Heterogenous Populations of Tissue-Resident CD8+ T Cells Are Generated in Response to Infection and Malignancy.

Tissue-resident memory CD8+ T cells (Trm) provide host protection through continuous surveillance of non-lymphoid tissues. Using single-cell RNA-sequencing (scRNA-seq) and genetic reporter mice, we identified discrete lineages of intestinal antigen-specific CD8+ T cells, including a Blimp1hiId3lo tissue-resident effector cell population most prominent in the early phase of acute viral and bacterial infections and a molecularly distinct Blimp1loId3hi tissue-resident memory population that subsequently accumulated at later infection time points. These Trm populations exhibited distinct cytokine production, secondary memory potential, and transcriptional programs including differential roles for transcriptional regulators Blimp1, T-bet, Id2, and Id3 in supporting and maintaining intestinal Trm. Extending our analysis to malignant tissue, we also identified discrete populations of effector-like and memory-like CD8+ T cell populations with tissue-resident gene-expression signatures that shared features of terminally exhausted and progenitor-exhausted T cells, respectively. Our findings provide insight into the development and functional heterogeneity of Trm cells, which has implications for enhancing vaccination and immunotherapy approaches.

The junctional adhesion molecule-like protein (JAML) is important for the inflammatory response during contact hypersensitivity.

BACKGROUND: The junctional adhesion molecule-like protein (JAML) plays important roles in wound healing and activation of epidermal γδ T cells in mice. Whether JAML plays a role in contact hypersensitivity (CHS), the animal model of allergic contact dermatitis (ACD), is not known. METHODS: To examine the role of JAML in CHS, we used various mouse models of CHS in JAML knockout (KO) and wild-type (WT) mice. Furthermore, the expression of the JAML ligand coxsackievirus and adenovirus receptor (CXADR) on keratinocytes was accessed in vitro and in vivo. RESULTS: JAML KO mice had a diminished inflammatory response during both the sensitization and elicitation phase of CHS and had reduced numbers of CD8+ and CD4+ T cells in the epidermis. Furthermore, interferon γ (IFNγ), interleukin 1ß (IL-1ß) and CXCL10 production were significantly reduced in JAML KO mice during the elicitation phase. We found that CD8+ T cells express JAML and that JAML is essential for rapid flare-up responses to contact allergens. Finally, we show that keratinocytes up-regulate the JAML ligand CXADR following exposure to contact allergens. CONCLUSION: Our study is the first to show a central role of JAML in CHS and reveals a potential new target for the treatment of ACD in humans.

CD100 boosts the inflammatory response in the challenge phase of allergic contact dermatitis in mice.

BACKGROUND: Allergic contact dermatitis (ACD) is an inflammatory disease with a complex pathophysiology in which epidermal-resident memory CD8+ T (TRM ) cells play a key role. The mechanisms involved in the activation of CD8+ TRM cells during allergic flare-up responses are not understood. METHODS: The expression of CD100 and its ligand Plexin B2 on CD8+ TRM cells and keratinocytes before and after allergen exposure was determined by flow cytometry and RT-qPCR. The role of CD100 in the inflammatory response during the challenge phase of ACD was determined in a model of ACD in CD100 knockout and wild-type mice. RESULTS: We show that CD8+ TRM cells express CD100 during homeostatic conditions and up-regulate it following re-exposure of allergen-experienced skin to the experimental contact allergen 1-fluoro-2,4-dinitrobenzene (DNFB). Furthermore, Plexin B2 is up-regulated on keratinocytes following exposure to some contact allergens. We show that loss of CD100 results in a reduced inflammatory response to DNFB with impaired production of IFNγ, IL-17A, CXCL1, CXCL2, CXCL5, and IL-1ß and decreased recruitment of neutrophils to the epidermis. CONCLUSION: Our study demonstrates that CD100 is expressed on CD8+ TRM cells and is required for full activation of CD8+ TRM cells and the flare-up response of ACD.

The CD100 receptor interacts with its plexin B2 ligand to regulate epidermal γδ T cell function.

γδ T cells respond rapidly to keratinocyte damage, providing essential contributions to the skin wound healing process. The molecular interactions regulating their response are unknown. Here, we identify a role for interaction of plexin B2 with the CD100 receptor in epithelial repair. In vitro blocking of plexin B2 or CD100 inhibited γδ T cell activation. Furthermore, CD100 deficiency in vivo resulted in delayed repair of cutaneous wounds due to a disrupted γδ T cell response to keratinocyte damage. Ligation of CD100 in γδ T cells induced cellular rounding via signals through ERK kinase and cofilin. Defects in this rounding process were evident in the absence of CD100-mediated signals, thereby providing a mechanistic explanation for the defective wound healing in CD100-deficient animals. The discovery of immune functions for plexin B2 and CD100 provides insight into the complex cell-cell interactions between epithelial resident γδ T cells and the neighboring cells they support.

Origins of CD4+ circulating and tissue-resident memory T-cells.

In response to infection, naive CD4+ T-cells proliferate and differentiate into several possible effector subsets, including conventional T helper effector cells (TH 1, TH 2, TH 17), T regulatory cells (Treg ) and T follicular helper cells (TFH ). Once infection is cleared, a small population of long-lived memory cells remains that mediate immune defenses against reinfection. Memory T lymphocytes have classically been categorized into central memory cell (TCM ) and effector memory cell (TEM ) subsets, both of which circulate between blood, secondary lymphoid organs and in some cases non-lymphoid tissues. A third subset of memory cells, referred to as tissue-resident memory cells (TRM ), resides in tissues without recirculation, serving as 'first line' of defense at barrier sites, such as skin, lung and intestinal mucosa, and augmenting innate immunity in the earliest phases of reinfection and recruiting circulating CD4+ and CD8+ T-cells. The presence of multiple CD4+ T helper subsets has complicated studies of CD4+ memory T-cell differentiation, and the mediators required to support their function. In this review, we summarize recent investigations into the origins of CD4+ memory T-cell populations and discuss studies addressing CD4+  TRM differentiation in barrier tissues.

IL-1ß-dependent activation of dendritic epidermal T cells in contact hypersensitivity.

Substances that penetrate the skin surface can act as allergens and induce a T cell-mediated inflammatory skin disease called contact hypersensitivity (CHS). IL-17 is a key cytokine in CHS and was originally thought to be produced solely by CD4(+) T cells. However, it is now known that several cell types, including γδ T cells, can produce IL-17. In this study, we determine the role of γδ T cells, especially dendritic epidermal T cells (DETCs), in CHS. Using a well-established model for CHS in which 2,4-dinitrofluorobenzene (DNFB) is used as allergen, we found that γδ T cells are important players in CHS. Thus, more IL-17-producing DETCs appear in the skin following exposure to DNFB in wild-type mice, and DNFB-induced ear swelling is reduced by ∼50% in TCRδ(-/-) mice compared with wild-type mice. In accordance, DNFB-induced ear swelling was reduced by ∼50% in IL-17(-/-) mice. We show that DNFB triggers DETC activation and IL-1ß production in the skin and that keratinocytes produce IL-1ß when stimulated with DNFB. We find that DETCs activated in vitro by incubation with anti-CD3 and IL-1ß produce IL-17. Importantly, we demonstrate that the IL-1R antagonist anakinra significantly reduces CHS responses, as measured by decreased ear swelling, inhibition of local DETC activation, and a reduction in the number of IL-17(+) γδ T cells and DETCs in the draining lymph nodes. Taken together, we show that DETCs become activated and produce IL-17 in an IL-1ß-dependent manner during CHS, suggesting a key role for DETCs in CHS.

All hands on DE(T)C: Epithelial-resident γδ T cells respond to tissue injury.

Immunology has traditionally focused on the lymphocytes circulating among primary lymphoid organs while the large reservoir of tissue-resident T cells have received relatively less attention. In epithelia, these populations are comprised of significant, and sometimes exclusive, subsets of γδ T cells that are highly specialized in promoting tissue homeostasis. As the epithelial layers of the skin and gut are permanently exposed to the environment, they are continually subject to injury and therefore require highly efficient repair processes to maintain barrier functions. Here, we review the role of γδ T cells in promoting wound healing, a critical and complex process occurring in the skin and other barrier sites.

Molecular aspects of epithelial γδ T cell regulation.

γδ T cells lie at the interface between innate and adaptive immunity, sharing features with both arms of the immune system. The vast majority of γδ T cells reside in epithelial layers of tissues such as skin, gut, lung, tongue and reproductive tract where they provide a first line of defense against environmental attack. The existence of epithelium-resident γδ T cells has been known for over 20 years but our understanding of the molecular events regulating development and function of these cells is incomplete. We review recent advances in the field, with particular emphasis on the γδ T cell population resident in mouse epidermis. These studies have enhanced our knowledge and understanding of the life cycle of this enigmatic population of cells.

Cutting edge: dendritic epidermal γδ T cell ligands are rapidly and locally expressed by keratinocytes following cutaneous wounding.

TCR-specific activation is pivotal to dendritic epidermal T cell (DETC) function during cutaneous wound repair. However, DETC TCR ligands are uncharacterized, and little is known about their expression patterns and kinetics. Using soluble DETC TCR tetramers, we demonstrate that DETC TCR ligands are not constitutively expressed in healthy tissue but are rapidly upregulated following wounding on keratinocytes bordering wound edges. Ligand expression is tightly regulated, with downmodulation following DETC activation. Early inhibition of TCR-ligand interactions using DETC TCR tetramers delays wound repair in vivo, highlighting DETC as rapid responders to injury. To our knowledge, this is the first visualization of DETC TCR ligand expression, which provides novel information about how ligand expression impacts early stages of DETC activation and wound repair.

Anti-proliferative and anti-inflammatory effects of the application of baclofen cream, a GABAB receptor agonist, on skin inflammation in mice.

Previous studies have demonstrated the role of γ-aminobutyric acid type B (GABAB) receptors in skin-related conditions and pain. However, most studies have focused on the main effects of GABAB on the central nervous system. Therefore, this study has aimed to determine the potential topical anti-inflammatory and anti-proliferative effects of baclofen cream in an inflammatory skin disease model. The effects of the baclofen cream were evaluated using acute and chronic models of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin inflammation in mouse ears. Histological and immunohistochemical evaluations were performed using an ear oedema assay. The effect of baclofen on keratinocyte proliferation was assessed in PAM212, the murine keratinocyte cell line. The results demonstrate that a single topical application of 5% baclofen, 7.5% baclofen, and 1% dexamethasone each inhibited acute TPA-induced ear oedema (58.94 ± 6.14%, 47.73 ± 11.26%, and 87.33 ± 4.59%, respectively). These results were confirmed by histological analysis. In the chronic model, baclofen (5%) and dexamethasone (1%) each inhibited ear oedema and the maximum inhibitory effect was reached at the end of the experiment (9th day of TPA application) with a percentage inhibition of 54.60 ± 6.15% for baclofen and 71.68 ± 3.45% for dexamethasone, when compared to the vehicle. These results were confirmed by histological analysis. Baclofen and dexamethasone also reduced proliferating cell nuclear antigen expression by 62.01 ± 6.65% and 70.42 ± 6.11%, respectively. However, baclofen did not inhibit keratinocyte proliferation in PAM212 cells. In conclusion, these results demonstrate that baclofen exhibits notable topical antiproliferative and anti-inflammatory properties and could be a potential therapeutic alternative for treating inflammatory and proliferative skin diseases.

γδ T cell costimulatory ligands in antitumor immunity.

Antitumor immunity relies on the ability of T cells to recognize and kill tumor targets. γδ T cells are a specialized subset of T cells that predominantly localizes to non-lymphoid tissue such as the skin, gut, and lung where they are actively involved in tumor immunosurveillance. γδ T cells respond to self-stress ligands that are increased on many tumor cells, and these interactions provide costimulatory signals that promote their activation and cytotoxicity. This review will cover costimulatory molecules that are known to be critical for the function of γδ T cells with a specific focus on mouse dendritic epidermal T cells (DETC). DETC are a prototypic tissue-resident γδ T cell population with known roles in antitumor immunity and are therefore useful for identifying mechanisms that may control activation of other γδ T cell subsets within non-lymphoid tissues. This review concludes with a brief discussion on how γδ T cell costimulatory molecules can be targeted for improved cancer immunotherapy.

Hspa8 and ICAM-1 as damage-induced mediators of γδ T cell activation.

Tissue-resident γδ T cells form the first line of defense at barrier surfaces where they survey host tissue for signs of stress or damage. Following recognition of injury, γδ T cells play a crucial role in the wound-healing response through the production of growth factors and cytokines that promote proliferation in surrounding epithelial cells. To initiate this response, γδ T cells require interactions with a variety of epithelial-expressed costimulatory molecules in addition to primary signaling through their TCR. In the epidermis these signals include the coxsackie and adenovirus receptor (CAR), histocompatibility antigen 60c (H60c), and plexin B2, which interact with γδ T cell-expressed junctional adhesion molecule-like protein (JAML), NKG2D, and CD100, respectively. Here we identify heat shock protein family A member 8 (Hspa8) and ICAM-1 as two additional keratinocyte-expressed costimulatory molecules for epidermal resident γδ T cells (termed DETC). These molecules were rapidly up-regulated in the epidermis following wounding in both mouse and human tissue. Both Hspa8 and ICAM-1 had a costimulatory effect on DETC, inducing proliferation, CD25 up-regulation, and IL-2 production. We also provide evidence that DETC can be activated through the potential ICAM-1 and Hspa8 receptors LFA-1 and CD316. Finally, knockdown of Hspa8 in keratinocytes reduced their ability to activate DETC in culture and ICAM-1-/- mice exhibited impaired rates of healing in skin-organ culture suggesting a role for these proteins in the DETC-mediated damage response. Together with previous work on CAR, H60c, and plexin B2, these results add to a picture of a complex keratinocyte wound signature that is required for efficient DETC activation.

Gammadelta T cell-induced hyaluronan production by epithelial cells regulates inflammation.

Nonhealing wounds are a major complication of diseases such as diabetes and rheumatoid arthritis. For efficient tissue repair, inflammatory cells must infiltrate into the damaged tissue to orchestrate wound closure. Hyaluronan is involved in the inflammation associated with wound repair and binds the surface of leukocytes infiltrating damaged sites. Skin gammadelta T cells play specialized roles in keratinocyte proliferation during wound repair. Here, we show that gammadelta T cells are required for hyaluronan deposition in the extracellular matrix (ECM) and subsequent macrophage infiltration into wound sites. We describe a novel mechanism of control in which gammadelta T cell-derived keratinocyte growth factors induce epithelial cell production of hyaluronan. In turn, hyaluronan recruits macrophages to the site of damage. These results demonstrate a novel function for skin gammadelta T cells in inflammation and provide a new perspective on T cell regulation of ECM molecules.

JAML promotes CD8 and γδ T cell antitumor immunity and is a novel target for cancer immunotherapy.

T cells are critical mediators of antitumor immunity and a major target for cancer immunotherapy. Antibody blockade of inhibitory receptors such as PD-1 can partially restore the activity of tumor-infiltrating lymphocytes (TILs). However, the activation signals required to promote TIL responses are less well characterized. Here we show that the antitumor activity of CD8 and γδ TIL is supported by interactions between junctional adhesion molecule-like protein (JAML) on T cells and its ligand coxsackie and adenovirus receptor (CXADR) within tumor tissue. Loss of JAML through knockout in mice resulted in accelerated tumor growth that was associated with an impaired γδ TIL response and increased CD8 TIL dysfunction. In mouse tumor models, therapeutic treatment with an agonistic anti-JAML antibody inhibited tumor growth, improved γδ TIL activation, decreased markers of CD8 TIL dysfunction, and significantly improved response to anti-PD-1 checkpoint blockade. Thus, JAML represents a novel therapeutic target to enhance both CD8 and γδ TIL immunity.

Hypoxia-inducible factor activity promotes antitumor effector function and tissue residency by CD8+ T cells.

Adoptive T cell therapies (ACTs) hold great promise in cancer treatment, but low overall response rates in patients with solid tumors underscore remaining challenges in realizing the potential of this cellular immunotherapy approach. Promoting CD8+ T cell adaptation to tissue residency represents an underutilized but promising strategy to improve tumor-infiltrating lymphocyte (TIL) function. Here, we report that deletion of the HIF negative regulator von Hippel-Lindau (VHL) in CD8+ T cells induced HIF-1α/HIF-2α-dependent differentiation of tissue-resident memory-like (Trm-like) TILs in mouse models of malignancy. VHL-deficient TILs accumulated in tumors and exhibited a core Trm signature despite an exhaustion-associated phenotype, which led to retained polyfunctionality and response to αPD-1 immunotherapy, resulting in tumor eradication and protective tissue-resident memory. VHL deficiency similarly facilitated enhanced accumulation of chimeric antigen receptor (CAR) T cells with a Trm-like phenotype in tumors. Thus, HIF activity in CD8+ TILs promotes accumulation and antitumor activity, providing a new strategy to enhance the efficacy of ACTs.

Wendy L. Havran, PhD: 1955-2020.

Wendy Havran, Professor and Associate Dean of Graduate Studies at Scripps Research, passed away on January 20th, 2020 following a heart attack [...].

Get in Touch With Dendritic Epithelial T Cells!

Innate and adaptive immune systems continuously interchange information and orchestrate their immune responses to protect the host. γδT cells play crucial roles, as they incorporate both innate and adaptive immune characteristics. Dendritic epidermal T cells (DETC) are specialized γδT cells, which are uniquely positioned to rapidly respond to skin wounds and infections. Their elongated dendrite morphology allows them to be in continuous contact with multiple neighboring keratinocytes and Langerhans cells. Cellular interactions are fundamental to the formation, activation and maintenance of immune cell functions during steady state and pathology. Recent technological advances, especially in the field of cellular imaging, have contributed greatly to the characterization of complex cellular interactions in a spatiotemporally resolved manner. In this review, we will highlight the often-underappreciated function of DETC and other γδT cells during steady state and an ongoing immune response. More specifically, we discuss how DETC-precursors are shaped in the fetal thymus during embryogenesis as well as how direct cell-cell interactions of DETC with neighboring epidermal cells shape skin homeostasis and effector functions. Furthermore, we will discuss seminal work and recent discoveries made in the γδT cell field, which have highlighted the importance of γδT cells in the skin, both in humans and mice.

The Role of Tissue-resident T Cells in Stress Surveillance and Tissue Maintenance.

While forming a minor population in the blood and lymphoid compartments, T cells are significantly enriched within barrier tissues. In addition to providing protection against infection, these tissue-resident T cells play critical roles in tissue homeostasis and repair. T cells in the epidermis and intestinal epithelium produce growth factors and cytokines that are important for the normal turnover and maintenance of surrounding epithelial cells and are additionally required for the efficient recognition of, and response to, tissue damage. A role for tissue-resident T cells is emerging outside of the traditional barrier tissues as well, with recent research indicating that adipose tissue-resident T cells are required for the normal maintenance and function of the adipose tissue compartment. Here we review the functions of tissue-resident T cells in the epidermis, intestinal epithelium, and adipose tissue, and compare the mechanisms of their activation between these sites.