Iris K. Gratz

Response to self antigen imprints regulatory memory in tissues

Immune homeostasis in tissues is achieved through a delicate balance between pathogenic T-cell responses directed at tissue-specific antigens and the ability of the tissue to inhibit these responses. The mechanisms by which tissues and the immune system communicate to establish and maintain immune homeostasis are currently unknown. Clinical evidence suggests that chronic or repeated exposure to self antigen within tissues leads to an attenuation of pathological autoimmune responses, possibly as a means to mitigate inflammatory damage and preserve function. Many human organ-specific autoimmune diseases are characterized by the initial presentation of the disease being the most severe, with subsequent flares being of lesser severity and duration. In fact, these diseases often spontaneously resolve, despite persistent tissue autoantigen expression. In the practice of antigen-specific immunotherapy, allergens or self antigens are repeatedly injected in the skin, with a diminution of the inflammatory response occurring after each successive exposure. Although these findings indicate that tissues acquire the ability to attenuate autoimmune reactions upon repeated responses to antigens, the mechanism by which this occurs is unknown. Here we show that upon expression of self antigen in a peripheral tissue, thymus-derived regulatory T cells (Treg cells) become activated, proliferate and differentiate into more potent suppressors, which mediate resolution of organ-specific autoimmunity in mice. After resolution of the inflammatory response, activated Treg cells are maintained in the target tissue and are primed to attenuate subsequent autoimmune reactions when antigen is re-expressed. Thus, Treg cells function to confer ‘regulatory memory’ to the target tissue. These findings provide a framework for understanding how Treg cells respond when exposed to self antigen in peripheral tissues and offer mechanistic insight into how tissues regulate autoimmunity.

Memory regulatory T cells reside in human skin.

Regulatory T cells (Tregs), which are characterized by expression of the transcription factor Foxp3, are a dynamic and heterogeneous population of cells that control immune responses and prevent autoimmunity. We recently identified a subset of Tregs in murine skin with properties typical of memory cells and defined this population as memory Tregs (mTregs). Due to the importance of these cells in regulating tissue inflammation in mice, we analyzed this cell population in humans and found that almost all Tregs in normal skin had an activated memory phenotype. Compared with mTregs in peripheral blood, cutaneous mTregs had unique cell surface marker expression and cytokine production. In normal human skin, mTregs preferentially localized to hair follicles and were more abundant in skin with high hair density. Sequence comparison of TCRs from conventional memory T helper cells and mTregs isolated from skin revealed little homology between the two cell populations, suggesting that they recognize different antigens. Under steady-state conditions, mTregs were nonmigratory and relatively unresponsive; however, in inflamed skin from psoriasis patients, mTregs expanded, were highly proliferative, and produced low levels of IL-17. Taken together, these results identify a subset of Tregs that stably resides in human skin and suggest that these cells are qualitatively defective in inflammatory skin disease.

Tumor immune profiling predicts response to anti–PD-1 therapy in human melanoma

BACKGROUND Immune checkpoint blockade is revolutionizing therapy for advanced cancer, but many patients do not respond to treatment. The identification of robust biomarkers that predict clinical response to specific checkpoint inhibitors is critical in order to stratify patients and to rationally select combinations in the context of an expanding array of therapeutic options. METHODS We performed multiparameter flow cytometry on freshly isolated metastatic melanoma samples from 2 cohorts of 20 patients each prior to treatment and correlated the subsequent clinical response with the tumor immune phenotype. RESULTS Increasing fractions of programmed cell death 1 high/cytotoxic T lymphocyte-associated protein 4 high (PD-1hiCTLA-4hi) cells within the tumor-infiltrating CD8+ T cell subset strongly correlated with response to therapy (RR) and progression-free survival (PFS). Functional analysis of these cells revealed a partially exhausted T cell phenotype. Assessment of metastatic lesions during anti-PD-1 therapy demonstrated a release of T cell exhaustion, as measured by an accumulation of highly activated CD8+ T cells within tumors, with no effect on Tregs. CONCLUSIONS Our data suggest that the relative abundance of partially exhausted tumor-infiltrating CD8+ T cells predicts response to anti-PD-1 therapy. This information can be used to appropriately select patients with a high likelihood of achieving a clinical response to PD-1 pathway inhibition. FUNDING This work was funded by a generous gift provided by Inga-Lill and David Amoroso as well as a generous gift provided by Stephen Juelsgaard and Lori Cook.

Cutting Edge: Memory Regulatory T Cells Require IL-7 and Not IL-2 for Their Maintenance in Peripheral Tissues

Thymic Foxp3-expressing regulatory T cells are activated by peripheral self-antigen to increase their suppressive function, and a fraction of these cells survive as memory regulatory T cells (mTregs). mTregs persist in nonlymphoid tissue after cessation of Ag expression and have enhanced capacity to suppress tissue-specific autoimmunity. In this study, we show that murine mTregs express specific effector memory T cell markers and localize preferentially to hair follicles in skin. Memory Tregs express high levels of both IL-2Rα and IL-7Rα. Using a genetic-deletion approach, we show that IL-2 is required to generate mTregs from naive CD4+ T cell precursors in vivo. However, IL-2 is not required to maintain these cells in the skin and skin-draining lymph nodes. Conversely, IL-7 is essential for maintaining mTregs in skin in the steady state. These results elucidate the fundamental biology of mTregs and show that IL-7 plays an important role in their survival in skin.

Organ-specific and memory treg cells: specificity, development, function, and maintenance.

Foxp3+ regulatory T cells (Treg cells) are essential for establishing and maintaining self-tolerance, and also inhibit immune responses to innocuous environmental antigens. Imbalances and dysfunction in Treg cells lead to a variety of immune-mediated diseases, as deficits in Treg cell function contribute to the development autoimmune disease and pathological tissue damage, whereas overabundance of Treg cells can promote chronic infection and tumorigenesis. Recent studies have highlighted the fact that Treg cells themselves are a diverse collection of phenotypically and functionally specialized populations, with distinct developmental origins, antigen-specificities, tissue-tropisms, and homeostatic requirements. The signals directing the differentiation of these populations, their specificities and the mechanisms by which they combine to promote organ-specific and systemic tolerance, and how they embody the emerging property of regulatory memory are the focus of this review.

Treating Human Autoimmunity: Current Practice and Future Prospects

Recent advances in understanding immune regulation set the stage for new targeted therapies for autoimmune disease. Autoimmune diseases are caused by immune cells attacking the host tissues they are supposed to protect. Recent advances suggest that maintaining a balance of effector and regulatory immune function is critical for avoiding autoimmunity. New therapies, including costimulation blockade, regulatory T cell therapy, antigen-specific immunotherapy, and manipulating the interleukin-2 pathway, attempt to restore this balance. This review discusses these advances as well as the challenges that must be overcome to target these therapies to patients suffering from autoimmune disease while avoiding the pitfalls of general immunosuppression.

The life of regulatory T cells

Foxp3+ regulatory T (Treg) cells are essential for maintaining self‐tolerance and preventing autoimmune reactions. Treg cells arise as a consequence of self‐antigen recognition during the maturation of cells in the thymus, and also following self‐antigen recognition in the periphery. Both thymic and peripherally generated Treg cells respond to antigen recognition by expanding in number, increasing their suppressive activity, and accumulating in the tissue where the antigen is located. A fraction of these activated “effector” Treg cells survive even in the absence of antigen expression and continue to control inflammatory reaction in the tissues, thus functioning as a population of “memory” Treg cells. Antigen exposure and the presence of IL‐2 are key determinants in the generation of memory Treg cells. These results provide a foundation for studying the role of memory Treg cells in controlling and treating autoimmune disorders and for testing the hypothesis that defects in the generation and maintenance of these cells underlie chronic, relapsing inflammatory diseases.

Cutting Edge: Self-Antigen Controls the Balance between Effector and Regulatory T Cells in Peripheral Tissues

Immune homeostasis in peripheral tissues is achieved by maintaining a balance between pathogenic effector T cells (Teffs) and protective Foxp3+ regulatory T cells (Tregs). Using a mouse model of an inducible tissue Ag, we demonstrate that Ag persistence is a major determinant of the relative frequencies of Teffs and Tregs. Encounter of transferred naive CD4+ T cells with transiently expressed tissue Ag leads to generation of cytokine-producing Teffs and peripheral Tregs. Persistent expression of Ag, a mimic of self-antigen, leads to functional inactivation and loss of the Teffs with preservation of Tregs in the target tissue. The inactivation of Teffs by persistent Ag is associated with reduced ERK phosphorylation, whereas Tregs show less reduction in ERK phosphorylation and are relatively resistant to ERK inhibition. Our studies reveal a crucial role for Ag in maintaining appropriate ratios of Ag-specific Teffs to Tregs in tissues.

Spliceosome-Mediated RNA Trans-Splicing Facilitates Targeted Delivery of Suicide Genes to Cancer Cells

Patients suffering from recessive dystrophic epidermolysis bullosa (RDEB), a hereditary blistering disease of epithelia, show susceptibility to develop highly aggressive squamous cell carcinoma (SCC). Tumors metastasize early and are associated with mortality in the 30th–40th years of life in this patient group. So far, no adequate therapy is available for RDEB SCC. An approach is suicide gene therapy, in which a cell death-inducing agent is introduced to cancer cells. However, lack of specificity has constrained clinical application of this modality. Therefore, we used spliceosome-mediated RNA trans-splicing technology, capable of replacing a tumor-specific transcript with one encoding a cell death-inducing peptide/toxin, to provide tumor-restricted expression. We designed 3′ pre–trans-splicing molecules (PTM) and evaluated their efficiency to trans-splice an RDEB SCC-associated target gene, the matrix metalloproteinase-9 (MMP9), in a fluorescence-based test system. A highly efficient PTM was further adapted to insert the toxin streptolysin O (SLO) of Streptococcus pyogenes into the MMP9 gene. Transfection of RDEB SCC cells with the SLO-PTM resulted in cell death and induction of toxin function restricted to RDEB SCC cells. Thus, RNA trans-splicing is a suicide gene therapy approach with increased specificity to treat highly malignant SCC tumors. Mol Cancer Ther; 10(2); 233–41. ©2011 AACR.

Closure of a Large Chronic Wound through Transplantation of Gene-Corrected Epidermal Stem Cells

Figure 1. Regeneration of a transgenic functional epidermis on the skin wound of the JEB patient. (a) The long-standing ulceration on the lower right leg of the patient 2 days before transplantation. (b) Western blot analysis of cell lysates (20 mg protein, 30 seconds exposure time) from (lane 1) normal control and patient keratinocyte cultures (lane 2) before and (lane 3) after gene correction, probed with a monoclonal antibody against laminin 332-b3. (lane 4) Western blot analysis of a higher amount of loaded protein (65 mg, 5 seconds exposure time) of uncorrected patient keratinocytes, and (lane 5) normal keratinocyte cultures using the same laminin-332-b3 antibody. The 75-kD band in lane 4 is consistent with the truncated laminin-332-b3 generated by the c.1903C>T; p.R635X mutation. (c) Transplantation of cultured transgenic epidermal sheets (asterisks) on the prepared wound bed. Grafts are overlaid with petrolatum gauze. (d) Initial epidermal regeneration at 14 days. (e) Complete epidermal regeneration at 3.5 months. (f) Stable epidermal regeneration at 16 months. Note crusting and erosions outside of the grafted area. JEB, junctional epidermolysis bullosa. TO THE EDITOR Generalized junctional epidermolysis bullosa (JEB) is caused by mutations in LAMA3, LAMB3, or LAMC2, which together encode laminin-332, a heterotrimeric protein consisting of a3, b3, and g2 chains (Fine et al., 2014). In nonlethal generalized intermediate JEB, laminin332 is highly reduced, and hemidesmosomes are rudimentary or completely absent, leading to blister formation within the lamina lucida of the basementmembrane uponminor trauma. The resulting chronic skin wounds invariably develop recurrent infections and scarring, which greatly impair patients’ quality of life (Fine et al., 2014; Laimer et al., 2010; Nakano et al., 2002). There is no cure for JEB; treatments are symptomatic and aimed at relieving the devastating clinical manifestations (Carulli et al., 2013). The only published evidence for the possibility of a permanent local treatment of JEB was provided by a phase I/II trial showing that autologous epidermal cultures containing geneticallymodified epidermal stem cells were able to restore a normal epidermis on a JEB patient (De Rosa et al., 2014; Mavilio et al., 2006). However, the transgenic epidermis was applied in areas still covered by a diseased but apparently functional epidermis, which was surgically removed before grafting (Mavilio et al., 2006). Although it is clear that the ideal clinical application of transgenic epidermis would aim at preventing the development of devastating chronic lesions,manypatients suffer from therapyresistant chronic ulcerations that are highly predisposed to cancer development and need timely closure (Goldberg