Increase in Vδ1+γδ T cells in the peripheral blood and bone marrow as a selective feature of HIV-1 but not other virus infections

Dysregulation of T-cell receptor (TCR) αβ bearing lymphocytes and an increase in Vδ1⁺γδ T cells are typical features of HIV-1 infection. However, the role of γδ T cells remains unclear. Therefore, peripheral blood mononuclear cells (PBMC) of 103 HIV-1-infected patients were investigated with respect to expression of Vδ1. These results were compared to the Vδ1 expression of bone marrow mononuclear cells (BMMC). In contrast to healthy controls, Vδ1⁺ cells dominated among both PBMC and BMMC in HIV-1-infected patients. Analysis of the coexpression of CD25, CD8, HLA-DR and CD45RO revealed a high prevalence of Vδ1/CD45RO and Vδ1/HLA-DR double-positive PBMC only in HIV-1-infected patients but not in healthy donors. Furthermore, analysis of the γδ TCR repertoire in patients infected with hepatitis B virus, hepatitis C virus, herpes simplex virus (HSV)-1 and HSV-2 showed that the selective enhancement of Vδ1⁺ cells was restricted to HIV infection and not observed in other virus diseases. Our data provide further support for the involvement of γδ T cells in immunosuppression and progression of HIV infection.     

Magnitude of CD8+ T-cell responses against hepatitis C virus and severity of hepatitis do not necessarily determine outcomes in acute hepatitis C virus infection

Aim:  We investigated the relationship between the magnitude of comprehensive hepatitis C virus (HCV)-specific CD8⁺ T-cell responses and the clinical course of acute HCV infection.
Methods:  Six consecutive patients with acute HCV infection were studied. Analysis of HCV-specific CD8⁺ T-cell responses was performed using an interferon-γ-based enzyme-linked immunospot assay using peripheral CD8⁺ T-cells, monocytes and 297 20-mer synthetic peptides overlapping by 10 residues and spanning the entire HCV sequence of genotype 1b.
Results:  Five patients presented detectable HCV-specific CD8⁺ T-cell responses against a single and different peptide, whereas 1 patient showed responses against three different peptides. Neither the magnitude of HCV-specific CD8⁺ T-cell responses nor the severity of hepatitis predicts the outcome of acute hepatitis. The maximum number of HCV-specific CD8⁺ T-cells correlated with maximum serum alanine aminotransferase level during the course ( r  = 0.841, P  = 0.036).
Conclusions:  HCV-specific CD8⁺ T-cell responses were detectable in all 6 patients with acute HCV infection, and 6 novel HCV-specific CTL epitopes were identified. Acute HCV infection can resolve with detectable HCV-specific CD8⁺ T-cell responses, but without development of antibody against HCV.     

Quantitative and functional differences in CD8+ lymphocyte responses in resolved acute and chronic hepatitis C virus infection

CD8⁺ T lymphocyte responses are important in the clearance of viral infections. In chronic infections they may contribute to pathogenesis. To investigate the role of CD8⁺ T lymphocyte responses in viral clearance and chronic hepatitis C we have compared hepatitis C virus (HCV) specific cytotoxicity and interferon-gamma (IFN-γ) production in patients with resolved-acute, and chronic HCV infection. CD8⁺ T cell responses to a panel of 13 HCV T cell peptide epitopes were studied using Elispot assays of IFN-γ production and chromium release cytotoxicity assays. Responses of seven patients with resolved acute HCV infection were compared with those of 14 chronically infected patients. HCV-specific cytotoxicity differentiated the two populations of patients. The majority (71%) of patients with resolved acute infection tested positive to 42% of relevant peptides compared with the minority (28%) of patients with chronic hepatitis C ( P =0.03) who responded to only 8% of relevant peptides ( P =0.0009). In contrast, HCV-specific IFN-γ production was detected in 86% of patients with either resolved or chronic infection in response to 42% and 35%, respectively, of relevant peptides tested (not significant). In patients with chronic infection the magnitude of the HCV-specific IFN-γ production was inversely correlated to viral load ( R ²=0.52; P =0.042). Failure to clear HCV infection may be attributable to the presence of noncytolytic IFN-γ producing CD8⁺ T lymphocytes in chronically infected patients. However these CD8⁺ T cells may play a beneficial role in contributing to the control of viral load in chronic hepatitis C.     

Second transplantation with CD34+ bone marrow cells selected from a two-loci HLA-mismatched sibling for a patient with chronic myeloid leukaemia

A 43-year-old man with chronic myeloid leukaemia underwent a second transplant with CD34⁺ bone marrow cells selected from his two-loci HLA-mismatched sibling after rejection of the first graft from an HLA-matched unrelated donor. By immunomagnetic positive selection, CD34⁺ marrow cells at 0.95×10⁶/kg with 97% purity and CD3⁺ T lymphocytes at 1.3×10⁴/kg were collected and transplanted. Engraftment was confirmed to be of CD34⁺ cell-donor origin. The patient developed only grade I acute graft-versus-host disease (GVHD) and no chronic GVHD to date. These observations suggest that allogeneic CD34⁺ bone marrow cells are capable of reconstituting haemopoiesis and that CD34⁺ selection could be applicable to T-cell depletion.     

Hepatitis B e antigen negative chronic active hepatitis: hepatitis B virus core mutations occur predominantly in known antigenic determinants

Summary. In chronic hepatitis B virus (HBV) infection seroconversion from hepatitis B e antigen (HBeAg) to hepatitis B e antibody (HBeAb) may be followed either by remission of the disease with low-level viraemia, or by continuing inflammation with high-level viraemia. In both situations the virus may acquire a mutation in the precore sequence which prevents it from encoding HBeAg. We now show that the number of amino acid substitutions in the HBV core is low in viral sequences from patients with HBeAg positive chronic liver disease and HBeAg negative HBeAb positive patients in remission, but the frequency of substitutions is high in HBeAg, negative HBeAb positive patients with active liver disease. Furthermore we show that these substitutions cluster in the promiscuous CD4⁺ T-helper-cell epitope and in HBV core/e antibody binding determinants, but are not found in regions recognized by major histocompatability complex (MHC) restricted cytotoxic T lymphocytes. Sequential viral sequences from patients before and after HBeAg/HbeAb seroconversion shows that core mutations arise either at the same time or after the precore stop mutation which prevents the virus from encoding HBeAg. These results are consistent with the hypothesis that after clearance of HBeAg, mutations in regions of the virus recognized by CD4⁺ helper T cells and B cells allow persistence of the HBe negative virus in HBeAb positive patients with viraemia and active hepatitis.     

Expression of CD8β and alteration of cell surface phenotype in adult T-cell leukaemia cells

Typical adult T-cell leukaemia (ATL) cells have a CD4⁺CD8⁻ cell surface phenotype, but atypical phenotypes such as CD4⁺CD8⁺ and CD4⁻CD8⁺ have also been reported. The CD8 molecule is composed of α and β chains and commonly used monoclonal antibodies against CD8 molecule detect only CD8α. Since it has been reported that CD8α can be induced in mature CD4⁺ T cells by cell activation, but not CD8β, we studied whether ATL cells which express CD8α may also express CD8β. We found some cases of CD8α⁺ ATL were also positive for CD8β. Furthermore, we experienced a case whose ATL cell surface phenotype changed from CD4⁺CD8α⁺CD8β⁺ to CD4⁻CD8α⁺CD8β⁺ and finally to CD4⁺CD8α⁻CD8β⁻. Southern blot analysis revealed that the monoclonal integration of human T lymphotropic virus type I (HTLV-I) was identical throughout the course of the study, indicating that a single clone had demonstrated the alterations. These data suggest that peripheral CD4⁺CD8⁺ ATL cells can express not only CD8α, but also CD8β and that a single ATL cell clone has the potential to change its surface phenotype in vivo as well as in vitro .     

Identification of biclonal (duplex) leukaemic cells expressing either CD4+/CD8- or CD4-/CD8+ from a patient with adult T-cell leukaemia/lymphoma

A 24-year-old Japanese woman was admitted to our hospital in 1987 with a chief complaint of skin eruptions, and was diagnosed as having chronic ATLL. In 1993 the leucocyte count increased gradually to 126.0x10⁹/l with 91.5% abnormal lymphocytes expressing two different types of antigenicity, either CD4⁺/CD8^- or CD4^-/CD8⁺. Monoclonal integration of human T-cell lymphotropic virus type-I proviral DNA was detected at different sites of the genomic DNA in each cell type. These studies clearly indicate that CD4⁺/CD8^- and CD4^-/CD8^- leukaemic cells originated from two independent clones.     

Molecular correlates in AIDS patients following antiretroviral therapy: diversified T-cell receptor repertoires and in vivo control of cytomegalovirus replication

  ¹ Department of Immunology and   ² The Kobler Centre, Chelsea and Westminster Hospital, and   ³ Royal Free Hospital and University College Medical School, London, UK
Objectives   To evaluate whether successful, long-term immune reconstitution in vivo can be achieved in end-stage AIDS patients following antiretroviral therapy (ART).
Methods   A 1-year prospective study of changes of CD4+ and CD8+ T-cell surface phenotypes, T-cell receptor (TCR) repertoires and capacity to control in vivo replication of cytomegalovirus (CMV) was performed in five treatment-naive end-stage AIDS patients (median CD4+ T-cell counts of 19 cells/μL) following therapy. Proportions of CD45RA+, CD45RO+ and CD28+ cells within the CD4+ and CD8+ subsets, were determined by flow cytometry. Changes in TCR Vβ repertoires within the CD4+ and CD8+ T-cell compartments were evaluated using CDR3 spectratyping. CMV replication was determined by a sensitive polymerase chain reaction (PCR) assay using whole blood.
Results   Following ART, proportionate increases in 'naive' (CD45RA+) and 'memory' (CD45RO+) T cells were observed within both CD4+ and CD8+ T-cell subsets, while increased numbers of CD28+ T cells were mainly observed within the CD4+ subset. Diversification of CD4+ and CD8+ TCR repertoires was established concomitantly with renewed in vivo control of CMV replication.
Conclusions   An important degree of molecular and functional immune recovery is possible in end-stage AIDS patients introduced to therapy. Diversification of TCR repertoires and the in vivo restoration of immunocompetence to control opportunistic infections clearly show that an important degree of molecular immune reconstitution is established following the initiation of ART even in late-stage AIDS.     

Influence of bone marrow graft B lymphocyte subsets on outcome after HLA-identical sibling transplants

The potential role of the infused B cell subset after Hematopoietic Stem Cell Transplantation has not been yet studied. The present study analyzed the impact of B cells on transplant outcome in 254 patients who received a bone marrow graft from a human leucocyte antigen-identical sibling donor. The influence of B lineage-specific hematopoietic progenitor cells (CD34⁺ CD19⁺) and B cells (immature and mature B cells, CD34⁻ CD19⁺) was also analyzed. All included patients received a myeloablative regimen. The cumulative incidence function of acute graft-versus-host (GvHD) grade II to IV was 48% and was inversely associated with the number of CD34⁺ CD19⁺. There were no statistically significant associations between B cell subsets and chronic GvHD or survival. The CD34⁺ CD19⁺ B cell subset remained significantly associated with acute GvHD in multivariate analysis (Relative risk = 0·32, 95% confidence interval: 0·11–0·92, P  =   0·035). In conclusion, a higher B lineage-specific hematopoietic progenitor cells (CD34⁺ CD19⁺) cell dose is associated with a significant decrease incidence of acute GvHD.     

T-cell form of chronic lymphocytic leukaemia: a reaffirmation of its existence

Early in the 1980s three categories of T-cell chronic lymphocytic leukaemia were recognized: CD4⁺ CD8⁻ knobby type, CD4⁻ CD8⁺ azurophilic type and CD4⁺ CD8⁻ adult T-cell leukaemia (ATL) type. Both azurophilic and ATL types were later shown to be distinctive disorders, whereas the knobby type has been largely neglected and even considered non-existent by some authors. In this report we describe two patients with leukaemia of CD3⁺ CD4⁺ CD8⁻ post-thymic T lymphocytes presenting with marked lymphocytosis, generalized lymphadenopathy and hepatosplenomegaly. We believe that CLL of post-thymic T-lymphocytes is a distinct entity, and merits a separate designation from other T-cell leukaemias.     

Thymus-independent T-cell differentiation in vitro

The generatation of large quantities of novel human T-cell clones ex vivo would make a wide range of gene- and immuno-therapies for tumours and viral infections possible. Several techniques have been described to generate, in vitro and in vivo (using xenogenic hosts), mature T cells from fetal-neonatal and adult human CD34⁺ cells. All these techniques are cumbersome and cannot be easily translated into clinical protocols because they involve co-cultivation of CD34⁺ cells with thymic fragments from either human or murine fetuses. We report that the mononuclear cells of human cord blood contain a cell population that supports the differentiation of CD34⁺ cells into CD4⁺ or CD8⁺ naive T cells in serum-deprived cultures stimulated with stem cell factor and interleukin 7. CD4⁺ or CD8⁺ CD45RA⁺ TCRαβ⁺ T cells were continuously produced in vitro over a period of 20 d under these conditions. The generation of T cells in these cultures was a dynamic process and clones of T cells expressing new T-cell receptor β-chain rearrangments were generated over time. These results pave the way for the development of very simple culture conditions for ex-vivo production of naive helper or cytotoxic T cells which could be very useful for gene- and immuno-therapy of human diseases.     

Clinical significance of CD45RO expression on peripheral blood mononuclear cells in HTLV-I-infected individuals

The phenotype of peripheral blood mononuclear cells (PBMC) was examined in 13 healthy volunteers, 26 HTLV-I carriers, and 58 ATL patients (22 smouldering, five chronic, 24 acute, and seven lymphoma type). The percentage of CD4⁺, CD25⁺, CD28⁺ and CD45RO⁺ cells in the PBMC of the chronic and acute type patients was significantly higher than that of the volunteers, whereas the percentage of CD8⁺ and CD45RA⁺ cells in these patients was significantly low. The histogram for CD45RO fluorescence intensity (FI) revealed two patterns: pattern A consisted of CD45RO⁺ cells with high FI (CD45RO^high) and intermediate FI (CD45RO^int). Pattern B consisted exclusively of CD45RO^high. Pattern A was evident in all volunteers. The percentage of subjects showing pattern B was increased in an order that reflected disease progression. In the patients with pattern A, the CD45RO^int cells were CD4⁺ and CD8⁻, and the FI of CD2, CD3, and Fas within the CD45RO^int cells appeared to be lower than that within the CD45RO^high cells. The acute type patients with pattern A had a significantly longer survival curve than that of these patients with pattern B. These results suggest that the presence of CD45RO^int cells may be related to protection against disease progression in HTLV-I-infected individuals.     

Interferon-γ Production Specific for Hepatitis B Virus Antigen by Intrahepatic T Lymphocytes in Patients with Acute and Chronic Hepatitis B

Objectives : Studies were undertaken to determine whether intrahepatic lymphocytes have a greater cellular immune response specific for hepatitis B virus (HBV) antigen than peripheral blood lymphocytes in HBV- infected man. Methods : HB nucleocapsid antigen-stimulated interferon-γ (IFN-γ) production of lymphocytes was measured in acute self-limited hepatitis (AH) (eight cases) and chronic hepatitis (CH) (14 cases). Results : In both patient groups, basal IFN-γ levels without any stimulation were higher in the hepatic T cells than in the peripheral T cells. The values in cultures from blood and liver were larger in AH than in CH. Alternatively, IFN-γ response to HBcAg and HBeAg was less in hepatic T cells than in the corresponding blood cultures; T cell response was HLA class II restricted. Cell flow cytometry study showed that in all patient groups, percentages of CD3⁺, CD8⁺, and HLA-DR⁺ cells were significantly greater in liver than in peripheral blood, whereas the proportion of hepatic CD4⁺ cells was decreased, compared with that in peripheral CD4⁺ cells. Conclusions : These findings suggest that liver infiltrates are already activated in vivo to produce IFN-γ, particularly in patients with AH. The changes in the proportion of lymphocyte subsets may be responsible for the altered IFN-γ response of hepatic T cells.     

HBcAg-pulsed dendritic cell vaccine induces Th1 polarization and production of hepatitis B virus-specific cytotoxic T lymphocytes

Aim:  Dendritic cells (DCs) pulsed with HBsAg efficiently reverse the immune tolerance to hepatitis B virus (HBV) and induce HBV-specific cytotoxic T lymphocyte (CTL) responses in transgenic mice and healthy volunteers. However, it is not clear whether HBV core antigen (HBcAg)-pulsed DCs can effectively induce CD4⁺ helper T cells polarization into Th1, which contribute to the induction and maintenance of HBV-specific CD8⁺ T cells in chronic hepatitis B (CHB) patients. To address this issue, we conducted this study and investigated whether HBcAg-pulsed DCs could polarize Th1 cells and induce an HBcAg-specific CTL response.
Methods:  HBcAg-pulsed DCs were generated from 21 CHB patients. The capacity of the HBcAg-pulsed DC vaccine to stimulate CD4⁺ and CD8⁺ T cells to produce IFN-γ and IL-4 was estimated by intercellular cytokine staining, and the HBcAg-pulsed DCs derived from 10 humam leucocyte antigen (HLA)-A2⁺ CHB patients were tested for the induction of HBV-specific CTLs from autologous T cells by pentamer staining. The cytotoxicity of these CTLs was evaluated in vitro by flow cytometry.
Results:  The HBcAg-pulsed DCs derived from CHB patients exhibited a stronger capacity to stimulate autologous CD4⁺ and CD8⁺ T cells to release IFN-γ rather than IL-4, which could induce HBV core 18-27 specific CTLs, suggesting a specific cytotoxicity against T2 cells that had been loaded with the HBV core 18-27 peptide in vitro .
Conclusion:  HBcAg-pulsed DC vaccine derived from CHB patients efficiently induced autologous T cell polarization to Th1 and generation of HBV core 18-27 specific CTLs.     

Large granular lymphocyte leukaemia occurring after renal transplantation

Post-transplantation lymphoproliferative disorders (PTLD) are a clinicopathologically heterogenous group of lymphoid proliferations. The majority are of B-cell origin and associated with Epstein-Barr virus (EBV) infection. In contrast, the development of T-cell PTLD is much less common and EBV does not appear to be involved in pathogenesis. In this report we describe three patients who developed large granular lymphocyte (LGL) leukaemia after renal transplantation. These patients had clonal expansion of CD3⁺, CD8⁺, CD57⁺, CD56⁻ LGL. We were unable to detect CMV antigen or find evidence for EBV or human T-cell leukaemia/lymphoma virus genome in the LGL from these patients. These data show that LGL leukaemia should be included as one of the types of T-cell proliferations which can occur post transplant.     

Immunophenotypic and clinical features of T-cell receptor γδ+ T-lineage acute lymphoblastic leukaemia

The immunophenotypic and clinical features of TCR-γδ⁺ T-lineage acute lymphoblastic leukaemia (T-ALL) were prospectively analysed in 52 children with membrane CD3⁺ T-ALL. We observed a relatively high incidence of TCR-γδ⁺ T-ALL (26/52 patients). Leukaemic blasts from 22 children demonstrated TCR-αβ positivity, and simultaneous expression of the TCR-β and -δ chain was found in four children. Clinical and haematological features of TCR-αβ and γδ⁺ T-ALL did not differ significantly, except that haemoglobin levels were significantly lower in TCR-γδ⁺ cases. Event-free survival at 4 years was significantly better in TCR-γδ⁺ compared with TCR-αβ⁺ T-ALL, but expression of TCR molecules did not emerge as an independent prognostic factor in multivariate analysis.     

Eosinophilia associated with proliferation of CD3+4−8−αβ+ T cells with chromosome 16 anomalies

We describe a patient with eosinophilia and an abnormal CD3⁺4⁻8⁻αβ⁺ T-cell population. Chromosomal analysis of sorted CD3⁺4⁻8⁻ cells revealed abnormal karyotypes on chromosome 16. In the presence of IL-2 the production of IL-5 from CD3⁺4⁻8⁻ cells was higher than that from CD3⁺4⁺/8⁺ cells. Eosinophil survival-enhancing activity in the patient serum was inhibited by a combination of anti-IL-5 and anti-GM-CSF monoclonal antibodies. These data suggest that increased production of IL-5 and GM-CSF from the abnormal CD3⁺4⁻8⁻ cells might cause eosinophilia.     

Sustained response to interferon-α plus ribavirin therapy for chronic hepatitis C is closely associated with increased dynamism of intrahepatic natural killer and natural killer T cells

Aim:  Previous studies have revealed that functional impairment of innate immune cells, including natural killer (NK) and natural killer T (NKT) cells, might be associated with the persistence of hepatitis C virus (HCV) infection. However, the involvement of innate immune cells, which predominate in the liver, in therapeutic HCV clearance is still unclear.
Methods:  To clarify the role of intrahepatic innate immune cells in the clinical outcome of patients with chronic hepatitis C (CHC) treated with interferon-α plus ribavirin (IFN/RBV), we prospectively investigated the status of NK and NKT cells in paired liver biopsy and peripheral blood (PB) samples obtained from 21 CHC patients before and immediately after IFN/RBV treatment by flow cytometry. Normal liver and PB samples were obtained from 10 healthy donors for living donor liver transplantation.
Results:  Before treatment, intrahepatic NK and NKT cells constituted a significantly lower proportion in CHC patients than in healthy individuals ( P  < 0.05). After IFN/RBV treatment, the proportions and absolute numbers of CD3^-CD161⁺ NK and CD3⁺CD56⁺ NKT cells in the liver, but not in PB, were significantly increased in sustained responders (SR) as compared with poor responders ( P  < 0.05). The proportion of CD3⁺CD161⁺ NKT cells was also increased in the liver of SR after the treatment. Moreover, there was a striking increase of activated CD152⁺ cells among CD3⁺CD56⁺ NKT cells in the liver of SR ( P  = 0.041).
Conclusion:  These findings demonstrate that sustained response to IFN/RBV treatment for patients with CHC is closely associated with increased dynamism of NK and NKT cells in the liver.     

Thymic function in adults: evidence derived from immune recovery patterns following myeloablative chemotherapy and stem cell infusion

We studied 45 patients aged 14–66 years who had undergone stem cell transplantation for a variety of malignant conditions at least 12 months previously. Compared to normal controls, they had significantly reduced absolute numbers of CD4⁺, CD4⁺CD45RA⁺ and CD4⁺CD45RO⁺ T cells and a reduced CD4⁺CD45RA⁺: CD4⁺CD45RO⁺ ratio. In all subsets T-cell numbers were significantly greater 24 months, compared to 12–24 months, after transplantation and there was a non-significant trend towards lower T-cell numbers with increasing age. We conclude that the thymus, or putative thymic-equivalent tissue, remains functional in older adults.