J. Teague

Skin Effector Memory T Cells Do Not Recirculate and Provide Immune Protection in Alemtuzumab-Treated CTCL Patients

Alemtuzumab depletes malignant T cells but spares skin resident effector memory T cells, treating L-CTCL without increased risk of infection. Resolving Cutaneous T Cell Lymphoma Becoming more active figures prominently in many New Year’s resolutions. We vow to get off the couch, or up from the lab bench, and get in shape. Yet, after a 2- to 3-week frenzy, spaces appear in gym parking lots as most people resume their sedentary life-styles. Some but not all memory T cells mimic this cyclic action. Clark et al. now exploit this difference to successfully treat cutaneous T cell lymphoma (CTCL) without increasing patients’ risk of infection. CTCL is a cancer of skin-homing T cells that can either be restricted to the skin—mycosis fungoides (MF)—or extend to the blood—leukemic CTCL (L-CTCL). MF and L-CTCL are caused by malignancies in two different types of memory T cells: skin resident effector memory T cells (TEM) and central memory T cells (TCM), respectively. Clark et al. found that treating patients with a low dose of alemtuzumab—a monoclonal antibody to CD52, a protein found on the surface of some mature lymphocytes—has a therapeutic effect on L-CTCL but not MF. Indeed, alemtuzumab depleted all T cells, both benign and malignant, from the blood; however, in the skin, only TCM were depleted with alemtuzumab treatment. This effect depended on the presence of neutrophils—phagocytic cells found in the blood but rare in normal skin—which suggests that TCM but not TEM recirculate in the blood. Importantly, the remaining skin resident TEM in the L-CTCL patients may have a protective function, because risk of infection was not heightened in the treated patients. Thus, alemtuzumab destroys the more active malignant cells while sparing the more sessile population, resulting in cancer therapy with retained immunity and, hopefully, the chance for these patients to break many more New Year’s resolutions. Cutaneous T cell lymphoma (CTCL) is a cancer of skin-homing T cells with variants that include leukemic CTCL (L-CTCL), a malignancy of central memory T cells (TCM), and mycosis fungoides (MF), a malignancy of skin resident effector memory T cells (TEM). We report that low-dose alemtuzumab (αCD52) effectively treated patients with refractory L-CTCL but not MF. Alemtuzumab depleted all T cells in blood and depleted both benign and malignant TCM from skin, but a diverse population of skin resident TEM remained in skin after therapy. T cell depletion with alemtuzumab required the presence of neutrophils, a cell type frequent in blood but rare in normal skin. These data suggest that TCM were depleted because they recirculate between the blood and the skin, whereas skin resident TEM were spared because they are sessile and non-recirculating. After alemtuzumab treatment, skin T cells produced lower amounts of interleukin-4 and higher amounts of interferon-γ. Moreover, there was a marked lack of infections in alemtuzumab-treated L-CTCL patients despite the complete absence of T cells in the blood, suggesting that skin resident TEM can protect the skin from pathogens even in the absence of T cell recruitment from the circulation. Together, these data suggest that alemtuzumab may treat refractory L-CTCL without severely compromising the immune response to infection by depleting circulating TCM but sparing the skin resident TEM that provide local immune protection of the skin.

Human skin is protected by four functionally and phenotypically discrete populations of resident and recirculating memory T cells

Four different T cell populations with different functions and migration patterns protect human skin. T cells are more than skin deep Skin is more than just a passive barrier to infection—it’s a dynamic immune microenvironment. Indeed, skin in a human adult is home to around 20 billion memory T cells; however, little is known about the composition and function of these cells. Now, Watanabe et al. characterize four different populations of T cells in the skin, two resident and two recirculating. The resident memory T cells had more potent effector functions than recirculating cells as well as different proliferative capacities. In addition, the recirculating cells returned to the skin at different rates. These data suggest that the different T cell populations in the skin may each provide a singular function in protecting the body from infection. The skin of an adult human contains about 20 billion memory T cells. Epithelial barrier tissues are infiltrated by a combination of resident and recirculating T cells in mice, but the relative proportions and functional activities of resident versus recirculating T cells have not been evaluated in human skin. We discriminated resident from recirculating T cells in human-engrafted mice and lymphoma patients using alemtuzumab, a medication that depletes recirculating T cells from skin, and then analyzed these T cell populations in healthy human skin. All nonrecirculating resident memory T cells (TRM) expressed CD69, but most were CD4+, CD103−, and located in the dermis, in contrast to studies in mice. Both CD4+ and CD8+ CD103+ TRM were enriched in the epidermis, had potent effector functions, and had a limited proliferative capacity compared to CD103− TRM. TRM of both types had more potent effector functions than recirculating T cells. We observed two distinct populations of recirculating T cells, CCR7+/L-selectin+ central memory T cells (TCM) and CCR7+/L-selectin− T cells, which we term migratory memory T cells (TMM). Circulating skin-tropic TMM were intermediate in cytokine production between TCM and effector memory T cells. In patients with cutaneous T cell lymphoma, malignant TCM and TMM induced distinct inflammatory skin lesions, and TMM were depleted more slowly from skin after alemtuzumab, suggesting that TMM may recirculate more slowly. In summary, human skin is protected by four functionally distinct populations of T cells, two resident and two recirculating, with differing territories of migration and distinct functional activities.

Human TH9 Cells Are Skin-Tropic and Have Autocrine and Paracrine Proinflammatory Capacity

Aberrant activation of human TH9 cells may contribute to inflammatory diseases of the skin. TH9 Cells: Immune Cell Specialists One of the main strengths of the immune system is its diverse strategies for fighting off infection. There are the stalwart innate cells and more flexible adaptive cells. However, even among these groups are many more specialized subsets, adapted to fight particular foes. Schlapbach et al. describe a subset of proinflammatory human helper T cells—TH9 cells—that may contribute to inflammatory diseases of the skin. Although TH9 cells had been described in mouse models, little was known about the role of TH9 cells in humans. The authors found that most of these cells were either skin-tropic or skin-resident. These cells produced interleukin-9 but not cytokines common to other T helper cell subsets, and many were specific for Candida albicans, suggesting a protective role against infection. However, these cells were also increased in psoriasis lesions, suggesting that they may contribute to inflammatory disease as well. T helper type 9 (TH9) cells can mediate tumor immunity and participate in autoimmune and allergic inflammation in mice, but little is known about the TH9 cells that develop in vivo in humans. We isolated T cells from human blood and tissues and found that most memory TH9 cells were skin-tropic or skin-resident. Human TH9 cells coexpressed tumor necrosis factor–α and granzyme B and lacked coproduction of TH1/TH2/TH17 cytokines, and many were specific for Candida albicans. Interleukin-9 (IL-9) production was transient and preceded the up-regulation of other inflammatory cytokines. Blocking studies demonstrated that IL-9 was required for maximal production of interferon-γ, IL-9, IL-13, and IL-17 by skin-tropic T cells. IL-9–producing T cells were increased in the skin lesions of psoriasis, suggesting that these cells may contribute to human inflammatory skin disease. Our results indicate that human TH9 cells are a discrete T cell subset, many are tropic for the skin, and although they may function normally to protect against extracellular pathogens, aberrant activation of these cells may contribute to inflammatory diseases of the skin.

Intramuscular Therapeutic Vaccination Targeting HPV16 Induces T Cell Responses That Localize in Mucosal Lesions

T helper 1 (TH1) immune responses are detectable in target lesions after therapeutic vaccination. Putting Cancer Vaccines in Context Despite the notable success of vaccines for infectious diseases, cancer vaccines remain a challenge. Cancer vaccines must overcome many hurdles, including an immunosuppressive tumor microenvironment, antigenic similarity to healthy cells, and the vast diversity of cancer types and origins. Even vaccines that target infection-induced cancer, such as for cervical intraepithelial neoplasias (CINs) caused by human papillomavirus (HPV), have had limited success at inducing peripheral blood T cell responses. Now, Maldonado et al. suggest that peripheral therapeutic vaccination to HPV can induce a tissue-localized effector immune response. The authors hypothesized that context was important when looking for immune responses to the therapeutic HPV vaccine. Although CIN patients had only modest changes in the immune response in the blood, these changes were much more pronounced in the target lesion microenvironment. They found evidence of immune proliferation and activation in the CIN lesions that was not detectible in peripheral blood. These data suggest that some early vaccine “failures,” which are often determined by peripheral blood T cells response, may not be failures at all and that looking in the target lesion may be the best place to determine vaccine response. About 25% of high-grade cervical intraepithelial neoplasias (CIN2/3) caused by human papillomavirus serotype 16 (HPV16) undergo complete spontaneous regression. However, to date, therapeutic vaccination strategies for HPV disease have yielded limited success when measured by their ability to induce robust peripheral blood T cell responses to vaccine antigen. We report marked immunologic changes in the target lesion microenvironment after intramuscular therapeutic vaccination targeting HPV16 E6/E7 antigens, in subjects with CIN2/3 who had modest detectable responses in circulating T lymphocytes. Histologic and molecular changes, including markedly (average threefold) increased intensity of CD8+ T cell infiltrates in both the stromal and epithelial compartments, suggest an effector response to vaccination. Postvaccination cervical tissue immune infiltrates included organized tertiary lymphoid-like structures in the stroma subjacent to residual intraepithelial lesions and, unlike infiltrates in unvaccinated lesions, showed evidence of proliferation induced by recognition of cognate antigen. At a molecular level, these histologic changes in the stroma were characterized by increased expression of genes associated with immune activation (CXCR3) and effector function (Tbet and IFNβ), and were also associated with an immunologic signature in the overlying dysplastic epithelium. High-throughput T cell receptor sequencing of unmanipulated specimens identified clonal expansions in the tissue that were not readily detectable in peripheral blood. Together, these findings indicate that peripheral therapeutic vaccination to HPV antigens can induce a robust tissue-localized effector immune response, and that analyses of immune responses at sites of antigen are likely to be much more informative than analyses of cells that remain in the circulation.

Human Papillomavirus 16-Associated Cervical Intraepithelial Neoplasia in Humans Excludes CD8 T Cells from Dysplastic Epithelium

High-grade cervical dysplasia caused by human papillomavirus (HPV) type 16 is a lesion that should be susceptible to an HPV-specific immune response; disease initiation and persistence is predicated on expression of two viral Ags, E6 and E7. In immune-competent subjects, at least 25% of HPV16+ high-grade cervical dysplasia lesions undergo complete regression. However, in the peripheral blood, naturally occurring IFN-γ T cell responses to HPV E6 and E7 are weak, requiring ex vivo sensitization to detect, and are not sufficiently sensitive to predict regression. In this study, we present immunologic data directly assessing cervical lymphocytes from this cohort. We found that nearly all cervical tissue T cells express the mucosal homing receptor, α4β7 surface integrin. T cells isolated from dysplastic mucosa were skewed toward a central memory phenotype compared with normal mucosal resident T cells, and dysplastic lesions expressed transcripts for CCL19 and CCL21, raising the possibility that the tissue itself sustains a response that is not detectable in the blood. Moreover, lesion regression in the study window could retrospectively be predicted at study entry by the ability of CD8+ T cells to gain access to lesional epithelium. Vascular endothelial expression of mucosal addressin cell adhesion molecule-1, the ligand that supports entry of α4β7+ T cells into tissues, colocalized tightly with the distribution of CD8 T cells and was not expressed in persistent dysplastic epithelium. These findings suggest that dysregulated expression of vascular adhesion molecules plays a role in immune evasion very early in the course of HPV disease.

Survival of tissue-resident memory T cells requires exogenous lipid uptake and metabolism

Tissue-resident memory T (TRM) cells persist indefinitely in epithelial barrier tissues and protect the host against pathogens. However, the biological pathways that enable the long-term survival of TRM cells are obscure. Here we show that mouse CD8+ TRM cells generated by viral infection of the skin differentially express high levels of several molecules that mediate lipid uptake and intracellular transport, including fatty-acid-binding proteins 4 and 5 (FABP4 and FABP5). We further show that T-cell-specific deficiency of Fabp4 and Fabp5 (Fabp4/Fabp5) impairs exogenous free fatty acid (FFA) uptake by CD8+ TRM cells and greatly reduces their long-term survival in vivo, while having no effect on the survival of central memory T (TCM) cells in lymph nodes. In vitro, CD8+ TRM cells, but not CD8+ TCM cells, demonstrated increased mitochondrial oxidative metabolism in the presence of exogenous FFAs; this increase was not seen in Fabp4/Fabp5 double-knockout CD8+ TRM cells. The persistence of CD8+ TRM cells in the skin was strongly diminished by inhibition of mitochondrial FFA β-oxidation in vivo. Moreover, skin CD8+ TRM cells that lacked Fabp4/Fabp5 were less effective at protecting mice from cutaneous viral infection, and lung Fabp4/Fabp5 double-knockout CD8+ TRM cells generated by skin vaccinia virus (VACV) infection were less effective at protecting mice from a lethal pulmonary challenge with VACV. Consistent with the mouse data, increased FABP4 and FABP5 expression and enhanced extracellular FFA uptake were also demonstrated in human CD8+ TRM cells in normal and psoriatic skin. These results suggest that FABP4 and FABP5 have a critical role in the maintenance, longevity and function of CD8+ TRM cells, and suggest that CD8+ TRM cells use exogenous FFAs and their oxidative metabolism to persist in tissue and to mediate protective immunity.

TH2 Cytokines from Malignant Cells Suppress TH1 Responses and Enforce a Global TH2 Bias in Leukemic Cutaneous T-cell Lymphoma

Purpose: In leukemic cutaneous T-cell lymphoma (L-CTCL), malignant T cells accumulate in the blood and give rise to widespread skin inflammation. Patients have intense pruritus, increased immunoglobulin E (IgE), and decreased T-helper (TH)-1 responses, and most die from infection. Depleting malignant T cells while preserving normal immunity is a clinical challenge. L-CTCL has been variably described as a malignancy of regulatory, TH2 and TH17 cells. Experimental Design: We analyzed phenotype and cytokine production in malignant and benign L-CTCL T cells, characterized the effects of malignant T cells on healthy T cells, and studied the immunomodulatory effects of treatment modalities in patients with L-CTCL. Results: Twelve out of 12 patients with L-CTCL overproduced TH2 cytokines. Remaining benign T cells were also strongly TH2 biased, suggesting a global TH2 skewing of the T-cell repertoire. Culture of benign T cells away from the malignant clone reduced TH2 and enhanced TH1 responses, but separate culture had no effect on malignant T cells. Coculture of healthy T cells with L-CTCL T cells reduced IFNγ production and neutralizing antibodies to interleukin (IL)-4 and IL-13 restored TH1 responses. In patients, enhanced TH1 responses were observed following a variety of treatment modalities that reduced malignant T-cell burden. Conclusions: A global TH2 bias exists in both benign and malignant T cells in L-CTCL and may underlie the infectious susceptibility of patients. TH2 cytokines from malignant cells strongly inhibited TH1 responses. Our results suggest that therapies that inhibit TH2 cytokine activity, by virtue of their ability to improve TH1 responses, may have the potential to enhance both anticancer and antipathogen responses. Clin Cancer Res; 19(14); 3755–63. ©2013 AACR.

TCR sequencing facilitates diagnosis and identifies mature T cells as the cell of origin in CTCL

High-throughput TCR sequencing can accurately diagnose and discriminate CTCL cells in skin. Discriminating taste for CTCL Cutaneous T cell lymphoma (CTCL) is a potentially debilitating disease, but early stages resemble rashes of less dangerous inflammatory skin diseases. Now, Kirsch et al. report that high-throughput TCR sequencing (HTS) can be used to distinguish CTCL from benign inflammatory disease by identifying T cell clones. This diagnostic was more sensitive and specific than the current standard of care and was also able to determine therapeutic response and identify early recurrence. The authors then used HTS to gain insight into CTCL pathogenesis, reporting that the malignancy derived from mature T cells that may have a specialized niche in the skin. Early diagnosis of cutaneous T cell lymphoma (CTCL) is difficult and takes on average 6 years after presentation, in part because the clinical appearance and histopathology of CTCL can resemble that of benign inflammatory skin diseases. Detection of a malignant T cell clone is critical in making the diagnosis of CTCL, but the T cell receptor γ (TCRγ) polymerase chain reaction (PCR) analysis in current clinical use detects clones in only a subset of patients. High-throughput TCR sequencing (HTS) detected T cell clones in 46 of 46 CTCL patients, was more sensitive and specific than TCRγ PCR, and successfully discriminated CTCL from benign inflammatory diseases. HTS also accurately assessed responses to therapy and facilitated diagnosis of disease recurrence. In patients with new skin lesions and no involvement of blood by flow cytometry, HTS demonstrated hematogenous spread of small numbers of malignant T cells. Analysis of CTCL TCRγ genes demonstrated that CTCL is a malignancy derived from mature T cells. There was a maximal T cell density in skin in benign inflammatory diseases that was exceeded in CTCL, suggesting that a niche of finite size may exist for benign T cells in skin. Last, immunostaining demonstrated that the malignant T cell clones in mycosis fungoides and leukemic CTCL localized to different anatomic compartments in the skin. In summary, HTS accurately diagnosed CTCL in all stages, discriminated CTCL from benign inflammatory skin diseases, and provided insights into the cell of origin and location of malignant CTCL cells in skin.

Tumor-Specific T Cells in Human Merkel Cell Carcinomas: A Possible Role for Tregs and T-Cell Exhaustion in Reducing T-Cell Responses

Merkel cell carcinomas (MCC) are rare but highly malignant skin cancers associated with a novel polyomavirus. MCC tumors were infiltrated by T cells, including effector, central memory and regulatory T cells. Infiltrating T cells showed markedly reduced activation as evidenced by reduced expression of CD69 and CD25. Treatment of MCC tumors in vitro with IL-2 and IL-15 led to T cell activation, proliferation, enhanced cytokine production and loss of viable tumor cells from cultures. Expanded tumor-infiltrating lymphocytes showed TCR repertoire skewing and upregulation of CD137. MCC tumors implanted into immunodeficient mice failed to grow unless human T cells in the tumor grafts were depleted with denileukin diftitox, suggesting tumor-specific T cells capable of controlling tumor growth were present in MCC. Both CD4+ and CD8+ FOXP3+ regulatory T cells were frequent in MCC. 50% of non-activated T cells in MCC expressed PD-1, a marker of T-cell exhaustion, and PD-L1 and PD-L2 were expressed by a subset of tumor dendritic cells and macrophages. In summary, we observed tumor-specific T cells with suppressed activity in MCC tumors. Agents that stimulate T cell activity, block Treg function or inhibit PD-1 signaling may be effective in the treatment of this highly malignant skin cancer.

Galectin-1 inhibits the viability, proliferation, and Th1 cytokine production of nonmalignant T cells in patients with leukemic cutaneous T-cell lymphoma

Tumor-derived galectin-1 (Gal-1), a β-galactoside-binding S-type lectin, has been shown to encourage T-cell death and promote T cell-mediated tumor immune escape. In this report, we show that patients with leukemic cutaneous T-cell lymphomas, known to have limited complexity of their T-cell repertoires, have a predominant T helper type-2 (Th2) cytokine profile and significantly elevated plasma levels of Gal-1 compared with healthy controls. Circulating clonal malignant T cells were a major source of Gal-1. The conditioned supernatant of cultured malignant T cells induced a β-galactoside-dependent inhibition of normal T-cell proliferation and a Th2 skewing of cytokine production. These data implicate Gal-1 in development of the Th2 phenotype in patients with advanced-stage cutaneous T-cell lymphoma and highlight the Gal-1-Gal-1 ligand axis as a potential therapeutic target for enhancing antitumor immune responses.