Grzegorz K. Przybylski

Two subsets of naive T helper cells with distinct T cell receptor excision circle content in human adult peripheral blood

During ageing thymic function declines and is unable to meet the demand for peripheral T helper (Th) cell replenishment. Therefore, population maintenance of naive Th cells must be at least partly peripherally based. Such peripheral postthymic expansion of recent thymic emigrants (RTEs) during ageing consequently should lead to loss or dilution of T cell receptor excision circles (TRECs) from a subset of naive T cells. We have identified two subsets of naive Th cells in human adult peripheral blood characterized by a striking unequal content of TRECs, indicating different peripheral proliferative histories. TRECs are highly enriched in peripheral naive CD45RA+ Th cells coexpressing CD31 compared with peripheral naive CD45RA+ Th cells lacking CD31 expression, in which TRECs can hardly be detected. Furthermore we show that CD31−CD45RA+ Th cells account for increasing percentages of the naive peripheral Th cell pool during ageing but retain phenotypic and functional features of naive Th cells. As CD31 is lost upon T cell receptor (TCR) engagement in vitro, we hypothesize that TCR triggering is a prerequisite for homeostatically driven peripheral postthymic expansion of human naive RTEs. We describe here the identification of peripherally expanded naive Th cells in human adult blood characterized by the loss of CD31 expression and a highly reduced TREC content.

Subcutaneous panniculitis-like T-cell lymphoma : Clinicopathologic, immunophenotypic, and genotypic analysis of alpha/beta and gamma/delta subtypes

Subcutaneous panniculitis-like T-cell lymphoma (SPTCL) is an uncommon cutaneous lymphoma that has been proposed as a distinct clinicopathologic entity, but studies of SPTCL are limited. We studied the clinicopathologic, immunophenotypic, and genetic features of 11 SPTCLs. All cases had a variable admixture of pleomorphic small, medium, or large lymphocytes and histiocytes infiltrating the subcutis in a lobular panniculitis-like pattern. A granulomatous reaction was seen in three cases and erythrophagocytosis in four. Karyorrhexis and fat necrosis were present in all cases. Angioinvasion was seen in seven SPTCLs; four had areas of coagulation necrosis. All cases expressed T-cell-associated antigens (CD3epsilon, CD45RO, or CD43) and T-cell receptors (TCR); nine expressed alphabeta TCRs and two expressed gammadelta TCRs. T-cell receptor-gamma, TCRbeta, or TCRdelta genes were clonally rearranged in 8 of 10 cases studied. Both gammadelta SPTCLs expressed Vdelta2+ TCRs and were CD4-, CD8- and CD56+. CD56 was negative in seven of nine alphabeta SPTCLs and inconclusive in the other two. Six of nine alphabeta SPTCLs were CD8+; the CD4/CD8 phenotypes were indeterminate in the other three. Cytolytic granule-associated proteins were expressed by all SPTCLs (11 of 11 were TIA-1+, 4 of 4 were perforin+). In situ hybridization for Epstein-Barr virus-encoded RNA (EBER-1) was negative in all cases. Most patients responded to systemic chemotherapy or local radiation therapy. Seven patients are alive: four without disease (19-73 months) and three with disease (32-72 months); four died: three of disease (3-25 months) and one without disease (42 months). We conclude that SPTCLs are clonal, EBV-, cytotoxic T-cell lymphomas derived from alphabeta T-cells or gammadelta T-cells. The gammadelta SPTCLs appear to be preferentially derived from the Vdelta2+ subset. Subcutaneous panniculitis-like T-cell lymphoma may be rapidly fatal or indolent; local therapy may be appropriate for some patients.

Disruption of the BCL11B gene through inv(14)(q11.2q32.31) results in the expression of BCL11B-TRDC fusion transcripts and is associated with the absence of wild-type BCL11B transcripts in T-ALL

T-cell acute lymphoblastic leukemia (T-ALL) is associated with chromosomal aberrations characterized by juxtaposition of proto-oncogenes to T-cell receptor gene loci (TCR), resulting in the deregulated transcription of these proto-oncogenes. Here, we describe the molecular characterization of a novel chromosomal aberration, inv(14)(q11.2q32.31), in a T-ALL sample, involving the recently described BCL11B gene and the TCRD locus. The inversion joined the 5′ part of BCL11B, including exons 1–3, to the TRDD3 gene segment of the TCRD locus, whereas the reciprocal breakpoint fused the TRDV1 gene segment to the fourth exon of BCL11B. The TRDV1-BCL11B joining region was 1344 bp long and contained fragments derived from 20q11.22, 3p21.33 and from 11p12, indicating the complex character of this aberration. A strong expression of in-frame transcripts with truncated BCL11B and TCRD constant region (TRDC) were observed, but in contrast to normal T cells and other T-ALL samples, no wild-type BCL11B transcripts were detected in the T-ALL sample. Screening of 37 other T-ALLs revealed one additional case with expression of the BCL11B-TRDC fusion transcript. As BCL11B appears to play a key role in T-cell differentiation, BCL11B disruption and disturbed expression may contribute to the development of T-cell malignancies in man.

Hepatosplenic and Subcutaneous Panniculitis-Like γ/δ T Cell Lymphomas Are Derived from Different Vδ Subsets of γ/δ T Lymphocytes

Gamma/delta T cell lymphomas (γ/δ TCL) represent rare, often aggressive types of T cell malignancy that are clinically and pathologically diverse. Most γ/δ TCL occur as a hepatosplenic or subcutaneous type. To date, analysis of the T cell receptor δ (TCRδ) gene repertoire of hepatosplenic γ/δ TCL (γ/δ HSTCL) and subcutaneous panniculitis-like γ/δ TCL (γ/δ SPTCL) has been reported only in a limited number of cases. In this study we analyzed 11 γ/δ HSTCL and 4 γ/δ SPTCL by polymerase chain reaction and immunostaining to determine their usage of the Vδ subtypes (Vδ1–6). It is noteworthy that 10 of 11 γ/δ HSTCL expressed the Vδ1 gene. The remaining case also expressed T cell receptor δ (TCRδ) as determined by flow cytometry and TCRδ rearrangement in Southern blot. However, the Vδ gene expressed by this lymphoma could not be determined, which suggests usage of an as yet unidentified Vδ gene. In striking contrast to the γ/δ HSTCL, all 4 γ/δ SPTCL expressed the Vδ2 gene. Our data demonstrate that γ/δ HSTCL are preferentially derived from the Vδ1 subset of γ/δ T lymphocytes, whereas γ/δ SPTCL are preferentially derived from the Vδ2 subset. The pattern of Vδ gene expression in HSTCL and SPTCL corresponds to the respective, predominant γ/δ T cell subsets normally found in the spleen and skin. This finding suggests that γ/δ TCL are derived from normal γ/δ T lymphocytes which reside in the affected tissues. Furthermore, the selective, lymphoma type-specific Vδ gene segment usage may provide a molecular tool to distinguish better among various types of γ/δ TCL lymphoma particularly in the clinically advanced, widely disseminated cases.

Activation of miR-17-92 by NK-like homeodomain proteins suppresses apoptosis via reduction of E2F1 in T-cell acute lymphoblastic leukemia

The NK-like family of homeobox genes includes TLX1, TLX3 and NKX2-5, which are ectopically activated in distinct subsets of T-cell acute lymphoblastic leukemia (T-ALL) cells. Here we analysed their effect on the miR-17-92 cluster overexpressed in several types of cancer, including T-ALL. The pri-miR-17-92 polycistron encodes micro-RNAs (miRNAs), which decrease E2F1 protein expression, regulating proliferation and/or apoptosis. Quantification of pri-miR-17-92 in T-ALL cell lines suggested an implication of the NK-like homeodomain proteins in transcriptional regulation. Lentiviral-mediated overexpression of NKX2-5 in the T-ALL cell line MOLT-4 consistently resulted in increased miR-17-92 pri-miRNA levels and decreased amounts of E2F1 protein. Induction of apoptosis by treating miR17-92 or E2F1 transduced T-ALL cells with etoposide led to reduced or enhanced cell viability, respectively. Furthermore, analysis of pri-miR-17-92 in T-ALL patients indicated elevated expression in those bearing TLX1/3 positive cells. These data support an activatory effect of NK-like homeodomain proteins on pri-miR-17-92 expression and concomitantly reduced E2F1 protein levels, thereby enhancing survival of leukemic T-cells.

Hepatosplenic gamma-delta T-cell lymphoma as a late-onset posttransplant lymphoproliferative disorder in renal transplant recipients

We report 2 cases of renal transplant recipients in whom hepatosplenic gamma-delta T-cell lymphoma (gamma-delta HSTCL) developed 5 and 10 years after transplantation. Both patients had marked hepatosplenomegaly, B symptoms (weight loss, fever, and night sweats), and abnormal peripheral blood findings, including anemia in both, thrombocytopenia and leukoerythroblastic changes in 1, and leukocytosis in the other. Markedly atypical lymphoid infiltrate of intermediate to large cells was observed in the spleen, liver, and bone marrow. The malignant cells showed typical immunophenotype of gamma-delta T cells (CD2+, CD3+, CD4-, CD8-, CD7+, gamma-delta T-cell receptor-positive, and alpha-beta T-cell receptor-negative) with clonal T-cell receptor gene rearrangement and were of the V-delta-1 subset. In addition, the cells contained a cytolytic granule-associated protein, TIA-1, and Fas ligand, indicating cytotoxic T-cell differentiation. The malignant T cells in both cases were of host tissue origin. Both cases were negative for Epstein-Barr virus genome using Southern blot analysis. The patients did not respond to reduction of immunosuppression. Despite initial response to chemotherapy, both patients died within 6 months of diagnosis. Our findings indicate that gamma-delta HSTCL can occur as a late complication in transplant recipients.

Inhibition of BCL11B expression leads to apoptosis of malignant but not normal mature T cells

The B-cell chronic lymphocytic leukemia (CLL)/lymphoma 11B gene (BCL11B) encodes a Krüppel-like zinc-finger protein, which plays a crucial role in thymopoiesis and has been associated with hematopoietic malignancies. It was hypothesized that BCL11B may act as a tumor-suppressor gene, but its precise function has not yet been elucidated. Here, we demonstrate that the survival of human T-cell leukemia and lymphoma cell lines is critically dependent on Bcl11b. Suppression of Bcl11b by RNA interference selectively induced apoptosis in transformed T cells whereas normal mature T cells remained unaffected. The apoptosis was effected by simultaneous activation of death receptor-mediated and intrinsic apoptotic pathways, most likely as a result of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) upregulation and suppression of the Bcl-xL antiapoptotic protein. Our data indicate an antiapoptotic function of Bcl11b. The resistance of normal mature T lymphocytes to Bcl11b suppression-induced apoptosis and restricted expression pattern make it an attractive therapeutic target in T-cell malignancies.

Tumor suppressor TNFAIP3 (A20) is frequently deleted in Sézary syndrome

Despite recent therapeutic improvements, the prognosis for patients suffering from Sézary syndrome (SS), a disseminated form of cutaneous T-cell lymphomas, is still poor. We identified bi- and monoallelic deletions of the tumor necrosis factor-α-induced protein 3 gene (TNFAIP3; A20) in a high proportion of SS patients as well as biallelic A20 deletion in the SS-derived cell line SeAx. Furthermore, we demonstrate that inhibition of A20 activates the NF-κB pathway thereby increasing the proliferation of normal T lymphocytes. On the other hand, the reconstitution of A20 expression slowed down the cell cycle in SeAx cells. Recently A20 inactivation has been reported in various B-cell lymphomas. In this study, we show that A20 is also a putative tumor suppressor in the T-cell malignancy—SS.

Down regulation of BCL11B expression inhibits proliferation and induces apoptosis in malignant T cells by BCL11B-935-siRNA

Abstract To screen the highly efficient and specific B-cell chronic lymphocytic leukemia/lymphoma 11B (BCL11B) small interfering RNA (siRNA) which are able to downregulate the BCL11B gene expression in human T-cell acute lymphoblastic leukemia, thereby inhibiting the leukemic T-cell proliferation and inducing apoptosis, four BCL11B-siRNAs and the scrambled non-silencing siRNA control (sc) were designed and obtained by chemosynthesis. After nucleofection, BCL11B expression in the mRNA and the protein levels were measured by qRT–PCR and immunoblotting, respectively. The biological consequences based on the highly efficient and specific BCL11B-siRNA were demonstrated by CCK-8 kit, morphological changes (Hoechst 33258 staining), high-resolution imaging, and flow cytometry. Reduction in the BCL11B mRNA level was observed at 24 or 48 hours in molt-4 T cells with BCL11B-935-siRNA, BCL11B-434-siRNA, or BCL11B-748-siRNA, respectively. BCL11B protein expression levels were reduced by 34·77% and 41·73% in the BCL11B-935-siRNA- and BCL11B-434-siRNA-treated cells, compared with the control level at 72 hours. In comparison with BCL11B-434-siRNA treatment group, the Molt-4 cells transfected with the BCL11B-935-siRNA showed significantly inhibited proliferation and effectively induced apoptosis (P<0·05). When highly efficient and specific BCL11B-935-siRNA was used to analyze the inhibition of BCL11B mRNA level in primary T-cell acute lymphoblastic leukemia (T-ALL) cells, similar result was obtained. In conclusion, siRNAs targeting the different exon domains resulted in different silencing effects and biological consequences. Suppression of BCL11B by RNA interference could inhibit the proliferation and induce the apoptosis effectively in leukemic T cells, which might be considered as a new target therapeutic strategy in T-cell malignancies.

Acute myeloid and T-cell acute lymphoblastic leukaemia with aberrant antigen expression exhibit similar TCRdelta gene rearrangements.

TCRδ gene recombination patterns were analysed by Southern blot, polymerase chain reaction and nucleotide sequencing in acute myeloid leukaemias with coexpression of lymphoid antigens (Ly+ AML, n = 10) as well as in early T‐cell acute lymphoblastic leukaemias with (My+ T‐ALL, n = 10) and without coexpression of myeloid antigens (My− T‐ALL, n = 9). These 29 acute leukaemias exhibiting TCRδ gene rearrangements were selected from 66 Ly+ AML, 14 My+ T‐ ALL and 12 My− T‐ALL cases.