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.     

Increased frequency of CD4+ cells expressing CD161 in cancer patients.

PURPOSE: Although the function of natural killer receptors on T cells infiltrating tumors and their potential effect on antitumor immunity has been investigated, little is known about T cells expressing NKR-P1A (CD161) in cancer patients. In the present study, we examined T cells expressing CD161 in the peripheral blood, the tumor tissue and in malignant effusions of patients with several types of malignancies. EXPERIMENTAL DESIGN: Expression of CD161 in CD4(+) or CD8(+) (lacking CD56) T cells isolated from peripheral blood (n = 61), tumor specimens (n = 8), and malignant effusions (n = 37) of cancer patients was examined using four-color flow cytometry. Proliferative capacity and cytokine production of purified CD4(+)CD161(+)CD56(-) cells were studied after weak or strong stimulation, with or without costimulation, in the presence or absence of interleukin 2. The possible regulatory function of activated CD4(+)CD161(+)CD56(-) cells on T-cell alloresponses was also investigated. RESULTS: CD4(+) cells expressing CD161 were increased in cancer patients, compared with healthy individuals. This increase in the peripheral blood of cancer patients positively correlated with disease stage and was augmented at the tumor site. Phenotypic analysis revealed that CD4(+)CD161(+) cells are memory T cells, with low expression of activation markers. CD4(+)CD161(+) cells play an immunoregulatory role through cytokine production, because upon receiving costimulatory signals via CD28, they exert suppressive activity on autologous peripheral blood mononuclear cell alloresponses. CONCLUSIONS: CD4(+)CD161(+)CD56(-) cells represent a distinct memory T-cell population significantly increased in cancer patients. Depending on the type of signals provided by the tumor microenvironment, CD4(+)CD161(+) cells may regulate the immune response.     

Alterations in the T-cell receptor variable beta gene-restricted profile of CD8+ T lymphocytes in the peripheral circulation of patients with squamous cell carcinoma of the head and neck.

PURPOSE: Apoptosis of activated CD8(+) T cells is often seen in tumor-infiltrating lymphocytes and circulating peripheral blood mononuclear cells (PBMC) in patients with squamous cell carcinoma of the head and neck (SCCHN). We investigated whether T-cell receptor (TCR) variable beta chain (Vbeta)-restricted T cells were more sensitive to apoptosis than non-TCR Vbeta-restricted T cells. EXPERIMENTAL DESIGN: Flow cytometry analysis with anti-TCR Vbeta antibodies was used to define expansions and contractions of Vbeta-restricted T cells in patients with SCCHN relative to normal donors. This staining was combined with Annexin V binding to indicate early T-cell apoptosis. RESULTS: The TCR Vbeta profiles of CD3(+) T cells in tumor-infiltrating lymphocytes and PBMCs of patients with SCCHN were altered relative to controls, with one to five expansions and numerous contractions of TCR Vbeta-restricted T cells detected. These types of alterations were significantly greater in CD8(+) than CD4(+) T cells. Enhanced Annexin V binding to CD8(+) T cells was evident in PBMCs obtained from all patients, with 3 of 13 showing preferential targeting for apoptosis of TCR Vbeta-restricted T cells. CONCLUSIONS: TCR Vbeta profiles of CD8(+) T cells were altered in patients with SCCHN relative to normal controls. This may reflect increased apoptosis of expanded or contracted CD8(+) T cells, which define the TCR Vbeta profile of antigen-responsive T-cell populations in patients with cancer.     

Diminished production of interleukin 2 and gamma-interferon by cloned "T" cells from patients with the acquired immunodeficiency syndrome (AIDS)

A total of 76 T-cell clones established from peripheral blood (PB) of 2 patients with the acquired immune deficiency syndrome (AIDS) and of 141 T-cell clones established from PB of 3 normal donors were compared for their ability to produce interleukin 2 (IL-2) and gamma-interferon (gamma-IFN). Twenty-seven clones from AIDS patients and 85 clones from controls expressed the CD4 phenotype, whereas 49 clones from AIDS patients and 56 clones from controls expressed the CD8 phenotype. There were no significant differences in the proportions of IL-2-producing CD4 T-cell clones established from PB of patients with AIDS and controls, but the mean concentration of IL-2 produced by CD4 clones from AIDS patients was significantly lower than that produced by CD4 clones from controls. Both the proportion of gamma-IFN-producing CD4 clones and the mean concentration of gamma-IFN produced by CD4 clones were significantly lower in AIDS patients than in controls. In contrast, there were no differences between AIDS patients and normal individuals in the proportion of IL-2- or gamma-IFN-producing CD8 clones, or in the mean concentration of IL-2 and gamma-IFN produced by CD8 clones. These data suggest that the reduced ability of PB T-cells from patients with AIDS to produce IL-2 and gamma-IFN is not simply due to altered proportions or numbers of T-cell subpopulations, but also reflects intrinsic abnormalities of individual CD4 T lymphocytes.     

Analysis of lymphocyte surface markers in childhood acute lymphoblastic leukemia during maintenance chemotherapy]

In this study we investigated T-cell subsets, HLA-DR and IL2 receptor (IL2R, CD25) expression on T-cells in peripheral blood lymphocytes from the patients with childhood acute leukemia undergoing maintenance chemotherapy with 6-MP and MTX, and compared them with those in the healthy controls. There were decrease of relative number of CD4 positive (CD4+) cells and the increase of relative number of CD8 positive (CD8+) cells in comparison with those in controls; thus the ratio of CD4+ cells to CD 8+ cells decreased. HLA-DR and IL2R expression on T-cells increased in the patients. Increased HLA-DR expression was detected on both CD4+ cells and CD8+ cells, but the increased IL2R expression was related only to CD4+ cells. IL2R expression in the patients with side effects was higher than in controls. Methotrexate (MTX)-induced increase of HLA-DR and IL2R expression was observed in 2 of every 3 patients undergoing intensive chemotherapy.     

Elevated frequencies of CD4⁺ CD25⁺ CD127lo regulatory T cells is associated to poor prognosis in patients with acute myeloid leukemia

Regulatory T cells (Treg) mediate amelioration of disease and immune homeostasis by inhibiting immune activation and maintaining peripheral immune tolerance. The suppressive mechanisms and clinical significance of Treg have not been completely elucidated in patients with acute myeloid leukemia (AML). Here, we demonstrated that CD127 in combination with CD4 and CD25 can identify FoxP3(+) Treg in peripheral blood (PB) and bone marrow (BM) using multicolor flow cytometry. We showed that the CD4(+) CD25(+) CD127(lo) Treg frequencies were significantly increased and their phenotypes were different in PB from newly diagnosed AML patients compared to those from healthy volunteers (HVs). Moreover, the Treg frequencies were significantly higher in BM than those from PB in the same patients. The Treg frequencies were reduced when patients achieved complete remission (CR) and were increased when patients relapsed. The Treg frequencies at diagnosis in PB and BM of patients who had achieved CR were lower than those of patients who had persistent leukemia or died, respectively. CD4(+) CD25(+) Treg were isolated by magnetic-activated cell sorting and tested for suppressive functions in coculture with allogeneic carboxyfluorescein diacetate succinimidylester-labeled CD4(+) CD25(-) responder cells. Suppression mediated by Treg was higher in AML patients compared to HVs. No significant differences were observed in the cytokines production of Treg, including interferon-gamma (IFN-γ), interleukin (IL)-4,IL-2 and IL-10, between patients with AML and HVs. Our study suggests that Treg may play a role in the pathogenesis of AML, and sequential measurements of Treg frequency may have clinical value in the evaluation of therapeutic effects and clinical outcome.     

Naturally occurring T-cell response against mutated p21 ras oncoprotein in pancreatic cancer.

Mutated p21 ras proteins (muRas) are present in approximately 90% of pancreatic adenocarcinomas and express mutants which can function as cancer-specific antigens. To evaluate the frequency and magnitude of the natural T-cell response against muRas in 19 HLA-A2-positive patients with muRas-positive pancreatic carcinomas, antigen-experienced T lymphocytes in fresh peripheral blood mononuclear cells were shown by IFN-gamma enzyme-linked immunospot using muRas peptides (5-21) that encompass both HLA class I (HLA-A2)- and class II-restricted (HLA-DRB1) epitopes. Six of 19 patients (32%) were found to have a specific T-cell response against individual mutation-specific ras(5-21) but not against other ras mutations or wild-type ras. In contrast, none of 19 healthy subjects had T cells specifically secreting IFN-gamma (P = 0.004). The T-cell response consisted of both CD8(+) and CD4(+) T cells but was dominated by CD8 T cells in three of four patients. MuRas(5-14) and muRas(6-14) were shown to specifically induce CD8(+) T-cell mediated cytotoxicity against HLA-A2-positive, muRas-bearing pancreatic carcinoma cells. The T-cell response was not correlated with prognostic or clinical variables such as tumor-node-metastasis status, stage, or survival. In conclusion, a natural T-cell response against muRas proteins that could be exploited for immunostimulatory therapeutic approaches has been shown in a significant proportion of patients with pancreatic cancer.     

Peripheral blood and bone marrow immunophenotypic and functional modifications induced in acute leukemia patients treated with interleukin 2: evidence of in vivo lymphokine activated killer cell generation

The effect of treatment with interleukin 2 (IL2) on the phenotypic and functional immune system of acute leukemia patients was investigated. Fifteen acute myeloid leukemia and acute lymphoid leukemia patients with evidence of persistent disease were further subdivided into two groups according to the percentage of bone marrow (BM) blasts: group a had 6-15% blasts and group b had 30-65%. Following two cycles of IL2 (Glaxo Imb, Geneva, Switzerland) given i.v. by continuous infusion at escalating doses, no major changes in the proportion of CD3-, CD4-, and CD8-positive cells were encountered in the blood or in the marrow of either group of patients. When these could be retested after four cycles of IL2, a significant increase of CD3+ and CD4+ cells was documented in the peripheral blood (PB), as well as a significant increase of CD3+ cells in the BM. Irrespective of the number of cycles administered, the proportion of CD16+ cells increased significantly in the blood in both groups of patients and in the marrow of group a patients only. The expression of CD25 was significantly enhanced in all samples tested. Following IL2 administration, an enhancement of the natural killer compartment was documented. This was consistently more evident in patients with more limited disease. A significant amplification of the in vitro-induced lymphokine-activated killer function was noted in the BM of the treated patients. Furthermore, we documented the presence both in the PB and in the BM of "spontaneous" lymphokine-activated killer cells generated in vivo following IL2 administration. These results demonstrate that in acute leukemia of both myeloid and lymphoid origin, treatment with IL2 is capable of inducing profound immunophenotypic and functional modifications in PB and in BM lymphocytes, particularly in patients with more limited disease. The evidence of the in vivo activation of cytotoxic cells, particularly in the BM, may help to explain the clinical responses preliminarily observed in individual acute leukemia patients.     

The effect of peripheral blood lymphocyte stimulation on zeta chain expression and IL-2 production in Hodgkin's disease

It has been reported that peripheral blood T cells and NK cells express reduced levels of the T-cell receptor signal-transducing zeta chain in Hodgkin's disease (HD). The zeta chain has emerged as a key subunit of the T-cell antigen receptor, which plays a central role in the signal-transducing events leading to T and NK-cell activation. We were interested in determining whether the low zeta chain expression in HD could be corrected by anti-CD3, anti-CD3-rIL-2 ex vivo stimulation. Zeta chain expression was analysed by dual immunofluorescence on permeabilized cells before and after 72 hours of culture. The IL-2 concentration in the culture supernatants was measured by ELISA. Zeta chain was significantly reduced on unstimulated CD4+, CD8+ and CD56+ cells from patients in active disease compared with normal subjects. In patients in complete remission, the values were normal except for CD8+ cells, on which zeta expression remained significantly reduced. Stimulation with anti-CD3 did not change zeta expression. Co-stimulation with rIL-2 increased but did not normalize the proportions of CD4(+)/zeta(+), CD8(+)/zeta(+)and CD56(+)/zeta(+)cells and IL-2 production in active disease. Stimulation of cells from patients in clinical remission with anti-CD3(+)rIL-2 increased the proportion of CD8(+)zeta(+)cells and normalized IL-2 production levels. Considering the pivotal role of CD3-zeta in immune response, our data suggest that successful immunotherapy approaches in active HD should consider inclusion of other potent cytokines, as well as genetically engineered tumour vaccines.     

Fas-FasL-mediated CD4+ T-cell apoptosis following stem cell transplantation.

We report the preferential expression of Fas on CD4+ T cells and Fas ligand (FasL) on monocytes and their potential role in the selective loss of CD4+ T cells in breast cancer patients undergoing high-dose chemotherapy and peripheral blood stem cell transplantation (PSCT). A high frequency of apoptotic CD4+ T cells (28-51%) is observed during the first 100 days after PSCT concomitant with a significant increase in monocyte frequency and FasL expression (11.6-23%) on monocytes. The preferential expression of Fas on CD4+ T cells (73-92%) in the peripheral blood (PB) of these patients is associated with a significantly higher frequency of CD4+ T-cell apoptosis compared with CD8+ T cells (28-47%) and CD4+ T cells (46 +/- 5.7%) in normal PB. These data suggest that "primed" Fas+ CD4+ lymphocytes interact with activated monocytes that express FasL, resulting in apoptosis, leading to deletion of CD4+ T cells, an inversion in the CD4:CD8 T-cell ratio, and immune dysfunction. The prevention of CD4+ T-cell apoptosis and improved immune reconstitution by the manipulation of PB stem cell products, blockade of Fas-FasL interactions, or cytokine support after transplantation may be important adjuvant immunotherapeutic strategies in patients undergoing high-dose chemotherapy and PSCT.     

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.     

Altered cytokine levels and increased CD4+CD57+ T cells in the peripheral blood of hepatitis C virus-related hepatocellular carcinoma patients

Although CD57+ lymphocytes are closely correlated with prognosis in various cancers, the role of subsets of CD57+ cells in hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC) is unclear. In the present study, peripheral blood (PB) from HCV-related HCC patients was analyzed. Plasma cytokine levels and in vitro cytokine-producing capabilities were analyzed with enzyme-linked immunosorbent assays, and CD57+ cell subsets were studied using a multi-color FACS system. Interferon (IFN)-γ was undetectable in the plasma of patients with tumors at any stage, whereas the plasma levels of tumor necrosis factor (TNF)-α, interleukin (IL)-10 and IL-18, but not that of IL-12, were significantly higher in stage IV patients compared to patients with earlier-stage tumors. In contrast, the IFN-γ-producing capability of PB was highest in stage I patients and gradually decreased with tumor progression. The IL-10-, IL-18- and IL-12-producing capabilities of PB increased from stage I to III. However, PB-TNF-α, IL-10- and IL-18-producing capabilities were reduced in stage IV patients, probably due to repeated anti-cancer treatments. The percentage of CD4+CD57+αβTCR+ cells (CD4+CD57+ T cells) in peripheral blood lymphocytes (PBLs) increased with tumor progression. Moreover, the percentage of CD4+CD57+ T cells in PBLs and the ratio of CD4+CD57+ T cells to CD4+αβTCR+ cells (CD4+ T cells), but not that of CD4+CD57+ T cells to CD57+αβTCR+ cells (CD57+ T cells), showed a significant inverse correlation with PB-IFN-γ-producing capability. The present results suggest that an increase in CD4+CD57+ T cells controls the capability of PB to produce the anti-tumor cytokine IFN-γ and that PB-IFN-γ production is impaired with HCC tumor progression.     

NKT and CD8 lymphocytes mediate suppression of hepatocellular carcinoma growth via tumor antigen-pulsed dendritic cells

Dendritic cells (DCs) are antigen presenting cells that play a role in T-cell activation. Liver-associated natural killer T lymphocytes (NKTs) are a unique subset of lymphocytes that may be important in antitumor immunity. Hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC) expresses hepatitis B virus surface antigen (HBsAg) on its cell surface and may serve as a tumor-associated antigen. The aim of the study was to evaluate the antitumor effect of DC pulsed with tumor or viral-associated antigens in HBV-expressing HCC in mice and to determine the role of NKT lymphocytes in this process. Balb/c mice were sublethally irradiated and transplanted with Hep3b HCC cell line, followed by transplantation of naive splenocytes. DCs were separated using CD11c beads and pulsed with HBV-enveloped proteins (group A), HCC cell lysate (group B), or BSA (control group C). Mice were followed for survival and tumor size. To determine the mechanism of the antitumor effect, intrasplenic and intrahepatic lymphocyte subpopulations were analyzed by FACS for NKT, CD4 and CD8 markers. Tumor-associated antigens-specific IFNgamma ELISPOT, T-cell proliferation assays and serum cytokine analysis were performed. Treatment with tumor-associated antigen-pulsed DC significantly improved survival (40% and 50% as compared with 0% in groups A, B, and control group C, respectively; p < 0.005). Tumor size decreased to 12.8 +/- 0.4 and 0 from 60.4 +/- 0.9 mm(3) in groups A, B, and control group C, respectively (p < 0.005). Adoptive transfer of HBV or Hep3b-associated antigens-pulsed DC induced a 6-fold increase in peripheral CD8(+) lymphocytes (from 1% in control mice to 6% and 5.5% in groups A and B, respectively), along with a decrease in CD4(+) lymphocytes (from 3.5% in controls to 1.4% and 2.3% in A and B, respectively; p < 0.005). The CD8(+)/CD4(+) ratio increased from 0.28 in controls to 4.28 and 2.39 in groups A and B, respectively (p < 0.005). Intrasplenic NKT cells increased from 7% in control mice to 7.98% and 14.6% in groups A and B, respectively. In contrast, an opposite shift was observed inside the liver. Intrahepatic lymphocyte analysis showed a marked increase in CD4(+) and a decrease in CD8(+) lymphocytes in treated groups. The intrahepatic CD4(+) number increased from 0.5% in controls to 2.15% and 25.8% in groups A and B, respectively (p < 0.005). In contrast, a significant decrease in the intrahepatic CD8(+) numbers was observed (from 7% in controls to 1.0% and 2.4% in groups A and B, respectively; p < 0.005). A significant increase was noted in HBV-specific IFNgamma spot-forming T-cell colonies from 0.0 to 8.8 +/- 1.7 and 1.8 +/- 2.9 in groups C, A, and B, respectively (p < 0.005). Similarly, a significant increase in the HBV-specific T-cell stimulation index, from 0.8 +/- 0.2 to 7.2 +/- 0.4, in groups C and B, respectively, was noted (p < 0.002). IFNgamma and IL12 serum levels increased significantly in treated groups. IFNgamma and IL12 serum levels increased to 380 +/- 30 and 400 +/- 20, and 960 +/- 40 and 760 +/- 60 in groups A and B, compared with 150 +/- 16 and 490 +/- 40 pg/ml in control mice (p < 0.005). Tumor antigen-pulsed DCs effectively suppressed the growth of hepatocellular carcinoma in mice. This effect was associated with enhanced NKT and CD8(+) lymphocyte function and augmentation of the antitumor/antiviral-specific IFNgamma production.     

Tumor antigen-specific T-cell expansion is greatly facilitated by in vivo priming.

PURPOSE: Adoptive T-cell therapy is a promising strategy for the treatment of patients with established tumors but is often limited to specific cancers where tumor-infiltrating lymphocytes, the source of T cells for ex vivo culture, can be obtained. In this study, we evaluated the feasibility of expanding HER-2/neu-specific T cells derived from peripheral blood ex vivo following in vivo priming with a HER-2/neu peptide vaccine. EXPERIMENTAL DESIGN: Peripheral blood mononuclear cells from cytomegalovirus (CMV)-seronegative and CMV-seropositive donors as well as HER-2/neu-positive cancer patients who had or had not been vaccinated with a HER-2/neu peptide-based vaccine was used as a source of T lymphocytes. Antigen-specific T-cell lines were generated by in vitro stimulation with antigen followed by nonspecific expansion on CD3/CD28 beads. The ability to expand antigen-specific T cells was assessed using IFN-gamma and granzyme B enzyme-linked immunosorbent spot. The phenotype of the resultant T-cell lines was evaluated by flow cytometry, including the presence of FOXP3-expressing CD4(+) T cells. RESULTS: The frequencies of CMV-specific T cells generated from CMV(+) donors were >11-fold higher than the frequencies from CMV(-) donors (P = 0.001), with 22-fold increase of total number of CD3(+) T cells. The frequencies of HER-2/neu-specific T cells generated from the primed patients were >25-fold higher than the frequencies from unvaccinated patients (P = 0.006), with an average of a 19-fold increase of total number of CD3(+) T cells. Using peripheral blood as the source of T cells did not result in concurrent expansion of FOXP3(+)CD4(+) regulatory T cells despite the use of interleukin-2 in in vitro culture. Both CD4(+) and CD8(+) HER-2/neu-specific T cells could be expanded. The extent of ex vivo expansion correlated with the magnitude of immunity achieved during immunization (P = 0.008). CONCLUSION: Tumor-specific T cells can be efficiently expanded from the peripheral blood ex vivo following in vivo priming with a vaccine. This approach provides an effective method to generate tumor-specific polyclonal T cells for therapeutic use that could be applied to cancer patients with any tumor type.     

Spontaneous apoptosis of circulating T lymphocytes in patients with head and neck cancer and its clinical importance.

Spontaneous apoptosis was observed in a proportion of peripheral blood mononuclear cells obtained from patients with head and neck cancer (HNC) but not from normal healthy donors (T. Saito et al., Clin. Cancer Res., 5: 1263-1273, 1999). To further investigate this phenomenon, peripheral blood mononuclear cells were obtained from patients with HNC or normal controls (NCs) and evaluated for expression of apoptosis markers (annexin V binding and caspase-3 activation), T-cell receptor-associated zeta chain, and the death receptor Fas (APO-1, CD95) in CD3(+) T cells by multicolor flow cytometry. Soluble Fas ligand (sFasL) in the sera of these individuals was quantitated by ELISA. In patients with HNC, 74 +/- 15% (mean +/- SD) of CD3(+) T cells were Fas(+) compared with 52 +/- 13% in NCs (P < 0.0001). Furthermore, 29 +/- 16% of the Fas(+) CD3(+) T cells bound annexin V in patients and only 14% +/- 7% of the Fas(+) CD3(+) T cells bound annexin V in NCs (P < 0.0001). In patients, Fas(+) CD3(+) cells preferentially underwent apoptosis and showed a loss of zeta chain expression. Significantly greater proportions of CD8(+) T cells than CD4(+) T cells were apoptotic (P < 0.0002), which indicates that CD8(+) T cells were especially sensitive to apoptosis. Serum levels of sFasL were lower in HNC patients with active disease than in NCs or in patients with no evident disease (P < 0.0183). This suggested utilization of sFasL produced in vivo and activation of the Fas/Fas ligand (FasL) pathway in Fas(+) T cells. Proportions of apoptotic T cells were higher in HNC patients than in NCs (P < 0.0001), and a subset of HNC patients with active disease had the highest proportions of circulating Fas(+) annexin V(+) T lymphocytes. The data indicate that the Fas/FasL pathway is involved in spontaneous apoptosis of circulating Fas(+) T lymphocytes in cancer patients. Fas/FasL interactions might lead to excessive turnover of T cells in the circulation and, consequently, to reduced immune competence in patients with HNC.     

Changes in peripheral lymphocyte subsets in patients after partial microwave ablation of the spleen for secondary splenomegaly and hypersplenism: a preliminary study.

PURPOSE: Microwave ablation therapy for secondary splenomegaly and hypersplenism has been shown to be effective from pre-clinical animal models and clinical investigations. This study was performed to determine its effects on the status of peripheral lymphocyte subsets in patients receiving microwave ablation of the spleen. MATERIALS AND METHODS: Ten patients with secondary splenomegaly and hypersplenism received microwave ablation of the spleen during laparoscopy or percutaneously under ultrasound guidance. The percentage peripheral blood T cells, B lymphocytes and NK cells were measured using flow cytometry before and on days 1, 3 and 7 after therapy, as well as 1 and 3 months afterwards. RESULTS: Percentages of CD3(+) and CD4(+) cells increased rapidly 1 month after therapy. There was no significant change in CD8(+), CD4(+)/CD8(+) or NK cells of the pre- and post-therapy levels and B lymphocytes increased significantly after therapy. In patients with an ablation volume (AV) less than 20% (group A), T cells increased 1 month after ablation but decreased 3 months after ablation. B lymphocytes increased significantly after surgery. Levels of NK cells were lower than that before therapy on each testing. In patients with 20-40% AV (group B), levels of T cells, B lymphocytes and NK cells showed an increase. Levels of CD4(+) cells were significantly higher in group B than in group A, 3 months after therapy. CONCLUSIONS: Microwave ablation therapy for splenomegaly and hypersplenism appears to have a favourable effect on peripheral lymphocyte subsets. A relationship may exist between the ablation volume and the level of peripheral lymphocyte subsets.     

Regulatory CD4(+)CD25(+) T cells in tumors from patients with early-stage non-small cell lung cancer and late-stage ovarian cancer.

Immunosuppression may contribute to the progression of cancer. In this study we assessed the structural and functional status of T cells from tumor specimens obtained from patients with early stage non-small cell lung cancer and late-stage ovarian cancer. Although some groups have described structural alterations in the TCR in patients with other malignancies, we did not observe decreased expression of the CD3zeta subunit in the tumor-associated T cells. However, increased percentages of CD4(+)CD25(+) T cells were observed in the non-small cell lung cancer tumor-infiltrating lymphocytes and ovarian cancer tumor-associated lymphocytes. Furthermore, these CD4(+)CD25(+) T cells were found to secrete transforming growth factor-beta, consistent with the phenotype of regulatory T cells. Despite a generalized expression of lymphocyte activation markers in the tumor-associated T-cell populations, the CD8(+) T cells expressed low levels of CD25. To determine whether expression of CD25 could be restored on the CD8 cells, tumor-associated T cells were stimulated with anti-CD3 and anti-CD28 monoclonal antibodies. After stimulation, nearly all of the CD8 T cells expressed CD25. Furthermore, despite the low levels of interleukin 2, IFN-gamma, and tumor necrosis factor-alpha secretion by the tumor-associated and peripheral blood T cells at baseline, stimulation with anti-CD3 and anti-CD28 monoclonal antibodies significantly increased the fraction of cells producing these cytokines. Thus, tumor-associated T cells from patients with early and late-stage epithelial tumors contain increased proportions of CD4(+)CD25(+) T cells that secrete the immunosuppressive cytokine transforming growth factor-beta. Furthermore, our results are consistent with previous reports showing impaired expression of CD25 on CD8(+) T cells in cancer patients. Finally, increased lymphocyte costimulation provided by triggering the CD28 receptor is able to increase CD25 expression and cytokine secretion in tumor-associated T cells. These observations provide evidence for the contribution of regulatory T cells to immune dysfunction in cancer patients.     

Regulatory CD8+ T cells control thyrotropin receptor-specific CD4+ clones in healthy subjects

One of the mechanisms ensuring immunological unresponsiveness or tolerance depends on the action of CD8(+) lymphocytes. In this paper, we report that, in healthy subjects, a subset of CD8(+)CD28(-) T cells suppresses the specific response to TSH receptor (TSHR) of CD4(+) clones. Suppression was highly specific, required cell-cell interaction, and was not mediated by cytotoxicity. Co-incubation of CD8(+) and CD4(+) clones, followed by the removal of the CD8(+) cells from the cultures before testing CD4(+) responsiveness to TSHR, demonstrated that CD4(+) cells were anergic since they showed low response to the antigen and a significant impairment of IL-2 production. In CD8-mediated anergy induction, the T-cell receptor (TCR) on both CD4(+) and CD8(+) cells seems to play a role. Our results indicate that one of the mechanisms ensuring peripheral tolerance involve CD8(+)CD28(-) cells. A disregulation in the control of autoreactive clones by this subset might be important for the onset of autoimmune thyroid diseases.     

Association of increased levels of heavy-chain ferritin with increased CD4+ CD25+ regulatory T-cell levels in patients with melanoma.

We have shown previously that melanoma cells in culture release heavy-chain ferritin (H-Ferritin) into supernatants and that this is responsible for the suppression of responses of peripheral blood lymphocytes stimulated by anti-CD3. These effects were mediated by activation of regulatory T cells to produce interleukin (IL)-10. In the present study, we examined whether a similar relation might exist between levels of H-Ferritin and activation of regulatory T cells in patients with melanoma. Ferritin levels were evaluated by ELISA and regulatory T-cell numbers were assessed by three-color flow cytometry to identify CD4(+) CD25(+) CD69(-) T cells. CD69 positive cells were excluded to avoid inclusion of normal activated CD4, CD25 expressing T cells. Measurements of H- and light-chain (L)-Ferritin by ELISA revealed that H- but not L-Ferritin was elevated in the circulation of melanoma patients. In addition, these studies revealed a marked increase in the number of CD4(+) CD25(+) CD69(-) T cells in such patients, compared with age-matched controls. The ratio of H-Ferritin:L-Ferritin correlated with the levels of regulatory T cells consistent with a causal relation between unbound H-Ferritin levels and the activation of regulatory T cells. H-Ferritin or regulatory T cells did not, however, correlate with the stage of the melanoma. These results provide evidence for the importance of H-Ferritin in the induction of regulatory T cells in patients with melanoma and provide additional insight into the suppression of immune responses in such patients.