Phenotypic characterization of skin-infiltrating T cells in cutaneous T- cell lymphoma: comparison with benign cutaneous T-cell infiltrates

Using a panel of monoclonal antibodies, we have studied cell surface antigens of infiltrating mononuclear cells in skin biopsies from patients with cutaneous T-cell lymphoma (CTCL) and compared them with the T-cell surface phenotype seen in benign cutaneous T-cell infiltrations (e.g., contact dermatitis, delayed hypersensitivity skin tests, granuloma annulare) and in dermal infiltrates of lymphomatoid granulomatosis patients. We found that unlike circulating CTCL (Sezary) cells, CTCL cells infiltrating skin epidermis frequently expressed the T-cell antigen 3A1. Cutaneous infiltrates in 10 patients with mycosis fungoides (MF) and 1 patient with Sezary syndrome were OKT4 (inducer T cell), OKT8 (suppressor/cytotoxic T cell); 2 patients with MF were OKT4- , OKT8; and one MF patients's skin T cell were OKT4-, OKT8+. Similar to CTCL infiltrating cells, most of the benign skin T-cell infiltrates were usually 3A1+. OKT4+, and OKT8-. Our study shows the complex nature of T-cell antigen patterns in inflammatory and malignant skin T-cell infiltrations. We demonstrated that the CTCL the skin epidermal infiltrating T-cell phenotype is not invariate, and in many cases, is similar to the phenotype of clinically benign cutaneous T-cell infiltrations.     

T-cell prolymphocytic leukaemia: a clinical and immunological study

2 cases of T-cell prolymphocytic leukaemia (T-PLL) were investigated for their reactivity with a series of monoclonal antibodies (MoAbs) as well as for the cytochemical expression and functional activity of the pathological cells. Both patients showed morphological (large cells with abundant cytoplasm and eccentric and irregularly shaped nucleus with large and prominent nucleoli) and clinical (high leucocyte count and splenomegaly) features typical of T-PLL. The cells from 1 patient expressed a helper/inducer phenotype (T4+, T8-) and were reactive with the anti-Tac (interleukin-2 receptor) MoAb, while the other case co-expressed both the T4 and the T8 antigens. The response to phytohaemagglutinin and the natural killer activity (assessed by 51chromium release) were significantly reduced in both cases, while the helper capacity, tested in a pokeweed mitogen-driven system, was maintained only in the 1st case. This latter case which expressed a more mature phenotype (T4+, T8-) responded well to chemotherapy.     

CD56- and CD4-positive non-Hodgkin's lymphoma of probable T-cell origin]

A 65-year-old man was admitted with swelling of the right neck and bilateral inguinal lymph nodes. Endoscopic examination revealed no nasal infiltration. Pathological examination of a neck lymph node biopsy specimen revealed peripheral T-cell lymphoma according to the Revised European-American Classification of Lymphoid Neoplasms (REAL). The phenotype of the lymphoma cells was CD56+, CD16-, CD2+, surface CD3-, cytoplasmic CD3+, CD4+, CD8-, CD5+, CD7- and CD45RO+. May-Giemsa staining demonstrated no azurophilic granules in the lymphoma cells. Immunohistopathologic examination showed negativity for TIA-1 and granzyme B, and rearrangement of the TCR C beta 1 gene was also noted. These findings strongly suggested that this was a T-cell lymphoma. The patient received 8 courses of CHOP chemotherapy plus sobuzoxane. This led to a marked decrease of lymph node swelling, and currently the patient is still in remission. According to the REAL classification, T/NK-cell lymphomas are included among the peripheral T cell tumours, and seem to constitute a heterogeneous group of neoplasms. Although some cases of CD4+ CD56+ lymphoma have been reported, the present case appears to be the first example to show TCR gene rearrangement and negativity for TIA-1 and granzyme B. Since the classification of T/NK-cell lymphoma is still controversial, accumulation of such cases may help to better define T/NK-cell neoplasms.     

Monoclonal antibody characterization of surface antigens in childhood T-cell lymphoid malignancies

Although childhood T-cell acute lymphocytic leukemia (T-ALL) and T-cell non-Hodgkin's lymphoma (T-NHL) have certain clinical features in common, T-ALL carries a notably poorer prognosis than does T-NHL. To determine whether the malignant cells from patients with these disorders are distinguishable, we examined bone marrow and/or blood from 51 children with T-ALL and tumor biopsy specimens from 17 with T-NHL, using a panel of monoclonal antibodies directed against T-cell differentiation antigens. We found considerable phenotypic heterogeneity in both T-ALL and T-NHL. Of the T-ALL (defined by greater than 25% blasts in the bone marrow) patients, 33% demonstrated a surface antigen pattern consistent with the earliest thymocyte stage of T-cell development (T9+ and/or T10+, or T6-/T4-/T8-/T3-), 37% were of a midthymocyte stage (T6+, and/or simultaneous expression of T4-helper and T8-suppressor antigens), and 30% expressed surface antigen patterns found on mature thymocytes (T3+, variable expression of other antigens). In contrast, tumor cell phenotypes in the 17 T-NHL patients were approximately equally distributed between mid- and mature thymocyte phenotypes. No NHL samples were classified as the early thymocyte phenotype. Clinical features as related to specific T-ALL immunophenotypes are presented, and the implications of these findings in regard to the current understanding of the differences in tumor biology between T-ALL and T-NHL are discussed.     

Hepatosplenic T-cell lymphoma: a distinct clinicopathologic entity of cytotoxic gamma delta T-cell origin

We identified eight cases of T-cell lymphoma with evidence of a gamma delta phenotype over a 13-year period. Seven of these cases conformed to a distinct clinicopathologic entity of hepatosplenic gamma delta T- cell lymphoma. Nearly all of these patients were young adult males (five of seven), with a median age at presentation of 20 years. They presented with marked hepatosplenomegaly, without lymphadenopathy or significant peripheral blood lymphocytosis. Thrombocytopenia was seen in all patients, and five of seven were mildly anemic. The clinical course was aggressive, and despite multiagent chemotherapy, the median survival duration was less than 1 year. The morphologic findings were uniform; a monomorphic population of medium-sized lymphoid cells with moderately clumped chromatin and a rim of pale cytoplasm infiltrated the sinusoids of the spleen, liver, and bone marrow. The cells had a characteristic immunophenotype: CD2+, CD3+, CD4-, CD5-, CD7+, CD16+, CD57-, CD25-, T-cell receptor (TCR)delta +, beta F1-. CD8 was positive in four of seven cases tested, and CD56 was positive in five of six. All cases expressed the cytotoxic granule-associated protein, TIA1, but perforin was detected in only one case. All cases with assessable DNA had a TCR gamma gene rearrangement, and lacked Epstein-Barr virus sequences. Isochromosome 7q was identified in two cases with cytogenetic information. The one case of cutaneous gamma delta T-cell lymphoma differed in its clinical manifestations, histologic appearance, and immunophenotype. We conclude that hepatosplenic gamma delta T-cell lymphoma is a distinct clinicopathologic entity derived from cytotoxic gamma delta T cells, and should be distinguished from other lymphomas of T-cell and natural-killer cell (NK)-like T-cell derivation.     

CD3+, CD4-, CD8- large granular T-cell lymphoproliferative disorder.

Large granular T-cell lymphoproliferative disorder (LGTLD) is a heterogeneous disorder covering a broad spectrum of diseases and requiring further subdivision. Most reported cases emphasized its suppressor phenotype (T gamma cell or CD8+), but we encountered two cases of CD3+, CD4-, CD8- LGTLD. Both cases had a benign clinical course and required no chemotherapy despite persistent lymphocytosis. This unique phenotype has been reported in a few cases of acute lymphoblastic leukemia expressing the T-cell receptor (TcR) gamma chain gene and is considered the counterpart of thymocytes at the intermediate stage between early precursors and mature thymocytes. Our case 1 provides further evidence that the CD3+, CD4-, CD8- phenotype, indeed, expresses the TcR gamma chain gene. However, the negative reaction to terminal deoxynucleotidyl transferase in our case 1 indicates that this phenotype represents proliferation of peripheral T-cells, in which about 2% bear the CD3+, CD4-, CD8- phenotype in the normal population. The selective use of CD3, CD4, CD8, HNK-1 monoclonal antibodies and of cytochemical stains (acid phosphatase and alpha-naphthyl butyrate esterase) for characterization of this disorder is discussed.     

Phenotypical heterogeneity of Japanese adult T-cell leukaemia

The leukaemic cells of 11 patients with Japanese adult T-cell leukaemia (ATL) were characterized in terms of phenotype employing various monoclonal antibodies against T-cell surface antigens. The leukaemic cells of all ATL patients except patient no. 11 displayed the phenotype OKT4+/OKT8-. However, the % of OKT3+ cells varied from 3 to 98. In 1 case, cells with phenotype OKT4+/OKT8+, which is characteristic of T-cells at an early stage of differentiation, were found. These results suggest that some ATL cells may be in an immature differentiation stage. Among the 11 patients, 2 with unusual ATL cell phenotypes (1 case with OKT3- cells and 1 case with Ia+/EAC+ cells) showed spontaneous regression of leukaemic cells. Characterization of leukaemic cells from a larger number of ATL patients may demonstrate a significant relationship between cell phenotype and prognosis.     

T-cell prolymphocyte leukaemia: A clinical and immunological study

2 cases of T-cell prolymphocytic leukaemia (T-PLL) were investigated for their reactivity with a series of monoclonal antibodies (MoAbs) as well as for the cytochemical expression and functional activity of the pathological cells. Both patients showed morphological (large cells with abundant cytoplasm and eccentric and irregularly shaped nucleous with large and prominent nucleoli) and clinical (high leucocyte count and splenomegaly) features typical of T-PLL. The cells from 1 patient expressed a helper/inducer phenotype (T4+, T8^-) and were reactive with the anti-Tac (interleukin-2 receptor) MoAb, while the other case co-expressed both the T4 and the T8 antigens. The response to phytohaemagglutinin and the natural killer activity (assessed by ⁵¹chromium release) were significantly reduced in both cases, while the helper capacity, tested in a pokeweed mitogen-driven system, was maintained only in the 1st case. This latter case which expressed a more mature phenotype (T4+, T8^-) responded well to chemotherapy.     

Recombinant human interleukin-2 restores in vitro T-cell colony formation by peripheral blood mononuclear cells after autologous bone marrow transplantation

Immune reconstitution after autologous bone marrow transplantation (ABMT) was studied in peripheral blood by phytohemagglutinin stimulated T-cell colony formation (CFU-TL) and by surface phenotype analysis of T-lymphocytes with monoclonal antibodies. Twenty-six patients (15 small-cell lung cancer, 5 lymphoma, 3 acute myeloid leukemia [AML], 2 germ cell cancer, and 1 melanoma) were conditioned with high-dose multiple drug combinations (plus total body irradiation in AML patients). No maintenance chemotherapy was given following treatment. Despite a rapid return to normal values of peripheral T3+, T11+ lymphocytes, the T4/T8 ratio remained below 1.0 up to 24 months after transplant, regardless of infection by cytomegalovirus (CMV). A high percentage (26% +/- 3%) of lymphocyte cells with immature phenotype (T8+, Ia+) was found during the first 6 months after transplant. Out of 84 cultures, performed in 26 patients, no growth was observed in 47 instances (22 patients) up to 28 months after grafting. Growth occurred in 37 cultures (11 patients, from 1 to 51 months after transplant) although it never reached the colony numbers of normal controls. Recombinant human interleukin-2 (rIL-2) added to lymphocyte culture induced proliferation in 8 (4 CMV-positive and 4 CMV-negative patients) out of 12 instances of no growth. In cases of depressed CFU-TL (20 cultures), rIL-2 induced a 48% and 92% increase in six CMV-positive patients and nine CMV-negative patients, respectively. These observations show that after ABMT and regardless of CMV status, defects in CFU-TL can be partially corrected by rIL-2.     

α, β and γ T-cell receptor genes: rearrangements correlate with haematological phenotype in T cell leukaemias

We have studied the arrangement of the alpha, beta and gamma T cell receptor (TCR) genes in 27 patients with T cell lymphoproliferative disorders. Nine patients had acute lymphoblastic leukaemia (T-ALL), nine patients had prolymphocytic leukaemia (PLL), six patients presented with a T-CLL/T-lymphocytosis syndrome, two patients had Sezary syndrome (SS) and one patient had HTLV-I positive T-cell leukaemia/lymphoma (ATLL). alpha TCR gene rearrangement could be demonstrated by the use of three available probes in only one case. By contrast, both beta and gamma TCR gene rearrangement could be demonstrated by Southern blot analysis of DNA samples digested with appropriate restriction enzymes in the majority of cases. In general, when rearrangements were present they involved both alleles. The proportion of rearranged chromosomes was lower in T-ALL than in other forms of T-cell leukaemia and it was lower in cases with the CD4-/CD8+ phenotype than in those with a CD4+/CD8- phenotype. In three out of 34 cases of B-cell leukaemia the TCR beta-gene but not the TCR gamma-gene was rearranged, just as in two out of 26 cases of T-cell leukaemia the immunoglobulin (Ig) heavy chain but not the light chain genes were rearranged. These data suggest that development of the machinery required for gene rearrangement may precede commitment to B or T cell lineage. The use of this technique is especially useful for the classification of cases of ALL in which the cells are negative with respect to most current phenotypic markers and in cases of T cell lymphocytosis in which the finding of a gene rearrangement identifies a monoclonal cell population.     

Establishment and characterization of 10 cell lines derived from patients with adult T-cell leukemia.

By using human T-cell growth factor (TCGF), 10 cell lines were established from tissue samples of 10 patients with adult T-cell leukemia (ATL). Three cell lines were adapted to growth in medium lacking TCGF. The surface markers of all cell lines were characteristic of inducer/helper T cells, i.e., OKT3+, OKT4+, OKT6-, OKT8-, OKIa1+, and human Lyt2+ and Lyt3+, except that one cell line was OKT3-. The expression of the viral antigen was examined during establishment of 8 of the 10 cell lines. The viral antigen was not expressed in leukemic cells before cultivation. In 5 lines, the viral antigen was detected by immunofluorescent staining after a short period of cultivation. However, 3 cell lines, ATL-6A, ATL-9Y, and ATL-1K did not express the viral antigen during short-term culture: the ATL-6A and ATL-9Y cell lines became positive for the viral antigen after 5 and 2 months of cultivation, respectively; the ATL-1K cell line remained antigen-negative throughout a culture period of 13 months. Southern blot hybridization assay showed that all of the cell lines, including the viral antigen-negative ATL-1K cell line, contained the viral genome. Thus, the retrovirus was associated with all 10 cell lines established from ATL patients, but there was a heterogeneity in the expression time of the retroviral antigen in leukemic cells maintained in vitro. Our findings suggested that the expression of the viral antigen was not required for maintenance of the leukemic state in vivo and for growth of leukemic cells in vitro.     

Phenotypic heterogeneity of B- and T-cell differentiation antigens in B-CLL

Peripheral blood from 38 B-CLL patients was studied by flow cytometry with 25 clustered or non-clustered monoclonal antibodies (moAbs) in order to characterize the cell surface phenotype of lymphoid cells. All moAbs chosen detected B or CD1-8 T-cell differentiation antigens or MHC class II antigens. The results showed a heterogeneity in the leukemic cell reactivity with the various moAbs and between patients. The restricted B-cell antigens recognized by BL14, Y2955 and anti-class II moAbs were constantly expressed in leukemic cells, while B-cell antigens reacting with FMC7 and BL13 moAbs were variably detected. In addition to CD5 antigens, other T-cell markers, including several epitopes of the CD1 group, were also found to be present on the leukemic cell surface in several cases. The relationship between the various clinical and/or biological features of the disease and the cell surface phenotype was studied. Our results indicated a correlation between the lymphoid cell reactivity with M241 (CD1) and BL13 (CD21) and the severity of the disease.     

A new monoclonal antibody (CH-F42) recognizes a CD7- subset of normal T lymphocytes and circulating malignant cells in adult T-cell lymphoma-leukemia and Sézary syndrome.

We describe a new rat immunoglobulin M monoclonal antibody (CH-F42) that recognizes a subset (1.5% to 8%) of normal peripheral blood T lymphocytes. The phenotype of these cells was determined, using dual-color immunofluorescence, to be CD2+, CD3+, CD4+, CD5+, CD7-, CD8-. They do not express T-cell activation markers, and are positive for UCHL1 (CD45RO), but negative for 2H4 (CD45RA). The antigen was expressed on circulating malignant cells in Sézary syndrome (four of four cases) and adult T-cell lymphoma-leukemia (ATLL) (four of six cases) and negative in a variety of other hematologic malignancies tested. These included chronic and acute lymphoid leukemias of B and T lineage, together with chronic and acute myeloid leukemias. However, normal CH-F42+ cells do not display any of the ultrastructural features associated with Sézary or ATLL cells. The marked similarities between these conditions together with the shared expression of an otherwise very restricted surface antigen (CH-F42) provide strong evidence for the existence of a common normal counterpart. Preliminary characterization studies of the antigen, which is also expressed by K562 and Jurkat cells, suggest the CH-F42 antigen is an O-linked, sialated glycan on a glycoprotein.     

Rearrangement of the T-cell receptor delta genes in human T-cell leukemias

Two distinct types of T-cell receptors (TCR), designated alpha beta and gamma delta, have been identified on the surface of T cells. In the adult, T cells bearing the gamma delta TCR are a minority and they have the phenotype CD3+, CD4-, CD8-/+. By using appropriate probes, rearrangements of the TCR alpha, beta, and gamma genes have been extensively investigated in a variety of lymphoproliferative disorders. Because the TCR delta gene has been cloned only recently, no comparable information exists with respect to this in human leukemias. We report the analysis of the TCR delta gene configuration in 21 T-cell acute and chronic leukemias, 40 B-cell leukemias, 4 acute myeloid leukemias of difficult classification, and 12 normal controls. The TCR delta genes were structurally modified in all T-cell disorders and in germ-line configuration in all controls and all but one case of non-T-cell leukemias tested. In one case of T-chronic lymphocytic leukemia (CD3+, CD4-, CD8+) we found rearrangement and expression of TCR gamma and delta (but not alpha and beta), suggesting that leukemic transformation took place in a cell bearing a TCR gamma delta rather than a TCR alpha beta. In two cases of pre-T-acute lymphoblastic leukemia, only delta was rearranged out of the three TCR genes tested. This finding is in keeping with the suggestion that the TCR delta gene might be the first to rearrange in T cell ontogeny, and that its mode of rearrangement may play a role in the subsequent choice of the cell between production of a TCR alpha beta or gamma delta. Thus, TCR delta chain gene analysis can provide novel information of the clonal nature of T-cell disorders, particularly if the analysis of the beta and gamma genes has not been helpful.     

Dipeptidylaminopeptidase IV (DAP IV) activity in normal and malignant T-cell subsets as defined by monoclonal antibodies

The activity of dipeptidylaminopeptidase IV (DAP IV) was investigated by a cytochemical method in isolated fractions of normal peripheral blood mononuclear cells and malignant cells from 9 patients with chronic T-cell leukaemia. A positive DAP IV reaction was observed in 52% of normal T cells, a consistent negative reaction in normal B cells and monocytes. 2 distinct T-cell subsets, helper/inducer cells (T4+/T8-) and cytotoxic/suppressor cells (T4-/T8+), were negatively selected by complement-mediated cytolysis utilizing the monoclonal antibodies OKT4 and OKT8. On examination of these T-cell subsets a positive DAP IV reaction was restricted to the majority of normal T4+/T8- cells. The malignant cells from 3 patients with T-chronic lymphocytic leukaemia, expressing the cytotoxic/suppressor phenotype (T4-/T8+) lacked DAP IV activities. In contrast, almost all leukaemic cells from 3 other cases, expressing the helper/inducer phenotype (T4+/T8-), were strongly positive. Despite their T4+/T8- phenotype, the malignant cells from 3 patients with Sézary syndrome were completely DAP IV negative. These findings suggest that the DAP IV reaction may be helpful in the further characterization of normal and malignant T-cell subsets.     

Interleukin-2-dependent T-cell lines established from paroxysmal nocturnal hemoglobinuria patients

Peripheral blood T lymphocytes obtained from two patients with paroxysmal nocturnal hemoglobinuria (PNH) were immortalized with human T-lymphotropic virus type 1 (HTLV-1). These cells showed interleukin-2 (IL-2)-dependent cell growth in culture. Cell surface analysis showed that they had the phenotype of a helper/inducer T subset that was positive for CD2, CD3, and CD4, but negative for CD8, similar to adult T-cell leukemia cells induced by HTLV-1. These cell lines lacked glycosylphosphatidylinositol (GPI)-anchored proteins, CDw52, CD55 (decay-accelerating factor; DAF), and CD59 on the cell surface, whereas intracellular DAF protein was detected. These T-subset cell lines with a PNH phenotype did not synthesize GPI anchor, whereas a control cell line, similarly prepared from the T cells of a healthy volunteer, produced the anchor. The control cells expressed CDw52, DAF, and CD59 on the cell surface and showed the phenotype of a helper/inducer subset. Southern blot analysis confirmed the clonality of each cell line. These CD4+ T-cell lines with a PNH phenotype and a subset-matched control counterpart could be a useful model for PNH investigation.     

Acquired immunodeficiency syndrome-associated T-cell lymphoma: evidence for human immunodeficiency virus type 1-associated T-cell transformation.

The majority of lymphomas in the setting of acquired, iatrogenic, or congenital immunodeficiencies are B-cell lymphoproliferations. We describe a rare T-cell lymphoma in a fulminantly ill patient infected with human immunodeficiency virus type 1 (HIV-1). The T-cell nature of the process was defined genotypically (monoclonal T-cell receptor beta-chain [CT beta] rearrangement) and phenotypically (CD45RO+, CD4+, CD5+, CD25+, CD8-, CD3- and negative for a variety of B-cell and monocyte markers). The CD4+, CD25+ (interleukin-2 receptor [IL-2R]) phenotype with production of IL-2 and IL-2R RNA is analogous to human T-lymphotropic virus type I (HTLV-I)-associated adult T-cell leukemia/lymphoma (ATLL); however, no HTLV-1 could be detected. Southern blot analysis did demonstrate monoclonally integrated HIV-1 within the tumor genome. Furthermore, the tumor cells were producing HIV p24 antigen as shown by immunohistochemistry. This is the first case of acquired immunodeficiency syndrome (AIDS)-associated non-Hodgkin's lymphoma in which HIV-1 infection may have played a central role in the lymphocyte transformation process.     

Oligodendrocyte-specific autoreactive T cells using an alpha/beta T-cell receptor kill their target without self restriction.

The oligodendrocyte (Od), a glial cell that produces myelin in the central nervous system, may be a target for autoreactive T cells in autoimmune demyelinating processes, although not expressing major histocompatibility complex (MHC) products. To analyze Od-T-cell interactions, we selected from normal SJL/J mouse splenocytes sensitized in vitro by Lewis rat Od a T-cell clone, named C2, exhibiting a surface phenotype of mature T cell (Thy 1+, CD3+, CD8+, CD4-, asialo-GM1-). C2 T cells displayed a specific cytotoxicity to syngeneic Od as well as to rat Od, but not to astrocytes or lymphoblasts, or to YAC-1 cells, a target for natural killer and lymphokine-activated killer activity. The T-cell receptor of clone C2 was found to be a CD3-associated alpha/beta-chain heterodimer similar to that usually expressed by antigen-specific MHC-restricted mature T cells. Attempts to block the C2-mediated cytolysis by a series of monoclonal antibodies showed that both the CD3-T-cell receptor complex and the CD8 accessory molecule were required for OD-T-cell interaction and confirmed the lack of involvement of polymorphic MHC products as epitope-presenting structures. Antibodies directed against a surface Od glycoprotein, previously shown to elicit demyelinating autoantibodies in experimental autoimmune encephalomyelitis, fully blocked the cytotoxicity of T-cell clone C2 to its Od target. These data suggest that an epitope of a surface Od glycoprotein may be directly and specifically recognized and killed by autoreactive T cells expressing an alpha/beta receptor without conventional MHC restriction.     

The use of anti-T-cell receptor-Vbeta antibodies for the estimation of treatment success and phenotypic characterization of clonal T-cell populations in cutaneous T-cell lymphomas

Sézary syndrome and Mycosis fungoides are the most common forms of cutaneous T-cell lymphomas. To assess the response to different therapies especially in Sézary syndrome, it is helpful to monitor the percentage of circulating tumour cells in the blood. The use of T-cell receptor (TCR)-Vbeta specific monoclonal antibodies provides a suitable tool for detecting Sézary cells. In this study, we analysed the levels of clonal CD4+Vbeta+ cells of seven patients with various treatment modalities using flow cytometry and investigated the immunophenotype of the clonal cells by double staining with a panel of antibodies recognizing lymphatic surface markers. Additionally, a polymerase chain reaction-denaturing gradient gel electrophoresis assay was performed on clonal CD4+Vbeta2+ cells, showing that these cells carry a Vgamma10/11, JgammaP1/2 TCR rearrangement. Follow-up studies revealed close association of the Vbeta+ clone developmentwith the clinical response to different therapiesinsixpatients. Intwo cases, the CD4+Vbeta+ cells decreased accompanied by partial regression or even complete remission. In four cases, a stable or increasing clonal CD4+Vbeta+ population reflected well a stable or progressing course of the disease. Double staining of Vbeta+ cells revealed the following pattern, CD3+, CD5+, CD7+, CD28+, CD80-, CD86+ and human leucocyte antigen (HLA) class I+. In contrast, HLA-DR was heterogeneously expressed. We conclude that identification and monitoring of CD4+Vbeta+ clonal T cells by fluorescence-activated cell sorting with double staining is a suitable method to assess clinical responses to different therapies.     

Heterogeneity of large granular lymphocyte proliferations: morphological, immunological and molecular analysis in seven patients

The clinical, morphological, immunological and molecular features of seven patients with a stable picture of chronic granular lymphocytosis, observed over a period of up to 4 years, were studied. Mild splenomegaly was detected in one patient, while lymphoadenopathy and hepatomegaly were absent. Surface marker analysis showed in five patients the common membrane phenotype of granular T-cell lymphocytosis (T3+, T4-, T8+, Leu-7+); of the remaining two, one presented an unusual phenotype (T3+, T4+, T8+) and the other showed a marked positivity with the Leu-11 and M1 monoclonal antibodies, but lacked the T3, T4, T8 antigens. Three cases had a low (less than 30%) expression of the T1 antigen. Functional studies showed that the proliferative response to PHA and the NK function were reduced in four of the seven cases. Molecular analysis, performed in six cases, revealed a monoclonal rearrangement of the T-cell receptor beta-chain gene in three, a polyclonal T-cell configuration in two and a germ-line arrangement in the last. All three monoclonal cases showed a depressed NK activity and two a reduced PHA response. The results of this study document the heterogeneity of granular lymphocyte expansions and suggest that the clonal or reactive nature of these often indolent proliferations, suspected on the basis of immunologic functional studies, may be recognized at the DNA level.