Spectrum of mutational signatures in T-cell lymphoma reveals a key role for UV radiation in cutaneous T-cell lymphoma.

T-cell non-Hodgkin's lymphomas develop following transformation of tissue resident T-cells. We performed a meta-analysis of whole exome sequencing data from 403 patients with eight subtypes of T-cell non-Hodgkin's lymphoma to identify mutational signatures and associated recurrent gene mutations. Signature 1, indicative of age-related deamination, was prevalent across all T-cell lymphomas, reflecting the derivation of these malignancies from memory T-cells. Adult T-cell leukemia-lymphoma was specifically associated with signature 17, which was found to correlate with the IRF4 K59R mutation that is exclusive to Adult T-cell leukemia-lymphoma. Signature 7, implicating UV exposure was uniquely identified in cutaneous T-cell lymphoma (CTCL), contributing 52% of the mutational burden in mycosis fungoides and 23% in Sezary syndrome. Importantly this UV signature was observed in CD4 + T-cells isolated from the blood of Sezary syndrome patients suggesting extensive re-circulation of these T-cells through skin and blood. Analysis of non-Hodgkin's T-cell lymphoma cases submitted to the national 100,000 WGS project confirmed that signature 7 was only identified in CTCL strongly implicating UV radiation in the pathogenesis of cutaneous T-cell lymphoma.

Frequent and Persistent PLCG1 Mutations in Sézary Cells Directly Enhance PLCγ1 Activity and Stimulate NFκB, AP-1, and NFAT Signaling.

Phospholipase C Gamma 1 (PLCG1) is frequently mutated in primary cutaneous T-cell lymphoma (CTCL). This study functionally interrogated nine PLCG1 mutations (p.R48W, p.S312L, p.D342N, p.S345F, p.S520F, p.R1158H, p.E1163K, p.D1165H, and the in-frame indel p.VYEEDM1161V) identified in Sézary Syndrome, the leukemic variant of CTCL. The mutations were demonstrated in diagnostic samples and persisted in multiple tumor compartments over time, except in patients who achieved a complete clinical remission. In basal conditions, the majority of the mutations confer PLCγ1 gain-of-function activity through increased inositol phosphate production and the downstream activation of NFκB, AP-1, and NFAT transcriptional activity. Phosphorylation of the p.Y783 residue is essential for the proximal activity of wild-type PLCγ1, but we provide evidence that activating mutations do not require p.Y783 phosphorylation to stimulate downstream NFκB, NFAT, and AP-1 transcriptional activity. Finally, the gain-of-function effects associated with the p.VYEEDM1161V indel suggest that the C2 domain may have a role in regulating PLCγ1 activity. These data provide compelling evidence to support the development of therapeutic strategies targeting mutant PLCγ1.

Contribution of STAT3 and RAD23B in Primary Sézary Cells to Histone Deacetylase Inhibitor FK228 Resistance.

FK228 (romidepsin) and suberoylanilide hydroxamic acid (vorinostat) are histone deacetylase inhibitors (HDACi) approved by the US Food and Drug Administration for cutaneous T-cell lymphoma (CTCL), including the leukemic subtype Sézary syndrome. This study investigates RAD23B and STAT3 gene perturbations in a large cohort of primary Sézary cells and the effect of FK228 treatment on tyrosine phosphorylation of STAT3 (pYSTAT3) and RAD23B expression. We report RAD23B copy number variation in 10% (12/119, P ≤ 0.01) of SS patients, associated with reduced mRNA expression (P = 0.04). RAD23B knockdown in a CTCL cell line led to a reduction in FK228-induced apoptosis. Histone deacetylase inhibitor treatment significantly reduced pYSTAT3 in primary Sézary cells and was partially mediated by RAD23B. A distinct pattern of RAD23B-pYSTAT3 co-expression in primary Sézary cells was detected. Critically, Sézary cells harboring the common STAT3 Y640F variant were less sensitive to FK228-induced apoptosis and exogenous expression of STAT3 Y640F, and D661Y conferred partial resistance to STAT3 transcriptional inhibition by FK228 (P ≤ 0.0024). These findings suggest that RAD23B and STAT3 gene perturbations could reduce sensitivity to histone deacetylase inhibitors in SS patients.

Candidate driver genes involved in genome maintenance and DNA repair in Sézary syndrome.

Sézary syndrome (SS) is a leukemic variant of cutaneous T-cell lymphoma (CTCL) and represents an ideal model for study of T-cell transformation. We describe whole-exome and single-nucleotide polymorphism array-based copy number analyses of CD4(+) tumor cells from untreated patients at diagnosis and targeted resequencing of 101 SS cases. A total of 824 somatic nonsynonymous gene variants were identified including indels, stop-gain/loss, splice variants, and recurrent gene variants indicative of considerable molecular heterogeneity. Driver genes identified using MutSigCV include POT1, which has not been previously reported in CTCL; and TP53 and DNMT3A, which were also identified consistent with previous reports. Mutations in PLCG1 were detected in 11% of tumors including novel variants not previously described in SS. This study is also the first to show BRCA2 defects in a significant proportion (14%) of SS tumors. Aberrations in PRKCQ were found to occur in 20% of tumors highlighting selection for activation of T-cell receptor/NF-κB signaling. A complex but consistent pattern of copy number variants (CNVs) was detected and many CNVs involved genes identified as putative drivers. Frequent defects involving the POT1 and ATM genes responsible for telomere maintenance were detected and may contribute to genomic instability in SS. Genomic aberrations identified were enriched for genes implicated in cell survival and fate, specifically PDGFR, ERK, JAK STAT, MAPK, and TCR/NF-κB signaling; epigenetic regulation (DNMT3A, ASLX3, TET1-3); and homologous recombination (RAD51C, BRCA2, POLD1). This study now provides the basis for a detailed functional analysis of malignant transformation of mature T cells and improved patient stratification and treatment.

Evaluation of Immunophenotypic and Molecular Biomarkers for Sézary Syndrome Using Standard Operating Procedures: A Multicenter Study of 59 Patients.

Differentiation between Sézary syndrome and erythrodermic inflammatory dermatoses can be challenging, and a number of studies have attempted to identify characteristic immunophenotypic changes and molecular biomarkers in Sézary cells that could be useful as additional diagnostic criteria. In this European multicenter study, the sensitivity and specificity of these immunophenotypic and recently proposed but unconfirmed molecular biomarkers in Sézary syndrome were investigated. Peripheral blood CD4(+) T cells from 59 patients with Sézary syndrome and 19 patients with erythrodermic inflammatory dermatoses were analyzed for cell surface proteins by flow cytometry and for copy number alterations and differential gene expression using custom-made quantitative PCR plates. Experiments were performed in duplicate in two independent centers using standard operating procedures with almost identical results. Sézary cells showed MYC gain (40%) and MNT loss (66%); up-regulation of DNM3 (75%), TWIST1 (69%), EPHA4 (66%), and PLS3 (66%); and down-regulation of STAT4 (91%). Loss of CD26 (≥80% CD4(+) T cells) and/or CD7 (≥40% CD4(+) T cells) and combination of altered expression of STAT4, TWIST1, and DNM3 or PLS3 could distinguish, respectively, 83% and 98% of patients with Sézary syndrome from patients with erythrodermic inflammatory dermatoses with 100% specificity. These additional diagnostic panels will be useful adjuncts in the differential diagnosis of Sézary syndrome versus erythrodermic inflammatory dermatoses.

Independent Loss of Methylthioadenosine Phosphorylase (MTAP) in Primary Cutaneous T-Cell Lymphoma.

Methylthioadenosine phosphorylase (MTAP) and the tumor suppressor genes CDKN2A-CDKN2B are frequently deleted in malignancies. The specific role of MTAP in cutaneous T-cell lymphoma subgroups, mycosis fungoides (MF) and Sézary syndrome (SS), is unknown. In 213 skin samples from patients with MF/SS, MTAP copy number loss (34%) was more frequent than CDKN2A (12%) in all cutaneous T-cell lymphoma stages using quantitative reverse transcription PCR. Importantly, in early stage MF, MTAP loss occurred independently of CDKN2A loss in 37% of samples. In peripheral blood mononuclear cells from patients with SS, codeletion with CDKN2A occurred in 18% of samples but loss of MTAP alone was uncommon. In CD4(+) cells from SS, reduced MTAP mRNA expression correlated with MTAP copy number loss (P < 0.01) but reduced MTAP expression was also detected in the absence of copy number loss. Deep sequencing of MTAP/CDKN2A-CDKN2B loci in 77 peripheral blood mononuclear cell DNA samples from patients with SS did not show any nonsynonymous mutations, but read-depth analysis suggested focal deletions consistent with MTAP and CDKN2A copy number loss detected with quantitative reverse transcription PCR. In a cutaneous T-cell lymphoma cell line, promoter hypermethylation was shown to downregulate MTAP expression and may represent a mechanism of MTAP inactivation. In conclusion, our findings suggest that there may be selection in early stages of MF for MTAP deletion within the cutaneous tumor microenvironment.

Regulation of T-plastin expression by promoter hypomethylation in primary cutaneous T-cell lymphoma.

T-plastin (PLS3) is an actin-bundling protein normally expressed in epithelial cells but absent in cells of hematopoietic origin. Aberrant PLS3 expression has been demonstrated in lymphocytes from Sézary syndrome (SS) patients and has been proposed as a biomarker for SS; however, the mechanism underlying dysregulation of PLS3 has not been determined. In this study, PLS3 mRNA expression was demonstrated in 21/35 (60%) SS patients and in 3/8 (38%) mycosis fungoides patients, all of whom had clonal blood involvement. No evidence for PLS3 mutations within coding or promoter regions was found, but significant hypomethylation of CpG dinucleotides 95-99 within the PLS3 CpG island was observed and this was restricted to the PLS3+ population. A polyclonal antibody specific to PLS3 was raised to examine coexpression of PLS3 with a panel of T-cell differentiation markers. All PLS3+ cells were CD3+CD4+ and CD26-, suggesting that loss of CD26 is consistently associated with gain of PLS3, whereas all other markers were distributed heterogeneously. However, a patient-specific TCR copy number assay also demonstrated heterogeneity in PLS3 expression in tumor cell populations. Importantly, our findings demonstrate PLS3 expression in the majority of SS patients and provide insight into the molecular regulation of PLS3 expression in CTCL.

Survival outcomes and prognostic factors in mycosis fungoides/Sézary syndrome: validation of the revised International Society for Cutaneous Lymphomas/European Organisation for Research and Treatment of Cancer staging proposal.

PURPOSE: We have analyzed the outcome of mycosis fungoides (MF) and Sézary syndrome (SS) patients using the recent International Society for Cutaneous Lymphomas (ISCL)/European Organisation for Research and Treatment of Cancer (EORTC) revised staging proposal. PATIENTS AND METHODS: Overall survival (OS), disease-specific survival (DSS), and risk of disease progression (RDP) were calculated for a cohort of 1,502 patients using univariate and multivariate models. RESULTS: The mean age at diagnosis was 54 years, and 71% of patients presented with early-stage disease. Disease progression occurred in 34%, and 26% of patients died due to MF/SS. A significant difference in survival and progression was noted for patients with early-stage disease having patches alone (T1a/T2a) compared with those having patches and plaques (T1b/T2b). Univariate analysis established that (1) advanced skin and overall clinical stage, increased age, male sex, increased lactate dehydrogenase (LDH), and large-cell transformation were associated with reduced survival and increased RDP; (2) hypopigmented MF, MF with lymphomatoid papulosis, and poikilodermatous MF were associated with improved survival and reduced RDP; and (3) folliculotropic MF was associated with an increased RDP. Multivariate analysis established that (1) advanced skin (T) stage, the presence in peripheral blood of the tumor clone without Sézary cells (B0b), increased LDH, and folliculotropic MF were independent predictors of poor survival and increased RDP; (2) large-cell transformation and tumor distribution were independent predictors of increased RDP only; and (3) N, M, and B stages; age; male sex; and poikilodermatous MF were only significant for survival. CONCLUSION: This study has validated the recently proposed ISCL/EORTC staging system and identified new prognostic factors.

Downregulation of Fas gene expression in Sézary syndrome is associated with promoter hypermethylation.

Sézary Syndrome (SS) is an aggressive leukemic variant of primary cutaneous T-cell lymphoma characterized by the presence of tumor or Sézary cells that generally display a mature memory T-cell immunophenotype. Sézary cells proliferate poorly and therefore their accumulation may be due to defective T-cell homeostasis involving resistance to apoptosis. In this study, we analyzed Fas expression in CD4+ lymphocytes at the mRNA and protein levels in a large cohort of SS patients as compared with healthy controls. Fas mRNA expression was dysregulated in 34/47 patients, with significant under- and overexpression of Fas mRNA detected in 21 and 13 patients respectively (P<0.01). Examination of cell-surface Fas expression showed correlation with the observed downregulation of mRNA in CD4+ T cells. Mutational analysis demonstrated that functional FAS gene mutations are rare. Moreover, 16 SS patients who showed significant under-expression of Fas mRNA also showed significant positional hypermethylation within the FAS CpG island, which was not present in healthy controls or SS patients determined to have normal or overexpression of Fas mRNA. These data demonstrate that dysregulation of Fas expression is a common feature of SS, and provide a rationale for targeted therapies to restore the extrinsic Fas-dependent apoptotic pathway in this malignancy.

Fine-mapping chromosomal loss at 9p21: correlation with prognosis in primary cutaneous diffuse large B-cell lymphoma, leg type.

Primary cutaneous diffuse large B-cell lymphoma, leg type (PCLBCL, LT) is the most aggressive type of primary cutaneous B-cell lymphoma. In a recent study on 12 patients it was found that inactivation of CDKN2A by either deletion of 9p21.3 or promoter hypermethylation is correlated with a worse prognosis. In the present EORTC multicenter study, skin biopsies of 64 PCLBCL, LT patients were analyzed by multiplex ligation-dependent probe amplification to validate these previous results and to fine-map the losses in this region. Although no minimal common region of loss could be identified, most homozygous loss was observed in the CDKN2A gene (43 of 64; 67%) encoding p16 and p14ARF. Promoter hypermethylation of p16 and p14ARF was found in six and zero cases, respectively. Survival was markedly different between patients with versus without aberrations in the CDKN2A gene (5-year disease-specific survival 43 versus 70%; P=0.06). In conclusion, our results confirm that deletion of chromosome 9p21.3 is found in a considerable proportion of PCLBCL, LT patients and that inactivation of the CDKN2A gene is associated with an unfavorable prognosis. In most patients the deletion involves a large area of at least several kilobase pairs instead of a small minimal common region.

A genomic and expression study of AP-1 in primary cutaneous T-cell lymphoma: evidence for dysregulated expression of JUNB and JUND in MF and SS.

Activator protein 1 (AP-1) consists of a group of transcription factors including the JUN and FOS family proteins with diverse biological functions. This study assessed the genomic and expression status of the AP-1 transcription factors in primary cutaneous T-cell lymphoma (CTCL) by using immunohistochemistry (IHC), Affymetrix expression microarray, real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and fluorescent in situ hybridization (FISH). IHC showed JUNB protein expression in tumor cells from 17 of 33 cases of Sezary syndrome (SS) and JUND protein expression in 16 of 23 mycosis fungoides cases. There was no correlation between JUNB and CD30 expression. However, 7 of 12 JUNB-positive SS cases expressed both phosphorylated and total extracellular signal-regulated kinase (ERK) 1/2 mitogen-activated protein kinase (MAPK) proteins. Expression microarray showed over threefold increased expression of JUNB in three of six SS patients and similar findings were also noted after re-analysis of previously published data. Real-time RT-PCR confirmed the overexpression of JUNB in four SS cases and of JUND in three of four cases. FISH showed increased JUNB copy number in four of seven SS cases. These findings suggest that deregulation of AP-1 expression in CTCL is the result of aberrant expression of JUNB and possible JUND resulting from genomic amplification and constitutive activation of ERK1/2 MAPK in this type of lymphoma.

Lack of suppressive CD4+CD25+FOXP3+ T cells in advanced stages of primary cutaneous T-cell lymphoma.

Mycosis fungoides and its leukemic variant, Sezary syndrome, are the most common primary cutaneous T-cell lymphomas (CTCLs). In an ex vivo study, we investigated the percentage, phenotype, and suppressive function of CD4+CD25+ regulatory T cells (Tregs) from peripheral blood of CTCL patients. The percentage of Tregs did not differ significantly between patients and controls. Functional assays demonstrated a dichotomy in Treg function: in four out of 10 patients CD4+CD25+ T cells were incapable of suppressing autologous CD4+CD25- T-cell proliferation, whereas suppressive function was intact in the other six patients. Suppressive activity of Tregs inversely correlated with the peripheral blood tumor burden. T-plastin gene expression, used as a Sezary cell marker, confirmed that Sezary cells were heterogeneous for CD25 expression. Mixed lymphocyte reactions demonstrated that CD4+CD25- T cells from patients who lacked functional Tregs were susceptible to suppression by Tregs from healthy controls, and had not become suppressive themselves. Furthermore, we found reduced expression of Foxp3 in the CD4+CD25+ Tregs of these patients relative to the other six CTCL patients and controls. Our findings thus indicate a dysfunction of peripheral Tregs in certain CTCL patients, which correlates with tumor burden.

Molecular analysis of T-cell receptor beta genes in cutaneous T-cell lymphoma reveals Jbeta1 bias.

Molecular characterization of T-cell receptor junctional region sequences in cutaneous T-cell lymphoma had not been previously reported. We have examined in detail the features of the T-cell receptor beta (TCRB) gene rearrangements in 20 individuals with well-defined stages of cutaneous T-cell lymphoma (CTCL) comprising 10 cases with early-stage mycosis fungoides (MF) and 10 cases with late-stage MF or Sezary syndrome. Using BIOMED-2 PCR primers, we detected a high frequency of clonally rearranged TCR gamma and TCRB genes (17/20 and 15/20 cases, respectively). We carried out sequencing analysis of each complete clonal variable (V)beta-diversity (D)beta-joining(J)beta fingerprint generated by PCR amplification, and determined the primary structure of the Vbeta-Dbeta-Jbeta junctional regions. We observed considerable diversity in the T-cell receptor Vbeta gene usage and complementarity-determining region 3 loops. Although we found that TCRB gene usage in CTCL and normal individuals share common features, our analysis also revealed preferential usage of Jbeta1 genes in all cases with advanced stages of disease.

Signal transducer and activator of transcription (STAT) signalling and T-cell lymphomas.

Interaction of cytokines with their cognate receptors leads to the activation of latent transcription factors - the signal transducers and activators of transcription (STAT) proteins - whose biological activities ultimately regulate many critical aspects of cell growth, survival and differentiation. Dysregulation of the JAK-STAT pathway is frequently observed in many primary human tumours, reflecting the importance of this pathway in the maintenance of cellular integrity. Here we review the current progress in STAT structure and function, and the contribution of STAT signalling to the pathogenesis of T-cell lymphomas.

Fine mapping of chromosome 10q deletions in mycosis fungoides and sezary syndrome: identification of two discrete regions of deletion at 10q23.33-24.1 and 10q24.33-25.1.

Previous cytogenetic studies in mycosis fungoides (MF) and Sezary syndrome (SS) have identified a large and poorly defined area of chromosomal deletion on chromosome 10q. We report an extensive fine-mapping allelotyping study using 19 microsatellite markers in the region 10q22.3-10q26.13. Allelic loss was identified by loss of heterozygosity analysis in 26 of 60 (43%) cases: 15 of 45 (33%) with MF and 11 of 15 (73%) with SS. MF and SS samples showed similar patterns of allelic loss with the identification of two discrete regions of deletion which were mutually exclusive in all but two cases. Within the first region of deletion at 10q23.33-10q24.1, around microsatellite marker D10S185 (2.77 Mb), 23 genes were identified, including three (KIF11, HHEX, and HELLS) with functions that, if dysregulated, could be critical in MF and SS. The second region of deletion, 10q24.33-10q25.1, around microsatellite marker D10S530 (3.92 Mb), encodes 11 genes, the majority of which have poorly identified functions. This extensive allelotyping study provides the basis for future highly selective candidate gene analyses.

Dysregulated expression of COOH-terminally truncated Stat5 and loss of IL2-inducible Stat5-dependent gene expression in Sezary Syndrome.

Sezary Syndrome (SzS) is a leukemic variant of cutaneous T-cell lymphoma characterized by the accumulation of clonal neoplastic CD4+ T cells. The signal transducers and activators of transcription (STAT) family members, Stat5a and Stat5b, play an important role in regulating T-cell activation. Recent studies have shown that inappropriate activation of STATs occurs frequently in a wide variety of human cancers. Here we examine the functional status of Stat5 proteins in SzS as compared with healthy donors. Western blotting demonstrates that in cytoplasmic extracts of unstimulated T cells from healthy controls two isoforms of Stat5, full-length and a COOH-terminal truncated isoform, termed Stat5(t), are present. However, bandshift assays demonstrate that only Stat5(t) translocates to the nucleus and binds DNA on IL-2 stimulation. In contrast, preactivated T cells express only full-length Stat5, which is functionally activated on IL-2 stimulation. Analysis of Stat5 protein isoforms from five of five SzS patients revealed predominant aberrant expression of Stat5(t) in preactivated peripheral blood mononuclear cell. Furthermore, patients showed preferential IL-2-induced DNA binding of Stat5(t). Consistent with the inappropriate activation of Stat5(t) in SzS patients, real-time PCR revealed that IL-2-induced mRNA expression of the Stat5 target genes, Bcl-2, PIM-1, and CISH were markedly reduced. These data indicate that functional Stat5 isoform expression is regulated by T-cell activation status and that dysregulated expression of Stat5(t) in malignant T cells in SzS can suppress Stat5-dependent gene expression. Thus, aberrant expression of Stat5(t) may be one mechanism that contributes to the cellular transformation of T cells in this disease.

Microsatellite instability is associated with hypermethylation of the hMLH1 gene and reduced gene expression in mycosis fungoides.

Fifty-one mycosis fungoides samples were analyzed for microsatellite instability (MSI) using the panel of markers recommended for hereditary nonpolyposis colorectal cancer kindred and a panel we designed for cutaneous T cell lymphoma in order to compare detection rates and determine if MSI is a genome-wide phenomenon. Samples demonstrating MSI were analyzed for abnormalities of the hMLH1 gene including loss of heterozygosity, mutations, and promoter hypermethylation. MSI was detected in 16% using the hereditary nonpolyposis colorectal cancer panel and 22% with the cutaneous T cell lymphoma panel. Overall, 27% demonstrated MSI and 73% had a stable phenotype. hMLH1 gene studies did not detect loss of heterozygosity or reveal any mutations. Promoter hypermethylation was detected in nine of 14 patients with MSI, however (64%). In addition hMLH1 and hMSH2 protein expression was studied using immunohistochemical techniques. Five of nine patients with MSI and hMLH1 promoter methylation showed abnormal hMLH1 protein expression with normal hMSH2 gene expression. All other patients tested demonstrated normal hMLH1 and hMSH2 protein expression. MSI was found to be more prevalent in tumor stage mycosis fungoides (47%) than early stage disease (20%) and was associated with an older age of onset of mycosis fungoides. MSI may be a consequence of hMLH1 promoter hypermethylation in mycosis fungoides patients and may prevent transcription in a subset of patients. This suggests that the development of a mutator phenotype may contribute to disease progression in mycosis fungoides.

Regulation of epithelial syndecan-1 expression by inflammatory cytokines.

Syndecan-1 is expressed on the basolateral surface of columnar epithelium and contributes to wound repair by facilitating increased growth factor binding. Inflammatory bowel disease (IBD) is associated with reduced syndecan-1 expression in areas of inflamed mucosa that is likely to impair mucosal healing. Reduced syndecan-1 expression in IBD may be related to the presence of increased inflammatory cytokines. To test this hypothesis, monolayers of HT29 and T84 colonic epithelial cells were stimulated with tumour necrosis factor (TNF)-alpha, interleukin (IL)-1beta or IL-6. Stimulation of HT29 cells with TNF-alpha and IL-1beta resulted in reversible down-regulation of syndecan-1 at both protein and mRNA levels but little effect was observed with IL-6. Loss of syndecan-1 expression was caused by shedding of the ectodomain as revealed by increased levels of soluble syndecan-1 measured in the conditioned medium of stimulated cells. No increase in cytoplasmic staining accompanied the loss of cell surface syndecan-1 expression. TNF-alpha and IL-1beta are capable of down-regulating syndecan-1 expression and may account in part for the reduced expression of syndecan-1 seen in IBD.

The identification, characterization, and distribution of guinea pig CCR4 and epitope mapping of a blocking antibody.

Th2 lymphocytes play a central role in the control and maintenance of allergic inflammation. The chemokine receptor CCR4 is preferentially expressed on the surface of Th2 lymphocytes polarised in vitro. However, CCR4 is found on the surface of a significant proportion of circulating memory T lymphocytes, some of which are capable of producing the Th1-associated cytokine interferon gamma. To investigate the function of CCR4 on guinea pig (gp) T lymphocytes, we identified the open-reading frame of gpCCR4, which encodes a 361-amino acid protein with 88 and 81% amino acid identity to human and murine CCR4 sequences, respectively. Cells transfected with gpCCR4 migrated toward the human and murine orthologues of the CCR4 ligands, macrophage-derived chemokine and thymus and activation-regulated chemokine. Surface expression of CCR4, using an anti-human CCR4 monoclonal antibody, 10E4, was detected on approximately 12% of guinea pig peripheral blood T helper cells, and CCR4(+) guinea pig thymocytes were detected in low numbers. However, CCR4(+) T helper cells constituted approximately 9% of the T lymphocyte population within the normal guinea pig lung and 52% of the guinea pig bronchoalveolar lavage fluid, which is consistent with a role for CCR4 in T lymphocyte development and trafficking through normal tissues. Subsequent analysis of chimeric chemokine receptors indicated that 10E4, a functional inhibitor of gpCCR4 responses, recognized the amino terminus of CCR4.