperresponsiveness in a non-antigen-specific fashion.
CD8 T-cell–mediated immune memory is long-lived and can retain its capacity for rapid cytokine release in a nonantigen-specific fashion. This novel type of memory enhances TH1 over TH2 immunity and prevents allergic sensitization after exposure to environmental antigens or infection.
CD8 T cell; immunologic memory; innate immunity; allergy; TH1/TH2; dendritic cell; IL-12
The conjunctiva contains a specialized population of lymphocytes that reside in the epithelium, named intraepithelial lymphocytes (IEL).
Here we characterized the IEL population prior to and after experimental desiccating stress (DS) for 5 or 10 days (DS5, DS10) and evaluated the effect of NK depletion on DS. The frequency of IELs in normal murine conjunctiva was CD3+CD103+ (∼22%), CD3+γδ+ (∼9.6%), CD3+NK+ (2%), CD3−NK+ (∼4.4%), CD3+CD8α (∼0.9%), and CD4 (∼0.6%). Systemic depletion of NK cells prior and during DS led to a decrease in the frequency of total and activated DCs, a decrease in T helper-17+ cells in the cervical lymph nodes and generation of less pathogenic CD4+T cells. B6.nude recipient mice of adoptively transferred CD4+T cells isolated from NK-depleted DS5 donor mice showed significantly less corneal barrier disruption, lower levels of IL-17A, CCL20 and MMP-3 in the cornea epithelia compared to recipients of control CD4+T cells.
Taken together, these results show that the NK IELs are involved in the acute immune response to desiccation-induced dry eye by activating DC, which in turn coordinate generation of the pathogenic Th-17 response.
The thymic medulla provides a specialized microenvironment for the negative selection of T cells, with the presence of autoimmune regulator (Aire)-expressing medullary thymic epithelial cells (mTECs) during the embryonic-neonatal period being both necessary and sufficient to establish long-lasting tolerance. Here we showed that emergence of the first cohorts of Aire+ mTECs at this key developmental stage, prior to αβ T cell repertoire selection, was jointly directed by Rankl+ lymphoid tissue inducer cells and invariant Vγ5+ dendritic epidermal T cell (DETC) progenitors that are the first thymocytes to express the products of gene rearrangement. In turn, generation of Aire+ mTECs then fostered Skint-1-dependent, but Aire-independent, DETC progenitor maturation and the emergence of an invariant DETC repertoire. Hence, our data attributed a functional importance to the temporal development of Vγ5+ γδ T cells during thymus medulla formation for αβ T cell tolerance induction and demonstrated a Rank-mediated reciprocal link between DETC and Aire+ mTEC maturation.
► Invariant Vγ5+ thymocytes regulate formation of Aire+ medullary thymic epithelium ► Generation of an invariant Vγ5+ T cell population requires thymus medulla development ► Skint-1-mediated Vγ5+ thymocyte development is Aire independent ► Dependency on Tnfrsf11a links γδ T cell and medullary epithelium development
γδ T cells mediate rapid tissue responses in murine skin and participate in cutaneous immune regulation including protection against cancer. The role of human γδ cells in cutaneous homeostasis and pathology is poorly characterized.
In this study we show in vivo evidence that human blood contains a distinct subset of pro-inflammatory cutaneous lymphocyte antigen (CLA) and C-C chemokine receptor (CCR) 6 positive Vγ9Vδ2 T cells, which is rapidly recruited into perturbed human skin. Vγ9Vδ2 T cells produced an array of pro-inflammatory mediators including IL-17A and activated keratinocytes in a TNF-α and IFN-γ dependent manner.
Examination of the common inflammatory skin disease psoriasis revealed a striking reduction of circulating Vγ9Vδ2 T cells in psoriasis patients compared to healthy controls and atopic dermatitis patients. Decreased numbers of circulating Vγ9Vδ2 T cells normalized after successful treatment with psoriasis-targeted therapy. Together with the increased presence of Vγ9Vδ2 T cells in psoriatic skin, this data indicates redistribution of Vγ9Vδ2 T cells from the blood to the skin compartment in psoriasis.
In summary, we report a novel human pro-inflammatory γδ T cell involved in skin immune surveillance with immediate response characteristics and with potential clinical relevance in inflammatory skin disease.
Frequent microbial and non-microbial challenges to epithelial cells trigger discrete pathways, promoting molecular changes, such as the secretion of specific cytokines and chemokines, and alterations to molecules displayed at the epithelial cell surface. In combination, these molecules impose major decisions on innate and adaptive immune cells. Depending on context, those decisions can be as diverse as those imposed by professional antigen presenting cells, benefitting the host by balancing immune competence with the avoidance of immunopathology. Nonetheless, this potency of epithelial cells is also consistent with the causal contribution of epithelial dysregulation to myriad inflammatory diseases. This pathogenic axis provides an attractive target for tissue-specific clinical manipulation. In this context, a research goal should be to identify all molecules used by epithelial cells to instruct immune cells. We term this the epimmunome.