Expression and Production of Interleukin 4 in B-Cell Chronic Lymphocytic Leukaemia

Interleukin (IL) 4 is a T-cell derived pleiotropic cytokine whose properties include alterations of B-cell function. In B-cell chronic lymphocytic leukaemia (B-CLL), IL4 is involved in the mechanism of survival of the leukaemic B-cells. The present study examines the expression and production of IL4 by B- and T-lymphocytes derived from patients with B-CLL and provides evidence that IL4 is not an autocrine factor in B-CLL. Freshly isolated B-CLL cells enriched for B- and T-cells did not express mRNA for IL4 but expressed mRNA for IL4 receptor (IL4R). Activation of B-cells with phorbol ester and calcium ionophore and of T-cells with phytohaemaglutinin (PHA) upregulated IL4 mRNA expression. However phorbol ester and calcium ionophore did not affect the mean level of IL4 production by either B-CLL or normal B-cells. Furthermore, in the presence or absence of activation, the amount of IL4 synthesised by B-CLL B-cells was not significantly different than that observed with peripheral blood B-cells isolated from normal individuals (with activation: P=0.239; without activation: P=0.565). Also, there was no significant difference between normal and B-CLL B-cells in the level of cytoplasmic IL4 (P=0.47).

PHA-activated enriched B-CLL T-cells produced significantly higher levels of IL4 compared to normal control T-cells (P=0.0136). In addition, in 47% of cases with B-CLL T-cells, a significant higher level of intracellular IL4 was observed (P=0.0027). The levels of production of IL4 by the T-cell-enriched preparations correlated positively with the intensity of cytoplasmic IL4 in CD4⁺ and CD8⁺ cells in tested samples (r=0.49 and r=0.76, respectively).

The significant differences observed in the production of IL4 by B-CLL B- and T-lymphocytes may suggest a paracrine function of IL4 in B-CLL.     

Bcl-2 and bax expression and chlorambucil-induced apoptosis in the T-cells and leukaemic B-cells of untreated B-cell chronic lymphocytic leukaemia patients

Chlorambucil and other cytotoxic drugs kill cells, non-selectively, by inducing apoptosis. In this study, we measured the apoptotic response to chlorambucil in T- and B-cells from untreated B-CLL patients and T-cells, from normal control subjects. We found increased chemosensitivity in the T-cells of B-CLL patients compared to the controls (P=0.0002). The chlorambucil ID(50) values for T-cells from B-CLL patients showed a direct correlation with Bcl-2 expression (P=0.002) and an inverse correlation with CD3 cell count (P<0.0001), suggesting a trend of increasing chemosensitivity and decreasing Bcl-2 expression with an elevated T-cell count. There was no differential expression of Bcl-2 or Bax between the CD4(+) and CD8(+) cells of B-CLL patients, isolated by immunomagnetic separation. We found correlations in the leukaemic B-cells between chlorambucil ID(50) values and both Bcl-2 expression (P=0.006), and Bcl-2/Bax ratios (P=0.002), suggesting a role for the Bcl-2/Bax ratio in predicting the response of untreated CLL patients to cytotoxic treatment. Chlorambucil produced almost identical changes in Bcl-2 and Bax expression in normal T-cells and leukaemic B-cells triggered to die by apoptosis, which together with the correlation between Bcl-2 and chemosensitivity confirms a pivotal role for Bcl-2 in regulating a distal step in the apoptotic pathway following cytotoxic cellular damage.     

Intracellular cytokine profiles by peripheral blood CD3+ T-cells in patients with classical Hodgkin lymphoma

The intracellular profiles of T helper type 1 (Th1) and T helper type 2 (Th2) T-cell cytokines by peripheral blood (PB) CD3+ T-cells in patients with classical Hodgkin lymphoma (HL) has not been investigated before. The present study examines the cytoplasmic production of interleukin (IL) 2, 4, 10, tumour necrosis factor alpha (TNFalpha), and interferon gamma (IFNgamma) by activated PB CD3+ T-cells and compares them with the profiles observed with normal individuals. We report a significantly lower mean level of intracellular IL2, TNFalpha and IFNgamma at any time post-cell activation in cells isolated from patients with HL compared with the normal control group. In contrast, the mean level of cytoplasmic IL4 was significantly higher in the HL compared with the control group. No significant difference between the two groups was observed with IL10. In the HL patient group, there was a significantly higher percentage of CD3+CD8+ T-cells that synthesised IL4 compared with the CD3+CD4+ subpopulation, no such difference was observed in normal controls. The intensity of IL4 (expressed as relative median fluorescence) was significantly higher in the CD3+CD8+ cells of the patients with HL compared with the CD3+CD4+ sub-population, or with normal CD3+CD8+ cells. In conclusion, there is reduced intracellular IL2, TNFalpha and IFNgamma and increased cytoplasmic IL4 production by activated PB T-cells in patients with HL. The CD3+CD8+ sub-population is responsible for the increased levels of IL4.     

Gamma-linolenic acid induces apoptosis in B-chronic lymphocytic leukaemia cells in vitro.

Gamma linolenic acid (GLA) is cytotoxic to many types of human cancer cells. Most chemotherapeutic agents are cytotoxic by inducing apoptosis. We examined the apoptotic activity of GLA on purified B-cells isolated from patients with B-cell chronic lymphocytic leukaemia (B-CLL) and from normal individuals. GLA significantly increased the degree of apoptosis in B-CLL B-cells after 24 hours of culture. The mean percentage of cells undergoing apoptosis when cultured in medium alone (spontaneous apoptosis) was 20% (range: 7 to 31%) (n=25) and in the presence of GLA (5 microg-60 microg) was: 42%-95%. In the presence of GLA 5 microg/ml and dexamethasone the degree of apoptosis was 86% (range: 72 to 100%). GLA induced apoptosis in B-CLL B-cells at a higher level than that observed with normal B-cells at all lower concentrations tested 5, 10 and 15 microg/ml: P=0.045; 0.027 and 0.022, respectively. At 30 microg/ml of GLA, no significant difference in the percentage of cells displaying apoptosis between B-CLL and normal B-cells was observed (P=0.075). GLA induced apoptosis in B-CLL T-cells at both 10 and 30 microg/ml. The degree of apoptosis in normal T-cells with GLA was also significant at the higher concentration of 30 microg/ml. Interleukin 4 (IL4), a viability factor in B-CLL, and vitamin E, an anti-oxidant, protected B-CLL B-cells against GLA (20 microg/ml)-induced apoptosis. These results demonstrate that GLA induces apoptosis in B-CLL B- and T-cells cells in vitro and that they are more susceptible to GLA-induced apoptosis than normal peripheral blood B- and T-cells.     

γ-Linolenic Acid Induces Apoptosis in B-Chronic Lymphocytic Leukaemia Cells in Vitro

Gamma linolenic acid (GLA) is cytotoxic to many types of human cancer cells. Most chemotherapeutic agents are cytotoxic by inducing apoptosis. We examined the apoptotic activity of GLA on purified B-cells isolated from patients with B-cell chronic lymphocytic leukaemia (B-CLL) and from normal individuals. GLA significantly increased the degree of apoptosis in B-CLL B-cells after 24 hours of culture. The mean percentage of cells undergoing apoptosis when cultured in medium alone (spontaneous apoptosis) was 20% (range: 7 to 31%) (n=25) and in the presence of GLA (5μg-60μg) was: 42%-95%. In the presence of GLA 5μg/ml and dexamethasone the degree of apoptosis was 86% (range: 72 to 100%). GLA induced apoptosis in B-CLL B-cells at a higher level than that observed with normal B-cells at all lower concentrations tested 5, 10 and 15μg/ml: P=0.045; 0.027 and 0.022, respectively. At 30μg/ml of GLA, no significant difference in the percentage of cells displaying apoptosis between B-CLL and normal B-cells was observed (P=0.075). GLA induced apoptosis in B-CLL T-cells at both 10 and 30μg/ml. The degree of apoptosis in normal T-cells with GLA was also significant at the higher concentration of 30μg/ml. Interleukin 4 (IL4), a viability factor in B-CLL, and vitamin E, an anti-oxidant, protected B-CLL B-cells against GLA (20μg/ml)-induced apoptosis. These results demonstrate that GLA induces apoptosis in B-CLL B-and T-cells cells in-vitro and that they are more susceptible to GLA-induced apoptosis than normal peripheral blood B-and T-cells.     

Interleukin 4 production by peripheral blood lymphocytes in patients with classical Hodgkin lymphoma

Although the production of interleukin (IL) 2 and interferon (IFN) gamma by peripheral blood lymphocytes in patients with Hodgkin lymphoma (HL) is well documented, the synthesis of IL4 has not been investigated before. The present study examines the production of IL4 by 2-day phytohaemaglutinin (PHA)-stimulated peripheral blood (PB) cells in HL and correlates the cytokine levels with the proportion of the different T-cell sub-populations. We observed a significant increase in the mean level of production of IL4 in patients with HL when compared with normal controls. The increased amount of IL4 in patients with HL correlated significantly with the proportion of the CD3(+)CD8(+) cells but not with CD3(+)CD4(+). The intensity of cytoplasmic IL4 (expressed as relative median fluorescence (RMF)) was significantly higher in the CD3(+)CD8(+) cells of the patients with HL compared with the CD3(+)CD4(+) sub-population, or with the normal CD3(+)CD8(+) cells and correlated with the levels of IL4 release in culture supernatants. In conclusion, there is increased production of IL4 by PHA-activated PB lymphocytes in HL. The CD3(+)CD8(+) T-cell population appears to be responsible for this increased synthesis.     

Quantification and cytokine production of circulating lymphoid and myeloid cells in acute myelogenous leukaemia.

A simple assay was developed to assess the potential of patients with acute myelogenous leukaemia (AML) to respond to immunotherapy. Lymphocytes, monocytes and leukaemic blasts with their corresponding intracellular cytokine profiles were evaluated by four-colour flow cytometry. In 50 microl samples of whole blood, surface labelling for CD45, CD8 and CD3 was used for cell identification prior to intracellular staining for interleukin (IL)-4, IL-10, IL-12 and interferon (IFN)-gamma. Absolute numbers of CD8(+) and CD8(-) (putative CD4(+)) T-cells, NK cells (CD8(+)/CD3(-)) and monocytes were determined by reference to a fixed number of added fluorescent beads. The absolute numbers of CD8(-) and CD8(+) T-cells in the blood of patients with AML were similar to those of normal controls. More of the lymphocytes in the blood of leukaemic patients spontaneously produced cytokines compared with those of controls. Furthermore, primary AML blasts secreted predominantly IFN-gamma. After recovery from chemotherapy, lymphocyte counts tended to be lower than in normals and reduction of NK cells reached significance after the second chemotherapy (P=0.01). A prominent CD8(lo)/CD3(lo-int) lymphocyte subset appeared after recovery in some patients. This laboratory application of the study of cell subsets and intracellular cytokines in patients undergoing treatment may be helpful in monitoring immunological responses in AML.     

Exogenous interleukin 2 (IL 2) reconstitutes deficient in vitro T-cell growth of patients with chronic lymphocytic leukemia of B-cell lineage (B-CLL)

Mechanisms of tumor growth are concerned with the possibility that neoplasms are disturbed in their response to growth factors. Interleukin 2 (IL 2) is secreted by activated T-cells and is required for autocrine T-cell growth. Controversy exists as to the functional capacity of T-lymphocytes in patients with B-cell chronic lymphocytic leukemia (CLL). We studied 14 patients with previously untreated, early CLL. Data were compared to 16 healthy individuals. Immunophenotyping showed that T-cells in B-CLL are highly activated as evidenced by HLA-DR and IL 2 receptor expression. Membrane receptors for IL 2 on the patients' T-cells were overexpressed. The T4:T8 ratio was 1.5. T-cells for in vitro studies were isolated by their ability to form E-rosettes with AET and repeated Ficoll density gradient centrifugation. Purified T-cells were grown on semisolid agar and their capability to form colonies investigated using a variety of exogenous stimulants. It was found that patients with CLL showed a pronounced impairment in forming T-cell colonies. However, colony formation was restored to normal by adding exogenous recombinant IL 2, indicating that endogenous IL 2 production of T-cells is deficient. This finding might have implications for the therapeutical use of IL 2 in CLL patients.     

T-Cell Membrane CD45RA (2H4) and CD45RO (UCHL1) Determinants: II, Aberrant HLA-ABC Expression by CD45RA and CD45RO Cell Subpopulations of Mature CD4+ T-Cell Leukaemias

Six thymocyte suspensions, 10 normal blood CD4⁺ CD8^- lymphocyte-enriched fractions and leukaemic cells from 24 patients with CD4⁺ mature T-cell lymphoid malignancy (five Sezary Syndrome, six adult T-cell leukaemia-lymphoma and 13 cases of T-cell prolymphocytic leukaemia) were examined in this study for the expression of membrane HLA-ABC by CD45RA (2H4) and CD45RO (UCHL1) subpopulations. These analyses showed that the main increase in HLA-ABC expression by normal CD4⁺ CD8^- blood lymphocytes (mean 490 to 760 FITC units) paralleled the loss of membrane 2H4 whilst the acquisition of UCHL1 was not associated with any significant change in HLA-ABC staining intensity. The sequence of 2H4 differentiation by normal thymocytes, based on the observed increasing levels of HLA-ABC staining intensity appeared to be (a) CD1a ⁺ 2H4^- UCHL1⁺ (25 HLA-ABC fluorescent units), (b)CD1a^-2H4^intUCHL1⁺ (134 units), and (c) CD 1a^- 2H4 ⁺ UCHL1 ^- (197 units). Quantitative estimates of membrane HLA-ABC expression by leukaemic T-cells revealed marked heterogeneity between individual cases irrespective of diagnostic subgroup. Based on the lower observed limits for normal CD4⁺ 2H4⁺ (318 units) and CD4 ⁺ 2H4^- (478 units) fractions, 14% and 38% respectively of the leukaemic 2H4⁺ and 2H4^- components examined showed reduced HLA-ABC expression. Two cases showed very low membrane HLA-ABC levels that were within the range observed for normal CD1a^- thymocytes. In contrast, HLA-ABC staining intensities exceeding that of corresponding normal CD4⁺ 2H4⁺ (710 units) and CD4⁺ 2H4^- (1286 units) subpopulations were seen in a high proportion (65%) of leukaemic 2H4 ⁺ components, with only 14% of 2H4^- fractions showing raised levels and, in two cases, these staining intensities exceeded three times the normal observed limits. In addition to the quantitative differences in HLA-ABC expression, a remarkably consistent (81% of evaluable cases) feature of the leukaemic T-cells was that the 2H4^-UCHL1⁺ subpopulation in CD4⁺ malignancies had a lower HLA-ABC level than the 2H4⁺UCHL1 subpopulation. This was in marked contrast to normal post-thymic T-cells where increasing HLA-ABC expression was seen with increasing UCHL1 (or decreasing 2H4) staining. These results suggest that leukaemic T-cells have an aberrant intra-thymic and post-thymic sequence of 2H4/UCHL1 expression which has become 'uncoupled' from CD1a/HLA-ABC expression.     

Elevated levels of soluble CD44 are associated with advanced disease and in vitro proliferation of neoplastic lymphocytes in B-cell chronic lymphocytic leukaemia

PURPOSE: Increased expression of the adhesion molecule CD44 has been associated with an unfavourable clinical outcome in lymphomas. We evaluated the prognostic value of soluble CD44 in B-cell chronic lymphocytic leukaemia (B-CLL) and analysed the source and regulation of CD44 secretion in B-CLL clones in vitro. PATIENTS AND METHODS: Levels of soluble CD44 standard (sCD44s) and of the soluble variant isoform CD44v6 (sCD44v6) were analysed by enzyme linked immuno sorbent assay. Highly purified B-CLL cells (98% CD19 + CD3 - cells) were stimulated in vitro by different combinations of thioredoxin (Trx), Staphylococcus aureus Cowan strain 1 (SAC), IL-2, IL-4, IL-10, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and by anti-CD40 mAbs presented on irradiated CD32L cells. RESULTS: Serum levels of sCD44s and of sCD44v6 are significantly elevated in B-CLL patients (n = 90) in comparison with normal persons (n = 44) (P < 0.001). Elevated levels of sCD44s and sCD44v6 are associated with an advanced disease as reflected by an extended lymph node involvement (P < 0.02), an advanced Binet (P < 0.03) and Rai stage (P < 0.04) and chemotherapy requirement (P < 0.02). High levels of sCD44s are associated with high leukocyte counts (P < 0.04) and increased sCD44v6 is significantly associated with splenomegaly (P < 0.002). In B-CLL sCD44s as well as sCD44v6 is shed from leukaemia cells as shown by in vitro cultures. Stimulation of B-CLL clones results in a proliferation-associated increased secretion of sCD44s (rho = 0.7; P = 0.0001) and of sCD44v6 (rho = 0.5; P = 0.005). B-CLL clones from advanced stage patients are characterised by an increased capacity for proliferation and CD44 production in comparison with early stage patients. CONCLUSIONS: Both sCD44s and sCD44v6 represent a reliable prognostic marker in B-CLL and may be involved in the pathogenesis of B-CLL.     

Expansion of human T regulatory type 1 cells in the microenvironment of cyclooxygenase 2 overexpressing head and neck squamous cell carcinoma

Cyclooxygenase 2 (COX-2) overexpression and production of prostaglandin E(2) (PGE(2)) by head and neck squamous cell carcinomas (HNSCC) induce type 1 regulatory T (Tr1) cells and contribute to carcinogenesis by creating a tolerogenic milieu. To test this hypothesis, CD4(+)CD25(-) T cells obtained from the peripheral blood of 10 normal donors were cocultured with autologous dendritic cells, irradiated HNSCC cells and cytokines, interleukin 2 (IL-2), IL-10, and IL-15. HNSCC cells were either COX-2 negative, constitutively expressed COX-2, were transfected with COX-2, or had COX-2 expression knocked down by small interfering RNA. Other modifications included coculture plus or minus the COX-inhibitor, Diclofenac, or synthetic PGE(2) in the absence of HNSCC. Lymphocytes proliferating in 10-day cocultures were phenotyped by flow cytometry, studied for cytokine production by ELISA and for suppressor function in CFSE inhibition assays plus or minus anti-IL-10 or anti-transforming growth factor-beta(1) (TGF-beta(1)) monoclonal antibodies (mAb). COX-2(+) HNSCC or exogenous PGE(2) induced outgrowth of Tr1 cells with the CD3(+)CD4(+)CD25(-)IL2Rbeta(+)IL2Rgamma(+)FoxP3(+)CTLA-4(+)IL-10(+)TGF-beta(1)(+)IL-4(-) phenotype and high suppressor functions (range, 46-68%). Small interfering RNA knockout of COX-2 gene in HNSCC led to outgrowth of lymphocytes with decreased IL2Rgamma (P = 0.0001), FoxP3 (P = 0.05), and IL-10 (P = 0.035) expression and low suppressor activity (range, 26-34%). Whereas COX-2(+) cocultures contained IL-10 and TGF-beta(1) (medians, 615 and 824 pg/mL), cytokine levels were decreased (P < 0.0001) in COX-2(-) cocultures. Inhibition of COX-2 enzymatic activity in HNSCC abrogated outgrowth of Tr1 cells. Neutralizing mAbs to IL-10 and/or TGF-beta(1) abolished Tr1-mediated suppression. COX-2 overexpression in HNSCC plays a major role in the induction of Tr1 cells in the tumor microenvironment.     

Peripheral blood CD38 expression predicts survival in B-cell chronic lymphocytic leukemia

CD38 expression was investigated in 161 untreated patients with B-cell chronic lymphocytic leukemia (B-CLL). A score system, devised ad hoc by integrating the percentage and the mean fluorescence intensity (MFI) values of CD38(+) cells, indicated that B-CLL patients with a CD38 score < or =3 are characterized by a significantly longer survival compared to those with a CD38 score >3 (P=0.0026). Thirty-seven percent of patients with a CD38 score < or =3 and 58% of those with a score >3 were dead at 10 years. Multivariate analysis indicates that only the CD38 score successfully predicts survival (P=0.0028), with an estimated 3.8-fold greater risk of death for those cases with CD38 score >3.     

Gene transfer of CD154 and IL12 cDNA induces an anti-leukemic immunity in a murine model of acute leukemia.

IL12 is an essential cytokine for the generation of T helper 1 response, natural killer (NK) cells and cytotoxic T lymphocyte (CTL) stimulation. CD154 triggers CD40 on antigen-presenting cells, thus inducing antigen presentation to the immune system and production of IL12. As IL12 and CD154 share several pathways mediating immune response, we investigated in an aggressive murine model of acute leukemia the relative antileukemic efficiency of IL12, CD154 and IL12 + CD154 gene transfer. Live leukemic cells transduced by IL12, CD154, and IL12 + CD154 showed reduced leukemogenicity but CD154 protective effect was reduced when 10(6) leukemic cells were injected. Vaccines with lethally irradiated IL12-transduced cells were able to cure mice previously injected with 10(4) leukemic cells and adoptive transfer of IL12-induced antileukemic immunity protected recipient mice. NK cytotoxicity was enhanced in mice vaccinated with leukemic cells transduced by IL12, CD154, and CD154 + IL12. IL12 transduced cells induced IFN-gamma mRNA in CD4(+) and CD8(+) T cells isolated from the spleen of vaccinated animals, however, in vivo depletion experiments showed that IL12 vaccine effect was CD4(+) but not CD8(+) T cell dependent. We conclude that IL12 gene is a more potent candidate than CD154 for gene therapy of acute leukemia.     

Characterization of the role of CD8+T cells in breast cancer immunity following mammaglobin-A DNA vaccination using HLA-class-I tetramers

INTRODUCTION: Mammaglobin-A(mam-A) is expressed in over 80% of human breast tumors. We recently reported that mam-A DNA vaccination resulted in breast cancer immunity in a preclinical model. Here we investigated whether mam-A HLA-class-I tetramers could be used to monitor and define the role of CD8(+)cytotoxic T-lymphocytes(CTL) in mediating breast cancer immunity following mam-A DNA vaccination. STUDY DESIGN: Mam-A DNA vaccination was performed in HLA-A2(+)huCD8(+ )transgenic mice. HLA-A2 tetramers carrying the immunodominant mamA2.1 peptide were used to monitor CD8(+)CTL. Human breast cancer colonies were developed in immunodeficient SCID-beige mice. ELISPOT was used to correlate frequency of mamA2.1 tetramer(+)CD8(+)T cells and IFN-gamma production [spots per million cells (spm)] in human subjects. RESULTS: Vaccination of HLA-A2(+)huCD8(+) mice with mam-A DNA vaccine, but not empty vector, led to the expansion of mamA2.1 tetramer(+)CD8(+)T-cells in peripheral blood (<0.5% pre-vaccination compared to >2.0% post-vaccination). CD8(+)T cells from vaccinated mice specifically lysed UACC-812(HLA-A2(+)/mam-A(+), 25% lysis) but not MDA-MB-415(HLA-A2(-)/mam-A(+)) or MCF-7(HLA-A2(+)/mam-A(-)) breast cancer cells. Adoptive transfer of purified CD8(+)T cells from vaccinated mice into immunodeficient SCID-beige mice with established human breast cancer colonies led to tetramer(+)CD8(+ )T-cell infiltration with regression of UACC-812 but not MCF-7 tumors. HLA-A2(+) breast cancer patients revealed increased frequency of mamA2.1 tetramer(+)CD8(+ )T-cells compared to normal controls (2.86 +/- 0.8% vs. 0.71 +/- 0.1%, P = 0.01) that correlated with the IFN-gamma response to mamA2.1 peptide (48.1 +/- 20.9 vs. 2.9 +/- 0.8 spm, P = 0.03). CONCLUSIONS: CD8(+ )T-cells are crucial in mediating breast cancer immunity following mam-A DNA vaccination. Mam-A HLA-class-I tetramers can be effectively used to monitor development of CD8(+ )T-cells following mam-A vaccination.     

Down regulation of stem cell colony formation by purified CD8 lymphocytes and CD8 conditioned medium: potential importance for bone marrow transplantation in leukemia.

A methodology for selection of the CD8 cell subset from the peripheral blood and bone marrow mononuclear cells was developed using anti-T8 (CD8) antibody and magnetic microspheres coated with anti-mouse IgG. Following optimization of antibody:cell binding ratio and microsphere:cell ratios, CD8(+)-cells in the peripheral blood and bone marrow were effectively removed, with an overall final recovery of 34.9% +/- 8.6%, and 56% +/- 8.5% respectively with complete recovery of stem cells and very little contamination with effector cells. CD8(+)-depleted cell preparations demonstrated a 3-4 fold increase in CFU-C and CFU-E colony formation over non-depleted preparations when stimulated with G-CSF, GM-CSF or IL-3 and erythropoietin. The largest increase in colony formation was evident when IL-3 was used to stimulate colony formation. Purified autologous CD8+ T-cells or culture supernatant from in vitro cultures of purified autologous CD8+ T-cells added back to CD8 depleted preparations, induced 20%-90% suppression of CFU-C and CFU-E colony formation in a dose dependent manner. Colony formation by CD34+ cells, purified by anti CD34 antibodies and magnetic microspheres, were also inhibited by either pure CD8(+)-cell populations or CD8-culture supernatant. Preliminary fractionation studies indicate that the inhibitory factor is a protein of > 50 kd. In contrast, when purified autologous CD4+ cells were added to purified CD34+ stem cells, an increase (< 50%) in colony formation was observed. These results, taken together, suggest that CD8+ T-cells are negative effectors of normal hematopoiesis whereas CD4+ T-cells function as positive effectors.     

Augmented expression of programmed death-1 in both neoplastic and non-neoplastic CD4+ T-cells in adult T-cell leukemia/lymphoma

Adult T-cell leukemia/lymphoma (ATL) is a CD4(+)CD25(+) T-cell malignancy infected with human T-cell leukemia virus type-I (HTLV-I). HTLV-I infection causes the T-cell dysfunction, which contributes to the immunodeficient state of the patients. Programmed death-1 (PD-1) can negatively regulate T-cell response, when its ligand, PD-L1 or PD-L2 mainly expressed on antigen presenting cells, binds to this B7 family receptor. We investigated whether PD-1 is expressed on CD4(+) neoplastic (and/or non-neoplastic) cells or CD8(+) cytotoxic cells in peripheral blood mononuclear cells from 11 patients with ATL. By flow cytometry, we found that the levels of PD-1 expression on both CD4(+)CD25(+) and CD4(+)CD25(-) T-cell populations were increased in ATL patients compared to normal healthy volunteers, while PD-1 levels on CD8(+) T-cells were comparable between the patients and normal subjects. In stimulation with anti-CD3 antibody, the proliferation of PD-1-expressing T-cells from ATL patients was weak when compared to that of PD-1-nonexpressing normal T-cells. In addition to PD-1, PD-L1 was coexpressed on ATL cells in some patients, and PD-L1 expression was enhanced by stimulation with anti-CD3 antibody. Finally, the production of cytokines such as TNF-alpha by ATL cells was restored by blockade of PD-1/PD-L1 interaction. These findings suggest that CD4(+) T-cells are the main PD-1-expressing cells rather than CD8(+) T-cells in ATL patients, and both neoplastic and normal CD4(+) cells are exhausted as a result of PD-1 expression, and additionally PD-L1 expression on the neoplastic cell.