Expression Of Cutaneous Lymphocyte Antigen Research Articles

Cutaneous Lymphocyte Antigen Is a Potential Therapeutic Target in Cutaneous T-Cell Lymphoma

Cutaneous T-cell lymphoma (CTCL) such as Sézary syndrome or mycosis fungoides corresponds to an abnormal infiltration of T lymphocytes in the skin. CTCL cells have a heterogeneous phenotype and express cell adhesion molecules such as cutaneous lymphocyte antigen (CLA) supporting skin homing. The use of a mAb (HECA-452) against CLA significantly decreased transendothelial migration and survival of CTCL cells from patient samples and My-La cell line. The decrease of CLA expression by inhibition of its maturation enzyme, ST3 β-galactoside α-2,3-sialyltransferase 4, also impaired CTCL cell migration, proliferation, and survival. We confirmed invivo that treatment with anti-CLA mAb decreased the tumorigenicity as well as dissemination of CTCL cells in different tissues compared with the control group. Our findings provide evidence of the involvement of CLA in CTCL cell migration and survival, supporting that CLA inhibition could represent an actionable therapy in patients with CTCL.

Compartmentalized cytotoxic immune response leads to distinct pathogenic roles of natural killer and senescent CD8+ T cells in human cutaneous leishmaniasis.

Cytotoxic activity mediated by CD8+ T cells is the main signature of the immunopathogenesis of cutaneous leishmaniasis (CL). Here, we performed a broad evaluation of natural killer (NK) cell phenotypic and functional features during cutaneous leishmaniasis. We demonstrate for the first time that CL patients present the accumulation of circulating NK cells with multiple features of replicative senescence including low proliferative capacity and shorter telomeres, elevated expression of CD57, KLRG1 but diminished CD27 stimulatory receptor expression. Moreover, they exhibited higher cytotoxic and inflammatory potential than age-matched controls. The accumulation of circulating senescent NK cells (CD56dim CD57bright ) correlated positively with skin lesion size in the same patients, suggesting that they, like circulating senescent CD8+ T cells, may contribute to the immunopathology of CL. However, this senescent population had lower cutaneous lymphocyte antigen expression and so had diminished skin-homing potential compared with total or senescent CD8+ T cells. This was confirmed in CL skin lesions where we found a predominance of CD8+ T cells (both senescent and non-senescent) that correlated with the severity of the disease. Although there was also a correlation between the proportions of senescent NK cells (CD56+ CD57+ ) in the skin and lesion size, this was less evident. Collectively our results demonstrate first-hand that senescent cytotoxic cells may mediate skin pathology during human cutaneous leishmaniasis. However, as senescent cytotoxic CD8+ T cells predominate in the skin lesions, they may have a greater role than NK cells in mediating the non-specific skin damage in CL.

鞘氨醇1-磷酸盐受体拮抗剂（FTY720）抑制皮肤中分泌白细胞介素17的γδT细胞治疗银屑病的机制研究

Objective To evaluate therapeutic effect of the sphingosine-1-phosphate receptor antagonist FTY720 on the imiquimod-induced psoriasis-like mouse model, and to explore its molecular mechanism. Methods A total of 8 specific pathogen-free (SPF) female C57BL/6 mice were used in this study, and divided into experimental group (n=3) and control group (n=5) : each mouse ear was topically treated with 10 mg/d imiquimod for 7 consecutive days, and the experimental group and control group were intraperitoneally injected with FTY720 and phosphate buffer saline (PBS) respectively on days 2, 4 and 6. Ear skin thickness was measured every day. These mice were sacrificed on day 8, and histopathological changes of the ear skin were observed. The ear tissues and draining cervical lymph node cells were obtained. Flow cytometry was performed to analyze changes in the proportion of interleukin (IL) -17+ γδT cells in the mouse skin and draining lymph node tissues between the two above groups, and to determine the expression of sphingosine-1-phosphate receptor 1 (S1P1) and cutaneous lymphocyte antigen (CLA) on the surface of the γδT cells. The means of two independent samples were compared by using t test. Results After the 2-day topical application of imiquimod, the mouse ear thickness was significantly lower in the experimental group (0.217 mm ± 0.003 mm) than in the control group (0.232 mm ± 0.002 mm, t=4.23, P < 0.01) on day 3, and the significant difference existed till day 8 (all P < 0.01) . Histopathological examination of the ear skin revealed that the epidermal thickness was significantly lower in the experimental group (18.62 μm ± 0.19 μm) than in the control group (27.79 μm ± 1.58 μm, t=4.35, P < 0.01) . Immunofluorescence staining showed that the degree of neutrophil infiltration in the mouse ear tissue was lower in the experimental group than in the control group. As flow cytometry showed, the proportion of neutrophils was significantly lower in the experimental group (1.57% ± 0.12%) than in the control group (3.03% ± 0.33%, t=3.31, P=0.016) . In the ear tissues, the experimental group showed significantly decreased proportion of γδT cells or IL-17+ γδT cells (4.88% ± 0.42%, 40.53% ± 1.76% respectively) compared with the control group (9.45% ± 1.22%, 56.56% ± 0.66% respectively; t=2.75, 10.27 respectively, both P < 0.05) . In the draining lymph nodes, the experimental group showed significantly increased proportion of γδT cells or IL-17+ γδT cells compared with the control group (t=5.781, 4.140 respectively, both P < 0.05) , and the fluorescence intensity of S1P1 and CLA in the γδT cells was significantly lower in the experimental group than in the control group (P < 0.05) . Conclusion FTY720 can alleviate imiquimod-induced psoriasis-like manifestations in mouse models, likely by down-regulating the expression of S1P1 and CLA in γδT cells, increasing the proportion of γδT cells in the draining lymph nodes followed by the decrease of their proportion in the skin, and decreasing the production of IL-17 in skin tissues. Key words: Psoriasis; Mice, inbred C57BL; Receptors, antigen, T-cell, gamma-delta; Interleukin-17; Sphingosine-1-phosphate receptors

Ex vivo generation of umbilical cord blood T regulatory cells expressing the homing markers CD62L and cutaneous lymphocyte antigen.

Regulatory T cells (Tregs) are an important component of the immune system involved in regulation of immune cell proliferation and inflammatory responses and preventing autoimmune diseases. The use of Tregs in cellular therapy has recently been explored in clinical trials specifically evaluating the role of ex vivo expanded Tregs in the prevention of graft-versus-host disease during stem cell transplantation. The possibility of Treg use in the clinic requires clinical grade expansion of Tregs for development of cell therapy protocols and proper homing of Tregs to the intended target. Here we demonstrate a novel medium composition to expand CB Tregs, specifically upregulation the homing and activation markers CD62L and cutaneous lymphocyte antigen (CLA). CLA expression was uniquely acquired during activation of Tregs with subsequent loss or lack of expression with media change. This finding highlights the importance of proper growth conditions unique to Tregs that can alter expression of proteins and establishes a baseline for expanding marker specific Tregs that home and target unique tissues.

Vitamins A and D have antagonistic effects on expression of effector cytokines and gut-homing integrin in human innate lymphoid cells.

Retinoic acid (RA), the main biologically active metabolite of vitamin A, is known to promote gut homing of lymphocytes, as well as various regulatory and effector immune responses. In contrast, the active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25D3), is predominantly immunosuppressive. Little is known about the direct effects of these vitamins on the recently identified innate lymphoid cells (ILCs). We sought to characterize the effects of RA and 1,25D3 on human ILCs. Peripheral blood mononuclear cells were isolated from 27 non-selected blood donor buffy coats, and ILCs were sorted by FACS. ILC1, ILC2, and ILC3 cells were cultured for 5 days with RA, 1,25D3, and various cytokines known to activate ILCs (IL-2, IL-7, IL-12, thymic stromal lymphopoietin (TSLP), IL-25, and IL-33). Cytokines produced by ILCs were measured in culture supernatants, and surface receptor expression was analysed by flow cytometry. Retinoic acid acted synergistically with IL-2 and other activating cytokines to induce expression of the gut-homing integrin α4β7 in ILCs, as well as production of IL-5 and IL-13 in ILC2 cells, and IFN-γ in ILC1 and ILC3 cells. Expression of integrin α4β7 and cytokine production in ILCs stimulated with RA + IL-2 was increased at least fourfold as compared to ILCs cultured with RA or IL-2 alone. In contrast, RA completely inhibited the IL-2-induced expression of cutaneous lymphocyte antigen (CLA) in ILCs. Moreover, addition of 1,25D3 to ILCs cultured with RA + IL-2 inhibited cytokine production and expression of integrin α4β7 by at least 30%. Retinoic acid and 1,25D3 have antagonistic effects on the expression of effector cytokines and gut-homing integrin by human ILCs. The balance between these vitamins may be an important factor in the functioning of ILCs and the diseases in which ILCs are implicated, such as allergic inflammation.

Prognostic biomarkers in patients with localized natural killer/T-cell lymphoma treated with concurrent chemoradiotherapy.

Concurrent chemoradiotherapy has become one of the standard management approaches for newly diagnosed localized nasal natural killer (NK)/T-cell lymphoma (NKTCL). Few data are available on the prognostic biomarkers of NKTCL among patients treated with concurrent chemoradiotherapy. To evaluate the prognostic significance of immunophenotypic biomarkers for patients treated with concurrent chemoradiotherapy, latent membrane protein 1 (LMP1), cutaneous lymphocyte antigen (CLA) and cell origin were examined in samples from 32 patients who were enrolled in the Japan Clinical Oncology Group 0211 trial and treated with concurrent chemoradiotherapy. LMP1 and CLA were positive in 66% (19/29) and 29% (9/31) of the cases examined, respectively. The median follow-up duration was 68 months (range, 61–94). The patients with LMP1-positive tumors showed a better overall survival (OS) than the patients with LMP1-negative tumors (hazard ratio, 0.240; 95% confidence interval [CI], 0.057–1.013; 80% CI, 0.093–0.615; P = 0.035). All five patients with LMP1-negative tumors who experienced disease progression died of lymphoma, and both patients with local failure had LMP1-negative tumors. There was no significant difference in OS according to CLA expression. A total of 27 (84%) cases were of NK-cell origin, two were of αβ T-cell origin and three were of γδ T-cell origin. In contrast to those with tumors of NK-cell origin, all five patients with NKTCL of T-cell origin were alive without relapse at the last follow up. Our results indicate that LMP1 expression is a favorable prognostic marker and suggest that a T-cell origin of the tumor may be a favorable prognostic marker for patients with localized NKTCL treated with concurrent chemoradiotherapy.

Homing of Neural Stem Cells From the Venous Compartment Into a Brain Infarct Does Not Involve Conventional Interactions With Vascular Endothelium

Human neural stem cells (hNSCs) hold great potential for treatment of a wide variety of neurodegenerative and neurotraumatic conditions. Heretofore, administration has been through intracranial injection or implantation of cells. Because neural stem cells are capable of migrating to the injured brain from the intravascular space, it seemed feasible to administer them intravenously if their ability to circumvent the blood-brain barrier was enhanced. In the present studies, we found that interactions of hNSCs in vitro on the luminal surface of human umbilical vein endothelial cells was enhanced following enforced expression of cutaneous lymphocyte antigen on cell surface moieties by incubation of hNSCs with fucosyltransferase VI and GDP-fucose (fhNSCs). Interestingly, ex vivo fucosylation of hNSCs not only did not improve the cells homing into the brain injured by stroke following intravenous administration but also increased mortality of rats compared with the nonfucosylated hNSC group. Efforts to explain these unexpected findings using a three-dimensional flow chamber device revealed that transmigration of fhNSCs (under conditions of physiological shear stress) mediated by stromal cell-derived factor 1α was significantly decreased compared with controls. Further analysis revealed that hNSCs poorly withstand physiological shear stress, and their ability is further decreased following fucosylation. In addition, fhNSCs demonstrated a higher frequency of cellular aggregate formation as well as a tendency for removal of fucose from the cell surface. In summary, our findings suggest that the behavior of hNSCs in circulation is different from that observed with other cell types and that, at least for stroke, intravenous administration is a suboptimal route, even when the in vitro rolling ability of hNSCs is optimized by enforced fucosylation.

Prognostic Value Of LMP1, CLA, NK-Cell Origin, and Pretreatment Bone Marrow EBER-ISH In Localized NK/T-Cell Lymphoma Prospectively Treated With Concurrent Chemoradiotherapy: An Ancillary Study Of Japan Clinical Oncology Group (JCOG) 0211

Latent membrane protein 1 (LMP1), cutaneous lymphocyte antigen (CLA), NK-cell origin, and pretreatment bone marrow Epstein-Barr virus - encoded RNA by in situ hybridization (EBER-ISH) have been reported as prognostic biomarkers in patients with extranodal NK/T-cell lymphoma (NKTCL), nasal type when treated with conventional therapy (Kanemitsu N, et al. Clin Cancer Res 2012; Yoshino T, et al. Br J Haematol 2002; Kim GE, et al. Head Neck 2004; Lee J, et al. Clin Cancer Res 2007). Recently, concurrent chemoradiotherapy has become a standard management approach for newly diagnosed, localized, nasal NKTCL (Yamaguchi M, et al. J Clin Oncol 2009; Kim SJ, et al. J Clin Oncol 2009). Because it is a new treatment modality for lymphoma, little data are available on the prognostic biomarkers of NKTCL among patients treated with concurrent chemoradiotherapy. To evaluate the prognostic significance of known immunophenotypic biomarkers among patients treated with concurrent chemoradiotherapy, we conducted an ancillary clinicopathologic study of JCOG0211, which was a phase I/II trial of concurrent chemoradiotherapy (RT-DeVIC) for newly diagnosed, localized, nasal NKTCL (Yamaguchi M, et al. J Clin Oncol 2009 & 2012).The histological diagnoses of all 33 patients who were enrolled in JCOG0211 were confirmed as extranodal NKTCL, nasal type, by the Central Pathology Review. Pathological samples from 32 patients were available for the study. Patients received concurrent chemoradiotherapy comprising RT of 50 Gy and 3 cycles of DeVIC. The expression of LMP1 and CLA in tumor cells was examined using immunohistochemistry with paraffin sections of pretreatment lymphoma samples. Two monoclonal antibodies for TCRβ (βF1) and TCRγ (CγM1) were used to evaluate the cell of origin of lymphoma cells. Preliminary examination revealed that βF1 and CγM1 specifically recognized αβ and γδ T cells, respectively. Cases were considered to be of NK-cell origin if both TCRβ and TCRγ were negative. Cases showing positive staining for one or both of the antibodies (TCRβ and TCRγ) were determined to be of T-cell origin. Pretreatment bone marrow specimens from patients were examined for EBER-ISH.LMP1 and CLA were positive in 66% (19/29) and 29% (9/31) of the cases, respectively. Among 32 cases examined for cell lineage, 27 (84%) cases were of NK-cell origin. Two (6%) cases were of αβ T-cell origin on the basis of positivity for TCRβ but not TCRγ, and 3 (9%) cases were of γδ T-cell origin on the basis of positivity for TCRγ but not TCRβ. Pretreatment bone marrow EBER-ISH was positive in 2 (7%) out of 29 cases examined.The median follow-up was 68 months (range, 61-94). The overall survival (OS) was better in the LMP1-positive (LMP1+) group than in the LMP1-negative (LMP1-) group [hazard ratio (HR), 0.240; 95% confidence interval (CI), 0.057-1.013; 80% CI, 0.093-0.615; P=0.035]. The OS at 5 years was 84% (95% CI, 59-95%) in the LMP1+ patients and 50% (95% CI, 18-75%) in the LMP1- patients. The progression-free survival (PFS) at 5 years was 74% (95% CI, 48-88%) in the LMP1+ patients and 50% (95% CI, 18-75%) in the LMP1- patients. The HR of PFS in the LMP1+ patients was 0.421 (95% CI, 0.121-1.463; 80% CI, 0.187-0.950). There were no significant differences in the OS and PFS between the CLA+ and CLA- patients (5-yr OS, 56% vs. 77%; 5-yr PFS, 56% vs. 68%). All 5 patients of T-cell origin achieved complete response by RT-DeVIC and were alive without relapse at the last follow-up. The OS and PFS at 5 years in the 27 patients of NK-cell origin were 67% (95% CI, 46-81%) and 59% (95% CI, 39-75%), respectively. Two patients who were positive for pretreatment bone marrow EBER-ISH survived more than 5 years without disease progression (5-yr OS and PFS, 100%).Our current study, for the first time, validated a favorable impact on the OS of LMP1 expression in newly diagnosed, localized, nasal NKTCL in a cohort uniformly treated with concurrent chemoradiotherapy. Further evaluation with a larger number of patients is warranted. (Supported in part by The National Cancer Center Research and Development Funds #21-6-3 & #23-A-17) Disclosures:Ishizuka:Sanofi: Employment. Oshimi:Eisai: Employment.

Peptide Vaccination in Montanide Adjuvant Induces and GM-CSF Increases CXCR3 and Cutaneous Lymphocyte Antigen Expression by Tumor Antigen–Specific CD8 T Cells

T cell infiltration of melanoma is associated with enhanced clinical efficacy and is a desirable endpoint of immunotherapeutic vaccination. Infiltration is regulated, in part, by chemokine receptors and selectin ligands on the surface of tumor-specific lymphocytes. Therefore, we investigated the expression of two homing molecules--CXCR3 and CLA - on vaccine-induced CD8 T cells, in the context of a clinical trial of a melanoma-specific peptide vaccine. Both CXCR3 and CLA have been associated with T cell infiltration of melanoma. We demonstrate that a single subcutaneous/intradermal administration of peptide vaccine in Montanide adjuvant induces tumor-specific CD8 T cells that are predominantly positive for CXCR3, with a subpopulation of CXCR3(+)CLA(+) cells. Addition of GM-CSF significantly enhances CXCR3 expression and increases the proportion of CLA-expressing cells. Concurrent with CXCR3 and CLA expression, vaccine-induced CD8 cells express high levels of Tbet, IFN-γ, and IL-12Rβ1. Collectively, these studies demonstrate that peptide vaccination in adjuvant induces CD8 T cells with a phenotype that may support infiltration of melanoma.

Tumor infiltrating lymphocytes derived from metastatic melanoma during intralesional immunostimulatory therapy exhibit a phenotype reflecting tumor progression (P2193)

Abstract Intralesional immunostimulatory therapy is a rational approach for in transit metastatic melanoma, as lesions are accessible and provide a source of matched tumor antigen to facilitate local and systemic immune responses. Moreover, the lesions are accessible for evaluation of the local immune response at the site of disease. To examine the immunological status at the site of disease during the course of intralesional therapy, T cells were extracted from lesions, cultured to preserve their in vivo phenotype, and expanded for functional analysis. T cells isolated from the skin lesions two weeks after initiation of intralesional therapy produced elevated Th1 cytokines relative to pre-therapy lesions. In contrast, T cells isolated at nine weeks produced decreased Th1 cytokines, but elevated Th2 cytokines, soluble IL-2Ra and IL-6, mediators associated with tumor progression. Expression of skin homing receptor cutaneous lymphocyte antigen (CLA) on lesion derived T cells correlated with Th1 cytokine production. Strikingly, lesion-derived Th1 cytokines and CLA expression were inversely correlated with tumor size during the course of therapy, whereas the phenotype of T cells derived from the blood did not change dramatically over the course of the therapy. Our studies indicate that T cells isolated from the site of disease in melanoma provide insight into the clinical state of the patient and suggest that they are critical regulators in the progression of tumor in malignant melanoma.

Regulatory T Cells Inhibit CD8+ T-Cell Tissue Invasion in Human Skin Graft-Versus-Host Reactions

Regulatory T cells (Tregs) effectively ameliorate graft-versus-host disease (GVHD). The mechanisms underlying Treg therapeutic effect on GVHD are not fully elucidated. This study investigates whether Treg prevention of GVH tissue damage is associated with blocking CD8 effector T-cell tissue invasion, a question not yet addressed in humans. Tissue-infiltrating T cells and histopathology scores were detected using an in vitro human GVHD skin explant model, together with immunohistochemistry, cytometric bead array, functional adhesion and migration assays, flow cytometry, and quantitative real-time polymerase chain reaction. Treg intervention during priming significantly decreased effector T-cell infiltration into target tissue (P<0.01) resulting in a striking reduction in the histopathology score of tissue injury (P<0.0001). These results were coupled with reduced CXCR3 and cutaneous lymphocyte antigen expression by effector T cells, together with decreased CXCL10 and CXCL11 expression in target tissue. Treg intervention also impaired the functional interaction of CXCR3 and cutaneous lymphocyte antigen with their specific ligands (P<0.01) and suppressed the secretion of CXCL9, CXCL10, and interferon-γ (P<0.01, P<0.05, and P<0.001, respectively). Late addition of Tregs into the effector phase abolished their ability to suppress effector T-cell tissue invasion, resulting in a total loss of their ability to ameliorate GVH tissue damage. Preventing effector T-cell tissue invasion is a critical mechanistic event leading to Treg attenuation of GVH tissue damage. This therapeutic effect is associated with a failure of CD8 T cells to increase tissue homing receptors after allo-stimulation, together with a breakdown of interferon-γ-induced chemoattractant expression in the target tissue.

Abstract 2858: A novel mouse model for cutaneous T-cell lymphoma reveals a role for IL-15 and activity of a new oral HDAC inhibitor

Abstract Cutaneous T-cell lymphoma (CTCL) is a type of non-Hodgkin's lymphoma representing a spectrum of diseases composed of malignant clonal CD4 (+) helper T lymphocytes. The etiology of CTCL remains to be elucidated and no animal models for CTCL are known. We quantified expression of IL-15 in patients with CTCL and demonstrated aberrant overexpression of IL-15 compared to normal CD4+ T cells (Fold increase = 7.476 ± 1.788 N=3, P=0.0073), that showed direct correlation with the stage of the disease (Fold increase in IL-15 in Stage I patients vs. Stage III patients = 3.283 ± 0.8246 vs. 7.476 ± 1.788, N=3 each, P=0.02). We engineered a transgenic (tg) mouse to constitutively and globally overexpress murine IL-15. These mice develop alopecia and cutaneous lesions in all mice within 6 weeks of birth, characterized by erythematous plaques/patches, erythroderma, pruritis and early death compared to wild type (WT) controls (49 weeks vs. 100 weeks, P=0.0003). Skin lesions showed an infiltration of atypical lymphocyte in the dermis and throughout the epidermis including the formation of classic Pautrier's microabscesses as seen in human CTCL. Immunophenotypic analysis of the skin T-cells from these mice revealed ∼25 fold increase in CD3+ T-cells compared to WT littermates. Intradermal CD4+ cells from CTCL mice were predominantly CD62L- and significantly higher than WT mice (P=0.007). Immunohistochemistry showed high expression of cutaneous lymphocyte antigen (CLA) and CCR4 on skin sections of CTCL mice, consistent with a skin-specific homing pattern as seen in human CTCL. Cells obtained from the skin of these IL-15tg CTCL mice could be successfully engrafted into skin of SCID recipient mice with reproducible histological lesions. Consistent with human studies, CTCL mice exhibited significantly increased levels of Hdac-1 in CD4+ T cells when compared to their WT counterparts (P = 0.0013). To investigate the importance of IL-15 in this process, we cultured sorted mouse WT (CD3+CD4+) T-cells in medium supplemented with 100ng/ml IL-15 and observed a significant increase in Hdac-1 expression at the mRNA and protein level within 10 days of culture, compared to freshly isolated WT CD4+ T cells (N=3 each, p =0.02). In a randomized, placebo-controlled trial of an orally administered novel HDAC inhibitor, AR-42 (Arno Therapeutics, Inc, Parsippany, NJ; Sargeant AM et al, Cancer Research, 2008) to IL-15 tg mice with CTCL, we observed an improvement in skin lesions and a marked reduction in T-cell dermal infiltrate as early as 12 days post-treatment. In conclusion, we provide evidence for a reproducible in vivo mouse model of spontaneous CTCL that appears to mimic the human condition. Further, we provide evidence for a role of aberrant IL-15 expression in the genesis of both human and experimental CTCL, and alterations in Hdac-1 expression that may to serve as an appropriate target for a novel oral HDAC inhibitor now in clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2858. doi:10.1158/1538-7445.AM2011-2858

Cutaneous Lymphocyte Antigen and TRAF-1 Are Useful in Differentiating Lymphomatoid Papulosis, Cutaneous ALCL and Systemic ALCL.

Lymphomatoid papulosis (LyP) and primary cutaneous anaplastic large cell lymphoma (pcALCL) comprise a spectrum of CD30+ lymphoproliferative disease with overlapping morphology and clinical behavior. The border between LyP and ALCL is often difficult to establish. Moreover, secondary skin lesions of systemic ALCL closely resemble pcALCL and confer a poor prognosis. Thus, it is important to develop new criteria to make these distinctions. This study uses tumor necrosis factor receptor (TNFR)-associated factor 1 (TRAF-1) and cutaneous lymphocyte antigen (CLA) in differentiating CD30-positive lymphoproliferations involving the skin. TRAF-1 is involved in the intracellular signal transduction of TNFR family members without a death domain. TRAF-1 is expressed mainly in activated lymphocytes and strongly induced by NF-kB. CLA is an E-selectin ligand that facilitates adhesion of T lymphocytes to cutaneous vascular endothelium, initiating egress of T lymphocytes to the skin from the systemic circulation. After obtaining SRC and IRB approval, formalin-fixed, paraffin-embedded biopsy specimens from confirmed cutaneous and systemic CD30+ lymphomas were selected from the pathology department of Moffitt Cancer database, 2000–2007. The selected cases included 13 LyP, 9 primary cutaneous ALCL, and 8 systemic ALCL. All cases were stained with TRAF-1 (Imgenex IMG 5757) and CLA (HECA 452) using manufacturer's protocols. Manual morphometry of 500 lymphocytes counted under high power view (40x HPF) and virtual flow cytometry were performed to quantitate the degree of antibody expression. Antigen expression was scored as negative (less than 5%), weak positive (5 to 50%), intermediate positive (50–80%) and strong positive (80–100%). TRAF-1 was expressed by > 5% of CD30+ large atypical cells in 12 out of 13 LyP cases and in all nine pcALCL. TRAF-1 expression was virtually absent in systemic ALCL with or without skin manifestations. There was no significant difference of TRAF-1 staining between LyP and pcALCL (p>0.05), but significant significant differences betweeen LyP and systemic ALCL (p<0.01), and between pcALCL vs systemic ALCL (p <0.001). Tumor cells of all cases of systemic ALCL case were negative for TRAF-1 (p= 0.0003). CLA expression showed significant differences between LyP and pcALCL (p<0.05) as well as LyP and systemic ALCL (p<0.001), and insignificant differences between pcALCL and systemic ALCL( p>0.05). Mean expression of TRAF-1 was 25% in LyP vs 32% in pcALCL and 0.87% in systemic ALCL. CLA had a mean of 52% for LyP, 28.5% for pcALCL and 6.5% for systemic ALCL. In conclusion, (1) there is significant differential expression of TRAF-1 and CLA in LyP, (2) pcALCL and systemic ALCL. CLA can differentiate LyP from pcALCL. Strong TRAF-1 expression is almost restricted to CD30+ cells in LyP and intermediate to high expression in primary cutaneous ALCL. Systemic ALCL is uniformly negative for TRAF-1. This study indicates potential utility of TRAF-1 and CLA expression data in separating systemic from pcCD30-positive lymphoproliferative disorders.

Cutaneous lymphocyte antigen expression in benign and neoplastic cutaneous B‐ and T‐cell lymphoid infiltrates

Cutaneous lymphocyte-associated antigen (CLA) is expressed in resident cutaneous T lymphocytes, high endothelial venules, peripheral monocytes, granulocytes and a small percentage of memory B cells. It has been postulated to be an important factor in homing of lymphocytes to the skin because of its function as a ligand for E-selectin expressed on cutaneous endothelial cells. We investigated the expression of CLA using the HECA-452 antibody on paraffin-embedded, formalin-fixed tissue in a variety of reactive, neoplastic and preneoplastic cutaneous lymphoid infiltrates of T- and B-cell derivation. CLA was expressed at high levels in various types of inflammatory dermatoses with the exception of lupus erythematosus. High levels of CLA expression were seen in epidermotropic T-cell dyscrasias and epidermotropic T-cell lymphomas including mycosis fungoides (MF) and adult T-cell leukemia/lymphoma (ATCLL). A loss of CLA in tumors normally positive for CLA was a feature of disease progression best exemplified by tumor-stage MF and acute ATCLL. There was a lack of CLA expression in those lymphocytic infiltrates manifesting subcutaneous localization including lupus profundus, panniculitis-like T-cell lymphoma and atypical lymphocytic lobular panniculitis. CLA expression was not only observed in primary cutaneous anaplastic large cell lymphoma but also seen in cases of nodal anaplastic large cell lymphoma secondarily involving the skin and was negative in cases of nodal anaplastic large cell lymphoma without any established skin involvement. An oligodot pattern defined a novel reaction pattern in those aggressive systemic dyscrasias with a proclivity to involve the skin. CLA was negative in the majority of B-cell lymphomas. CLA plays a role in the pattern of T-cell lymphocyte migration in the skin and subcutis in both reactive and neoplastic states. An alteration in the expression of this marker, whether it is in the context of the acquisition of expression in a cell that is normally CLA negative or its loss of expression, may define a key event in determining cutaneous and extracutaneous hematopoietic cell distribution.

The frequency of CLA+ CD8+ T cells in the blood of psoriasis patients correlates closely with the severity of their disease.

Psoriasis is thought to be a T cell-mediated skin disease and the cutaneous lymphocyte antigen (CLA) is an important skin homing epitope for T cells. We have studied the relationship between disease severity (PASI) and phenotypic analysis of T cells in the blood of 36 patients with psoriasis focusing on the expression of CLA, VLA-4 and CD25 on CD4+ and CD8+ T cells. The patients had a higher frequency of circulating CLA+ CD8+ cells than healthy controls. Furthermore, a much stronger correlation was observed between PASI and the frequency of CLA+ CD8+ than CLA+ CD4+ T cells. The frequency of CLA+D8+ T cells correlated more strongly with redness, thickness and scaling of the skin lesions than the total affected body surface area. In contrast to CLA the T cell expression of VLA-4 did not demonstrate any such correlation. Finally, the expression of the activation marker CD25 on CD8+ T cells showed a strong correlation with disease severity in patients with moderate to severe psoriasis (PASI > 10) but such correlation was not observed for CD4+ T cells. These findings support the notion that circulating CLA+ CD8+ T cells may play an important role in the pathogenesis of psoriasis.

Regulation of human T‐cell homing receptor expression in cutaneous bacterial infection

We investigated the regulation of T-cell homing receptors in infectious disease by evaluating the cutaneous lymphocyte antigen (CLA) in human leprosy. We found that CLA-positive cells were enriched in the infectious lesions associated with restricting the growth of the pathogen Mycobacterium leprae, as assessed by the clinical course of infection. Moreover, CLA expression on T cells isolated from the peripheral blood of antigen-responsive tuberculoid leprosy patients increased in the presence of M. leprae (2.4-fold median increase; range 0.8-6.1, n = 17), but not in unresponsive lepromatous leprosy patients (1.0-fold median increase; range 0.1-2.2, n = 10; P < 0.005). Mycobacterium leprae specifically up-regulated the skin homing receptor, CLA, but not alpha(4)/beta(7), the intestinal homing receptor, which decreased on T cells of patients with tuberculoid leprosy after antigen stimulation (2.2-fold median decrease; range 1.6-3.4, n = 3). Our data indicate that CLA expression is regulated during the course of leprosy infection and suggest that T-cell responsiveness to a microbial antigen directs antigen-specific T cells to the site of infection.

Intralymphatic Dendritic Cell Vaccination Induces Tumor Antigen–Specific, Skin-Homing T Lymphocytes

The identification of tumor antigens recognized by cytotoxic and T helper lymphocytes has led to the development of specific cancer vaccines. Immunization with tumor antigen-pulsed dendritic cells has proved effective at eliciting elevated levels of tumor antigen-specific T cells in patient blood, but objective clinical responses remain rare, suggesting that vaccine-induced T cells are not trafficking optimally to site(s) of tumor burden. Accumulating evidence from animal models suggests that route of immunization can have a substantial influence on the subsequent migration of primed, activated T cells in vivo. In a clinical trial designed to elicit more effective cytotoxic T-cell mediated antitumor responses, metastatic melanoma patients were immunized directly via a peripheral intralymphatic route with autologous dendritic cells pulsed with HLA-A*0201-restricted melanoma-associated peptide antigens derived from MART-1 and gp100. Within 10 days of intralymphatic dendritic cell vaccination, four of six patients developed dramatic and diffuse erythematous rashes in sun-exposed areas of skin that showed extensive T-cell infiltration. CTLs grown from rash biopsies were strongly enriched for tumor antigen-specific T cells that had elevated expression of cutaneous lymphocyte antigen and chemokine receptor-6, consistent with a skin-homing phenotype. Of note, the only patient in the study with cutaneously localized disease showed a significant regression of metastatic lesions following the development of a surrounding rash. The evidence presented here is consistent with immunization studies in animal models and supports the concept that T cells are "imprinted" in peripheral lymph node sites to express specific ligands and chemokine receptors that allow them to migrate to skin. Furthermore, the preferential migration of the T cells to sun-exposed cutaneous sites suggests that inflammation plays a critical role in this migration. These observations suggest that further study of the effects of immunization route and inflammation on T-cell migration in humans is warranted, and could lead to vaccination approaches that would more reliably direct trafficking of activated T cells to diverse sites of metastatic disease.

T-cell prolymphocytic leukemia: An aggressive T cell malignancy with frequent cutaneous tropism

T-cell prolymphocytic leukemia (T-PLL), formerly categorized as T-cell chronic lymphocytic leukemia, is a rare and aggressive hematologic malignancy. Although the skin is characteristically involved, it is not a well-recognized entity in the dermatologic literature. Six cases of cutaneous T-PLL are presented from a clinical, light microscopic, and phenotypic perspective. The patient population comprised 2 women and 4 men, with a mean age of 69.8 years. The disease was associated in all with skin involvement with facial preference; edema, purpura, and lesional symmetry were characteristic. The skin biopsies demonstrated a largely non-epidermotropic angiocentric lymphocytic infiltrate with accompanying hemorrhage. The cells showed irregular- to reniform-shaped nuclei with small nucleoli and eosinophilic rims of cytoplasm. Phenotypic studies revealed three prevailing profiles: CD4 dominant in 4, CD8 dominant in one, and co-expression of CD4 and CD8 in one. CD3 loss was seen in one case. All expressed T-cell leukemia 1 (TCL-1) and CD7; cutaneous lymphocyte antigen expression was discernible in a dot-like perinuclear array. All cases tested excluding one expressed TCL-1 and CD52. In two cases tested, T-cell receptor beta rearrangements were observed. Cytogenetic studies demonstrated a paracentromeric chromosome 14 inversion. Polysomy 8 and MYC amplification was seen in one case, manifesting an aggressive clinical course. Four patients died from their disease within 18 months of diagnosis. Cytogenetic MYC amplification, FISH, and TCR beta studies were conducted on each of 2 cases, respectively, due to limitations of tissue block samples and/or peripheral blood. cMYC translocation studies were conducted on 3 of the 6 cases, again due to limitations imposed by the tissue samples on the cases. The last case was recently diagnosed and, therefore, long-term follow-up is not possible. T-PLL is a distinctive post-thymic T-cell malignancy with frequent cutaneous tropism. A diagnosis is possible in almost all cases based on characteristic clinical, light microscopic, phenotypic, and cytogenetic features. While a chromosome 14 inversion is highly characteristic, additional inherent cytogenetic differences, such as trisomy 8 with CMYC over-amplification, may account for some case to case variation in clinical course.

Expression of Cutaneous Lymphocyte Antigen in t-Cells From Patients With Generalized Vitiligo

Expression of Cutaneous Lymphocyte Antigen in t-Cells From Patients With Generalized Vitiligo

Absence of CD26 expression on skin-homing CLA+ CD4+ T lymphocytes in peripheral blood is a highly sensitive marker for early diagnosis and therapeutic monitoring of patients with Sezary syndrome

Patients with Sézary syndrome (SS) show clonal expansion in the peripheral blood of skin-homing CD4+ T-helper cells expressing cutaneous lymphocyte antigen (CLA). However, an increase of CLA+ CD4+ T cells can also be observed in various inflammatory dermatoses. To facilitate early diagnosis and therapeutic monitoring of SS using flow cytometry, we evaluated the expression of CD7 and CD26 on the CLA+ CD4+ lymphocyte subset. Peripheral lymphocytes from 7 patients with SS, 16 patients with mycosis fungoides (MF) and 11 healthy controls were analysed by flow cytometry for the expression of CD4, CD7, CD26, CLA and CCR4. In addition, a longitudinal study was performed over 16 months in two patients with SS. Absence of CD7 and CD26 on CLA+ CD4+ T cells was highly specific for SS. Importantly, the absence of CD26 on CLA+ CD4+ T cells was very sensitive for SS, at 100% in our patient cohort. The number of CD26- CLA+ CD4+ T cells closely correlated with therapeutic interventions in the longitudinal analysis of two patients over more than 1 year. We conclude that the absence of CD26 expression on skin-homing CLA+ CD4+ T-helper cells is a very sensitive and highly specific parameter for early diagnosis and therapeutic monitoring of patients with SS.