Local Antigen Recognition Research Articles

PD-1 and ICOS coexpression identifies tumor-reactive CD4+ T cells in human solid tumors.

CD4+ Th cells play a key role in orchestrating immune responses, but the identity of the CD4+ Th cells involved in the antitumor immune response remains to be defined. We analyzed the immune cell infiltrates of head and neck squamous cell carcinoma and colorectal cancers and identified a subset of CD4+ Th cells distinct from FOXP3+ Tregs that coexpressed programmed cell death 1 (PD-1) and ICOS. These tumor-infiltrating lymphocyte CD4+ Th cells (CD4+ Th TILs) had a tissue-resident memory phenotype, were present in MHC class II–rich areas, and proliferated in the tumor, suggesting local antigen recognition. The T cell receptor repertoire of the PD-1+ICOS+ CD4+ Th TILs was oligoclonal, with T cell clones expanded in the tumor, but present at low frequencies in the periphery. Finally, these PD-1+ICOS+ CD4+ Th TILs were shown to recognize both tumor-associated antigens and tumor-specific neoantigens. Our findings provide an approach for isolating tumor-reactive CD4+ Th TILs directly ex vivo that will help define their role in the antitumor immune response and potentially improve future adoptive T cell therapy approaches.

PD-1 and ICOS co-expression identifies tumor-reactive CD4 Th cells in human solid tumors

Abstract CD4 T helper (Th) cells play a key role in orchestrating immune responses, but the identity of the CD4 Th cells involved in the immune response against cancer remains to be defined. We analyzed the immune cell infiltrates of head and neck squamous cell carcinoma and microsatellite stable colorectal cancers and identified a subset of CD4 Th cells distinct from FOXP3+ regulatory T cells that co-express PD-1 and ICOS. This cell population, which was present in the tumor but absent in the periphery, features of chronic stimulation and displays characteristics of tissue-resident memory T cells. PD-1+ICOS+ tumor-infiltrating (TIL) CD4 Th cells are located primarily in the tumor stroma in proximity to MHC class II+ cells and are proliferating, suggesting local antigen recognition. The T-cell receptor repertoire of the PD-1+ICOS+ CD4 Th TIL is oligoclonal, with T-cell clones expanded in the tumor, but present at low frequencies in the periphery. Finally, these PD-1+ICOS+ CD4 Th TIL were shown to recognize both tumor-associated antigens and tumor-specific neoantigens, which were distinct from the epitopes recognized by the CD8 T cells from the same patients. Our findings provide an approach for isolating tumor-reactive CD4 Th TIL directly ex vivo and imply that harnessing the cooperation between CD4 and CD8 T cells might lead to more efficient tumor recognition and control in cancer patients. Supported by the Providence Portland Medical Foundation and the Developmental Research Program for Head and Neck Cancer.

T Cell Receptor Is Required for Differentiation, but Not Maintenance, of Intestinal CD4+ Intraepithelial Lymphocytes

The gut epithelium is populated by intraepithelial lymphocytes (IELs), a heterogeneous Tcell population with cytotoxic and regulatory properties, which can be acquired at the epithelial layer. However, the role of Tcell receptor (TCR) in this process remains unclear. Single-cell transcriptomic analyses revealed distinct clonal expansions between cell states, with CD4+CD8αα+ IELs being one of the least diverse populations. Conditional deletion of TCR on differentiating CD4+ Tcells or of major histocompatibility complex (MHC) class II on intestinal epithelial cells prevented CD4+CD8αα+ IEL differentiation. However, TCR ablation on differentiated CD4+CD8αα+ IELs or long-term cognate antigen withdraw did not affect their maintenance. TCR re-engagement of antigen-specific CD4+CD8αα+ IELs by Listeria monocytogenes did not alter their state but correlated with reduced bacterial invasion. Thus, local antigen recognition is an essential signal for differentiation of CD4+ Tcells at the epithelium, yet differentiated IELs are able to preserve an effector program in the absence of TCR signaling.

Antigen recognition by CD8+ T cells in non-lymphoid tissues initiates a transcriptional program of tissue-resident memory differentiation

Abstract Tissue-resident memory (TRM) CD8+ T cells permanently reside within non-lymphoid tissues to provide a first-line of defense against invading pathogens. TRM T cells are largely derived from recently activated effector T cells, but the mechanisms that control the extent of TRM differentiation once they are recruited into non-lymphoid tissue microenvironments are largely undefined. To determine how T cell receptor (TCR) engagement promotes TRM formation, we utilized interferon-gamma (IFNγ)-YFP reporter T cells and found that only ~25% of the antigen-specific CD8+ T cells in the skin produced IFNγ during the course of a Vaccinia virus (VacV) infection. Strikingly, expression of IFNγ was strictly antigen-dependent and genome-wide transcriptional profiling revealed that IFNγ+ CD8+ T cells in the VacV-infected skin were becoming tissue-residents, suggesting that local antigen recognition identifies the dominant TRM precursor population. Mechanistically, TCR engagement caused effector CD8+ T cells to express Blimp1 and repress expression of the tissue egress receptor S1PR1 and the transcription factor KLF2, collectively preventing S1P-dependent migration. Interestingly, the S1PR1 antagonist CD69 was not transcriptionally upregulated by TCR stimulation. However, CD69 surface expression was Blimp1 dependent, suggesting that Blimp1-mediated repression of S1PR1 causes increased availability of CD69 protein to remain on the cell surface, a hallmark of TRM differentiation. Thus, antigen recognition by effector CD8+ T cells in non-lymphoid tissues is a critical factor that promotes Blimp1 expression, ultimately causing stable expression of CD69 and retention of TRM CD8+ T cells in the skin following viral infection.

Bystander IFN-γ activity promotes widespread and sustained cytokine signaling altering the tumor microenvironment.

The cytokine IFN-γ produced by tumor-reactive T cells is a key effector molecule with pleiotropic effects during anti-tumor immune responses. While IFN-γ production is targeted at the immunological synapse, its spatiotemporal activity within the tumor remains elusive. Here, we report that while IFN-γ secretion requires local antigen recognition, IFN-γ diffuses extensively to alter the tumor microenvironment in distant areas. Using intravital imaging and a reporter for STAT1 translocation, we provide evidence that T cells mediate sustained IFN-γ signaling in remote tumor cells. Furthermore, tumor phenotypic alterations required several hours of exposure to IFN-γ, a feature that disfavored local IFN-γ activity over diffusion and bystander activity. Finally, single-cell RNA-seq data from melanoma patients also suggested bystander IFN-γ activity in human tumors. Thus, tumor-reactive T cells act collectively to create large cytokine fields that profoundly modify the tumor microenvironment.

Peripheral and systemic antigens elicit an expandable pool of resident memory CD8+ T cells in the bone marrow.

BM has been put forward as a major reservoir for memory CD8+ T cells. In order to fulfill that function, BM should “store” memory CD8+ T cells, which in biological terms would require these “stored” memory cells to be in disequilibrium with the circulatory pool. This issue is a matter of ongoing debate. Here, we unequivocally demonstrate that murine and human BM harbors a population of tissue‐resident memory CD8+ T (TRM) cells. These cells develop against various pathogens, independently of BM infection or local antigen recognition. BM CD8+ TRM cells share a transcriptional program with resident lymphoid cells in other tissues; they are polyfunctional cytokine producers and dependent on IL‐15, Blimp‐1, and Hobit. CD8+ TRM cells reside in the BM parenchyma, but are in close contact with the circulation. Moreover, this pool of resident T cells is not size‐restricted and expands upon peripheral antigenic re‐challenge. This works extends the role of the BM in the maintenance of CD8+ T cell memory to include the preservation of an expandable reservoir of functional, non‐recirculating memory CD8+ T cells, which develop in response to a large variety of peripheral antigens.

Zymosan by-passes the requirement for pulmonary antigen encounter in lung tissue-resident memory CD8+ T cell development.

Tissue-resident memory T cells (Trm) in the lung provide a frontline defence against respiratory pathogens. Vaccination models that lodge CD8+ Trm populations in the lung have been developed, all of which incorporate the local delivery of antigen plus adjuvant into the airways; a necessary approach as local cognate antigen recognition is required for optimal lung Trm development. Although pulmonary delivery of antigen is important for lung Trm development, the impact the co-administered adjuvant has on Trm differentiation is unclear. We show that while altering the adjuvant co-administered with the pulmonary delivered antigen does not impact the size of the lung Trm population, a particular adjuvant, zymosan, when administered into the airways without antigen can drive effector CD8+ T cells to differentiate into lung Trm. Zymosan signalling via dectin-1 receptor was sufficient to promote antigen-independent lung Trm development. When combined with an injectable influenza vaccination regime, intranasal zymosan delivery significantly boosted the size of the influenza virus-specific lung Trm population. Our results highlight that eliciting the appropriate local inflammatory milieu can by-pass the requirement for local antigen recognition in lung Trm development and emphasises that the appropriate selection of adjuvant can greatly improve vaccines that aim to elicit pulmonary Trm.

Löfgren's Syndrome: Diagnosis, Management, and Disease Pathogenesis.

AbstractLöfgren's syndrome (LS), first described in 1946 by Swedish Professor of Medicine Sven Löfgren, is a clinically distinct phenotype of sarcoidosis. Patients typically experience an acute disease onset, usually with fever, and characteristic symptoms of bilateral hilar lymphadenopathy, erythema nodosum, and/or bilateral ankle arthritis or periarticular inflammation. LS patients are well documented to have a good prognosis, which is especially true for HLA-DRB1*03+ individuals. The presence of this allele correlates closely with an accumulation of clonal CD4+ T-cell populations in the lung, suggestive of local antigen recognition. Moreover, LS differs markedly from “non-LS” sarcoidosis regarding immune cell activation, differentiation, and regulation, which may influence clinical outcome and spontaneous disease resolution.This review offers an overview of the clinical characteristics, genetic background, and immunological characteristics of LS, as well as patient management, and reflections on future scientific challenges, emphasizing the concept of LS as a disease in its own right.

Visualizing mucosal resident memory CD8 T cell reactivation dynamics reveals in situ division and bystander resident memory T cell development

Abstract CD8 T cells survey tissues for the presence of abnormal cells. The parameters of CD8 T cell search patterns and immunosurveillance determines outcome of infections and cancer (elimination, controlled persistence, or death). We established a two-photon intravital microscopy model to visualize resident memory CD8 T cell (TRM) behavior and recall responses within the female reproductive tract (FRT) mucosa of living mice. Unlike what has been described in skin epidermis, FRT TRM were highly motile (10 μm/min). Local antigen recognition induced pausing and initiation of in situ TRM division, and this appeared CD4 T cell and dendritic cell independent. Expansion by dividing TRM dominated the local recall response compared to recruitment of recirculating cells or progeny of reactivated central memory T cells. TRM reactivation also triggered recruitment of Ag-specific and bystander recirculating memory cells, and these underwent a de novo TRM differentiation program in situ. These results demonstrate that 1) T cell migration patterns and differentiation may be permanently altered by unrelated infectious experience, 2) that cognate antigen within the FRT is not required to induce local TRM differentiation, 3) TRM undergo in situ division upon recall that 4) may dominate the local secondary effector and memory response.

Local antigen in nonlymphoid tissue promotes resident memory CD8+ T cell formation during viral infection.

Tissue-resident memory (Trm) CD8(+) T cells are functionally distinct from their circulating counterparts and are potent mediators of host protection against reinfection. Whether local recognition of antigen in nonlymphoid tissues during infection can impact the formation of Trm populations remains unresolved. Using skin infections with vaccinia virus (VacV)-expressing model antigens, we found that local antigen recognition had a profound impact on Trm formation. Activated CD8(+) T cells trafficked to VacV-infected skin in an inflammation-dependent, but antigen-independent, manner. However, after viral clearance, there was a subsequent ∼50-fold increase in Trm formation when antigen was present in the tissue microenvironment. Secondary antigen stimulation in nonlymphoid tissue caused CD8(+) T cells to rapidly express CD69 and be retained at the site of infection. Finally, Trm CD8(+) T cells that formed during VacV infection in an antigen-dependent manner became potent stimulators of localized antigen-specific inflammatory responses in the skin. Thus, our studies indicate that the presence of antigen in the nonlymphoid tissue microenvironment plays a critical role in the formation of functional Trm CD8(+) T cell populations, a finding with relevance for both vaccine design and prevention of inflammatory disorders.

Local antigen in non-lymphoid tissue promotes resident memory CD8+ T cell formation during viral infection.

Abstract Tissue-resident memory (TRM) CD8+ T cells are functionally distinct from their circulating counterparts and are potent mediators of host protection against re-infection. One largely accepted paradigm is that activated CD8+ T cells are able to freely migrate to non-lymphoid tissues and differentiate into TRM independent of local antigen recognition. In contrast, using skin infections with Vaccinia virus (VacV) expressing model antigens, we found that local antigen recognition had a profound impact on TRM formation. Activated CD8+ T cells trafficked to VacV-infected skin in an inflammation-dependent, but antigen-independent manner. However, following viral clearance, there was a subsequent ~50-fold increase in CD103+/CD69+ TRM formation when antigen was present in the tissue microenvironment. Secondary antigen stimulation in non-lymphoid tissue caused CD8+ T cells to rapidly express CD69 and be retained at the site of infection. Finally, TRM CD8+ T cells that formed during VacV infection in an antigen-dependent manner became potent stimulators of localized antigen-specific inflammatory responses in the skin. Thus, our studies indicate that the presence of antigen in the non-lymphoid tissue microenvironment plays a critical role in the formation of functional TRM CD8+ T cell populations, a finding with relevance for both vaccine design and prevention of inflammatory disorders.

CD4+ Th1 cells are effectors in lupus nephritis—but what are their targets?

Lupus nephritis involves both humoral and cellular effectors. Okamoto and colleagues isolated renal CD4+ cells from lupus-prone mice, selecting cells expressing T-cell receptors on the basis of their activity. After transfecting CD4+ cells with the relevant T-cell receptors, they transferred them into mice and induced more severe glomerulonephritis. This study supports a direct role for Th1 cells in lupus nephritis, although their antigenic specificity remains uncertain, as they did not recognize nucleosomes.

The Living Eye “Disarms” Uncommitted Autoreactive T Cells by Converting Them to Foxp3+ Regulatory Cells following Local Antigen Recognition

Immune privilege is used by the eye, brain, reproductive organs, and gut to preserve structural and functional integrity in the face of inflammation. The eye is arguably the most vulnerable and, therefore, also the most "privileged" of tissues; paradoxically, it remains subject to destructive autoimmunity. It has been proposed, although never proven in vivo, that the eye can induce T regulatory cells (Tregs) locally. Using Foxp3-GFP reporter mice expressing a retina-specific TCR, we now show that uncommitted T cells rapidly convert in the living eye to Foxp3(+) Tregs in a process involving retinal Ag recognition, de novo Foxp3 induction, and proliferation. This takes place within the ocular tissue and is supported by retinoic acid, which is normally present in the eye because of its function in the chemistry of vision. Nonconverted T cells showed evidence of priming but appeared restricted from expressing effector function in the eye. Pre-existing ocular inflammation impeded conversion of uncommitted T cells into Tregs. Importantly, retina-specific T cells primed in vivo before introduction into the eye were resistant to Treg conversion in the ocular environment and, instead, caused severe uveitis. Thus, uncommitted T cells can be disarmed, but immune privilege is unable to protect from uveitogenic T cells that have acquired effector function prior to entering the eye. These findings shed new light on the phenomenon of immune privilege and on its role, as well as its limitations, in actively controlling immune responses in the tissue.

The role of dendritic cells in CNS autoimmunity

Multiple sclerosis (MS) is a chronic immune-mediated, central nervous system (CNS) demyelinating disease. Clinical and histopathological features suggest an inflammatory etiology involving resident CNS innate cells as well as invading adaptive immune cells. Encephalitogenic myelin-reactive T cells have been implicated in the initiation of an inflammatory cascade, eventually resulting in demyelination and axonal damage (the histological hallmarks of MS). Dendritic cells (DC) have recently emerged as key modulators of this immunopathological cascade, as supported by studies in humans and experimental disease models. In one such model, experimental autoimmune encephalomyelitis (EAE), CNS microvessel-associated DC have been shown to be essential for local antigen recognition by myelin-reactive T cells. Moreover, the functional state and compartmental distribution of DC derived from CNS and associated lymphatics seem to be limiting factors in both the induction and effector phases of EAE. Moreover, DC modulate and balance the recruitment of encephalitogenic and regulatory T cells into CNS tissue. This capacity is critically influenced by DC surface expression of co-stimulatory or co-inhibitory molecules. The fact that DC accumulate in the CNS before T cells and can direct T-cell responses suggests that they are key determinants of CNS autoimmune outcomes. Here we provide a comprehensive review of recent advances in our understanding of CNS-derived DC and their relevance to neuroinflammation.

Lack of T cell oligoclonality in enzyme-digested synovial tissue and in synovial fluid in most patients with rheumatoid arthritis

The dominant presence of specific T-cell populations in the rheumatoid joint as detected by Southern blot analysis of T cell receptor (TCR) gene rearrangements would indicate local antigen recognition and T cell proliferation. We therefore studied TCR beta chain gene rearrangements using a C beta 2 probe in paired samples of T cell populations from synovial tissue and peripheral blood (n = 6) as well as synovial fluid (n = 16) and peripheral blood (n = 18) of patients with rheumatoid arthritis (RA). Peripheral blood mononuclear cells from healthy donors (n = 7) served as a control. T cells were studied directly after isolation or after non-specific expansion with OKT3 monoclonal antibody (MoAb) and T cell growth factor (TCGF). DNA samples were digested with EcoRI and HindIII to detect rearrangements to C beta 1 and C beta 2, respectively. Extra bands were detected in all EcoRI-digested DNA samples prepared from both freshly isolated and non-specifically expanded T cell populations of patients and healthy donors, possibly representing 'common' (V-) D-J rearrangements. Dominant rearrangements were found in only two out of 16 synovial fluid T cell populations (one freshly isolated and one expanded) and not in peripheral blood or synovial tissue derived T cell populations. No extra bands were detected in HindIII-digested DNA samples. To investigate the effect of in vitro culture techniques on rearrangement patterns we studied DNA samples prepared from synovial tissue T cells obtained both by outgrowth from tissue with TCGF or by enzyme digestion and subsequent expansion either with TCGF or with OKT3 MoAb and TCGF. Whereas the latter T cell population yielded 'common' rearrangements, the former T cell populations yielded different dominant rearrangements. These data indicate that oligoclonality of the T cell populations in synovial tissue and synovial fluid of patients with RA is a rare event. The data also show the influence of in vitro culture techniques on the result of TCR gene rearrangement analysis.

Architecture of minor salivary gland duct/lymphoid follicle associations and possible antigen-recognition sites in the monkey Macaca fascicularis

In Macaca fascicularis, lymphoid follicles with germinal centres related to minor salivary gland ducts are frequently found in the mucosa of lips, cheeks and the soft palate. Three semi-three-dimensional reconstructions, each based on 3 sets of 230–300 serial Epon sections, of such duct/follicle-assemblies (2 in the soft palate and 1 in the lip) and electron microscopic observations were made. These revealed that (1) these structures were about 0.05–0.1 mm 3 in size, (2) they were found preferentially at sites where small interlobular ducts fused to form a pelvis-like basin from which blind duct portions protruded into the surrounding lymphoid tissue and (3) they often included a germinal centre which partly embraced blind ducts. The walls of such blind ducts were heavily infiltrated by lymphocytes and in part blast-forming T-cells. Portions of the walls of interlobular and main secretory ducts, passing directly or peripherally through follicles, were also infiltrated with lymphocytes. The duct lumen within the follicles contained clusters of Gram-negative bacteria, probably rods. Based on these findings, it is argued that gland duct/follicle assemblies represent physiological entities, either formed and re-formed temporarily or of long-standing nature, which may provide the locus (i.e. tonsillar microcrypt-like pouches) and the structural matrix necessary for local antigen recognition. The antigens (bacteria, macromolecules in salivary fluid, etc.) might enter the above structure by way of the secretory ducts. Such matrices may also exist in man.

Structure of the soft palate and composition of the oral mucous membrane in monkeys

The soft palate of monkeys (M. irus, mulatta, fasciculata; 2.3 +/- 0.4 kg in weight) was studied morphologically and stereologically. After fixation by perfusion, the tissue was disected free, divided in halves and the halves into 5 segments each, and processed for light, transmission- and scanning electron microscopy. Morphometric techniques and a newly developed computer program for automatic data processing (Mumana) were employed in order to estimate, at the light microscopic level, the volumetric composition of the oral soft palate mucosa. The latter showed a different composition in anterior and uvular segments, without significant differences between male and female animals, but with slight changes possibly due to age. The main constituents of the oral soft palate mucosa were glandular and connective tissue, and lymph follicles, the latter being associated with the glandular duct system. The epithelial lining of the oral and nasal aspects were described, and taste buds embedded in island of orthokeratinizing stratified epithelium were demonstrated. The association between lymph follicles and the glandular duct system was discussed in relation to similar structures in man, and in relation to the hypothesis that they are concerned in local antigen recognition.