Blood T-cell Clones Research Articles

The Overlap of Skin and Blood T-Cell Clones in Early-Stage Mycosis Fungoides

Introduction: Mycosis fungoides (MF) is the most common subtype of cutaneous T-cell lymphoma that initially starts in the skin but can progress to involve blood with significant mortality in late stages. Even in early stages and in the absence of blood involvement, changes in blood T-cell receptor (TCR) repertoire have been observed in MF patients. While prior work has explored the significance of dominant blood T-cell clones in the prognosis of early MF, the relationship between TCR sequences and T-cell repertoires of blood and skin in early-stage MF patients has not been characterized. Objectives: To determine the relationship of blood and skin T-cell repertories and to reassess the impact of dominant blood T-cell clones on selected outcome endpoints in early-stage MF. Materials and Methods: We used high-throughput sequencing to interrogate TCR sequences in blood and skin of MF patients without blood involvement at the Jefferson Cutaneous Lymphoma Clinic (Adaptive Biotechnologies). ImmunoSEQ Analyzer provided T-cell repertoire overlap metric (Morisita's index) and diversity measure (Simpson's clonality score). Time to systemic treatment (TTST) was calculated as the time from initial diagnosis to initiation of first systemic therapy. Results: 60 MF patients with no blood involvement were enrolled. 28% had a dominant clone in blood; of these, 18% were identical to dominant skin clones and 82% were distinct from the dominant clones identified in skin. We found that MF patients with discordant dominant T-cell clones in blood and skin had a longer TTST when compared to the identical clone cohort (P=0.0057) (Figure 1A). Furthermore, patients with discordant dominant clones had a lower degree of T-cell repertoire overlap between blood and skin (P<0.0001) and higher blood T-cell repertoire diversity scores (P=0.013) when compared to the identical clone group (Figure 1B). In all patients, the degree of T-cell overlap in blood and skin did not significantly change over a period of months to years or among skin biopsies obtained from different anatomical locations. Finally, in a subset of patients with discordant clones, the dominant skin clone did not appear to be detected in blood even at low frequencies. Conclusion: Our data highlight the importance of establishing clonal associations between dominant T-cell clones of the skin and blood in MF. We propose that early-stage MF patients with dominant clones in blood segregate into two cohorts with distinct prognoses and T-cell repertoires based on the identity of their peripheral T-cell clones. In addition, our findings suggest that not all MF patients have reservoirs of malignant T-cell clones readily available in peripheral circulation to seed new lesions in skin. Figure 1: A: Kaplan-Meier analyses of time to systemic treatment (TTST) in skin-limited mycosis fungoides (MF) patients with an identical dominant T-cell clone in blood and skin, a discordant dominant T-cell clone in blood and skin, and no dominant blood clones. NS- not significant (P =0.5274), ** (P =0.0057), *** (P <0.0003). B: Average Morisita's index for blood and skin overlap for patients in indicated groups of I: identical dominant clones in blood and skin, D: discordant dominant clones in blood and skin, and N: no dominant clone in blood. **** (P <0.0001).

O-136 - The overlap of skin and blood T-cell clones in early-stage mycosis fungoides

O-136 - The overlap of skin and blood T-cell clones in early-stage mycosis fungoides

The overlap of skin and blood T-cell clones in early-stage mycosis fungoides

The overlap of skin and blood T-cell clones in early-stage mycosis fungoides

Hist-O-02 - High PD1 expression H score is associated with Sézary diagnosis: study of 91 patients with erythroderma

Erythroderma is a diagnostic challenge for dermatologists and pathologists. We aimed to identify clinical, histological and immunohistochemical markers that can be used to improve erythroderma diagnosis. This single-centre retrospective study included patients with erythroderma. To be included, erythroderma cases needed an accurate final diagnosis, at minimum one year of follow-up, available pictures and available skin biopsies to be reviewed. The demographic, clinical, biological (lactate deshydrogenase, eosinophilia, immunophenotype) and molecular features (T-cell clone in blood and skin) were recorded. All skin biopsies were reviewed for the following features: psoriasiform hyperplasia, parakeratosis, neutrophil microabscess, spongiosis, eosinophil infiltrate, lichenoid lesion, necrotic keratinocytes, epidermotropism, atypical lymphocytes, as well as routine immunostainings (CD3, CD4, CD8). Additional T follicular helper markers (PD1, ICOS and CXCL13), CD7 loss and Ki-67 proliferation index were also studied. For PD1 expression, percentage of positive lymphocytes as well as staining intensity (+ to +++) were recorded, all together combined in the semi-quantitative H score= [0 × (% cells 0) + 1 × (% cells 1+) + 2 × (% cells 2+) + 3 × (% cells 3+)]. Among 91 patients included with erythroderma, 21 were diagnosed as eczema, 17 as psoriasis, 20 as drug-induced erythroderma (DE), 13 as erythrodermic mycosis fungoides (E-MF) and 20 as Sézary syndrome (SS). Nail alterations, ear involvement and severe scaling were associated with psoriasis (p=0.044). Fever and hypereosinophilia were associated with DE diagnosis (p=0.044 and p=0.0003). Atypical lymphocytes in the skin were more frequent in E-MF and SS (p=0.044), as well as an abnormal blood immunophenotype and identical skin and blood T-cell clone. In SS, PD1 expression by T cells was significantly higher than in other aetiologies, as percentage of positive lymphocytes and intensity. PD1 H-score >50 was associated with SS (p<0.001, Se=75%; Sp=92%), with good reproducibility between observers, kappa coefficient of 0.66 (95% CI 0.33–1.00). CXCL13 expression was also associated with SS (p=0.044); CD7 loss was more frequent in E-MF and SS (p=0.022), and Ki-67 index was higher in SS and DE (p=0.041). In this large series erythroderma, we report the PD1 expression, and specifically the PD1 H-score combining percentage and intensity scoring, as a tool for differential diagnosis between SS and other aetiologies including E-MF. Interdisciplinary expertise combining clinical, pathological, and molecular data remain essential for erythroderma diagnosis. Erythroderma is a diagnostic challenge for dermatologists and pathologists. We aimed to identify clinical, histological and immunohistochemical markers that can be used to improve erythroderma diagnosis. This single-centre retrospective study included patients with erythroderma. To be included, erythroderma cases needed an accurate final diagnosis, at minimum one year of follow-up, available pictures and available skin biopsies to be reviewed. The demographic, clinical, biological (lactate deshydrogenase, eosinophilia, immunophenotype) and molecular features (T-cell clone in blood and skin) were recorded. All skin biopsies were reviewed for the following features: psoriasiform hyperplasia, parakeratosis, neutrophil microabscess, spongiosis, eosinophil infiltrate, lichenoid lesion, necrotic keratinocytes, epidermotropism, atypical lymphocytes, as well as routine immunostainings (CD3, CD4, CD8). Additional T follicular helper markers (PD1, ICOS and CXCL13), CD7 loss and Ki-67 proliferation index were also studied. For PD1 expression, percentage of positive lymphocytes as well as staining intensity (+ to +++) were recorded, all together combined in the semi-quantitative H score= [0 × (% cells 0) + 1 × (% cells 1+) + 2 × (% cells 2+) + 3 × (% cells 3+)]. Among 91 patients included with erythroderma, 21 were diagnosed as eczema, 17 as psoriasis, 20 as drug-induced erythroderma (DE), 13 as erythrodermic mycosis fungoides (E-MF) and 20 as Sézary syndrome (SS). Nail alterations, ear involvement and severe scaling were associated with psoriasis (p=0.044). Fever and hypereosinophilia were associated with DE diagnosis (p=0.044 and p=0.0003). Atypical lymphocytes in the skin were more frequent in E-MF and SS (p=0.044), as well as an abnormal blood immunophenotype and identical skin and blood T-cell clone. In SS, PD1 expression by T cells was significantly higher than in other aetiologies, as percentage of positive lymphocytes and intensity. PD1 H-score >50 was associated with SS (p<0.001, Se=75%; Sp=92%), with good reproducibility between observers, kappa coefficient of 0.66 (95% CI 0.33–1.00). CXCL13 expression was also associated with SS (p=0.044); CD7 loss was more frequent in E-MF and SS (p=0.022), and Ki-67 index was higher in SS and DE (p=0.041). In this large series erythroderma, we report the PD1 expression, and specifically the PD1 H-score combining percentage and intensity scoring, as a tool for differential diagnosis between SS and other aetiologies including E-MF. Interdisciplinary expertise combining clinical, pathological, and molecular data remain essential for erythroderma diagnosis.

PAK1-Dependent Antitumor Effect of AAC-11‒Derived Peptides on Sézary Syndrome Malignant CD4+ T Lymphocytes

Sézary syndrome is an aggressive form of cutaneous T-cell lymphoma characterized by the presence of a malignant CD4+ T-cell clone in both blood and skin. Its pathophysiology is still poorly understood, and the development of targeted therapies is hampered by the absence of specific target proteins. AAC-11 plays important roles in cancer cell progression and survival and thus has been considered as an anticancer therapeutic target. In this study, we show that a peptide called RT39, comprising a portion of AAC-11‒binding site to its protein partners coupled to the penetratin sequence, induces the specific elimination of the malignant T-cell clone both exvivo on the circulating cells of patients with Sézary syndrome and invivo in a subcutaneous xenograft mouse model. RT39 acts by direct binding to PAK1 that is overexpressed, located in the plasma membrane, and constitutively activated in Sézary cells, resulting in their selective depletion by membranolysis. Along with the absence of toxicity, our preclinical efficacy evidence suggests that RT39 might represent a promising alternative therapeutic tool for Sézary syndrome because it spares the nonmalignant immune cells and, contrary to antibody-based immunotherapies, does not require the mobilization of the cellular immunity that shows heavy deficiencies at advanced stages of the disease.

Clonality of CD4+ Blood T Cells Predicts Longer Survival With CTLA4 or PD-1 Checkpoint Inhibition in Advanced Melanoma.

Recognition of cancer antigens drives the clonal expansion of cancer-reactive T cells, which is thought to contribute to restricted T-cell receptor (TCR) repertoires in tumor-infiltrating lymphocytes (TILs). To understand how tumors escape anti-tumor immunity, we investigated tumor-associated T-cell repertoires of patients with advanced melanoma and after blockade of the cytotoxic T-lymphocyte-associated protein 4 (CTLA4) or programmed cell death 1 (PD-1). TCR Vβ-gene spectratyping allowed us to quantify restrictions of T-cell repertoires and, further, diversities of T-cell clones. In this study, we show that the blood TCR repertoires were variably restricted in CD4+ and extensively restricted in CD8+ T cells of patients with advanced melanoma, and contained clones in both T-cell fractions prior to the start of immunotherapy. A greater diversification especially of CD4+ blood T-cell clones before immunotherapy showed statistically significant correlations with long-term survival upon CTLA4 or PD-1 inhibition. Analysis of TILs and corresponding blood available in one patient indicated that blood clonality may at least partially be related to the clonal expansion in the tumor microenvironment. In patients who developed severe immune-related adverse events (IrAEs), CD4+ and CD8+ TCR spectratypes became more restricted during anti-CTLA4 treatment, suggesting that newly expanded oligoclonal T-cell responses may contribute to IrAEs. This study reveals diverse T-cell clones in the blood of melanoma patients prior to immunotherapy, which may reflect the extent to which T cells are able to react against melanoma and potentially control melanoma progression. Therefore, the T-cell clonality in the circulation may have predictive value for antitumor responses from checkpoint inhibition.

Coeliac disease: immunogenicity studies of barley hordein and rye secalin-derived peptides.

Coeliac disease (CD) is an inflammatory disorder of the small intestine. It includes aberrant adaptive immunity with presentation of CD toxic gluten peptides by HLA-DQ2 or DQ8 molecules to gluten-sensitive T cells. A ω-gliadin/C-hordein peptide (QPFPQPEQPFPW) and a rye-derived secalin peptide (QPFPQPQQPIPQ) were proposed to be toxic in CD, as they yielded positive responses when assessed with peripheral blood T-cell clones derived from individuals with CD. We sought to assess the immunogenicity of the candidate peptides using gluten-sensitive T-cell lines obtained from CD small intestinal biopsies. We also sought to investigate the potential cross-reactivity of wheat gluten-sensitive T-cell lines with peptic-tryptic digested barley hordein (PTH) and rye secalin (PTS). Synthesised candidate peptides were deamidated with tissue transglutaminase (tTG). Gluten-sensitive T-cell lines were generated by culturing small intestinal biopsies from CD patients with peptic-tryptic gluten (PTG), PTH or PTS, along with autologous PBMCs for antigen presentation. The stimulation indices were determined by measuring the relative cellular proliferation via incorporation of 3 H-thymidine. The majority of T-cell lines reacted to the peptides studied. There was also cross-reactivity between wheat gluten-sensitive T-cell lines and the hordein, gliadin and secalin peptides. PTH, PTS, barley hordein and rye secalin-derived CD antigen-sensitive T-cell lines showed positive stimulation with PTG. ω-gliadin/C-hordein peptide and rye-derived peptide are immunogenic to gluten-sensitive T-cell lines and potentially present in wheat, rye and barley. Additional CD toxic peptides may be shared.

Cutaneous Lymphoma International Consortium Study of Outcome in Advanced Stages of Mycosis Fungoides and Sézary Syndrome: Effect of Specific Prognostic Markers on Survival and Development of a Prognostic Model.

Advanced-stage mycosis fungoides (MF; stage IIB to IV) and Sézary syndrome (SS) are aggressive lymphomas with a median survival of 1 to 5 years. Clinical management is stage based; however, there is wide range of outcome within stages. Published prognostic studies in MF/SS have been single-center trials. Because of the rarity of MF/SS, only a large collaboration would power a study to identify independent prognostic markers. Literature review identified the following 10 candidate markers: stage, age, sex, cutaneous histologic features of folliculotropism, CD30 positivity, proliferation index, large-cell transformation, WBC/lymphocyte count, serum lactate dehydrogenase, and identical T-cell clone in blood and skin. Data were collected at specialist centers on patients diagnosed with advanced-stage MF/SS from 2007. Each parameter recorded at diagnosis was tested against overall survival (OS). Staging data on 1,275 patients with advanced MF/SS from 29 international sites were included for survival analysis. The median OS was 63 months, with 2- and 5-year survival rates of 77% and 52%, respectively. The median OS for patients with stage IIB disease was 68 months, but patients diagnosed with stage III disease had slightly improved survival compared with patients with stage IIB, although patients diagnosed with stage IV disease had significantly worse survival (48 months for stage IVA and 33 months for stage IVB). Of the 10 variables tested, four (stage IV, age > 60 years, large-cell transformation, and increased lactate dehydrogenase) were independent prognostic markers for a worse survival. Combining these four factors in a prognostic index model identified the following three risk groups across stages with significantly different 5-year survival rates: low risk (68%), intermediate risk (44%), and high risk (28%). To our knowledge, this study includes the largest cohort of patients with advanced-stage MF/SS and identifies markers with independent prognostic value, which, used together in a prognostic index, may be useful to stratify advanced-stage patients.

Abstract O.23: Lysyl Oxidase Expression In The Patients With Kawasaki Disease

Background: Histologic study of inflamed coronary arteries from the Kawasaki disease (KD) patients shows infiltration by immune effector cells, IL-17-producing myofibroblasts, and breaks in the internal elastic lamina. Transcripts from genes associated with cell trafficking (e.g. paxillin) are elevated in whole blood from acute KD patients. Lysyl oxidase (LOX) is a multifunctional protein affecting affecting “monocyte and smooth muscle cell migration, elastogenesis, and endothelial-to-mesenchymal transition. LOX promotes cell motility through paxillin phosphorylation, which influences T-cell trafficking via the calcium signaling pathway. We investigated the role of LOX in the pathogenesis of KD. Methods: Agilent microarray data using whole blood were analyzed for differential expression of enzymes in the LOX family (LOX, LOX-like (L)1-4) in acute and convalescent KD samples (n=19). LOXL1 results were validated using RT-PCR in an independent cohort (n=20). LOXL1 transcripts levels were measured in KD whole blood, peripheral blood mononuclear cells (PBMC), T-cell clones, B-cell lines, and myeloid dendritic cells from acute KD patients. Immunohistochemical staining for LOXL1 was performed on coronary arteries from a KD patient who died on illness day 7. Result: LOXL1 expression by microarray was 3.2-fold higher in acute than convalescent samples (p&lt;0.001) and was validated in an independent cohort by RT-PCR (5.4-fold higher, p&lt;0.001). LOXL1 transcripts levels were 2-35 times higher in PBMC compared to whole blood (n=8). KD T cell clones had higher LOXL1 transcript levels than B-cells and dendritic cells. Immunohistochemical staining showed LOXL1-expressing inflammatory cells and smooth muscle cells in the coronary artery walls. Conclusion: LOXL1 may increase inflammation in acute KD by promoting the motility of infiltrating T cells and smooth muscle cells through focal adhesion formation. However, LOXL1 may also participate in arterial wall repair by crosslinking collagen and elastin. Statins inhibit focal adhesion formation and thus cell trafficking but not the crosslinking mediated by LOXL1. Studies of LOXL1 protein levels and paxillin phosphorylation in PBMC may further our understanding of their role in KD pathogenesis.

Molecular detection of circulating Sezary cells in patients with mycosis fungoides: could it predict future development of secondary Sezary syndrome? A single-institution experience

While the majority of patients with early-stage mycosis fungoides (MF) have an excellent prognosis, a few cases progress to secondary Sezary syndrome (sSS), which carries a dismal clinical outcome. We retrospectively analyzed 135 cases of MF/SS and correlated molecular detection of T-cell clones in the skin and blood with other clinicopathologic findings. When stratified by the diagnoses, patients with MF demonstrated a 26.5% (31/117) positive rate for a blood T-cell clone, of which 50% (10/20) had an identical T-cell clone in the skin. Follow-up evaluation showed conversion into sSS or leukemic phase in 50% (5/10) of cases with a positive blood T-cell clone (estimated mean interval 41.8 months) in comparison to no cases in the group without a clone (0/31). Interestingly, 4/5 cases of sSS had an identical T-cell clone in the skin, while the remaining case did not have the test performed on skin for clonal comparison. Kaplan–Meier survival analysis demonstrated a poor clinical outcome in the group with a blood T-cell clone, in comparison with the group without, in overall survival (p < 0.0001) and progression-free survival (p < 0.0001; HR = 22.6). These findings suggest that molecular detection of a blood T-cell clone may have a role in predicting sSS. Due to amplification of non-neoplastic T-cell expansion in a significant number of cases, comparison of blood T-cell clones with skin may have confirmatory value.

“Wilms Tumor Protein 1” (WT1) Peptide Vaccination-induced Complete Remission in a Patient With Acute Myeloid Leukemia Is Accompanied by the Emergence of a Predominant T-cell Clone Both in Blood and Bone Marrow

Within the last few years, the first peptide vaccination trials for treatment of acute myeloid leukemia (AML) have been initiated. Athough the presence of epitope-specific T cells could be seen both in bone marrow (BM) and peripheral blood (PB), nothing is known about their clonal composition. In this study, we analyzed material from a patient with recurrent AML vaccinated with "Wilms Tumor Protein 1" (WT1) peptide, who achieved a complete remission (CR) lasting for 12 months. For identification of expanded WT1-specific T-cell clones, enrichment by tetramer and IFNγ secretion were followed by comparative quantitative reverse transcribed PCR (qRT PCR) quantification of all TCR Vβ-families. Vβ-families with increase in the enriched fraction were cloned and sequenced. A predominant clone was quantified by clonotypic qRT PCR from PB and BM. Quantity and functionality of WT1-specific cells were assessed by tetramer analyses and intracellular IFNγ staining. A specific predominant clone was identified during clinical remission. Clone-specific qRT PCR showed an increase both in PB and BM after 8 vaccinations. Six months after achieving CR, the transcript levels in BM decreased. Relapse was accompanied by secondary rise of the WT1-specific clone in PB but not in BM. In parallel, a lack of vaccine-induced WT1 specific IFNγ production was observed at that timepoint. In conclusion, we provide first data regarding evolution and compartmentalization of a peptide vaccine-induced T-cell clone in PB and BM of an AML patient. At the time of relapse, the same clone reappeared spontaneously in PB but not in BM showing impaired functionality.

Evolution of Clinical and Molecular Responses to Bexarotene Treatment in Cutaneous T-Cell Lymphoma

Background: The mechanisms of action of bexarotene are not well understood. Methods: A retrospective study on patients with cutaneous T-cell lymphoma (CTCL) treated with bexarotene was performed to see if bexarotene could act on the dominant T-cell clones. Thirty-five patients were included. Twenty-three were treated with bexarotene for more than 3 months (300 mg/m²). In 7 patients, phototherapy was given with bexarotene. Results: Dominant T-cell clones were observed in 11 patients in peripheral blood and in 19 patients in skin. Our results demonstrate no significant evolution of T-cell clones either in skin or peripheral blood. Furthermore, the detection of T-cell clones in peripheral blood before starting bexarotene was significantly associated with the progression of the disease. UV therapy given with bexarotene significantly improved therapeutic response without any correlation with T-cell clones. Conclusion: This is the first study on the evolution of the T-cell clone in blood and skin in CTCL patients during bexarotene therapy.

Molecular identity of skin and blood T‐cell clones in cutaneous T‐cell lymphoma patients as determined from the migration pattern of the T‐cell receptor‐γ gene by capillary electrophoresis

The presence of a dominant clonal T-cell population in skin lesions is an important clue in the diagnosis of cutaneous T-cell lymphoma (CTCL). However, it has never been determined whether dominant T-cell receptor (TCR) rearrangements identified in skin lesions and blood from CTCL patients, displaying strictly identical migration patterns by capillary electrophoresis, actually correspond to identical clones. As this information has potential clinical relevance, TCR-gamma (TCRG) gene-derived amplified fragments from dominant blood and skin T-cell clones featuring either identical or slightly different capillary electrophoresis migration patterns were analyzed in eleven CTCL patients. In nine patients with identical electrophoretic migration patterns, sequence analyses revealed the dominant skin and blood T-cell clones to be identical. In contrast, in two patients displaying slight migration differences between skin and blood samples, the TCRG sequences were distinct. Additionally, capillary electrophoresis appears more sensitive and accurate than heteroduplex analysis and in silico analysis of samples of different origins is possible a posteriori. These results demonstrate the efficacy of capillary electrophoresis in assessing molecular identity and discrepancy of dominant T-cell populations obtained from different tissues or at different times, facilitating diagnosis and follow-up.

Reactivity of in vitro activated human T lymphocytes to p‐phenylenediamine and related substances

Patch tests to p-phenylenediamine (PPD) and related substances often show concurrent reactions that can be attributed to separate sensitization or cross-reactivity. In order to understand the health risks associated with cross-reactivity, we studied cross-reactivity of eight chemicals in vitro by measurement of T-cell proliferation of peripheral blood mononuclear cells (PBMC), T-cell lines (TCL), and T-cell clones (TCC) of subjects with a positive patch test result to PPD. We studied PBMC from 13 patients and were able to generate TCL from seven and TCC from four patients. Their proliferative responses to the chemicals were estimated. Concurrent reactions to these compounds on the polyclonal and monoclonal level were found. A restricted T-cell receptor (TCR) Vbeta16-usage was observed (5/8 clones). A detailed analysis of 34 TCL showed broad cross-reactivity (64.7%) between PPD, p-toluenediamine, Bandrowski's Base, and p-aminoazobenzene. More restricted patterns were found in 8.8%, which responded only to compounds with two or three benzene rings, whereas 26.5% of the clones reacted specifically only to one compound. More than 60% of the clones showed a broad cross-reactivity pattern. Hence, clinically observed cross-reactivity between different para-amino compounds can be based on a TCR recognizing similar epitopes of these compounds with low specificity.

Gene expression analysis of the CD4+ T‐cell clones derived from gingival tissues of periodontitis patients

The function of T cells infiltrating periodontitis lesions is complex and has not been fully elucidated. Here, we established T-cell clones from the gingival tissues of periodontitis patients and examined their gene expression. A total of 57 and 101 T-cell clones were established by means of immobilized anti-CD3 antibody and IL-2 from gingival tissues and peripheral blood, respectively. The gingival T-cell clones were derived from three patients, and the peripheral blood T-cell clones from two of these patients and a further patient whose gingival T-cell clones were not established. Gingival tissues were also obtained from a further 19 periodontitis patients. The expression of cytokines and molecules related to both regulatory function and tissue destruction were examined by means of reverse-transcription polymerase chain reaction. All the gingival T-cell clones expressed mRNA for TGF-beta1, CTLA-4, and CD25, and all the T-cell clones from peripheral blood expressed IFN-gamma and TGF-beta1 mRNAs. Most but not all the T-cell clones from gingival tissues and peripheral blood expressed mRNA for IFN-gamma and, CD25 and CTLA-4, respectively. The frequency of T-cell clones and gingival tissues expressing FOXP3, a possible master gene for mouse CD4(+)CD25(+) regulatory T cells, was very high (97%, 93%, and 100% for gingival T-cell clones, peripheral blood T-cell clones, and gingival tissues, respectively). Whereas the frequency of IL-4-expressing T-cell clones was lower for gingival T-cell clones (70% vs. 87%), the frequency of the gingival T-cell clones expressing IL-10 and IL-17 was higher than peripheral blood T-cell clones (75% vs. 62% for IL-10, 51% vs. 11% for IL-17). A similar expression profile was observed for gingival T-cell clones compared with gingival tissue samples with the exception of IL-4 expression, where the frequency of positive samples was lower in the gingival tissues (70% vs. 11%). These results suggest that the individual T cells infiltrating gingival lesions can express mRNA for both Th1 and Th2 cytokines as well as regulatory cytokines simultaneously.

A randomized cross-over study to compare PUVA and extracorporeal photopheresis in the treatment of plaque stage (T2) mycosis fungoides

PUVA is a well-established and effective treatment for plaque stage mycosis fungoides (MF) but its use is limited on a long-term basis because of the risk of cutaneous carcinogenesis. A further disadvantage is that nonexposed areas (sanctuary sites) often develop persistent disease. Therefore it is important to find alternative methods of treatment. Extracorporeal photopheresis (ECP) is a form of photochemotherapy that involves exposure of white blood cells to UVA with psoralens and can be effective in Sézary syndrome and erythrodermic cutaneous T-cell lymphoma. The aim of this study was to compare the efficacy of PUVA and ECP in the treatment of patients with T2 plaque stage (Stage 1B) MF who had a detectable peripheral blood T-cell clone. The study was of a cross-over design. Sixteen patients were randomized to receive either PUVA twice weekly for 3 months followed by ECP once monthly for 6 months at relapse, or vice-versa. Response was assessed by monthly skin scores and peripheral blood T-cell clonality. Ten patients received PUVA initially and six ECP initially. Eight patients completed the study. Skin scores taken at the completion of each treatment arm in patients who completed the study were 113 units better (confidence interval, 42-184 units) following 3 months PUVA than 6 months ECP (P = 0.002). Peripheral blood T-cell clones were detectable in all patients post-treatment. This study indicates that ECP is not effective in the treatment of plaque stage (1B/T2) MF even in patients with molecular evidence of a peripheral blood T-cell clone. Although PUVA was more effective than ECP, neither treatment modality cleared malignant T-cells from the peripheral blood.

Peripheral blood T-cell clonality in mycosis fungoides and nonlymphoma controls.

In mycosis fungoides (MF), T-cell clonality is reported in about 90% of skin and 40% of blood samples. However, identity of blood and cutaneous T-cell clone and prognostic relevance of blood T-cell clonality remain controversial. By PCR/fluorescence fragment analysis with estimation of clonal fragment lengths and relative peak heights, we objectively identified T-cell clonality unrelated to malignant lymphoproliferation in healthy donors (5/38), autoimmune dermatoses (3/8), and nonlymphoma skin cancer (9/39). This T-cell expansion of undetermined significance (TEXUS) was also found in 8/64 MF patients. Dissemination of neoplastic cells into blood, as identified by identical clonal fragment lengths in blood and skin, was detected in 23/64 MF patients. When monitoring for progression at TNM stage for a mean of 45.7 months, univariate analysis identified age of >60 years and detection of a related blood T-cell clone to be of prognostic relevance, whereas detection of TEXUS, sex, TNM stage at initial diagnosis, and detection of a cutaneous T-cell clone were irrelevant. Although multivariate analysis was not possible, further stratification clearly indicated an age of >60 years to be the predominating prognostic factor. In conclusion, investigation of T-cell clonality in skin and blood samples at the initial diagnosis cannot predict the clinical course of MF and the occurrence of TEXUS should be considered when assessing blood T-cell clonality.

Extracorporeal photopheresis in Sézary syndrome: hematologic parameters as predictors of response

Data were analyzed from 23 patients with Sézary syndrome (defined by erythroderma, more than 10% circulating atypical mononuclear cells, and peripheral blood T-cell clone) undergoing monthly extracorporeal photopheresis as the sole therapy for up to 1 year. The cohort showed a significant reduction of skin scores during treatment (P =.001). Thirteen patients (57%) achieved a reduction in skin score greater than 25% from baseline at 3, 6, 9, or 12 months (responders). Reduction in skin score correlated with reduction in the Sézary cell count as a percentage of total white cell count (P =.03). Responders and nonresponders were compared. None of the measured parameters was significantly different between the 2 groups. It was assessed whether any of the baseline parameters predicted outcome. A higher baseline lymphocyte count was significantly associated with a decrease in skin score at 6 months (P <.05). A higher baseline Sézary cell count as a percentage of total white cell count predicted a subject was more likely to be a responder after 6 months of treatment (P =.021). No other parameters predicted responder status. These data show that the modest falls in CD4, CD8, and Sézary cell counts were seen in all patients and might have resulted from lymphocyte apoptosis. This mechanism could explain the more favorable response seen in patients with higher percentages of Sézary cells in the peripheral blood. Alternatively, minimum tumor burden might be required for the induction of a cytotoxic response. Analysis of tumor-specific cytotoxic T cells is needed to investigate these possibilities further.

Prognostic significance of tumor burden in the blood of patients with erythrodermic primary cutaneous T-cell lymphoma

AbstractErythrodermic cutaneous T-cell lymphoma (CTCL) includes patients with erythrodermic mycosis fungoides who may or may not exhibit blood involvement and Sézary syndrome and in whom hematological involvement is, by definition, present at diagnosis. These patients were stratified into 5 hematologic stages (H0-H4) by measuring blood tumor burden, and these data were correlated with survival. The study identified 57 patients: 3 had no evidence of hematologic involvement (H0), 8 had a peripheral blood T-cell clone detected by polymerase chain reaction (PCR) analysis of the T-cell receptor gene and less than 5% Sézary cells on peripheral blood smear (H1), and 14 had either a T-cell clone detected by Southern blot analysis or PCR positivity with more than 5% circulating Sézary cells (H2). Twenty-four patients had absolute Sézary counts of more than 1 × 109 cells per liter (H3), and 8 patients had counts in excess of 10 × 109 cells per liter (H4). The disease-specific death rate was higher with increasing hematologic stage, after correcting for age at diagnosis. A univariate analysis of 30 patients with defined lymph node stage found hematologic stage (P = .045) and lymph node stage (P = .013) but not age (P = .136) to be poor prognostic indicators of survival. Multivariate analysis identified only lymph node stage to be prognostically important, although likelihood ratio tests indicated that hematologic stage provides additional information (P = .035). Increasing tumor burden in blood and lymph nodes of patients with erythrodermic CTCL was associated with a worse prognosis.The data imply that a hematologic staging system could complement existing tumor-node-metastasis staging criteria in erythrodermic CTCL.

Defective chemokine production in T-leukemia cell lines and its possible functional role.

Peripheral blood lymphocytes and T-cell clones produced nanogram quantities of the chemokines RANTES, MIP-lα, MIP-lβ, MCP-l, IL-8 and GRO-α as well as the motogenic cytokine HGF. In contrast, various T-leukemia cell lines at different stages of differentiation did not produce the same chemokines/cytokines. In order to study the possible functional importance of the poor chemokine production different T-cell lines were compared with respect to development of motile forms and migration on extracellular matrix components in the absence and presence of various chemokines. RANTES, MIP-1α, MIP-1β, IL-8, GRO-α and lymphotactin did not augment the development of motile forms including the size and appearance of the pseudopodia activity of the T-leukemia cell lines. The T-cell lines migrated spontaneously on/to fibronectin in a Boyden chamber assay system. Chemokines augmented the migration of the T-leukemia cell lines on fibronectin in the Boyden system in a chemotactic fashion with peak responses at 10 to 50 ng/ml. Thus, the production of chemokines is defective, in neoplastic T-lymphocytes. The defective chemokine production does not seem to play any major role for the basic locomotor capacity of the cells but may modulate the responsiveness to exogenous chemokines.