Associations between levels of insulin-like growth factor 1 and sinusoidal obstruction syndrome after allogeneic haematopoietic stem cell transplantation.

Allogeneic myeloablative haematopoietic stem cell transplantation (HSCT) is challenged by severe adverse events, as cytotoxic effects of the conditioning may result in systemic inflammation, leaky epithelial barriers and organ toxicities, contributing to treatment-related morbidity and mortality. We hypothesised that insulin-like growth factor-1 (IGF-1), a mediator of growth and proliferation of various tissues, may attenuate chemotherapy-induced tissue damage after HSCT. We prospectively measured plasma levels of IGF-1 and its binding protein 3 (IGFBP-3) in 41 patients undergoing myeloablative HSCT. IGF-1 and IGFBP-3 levels were inversely correlated with C-reactive protein and interleukin-6 levels post HSCT. In multivariate analyses, low levels of IGF-1 and IGFBP-3 before conditioning were associated with increased risk of developing sinusoidal obstruction syndrome (SOS; OR=5.00 per 1 SDS decrease in IGF-1 (95% CI: 1.45-16.67), P=0.011 and OR=5.00 (1.37-20.00), P=0.015, respectively). Furthermore, low pre-transplant levels of IGF-1 and IGFBP-3 were associated with increased fluid retention during the first 21 days post transplant (OR=7.69 (95% CI: 1.59-33.33), P=0.012, and OR=2.94 (1.03-8.33), P=0.045). These data suggest that high levels of IGF-1 and IGFBP-3 may have a protective effect against fluid retention and SOS, possibly by attenuating systemic inflammation, and may prove useful as predictive biomarkers of SOS.

Children's white blood cell counts in relation to developmental exposures to methylmercury and persistent organic pollutants.

BACKGROUND: To explore possible markers of developmental immunotoxicity, we prospectively examined 56 children to determine associations between exposures to methylmercury and persistent organic pollutants since birth and the comprehensive differential counts of white blood cells (WBC) at age 5 years. MATERIALS AND METHODS: Extended differential count included: neutrophils, eosinophils, basophils, lymphocytes (includingT cells, NK cells, and B cells), and monocytes. Organochlorine compounds (OCs) including polychlorinated biphenyls (PCBs) and pesticides, five perfluoroalkyl substances (PFASs), and total mercury (Hg) were measured in maternal (n=56) and children's blood at 18 months (n=42) and 5 years (n=54). We constructed latent functions for exposures at three different ages using factor analyses and applied structural equation models adjusted for covariates. RESULTS: Prenatal mercury exposure was associated with depleted total WBC, especially for lymphocytes, where a one standard deviation (SD) increase in the exposure was associated with a decrease by 23% SD (95% CI: -43, -4) in the cell count. Prenatal exposure to OCs was marginally associated with decreases in neutrophil counts. In contrast, the 5-year PFASs concentrations were associated with higher basophil counts (B=46% SD, 95% CI: 13, 79). Significantly reduced subpopulations of lymphocytes such as B cells, CD4-positive T helper cells and CD4 positive recent thymic emigrants may suggest cellular immunity effects and dysregulation of T-cell mediated immunity. CONCLUSION: Developmental exposure to environmental immunotoxicants appears to have different impacts on WBC counts in childhood.

T cell reconstitution in allogeneic haematopoietic stem cell transplantation: prognostic significance of plasma interleukin-7.

Infections and acute graft-versus-host disease (aGVHD) are major causes of treatment-related mortality and morbidity following allogeneic haematopoietic stem cell transplantation (HSCT). Both complications depend on reconstitution of the T-lymphocyte population based on donor T cells. Although it is well established that Interleukin-7 (IL-7) is a cytokine essential for de novo T cell development in the thymus and homoeostatic peripheral expansion of T cells, associations between circulating levels of IL-7 and T cell reconstitution following HSCT have not been investigated previously. We prospectively measured IL-7 levels in 81 patients undergoing myeloablative HSCT with either sibling donor or an unrelated donor. Plasma IL-7 levels peaked at day +7 post-transplant (1.3-82.4 pg/ml), at the time of maximal lymphopaenia. In multivariate analysis, peak levels of IL-7 were significantly higher in patients treated with anti-thymocyte globulin (ATG) compared with those not treated with ATG (P = 0.0079). IL-7 levels at day +7 were negatively associated with T cell counts at day +30 to +60 (at day +60: CD3(+) : ß = -10.6 × 10(6) cells/l, P = 0.0030; CD8(+) : ß = -8.4 × 10(6) cells/l, P = 0.061; CD4(+) : ß = -2.1 × 10(6) cells/l, P = 0.062) in multivariate analyses. In adults, high IL-7 levels were associated with increased risk of grade II-IV aGVHD (OR = 5.4, P = 0.036) and reduced overall survival (P = 0.046). The present data indicate that high plasma levels of IL-7 in the early post-transplant period are predictive for slow T cell reconstitution, increased risk of aGVHD and increased mortality following HSCT.

The influence of interleukin-7 receptor α-chain haplotypes on outcome after allogeneic hematopoietic cell transplantation.

We investigated the influence of IL-7 receptor α-chain (IL-7Rα) gene haplotypes in donors on the outcome of haematopoietic cell transplantation (HCT). Unlike the association between single donor SNPs and HCT outcome found previously, only trends towards association were found here, due to 'dilution' of SNPs into haplotypes.

Polymorphism in the interleukin-7 receptor-alpha and outcome after allogeneic hematopoietic cell transplantation with matched unrelated donor.

Interleukin-7 (IL-7) is essential for T cell development in the thymus and maintenance of peripheral T cells. The α-chain of the IL-7R is polymorphic with the existence of SNPs that give rise to non-synonymous amino acid substitutions. We previously found an association between donor genotypes and increased treatment-related mortality (TRM) (rs1494555G) and acute graft versus host disease (aGvHD) (rs1494555G and rs1494558T) after hematopoietic cell transplantation (HCT). Some studies have confirmed an association between rs6897932C and multiple sclerosis. In this study, we evaluated the prognostic significance of IL-7Rα SNP genotypes in 590-recipient/donor pairs that received HLA-matched unrelated donor HCT for haematological malignancies. Consistent with the primary studies, the rs1494555GG and rs1494558TT genotypes of the donor were associated with aGvHD and chronic GvHD in the univariate analysis. The Tallele of rs6897932 was suggestive of an association with increased frequency of relapse by univariate analysis (P = 0.017) and multivariate analysis (P = 0.015). In conclusion, this study provides further evidence of a role of the IL-7 pathway and IL-7Rα SNPs in HCT.

The HLA-DP2 protein binds the immunodominant epitope from myelin basic protein, MBP85-99, with high affinity.

Myelin basic protein (MBP) is a candidate autoantigen in multiple sclerosis (MS). The immunodominant epitope for T-cell responses is assigned to the amino acid sequence MBP84-102, which binds to human leukocyte antigen (HLA)-DR2a (DRB5*0101) and HLA-DR2b (DRB1*1501) of the HLA-DR2 haplotype carrying the strongest genetic association with MS. In contrast with HLA-DR and -DQ molecules, HLA-DP molecules are poorly characterized with respect to the binding of self-peptides. We show here that HLA-DP2 binds MBP85-99 with high affinity, and that the amino acid residues in position MBP91, MBP92 and MBP93 are influencing the binding, as shown by alanine scans. We further used a series of truncated peptides to identify the core of the binding. Moving the frame along the peptide from residues 87-97 to 89-99 progressively decreased the binding affinity for HLA-DP2, while moving further towards the C-terminal completely abrogated the binding of peptides to HLA-DP2. The data suggest that the docking of the MBP85-99 peptide into the HLA-DP2 groove is dependent on MBP88V and MBP89V and may use either of them as primary anchor for the p1 position. HLA-DP2 might thus present the MBP85-99 peptide in the same register as the HLA-DRB1*1501, where the MBP89V is preferred as the p1 anchor. Notably, full-length MBP was able to compete for peptide binding with an affinity similar to that seen for the high-affinity binding peptides, DRα170-83 and IIP53-65. In summary, the HLA-DP2 molecule binds the immunodominant epitope in MS, MBP85-99, possibly in more than one register.

Expression of full-length and splice forms of FoxP3 in rheumatoid arthritis.

OBJECTIVE: The aim of our study was to compare the presence of full-length and alternative splice forms of FoxP3 mRNA in CD4 cells from rheumatoid arthritis (RA) patients and healthy controls. METHODS: A quantitative real-time polymerase chain reaction (QRT-PCR) method was used to measure the amount of FoxP3 mRNA full-length and splice forms. CD4-positive T cells were isolated from peripheral blood from 50 RA patients by immunomagnetic separation, and the FoxP3 mRNA expression was compared with the results from 10 healthy controls. RESULTS: We observed an increased expression of full-length FoxP3 mRNA in RA patients when compared to healthy controls, as well as an increase in CD25 mRNA expression, but no corresponding increase in CTLA-4 mRNA expression. The presence of an alternative splice form of FoxP3 lacking exon 2 was confirmed in both RA patients and healthy controls, but with no significant difference in expression between the two groups. There was a positive correlation between the amount of FoxP3 mRNA and the clinical inflammation parameters C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR), and a negative correlation between FoxP3 mRNA and the dose of methotrexate (MTX) given to the patients. CONCLUSION: RA patients express more full-length FoxP3 than healthy controls in peripheral blood CD4-positive cells, suggesting an increased number of regulatory T cells (Tregs). However, no concomitant increase in CTLA-4 expression was seen. We therefore propose that the Tregs are left unable to suppress the ongoing inflammation due to a deficiency in CTLA-4 needed for cell contact-dependent suppression.

Breakthrough disease during interferon-[beta] therapy in MS: No signs of impaired biologic response.

BACKGROUND: Disease activity is highly variable in patients with multiple sclerosis (MS), both untreated and during interferon (IFN)-beta therapy. Breakthrough disease is often regarded as treatment failure; however, apart from neutralizing antibodies (NAbs), no blood biomarkers have been established as reliable indicators of treatment response, despite substantial, biologically measurable effects. We studied the biologic response to treatment in a cohort of NAb-negative patients to test whether difference in responsiveness could segregate patients with and without breakthrough disease during therapy. METHODS: Gene expression in blood cells from 23 patients with relapsing-remitting MS was analyzed by microarray and PCR. Samples were collected pretreatment and 9-12 hours after IFNbeta injection at 3 and 6 months' treatment. Definition of breakthrough disease was based on the occurrence of relapses, disability progression, or subclinical activity on 3T MRI at 3 and 6 months. RESULTS: Sixteen patients had breakthrough disease and 7 patients were stable. Microarray and PCR showed marked effects of IFNbeta on gene expression profiles, but biologic responses did not differ between patients with breakthrough disease and stable patients. However, pretreatment variables did differ: patients with breakthrough disease had lower baseline IL10 expression, more gadolinium-enhancing lesions, and a higher number and volume of T2 lesions. CONCLUSIONS: Breakthrough disease during interferon (IFN)-beta treatment is not paralleled by differences in biologic responsiveness to treatment in NAb-negative patients; most likely, the spontaneously occurring variation in underlying disease activity between patients causes the varying level of breakthrough disease observed in IFNbeta-treated patients with multiple sclerosis.

Low thymic output in the 22q11.2 deletion syndrome measured by CCR9+CD45RA+ T cell counts and T cell receptor rearrangement excision circles.

Thymic hypoplasia is a frequent feature of the 22q11.2 deletion syndrome, but we know little about patients' age-related thymic output and long-term consequences for their immune system. We measured the expression of T cell receptor rearrangement excision circles (TREC) and used flow cytometry for direct subtyping of recent thymic emigrant (RTE)-related T cells in 43 patients (aged 1-54 years; median 9 years) from all over Norway and in age-matched healthy controls. Thymic volumes were estimated by ultrasound in patients. TREC levels correlated well with RTE-related T cells defined by co-expression of CD3, CD45RA and CCR9 (r=0.84) as well as with the CD4+ and CD8+ T cell subtypes. RTE-related T cell counts also paralleled age-related TREC reductions. CD45RA+ T cells correlated well with absolute counts of CD4+ (r=0.87) and CD8+ (r=0.75) RTE-related T cells. Apart from CD45RA- T cells, all T cell subsets were lower in patients than in controls. Thymic volumes correlated better with RTE-related cells (r=0.46) than with TREC levels (r=0.38). RTE-related T cells and TREC levels also correlated well (r=0.88) in patients without an identifiable thymus. Production of RTEs is impaired in patients with a 22q11.2 deletion, and CCR9 appears to be a good marker for RTE-related T cells.

HIV-infected patients with a large thymus maintain higher CD4 counts in a 5-year follow-up study of patients treated with highly active antiretroviral therapy.

CD4 recovery in HIV-infected patients treated with highly active antiretroviral therapy (HAART) is in part believed to be dependent on the degree of preserved thymic function. We investigated whether the thymus has a prolonged effect on CD4 recovery. Total and naïve CD4 counts as well as thymic output determined as the number of CD4 + cells containing T-cell receptor-rearrangement excision DNA circles were measured prospectively in 25 HIV-infected patients with known thymic size during 5 years of HAART. Patients with larger thymic size had at all time points of follow-up significantly higher CD4 counts than patients with minimal thymic size (P = 0.0036). The CD4 increase from time of initiation of HAART until 6 months of follow-up differed significantly between the two thymic groups (P = 0.045), but did not at later time points. Thymic output remained significantly higher in patients with larger thymic size at follow-up. However, no difference in the increase in thymic output was seen between thymic groups. In conclusion, the importance of the thymus to the rate of cellular restoration seems primarily to lie within the first two years of HAART. However, patients with larger thymic size are able to maintain higher CD4 counts even after 5 years of HAART.

Identification of new sensitive biomarkers for the in vivo response to interferon-beta treatment in multiple sclerosis using DNA-array evaluation.

OBJECTIVE: Neutralizing antibodies (NAbs) occur in a proportion of multiple sclerosis (MS) patients treated with interferon (IFN)-beta. NAbs impair the effect of treatment. The biological effect of IFN-beta can be measured as the induction of the myxovirus resistance protein A (MxA) molecule. However, other markers could be more sensitive for evaluating the response to IFN-beta. We used DNA array analysis to identify genes that are strongly induced in blood cells by IFN-beta, and measured their expression in MS patients with different NAb levels. METHODS: Gene expression was studied on DNA arrays in untreated patients, in NAb negative patients, and in MS patients with varying NAb levels 9-12 h and 36-48 h after IFN-beta administration. The expression of selected genes was measured by real-time PCR. NAb levels were assessed by a cytopathic effect assay. RESULTS: Several hundred genes were induced 9-12 h after an injection of IFN-beta. The molecules CXCL10, CCL2 and IFI27 were among the most strongly induced. Gene induction was generally much less pronounced after 36-48 h, but IFI27 remained strongly induced. The strong induction of these molecules and MxA was confirmed by real-time PCR. Induction of MxA, CCL2, CXCL10 and IFI27 was reduced in patients with low NAb levels and lost in patients with intermediate/high NAb levels. CONCLUSION: We identify IFI27, CCL2 and CXCL10 as sensitive biomarkers for the response to IFN-beta. The expression of these markers adequately reflects bioactivity of IFN-ss as documented by the decreased induction in low NAb-positive patients and the lost induction in patients with moderate/high NAb levels.

Limited impact of the thymus on immunological recovery during and after chemotherapy in patients with diffuse large B-cell lymphoma.

To investigate the impact of thymus on immunological recovery after dose-dense chemotherapy a prospective study of 17 patients diagnosed with diffuse large B-cell lymphoma (DLBCL) was conducted. Patients were monitored before, during and until 3 months after chemotherapy. The thymus was visualized using computer tomographic scans. Patients were divided into two groups according to thymic size, one group comprising of patients without detectable thymus and one group of patients with detectable thymus. Naïve CD4 and CD8 counts were measured by flow cytometry, and to measure thymic output determination of CD4+ cells containing T-cell receptor excision circles (TREC) was done. During chemotherapy, the naïve CD4 count decreased significantly as did the CD4-TREC%. Significant difference in recovery of naïve CD4 counts between patients with detectable and undetectable thymic tissue during treatment with chemotherapy was not found. CD4-TREC% was associated with lower age. It was not possible to demonstrate an association between thymic size and recovery of the naïve CD4+ cells. The study terminated 3 months after the last cycle of chemotherapy, and at that time point the naïve CD4 counts and the CD4-TREC% had not returned to pretreatment levels. However, patients with detectable thymic tissue had higher naïve CD4 counts after the first cycles of chemotherapy, suggesting that these patients may be less susceptible to infectious complications related to chemotherapy.

Increased levels of regulatory T cells (Tregs) in human immunodeficiency virus-infected patients after 5 years of highly active anti-retroviral therapy may be due to increased thymic production of naive Tregs.

This study determines levels of regulatory T cells (T(regs)), naive T(regs), immune activation and cytokine patterns in 15 adult human immunodeficiency virus (HIV)-infected patients receiving prolonged highly active anti-retroviral therapy (HAART) who have known thymic output, and explores if naive T(regs) may represent recent thymic emigrant T(regs). HIV-infected patients treated with HAART with a median of 1 and 5 years were compared with healthy controls. Percentages of T(regs) (CD3(+)CD4(+)CD25(+)CD127(low)), naive T(regs) (CD3(+)CD4(+)CD25(+)CD45RA(+)) and activation markers (CD38(+)human leucocyte antigen D-related) were determined by flow cytometry. Forkhead box P3 mRNA expression and T cell receptor excision circles (T(REC)) content in CD4(+) cells were determined by polymerase chain reaction and cytokines analysed with Luminex technology. Levels of T(regs) were significantly higher in HIV-infected patients compared with controls, both after 1 and 5 years of HAART (P<0.001), despite fully suppressed HIV-RNA and normalization of both CD4 counts, immune activation and cytokine patterns. Furthermore, levels of naive T(regs) were elevated significantly in HIV-infected patients (P<0.001) and were associated with thymic output measured as the T(REC) frequency in CD4(+) cells (P=0.038). In summary, T(reg) levels in HIV-infected patients are elevated even after 5 years of HAART. Increased thymic production of naive T(regs) may contribute to higher T(reg) levels in HIV-infection.

Regulatory T cells in human immunodeficiency virus-infected patients are elevated and independent of immunological and virological status, as well as initiation of highly active anti-retroviral therapy.

Infection with human immunodeficiency virus (HIV) causes a dysregulation of the immune system. This is caused by HIV-specific as well as non-specific mechanisms and has not been explained fully. In particular, knowledge is lacking about the potential role of host-mediated immunosuppressive mechanisms. During recent years it has become evident that a subpopulation of T cells [T regulatory (T(regs))] play a major role in sustaining tolerance to self-antigens. To investigate the influence of initiation of highly active anti-retroviral therapy (HAART) on the T(reg) level in HIV-infected patients we have conducted a prospective study enrolling treatment-naive HIV-infected patients just prior to starting treatment with HAART, measuring levels of T(regs) by flow cytometry and mRNA expression of forkhead box P3 (FoxP3) at weeks 0, 4, 12 and 24 of treatment. In this prospective study neither the percentage of CD4(+)CD25(high+) nor the expression of FoxP3 changed significantly during 24 weeks of HAART. Furthermore, HIV patients have higher T(regs) measured as percentages of CD4(+)CD25(high+) cells paralleled by higher levels of FoxP3 compared with healthy controls. The elevated level of T(regs) was found to be independent of both immunological and virological status, indicating that initiation of HAART has minor effects on the T(reg) level in HIV-infected patients.

Malignant Tregs express low molecular splice forms of FOXP3 in Sézary syndrome.

Sézary syndrome (SS) is an aggressive variant of cutaneous T-cell lymphoma. During disease progression, immunodeficiency develops; however, the underlying molecular and cellular mechanisms are not fully understood. Here, we study the regulatory T cell (Treg) function and the expression of FOXP3 in SS. We demonstrate that malignant T cells in 8 of 15 patients stain positive with an anti-FOXP3 antibody. Western blotting analysis shows expression of two low molecular splice forms of FOXP3, but not of wild-type (wt) FOXP3. The malignant T cells produce interleukin-10 and TGF-beta and suppress the growth of non-malignant T cells. The Treg phenotype and the production of suppressive cytokines are driven by aberrant activation of Jak3 independent of the FOXP3 splice forms. In contrast to wt FOXP3, the low molecular splice forms of FOXP3 have no inhibitory effect on nuclear factor-kappaB (NF-kappaB) activity in reporter assays which is in keeping with a constitutive NF-kappaB activity in the malignant T cells. In conclusion, we show that the malignant T cells express low molecular splice forms of FOXP3 and function as Tregs. Furthermore, we provide evidence that FOXP3 splice forms are functionally different from wt FOXP3 and not involved in the execution of the suppressive function. Thus, this is the first description of FOXP3 splice forms in human disease.

Gene expression analysis of interferon-beta treatment in multiple sclerosis.

Treatment with interferon-beta (IFN-beta) induces the expression of hundreds of genes in blood mononuclear cells, and the expression of several genes has been proposed as a marker of the effect of treatment with IFN-beta. However, to date no molecules have been identified that are stably induced by treatment with IFN-beta. We use DNA microarrays to study gene expression in 10 multiple sclerosis (MS) patients who began de novo treatment with IFN-beta. After the first injection of IFN-beta, the expression of 74 out of 3428 genes changed at least two-fold and statistically significantly (after Bonferroni correction). In contrast, we observed no persisting effects of IFN-beta on gene expression. Among the most strongly induced genes was MXA, which has been used in previous biomarker studies in MS. In addition, the study identified the induction of LGALS9 and TCIR1G, involved in negative regulation of T helper type I immunity and T-cell activation, as novel effects of IFN-beta therapy in MS.

Haematopoietic cell transplantation with non-myeloablative conditioning in Denmark: disease-specific outcome, complications and hospitalization requirements of the first 100 transplants.

We analysed the outcome and hospitalization requirements of the first 100 patients (Hodgkin's disease (HD), N=13; multiple myeloma (MM), N=14; CLL, N=12; non-Hodgkin's lymphoma (NHL), N=17; myelodysplastic syndrome (MDS), N=18; AML, N=24 and CML, N=2) treated in Denmark with haematopoietic cell transplantation after non-myeloablative conditioning with TBI 2 Gy+/-fludarabine. The cumulative incidence of acute GVHD grade II-IV and extensive chronic GVHD was 67 and 49%. After a median follow-up of 534 days, the overall survival, PFS, relapse-related mortality and treatment-related mortality were 59, 50, 25 and 17%, respectively. Patients with CLL, NHL, AML and MDS with <5% blasts at any time had a favourable outcome with a PFS of 61-71%. Patients with MM, HD and MDS and a history of > or =5% blasts had a less favourable outcome with a PFS of 19-38% (P=0.001). The cumulative incidence of discontinuation of immunosuppression was 37%. During the first and second year post transplant, patients experienced a mean of 41 and 13 outpatient clinic visits, and 53 and 16 days of hospitalization. Sixteen patients were admitted to the intensive care unit, of whom eight are still alive. In conclusion, transplantation outcomes were encouraging, but complications requiring admission and outpatient clinic visits occur frequently post transplant.

X chromosome inactivation in females with multiple sclerosis.

The aetiology of multiple sclerosis (MS) is unknown. Autoimmune mechanisms are most probably involved. Loss of immunological tolerance to self-antigens is a common feature of autoimmune disorders. Response to X-linked self-antigens could be influenced by X-chromosome inactivation, and contribute to the gender bias observed in autoimmune disorders. Previous studies have indicated an association between skewed X inactivation and autoimmune thyroid disease and scleroderma. To investigate a potential role of X inactivation in MS, we compared the X-inactivation pattern in 568 female MS patients with controls. We found no difference in degree of skewing between patients (median 64%) and controls (median 65%) (P = 0.474). The X-inactivation pattern did thus not explain the female predominance of MS patients in general. As the aetiology of different subgroups of MS may differ, patients were grouped according to disease course: relapsing-remitting (RR-MS), secondary progressive (SP-MS) and primary progressive (PP-MS). A comparison of the X-inactivation pattern between subgroups indicated a possible difference in degree of skewing between patients with a progressive versus a relapsing course (P = 0.05).

FOXP3+ regulatory T cells in cutaneous T-cell lymphomas: association with disease stage and survival.

FOXP3 is a unique marker for CD4+CD25+ regulatory T cells (Tregs). In solid tumours, high numbers of Tregs are associated with a poor prognosis. Knowledge about the implications of Tregs for the behaviour of haematological malignancies is limited. In this study, skin biopsies from 86 patients with mycosis fungoides (MF) and cutaneous T-cell lymphoma (CTCL) unspecified were analysed for the expression of FOXP3 on tumour cells and tumour-infiltrating Tregs. Labelling of above 10% of the neoplastic cells was seen in one case classified as an aggressive epidermotropic CD8+ cytotoxic CTCL. In the remaining 85 cases, the atypical neoplastic infiltrate was either FOXP3 negative (n=80) or contained only very occasional weakly positive cells (n=5). By contrast, all biopsies showed varying numbers of strongly FOXP3+ tumour-infiltrating Tregs. MF with early or infiltrated plaques had significantly higher numbers of FOXP3+ Tregs than CTCL unspecified or advanced MF with tumours or transformation to large cell lymphoma. An analysis of all patients demonstrated that increasing numbers of FOXP3+ Tregs were associated with improved survival in both MF and CTCL unspecified. In conclusion, our data indicate that the presence of FOXP3+ Tregs in CTCL is associated with disease stage and patient survival.