Established IL-2-dependent double-negative (CD4- CD8-) TCRαβ/CD3+ ATL cells: induction of CD4 expression

Summary. We established IL-2-dependent T cells from an adult T-cell leukaemia (ATL) patient whose leukaemic cells changed from CD4 single-p-positive in the initial phase to double-negative (CD4^- CD8^-) at the time of exacerbation. The cells termed SO-4 were of ATL cell origin and showed the double-negative TCRαβ/CD3⁺ T-cell phenotype. SO-4 cells acquired CD4 antigen expression following stimulation with concanavlin A (ConA) or immobilized anti-CD3 antibody. The induction was inhibited by herbimycin A, an inhibitor of protein tyrosine kinase (PTK) activity. No CD4 mRNA was detectable in unstimulated SO-4 cells but a 3.0 kb signal specific for CD4 mRNA was detected after stimulation. These findings indicate that SO-4 cells return to their original phenotype (CD4 single-positive) by stimulation involving PTK. The results indicate that there is a pathway of phenotypic cycling between CD4 single-positive and double-negative T cells.     

Cytokine activity after allogeneic bone marrow transplantation, V. Analysis of IL-2 and IFN production by isolated CD4+ and CD8+ cells

Summary. Previous studies from this laboratory have shown that PBMC from recipients of an HLA-identical sibling bone marrow transplant produce levels of IL-2 which are 10–100-fold lower than those produced by the same number of PBMC from healthy controls, whereas production of IFN-γ is normal. The present study examined IL-2 and IFN production over a range of cell numbers for PBMC and for isolated CD4⁺ and CD8⁺ cells for controls and marrow transplant recipients. There was a 5-fold lower IL-2 production in marrow transplant recipient CD8⁺ cells compared with equivalent numbers of control cells, whereas no difference was found in IL-2 production by CD4⁺ cells. In contrast, IFN production by CD4⁺ cells from marrow transplant recipients was 4-fold higher than in controls, whereas CD8⁺ cells from both populations produced similar amounts of IFN. When the observed production of cytokine by PBMC was compared with the expected production based on the CD4⁺ and CD8⁺ content of the PBMC, control values were similar, but the expected values for both cytokines were approximately 2-fold higher than the observed values for marrow transplant recipients. The results suggest that the capacity of T cells from marrow transplant recipients to produce IL-2 and IFN is not impaired, but that the frequency of cytokine-producing cells may be reduced, and that a negative interaction present in recipient PBMC, eliminated by isolating T-cell subsets, is responsible for the observed low levels of cytokine production.     

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.     

Monoclonal proliferation of CD4+ large granular lymphocytes with cytolytic activity

Summary. A rare case of monoclonal proliferation of CD3⁺4⁺8∼ T-cell receptor-αβ⁺ large granular lymphocytes (LGL) is presented. CD4⁺ LGL in the present case showed spontaneous cytotoxicity against herpes simplex virus-infected cells and antibody- and lectin-dependent cytotoxicity. Perforin, which is one of the important cytolytic mediators of cytotoxic T cells (CTL) and natural killer cells, was abundantly expressed in CD4⁺ LGL of this case. The present case suggests that perforin-positive CD4⁺ CTL, which have recently been shown in the in vitro studies, certainly exist in vivo.     

A biclonal large granular lymphocyte (LGL)/NK-associated (NKa) disorder of CD4+ and CD8+ lymphocyte subpopulations characterized by the simultaneous presence of distinct TCR rearrangements

Summary. This communication reports a patient with concomitant expansions of CD4⁺ and CD8⁺ large granular lymphocytes. Immunological analyses revealed that the abnomally increased CD4⁺ LGL fraction was phenotypically similar to other reported persistent CD4⁺ LGL expansions, whereas the phenotypic profile for the CD8⁺ LGL component was unusual. of particular note was the finding that both the CD4⁺ and CD8⁺ LGL fractions showed high membrane the CD45RO isoform expression, thus suggesting their 'primed' status. Molecular biology studies of immunomagnetically fractionated cells using a Tγ9 TCR gamma gene primer further revealed that the CD4⁺ and CD8⁺ components were both clonal but showed different patterns of rearrangement It is suggested that the simultaneous presence of CD4⁺ and CD8⁺ clonal populations are unlikely to have been derived from a common progenitor and that they reflect expansions of functionally restricted subpopulations     

CD3+ CD8+ CD56− clonal large granular lymphocyte leukaemia and HIV infection

High-grade malignant lymphomas associated with HIV infection are usually derived from B lymphocytes. Although a broad spectrum of T-cell-derived malignancies has been described, no case of monoclonal T large granular lymphocyte leukaemia has been reported to date. We report a case of clonal T-LGL (CD3⁺, CD4⁻, CD8⁺, CD56⁻, CD57⁺) in an HIV-infected, HTLV1/2-negative individual. Large granular lymphocytes are thought to represent activated cytotoxic T lymphocytes. HIV infection, as previously reported for HTLV1/2, may represent a pathway of antigen activation and lead to clonal expansion of T large granular lymphocytes.     

Major role for CD8+ T cells in the protection against Toxoplasma gondii following dendritic cell vaccination

Toxoplasma gondii is the causative agent of toxoplasmosis, a worldwide zoonosis for which an effective vaccine is needed. Vaccination with pulsed dendritic cells is very efficient but their use in a vaccination protocol is unconceivable. Nevertheless, unravelling the induced effector mechanisms is crucial to design new vaccine strategies. We vaccinated CBA/J mice with parasite extract-pulsed dendritic cells, challenged them with T. gondii cysts and carried out in vivo depletion of CD4⁺ or CD8⁺ T lymphocytes to study the subsequent cellular immune response and protective mechanisms. CD4⁺ lymphocytes were poorly implicated either in spleen and mesenteric lymph node (MLN) cytokine secretion or in mice protection. By contrast, the increasing number of intracerebral cysts and depletion of CD8⁺ cells were strongly correlated, revealing a prominent role for CD8⁺ lymphocytes in the protection of mice. Splenic CD8⁺ lymphocytes induce a strong Th1 response controlled by a Th2 response whereas CD8⁺ cells from MLNs inhibit both Th1 and Th2 responses. CD8⁺ cells are the main effectors following dendritic cell vaccination and Toxoplasma infection while CD4⁺ T cells only play a minor role. This contrasts with T. gondii infection which elicits the generation of CD4⁺ and CD8⁺ T cells that provide protective immunity.     

Blastogenic responses, interleukin-2 production and interleukin-2 receptor expression on CD4+ and CD8+ lymphocytes in rhesus monkeys experimentally inoculated with Loa loa

To better understand cellular responses in loiasis infection, in vitro blastogenesis of peripheral blood mononuclear cells (PBMC) to filarial antigen was assessed in 12 Loa loa -inoculated rhesus monkeys over a two-year period. Cellular reactivity to antigen was observed between 10–35 weeks postinoculation (WPI), but had declined by week 50. The roles of interleukin-2 (IL-2) and IL-2 receptor (IL-2R) expression on CD4⁺ and CD8⁺ T cells in regulating the response to antigen were examined during the initial (57 WPI) and late (92 WPI) time points of the observed diminished reactivity to antigen. The levels of IL-2 in antigen cultures at both time points were not significantly different from those in unstimulated cultures. Also, exogenous IL-2 partially reversed the PBMC response to antigen. The percentages of CD4⁺ and CD8⁺ T cells expressing IL-2R in antigen cultures at 57 WPI were not different from those of control animals. Likewise at 92 WPI, the percentage of CD4⁺ T cells expressing IL-2R in antigen cultures, were not increased above those of control animals. In contrast, the percentage of CD8⁺ T cells expressing IL-2R in antigen cultures were significantly increased above those of control animals ( P  < 0.0001), coinciding with an increase in CD8⁺ T cell numbers in these cultures. The data show that factors besides IL-2, and probably an imbalance in the percentages of CD4⁺ and CD8⁺ T cells bearing IL-2R in antigen cultures, may contribute to the diminished reactivity to antigen in L. loa -inoculated rhesus monkeys .     

CD3−CD4−CD8−CD56−CD19−CD14− cells and CD3+CD8 dull-positive cells produce IL-4 in AIDS patients

We used flow cytometry to identify the presence of intracellular cytokines (cytoflow) and analyse the production of IL-4 in peripheral blood from AIDS patients who have practically no CD4⁺ T cells. We found that IL-4 was produced by CD3⁻CD4⁻CD8⁻CD56⁻CD19⁻CD14⁻ cells and CD3⁺CD8 dull-positive cells in AIDS patients. Moreover, CD3⁻CD4⁻ CD8⁻CD56⁻CD19⁻CD14⁻ cells had helper activity for immunoglobulin synthesis. These findings indicate that instead of CD4⁺ T helper cells, C3⁻CD4⁻CD8⁻CD56⁻ CD19⁻CD14⁻ cells and CD3⁺CD8 dull-positive cells may be an important source of IL-4 in a variety of immune responses for AIDS patients.     

Recruitment of CD8+ T cells expressing granzyme A is associated with lesion progression in human cutaneous leishmaniasis

Human infection with Leishmania braziliensis leads to the establishment of cutaneous leishmaniasis (CL), characterized by the appearance of skin lesions that progress from nonulcerated to ulcerated forms. Our goal was to characterize the immunological kinetics associated with this progression, comparing the cellular composition, cytokines and granzyme expression between lesions of patients with early (E-CL) and late stages (L-CL) of CL. Histopathological analysis showed that lesions from L-CL had more exuberant inflammatory infiltrate as compared to E-CL. Although E-CL and L-CL lesions were predominantly mononuclear, lesions from E-CL patients presented higher neutrophil and eosinophil counts than L-CL. While percentages of CD4⁺ and of CD68⁺ cells were slightly higher in L-CL, a fivefold increase of CD8 ⁺ cells was observed in L-CL, as compared to E-CL. Moreover, CD8⁺ T-cells from L-CL expressed significantly higher levels of granzyme A than E-CL. Interestingly, granzyme A expression was positively correlated with intensity of the inflammatory infiltrate in L-CL but not E-CL. Lastly, percentages of IFN-γ ⁺ and IL-10⁺ cells were higher in L-CL as compared to E-CL, with CD4⁺ T-cells and CD68⁺ monocytes as the main sources of these cytokines, respectively. These results suggest that recruitment of CD8⁺ granzyme A⁺ T cells is involved in lesion progression in human CL.     

CD3-induced T-cell activation in the bone marrow of myeloma patients: major role of CD4+ cells

Summary. A large expansion of activated T cells (CD3⁺CD25⁺) with the potential to act as anti-tumour effector cells is inducible in multiple myeloma (MM) patients by culturing bone marrow mononuclear cells (BMMCs) with the anti-CD3 monoclonal antibody (mAb) OKT3. The aim of this study was to provide a greater characterization of CD3-activated T cells. On day 6, most T cells coexpressed the CD1 la, CD18, CD54, CD45R0 antigens and consisted of activated (CD25⁺) CD4⁺ and CD8⁺ cells in nearly equal proportions. Kinetics studies showed that CD4⁺CD25⁺ cells proliferated more rapidly and peaked earlier than CD8⁺CD25⁺ cells. When experiments were performed with purified subpopulations by removing CD4⁺ cells (resulting in CD8⁺ BMMCs) or by removing CD8⁺ cells (resulting in CD4⁺ BMMCs), T-cell activation and autologous plasma cell decrease were observed in CD4⁺ BMMCs only. Transwell cultures showed that CD4 help was necessary to make CD8⁺ BMMCs susceptible to CD3 stimulation. Relevant amounts of IL-2 were found in the supernatants of CD4⁺ BMMCs cultures, whereas no secretion of IL-4 was detected, indicating a Thl-like profile of CD3-activated CD4⁺ cells.
These data indicate that CD4⁺ cells proliferate earlier and provide optimal help to induce the subsequent expansion of CD8+ cells after CD3 stimulation of MM BMMCs. Adequate stimulation of CD4⁺ cells is therefore essential in any strategy aiming to recover T-cell-mediated immunity in MM.     

Combined negative and positive selection of mobilized CD34+ blood cells

We tested four negative and two positive selection methods for separation of CD34⁺ cells from mobilized blood cells, and analysed fold-enrichment, purity and recovery of CD34⁺ cells after selection procedures. The elimination of mature CD34⁻ cells was achieved by adhesion to nylon-wool fibre (5.9 ± 1.0 mean fold-enrichment and 65.2 ± 2.3 mean recovery of CD34⁺ cells). Standard or modified Ficoll-Hypaque and Percoll density gradients, as well as phagocytosis with magnetic beads, were less effective in eliminating CD34⁻ cells, both purity and fold-enrichment of CD34⁺ cells being lower than those obtained with separation by nylon-wool. Both positive selection methods tested, Ceprate and MiniMacs System, generated highly purified CD34⁺ cell populations ranging from 80% to 90%. The recovery of CD34⁺ cells was optimal with MiniMacs (77.9±3.6) and low with Ceprate (28.8±2.8). Based on these results, in two large-scale experiments we combined nylon-wool fibre and MiniMacs System in a two-step separation procedure obtaining a 36.9±2.6 mean fold-enrichment and a 50.5±0.3 mean recovery of CD34⁺ cells. In this way we achieved optimal enrichment and recovery of CD34⁺ cells, with a substantial saving of cost compared to either selection method alone.     

A CD4+ proliferation of large granular lymphocytes expresses the protease activated receptor-1

The platelet-type thrombin receptor was the first member to be identified in a family of protease activated receptors (PARs) and has been designated PAR-1. We recently reported that the large granular lymphocytes (LGLs) in patients with proliferations of CD8⁺ cells co-expressed PAR-1 and the expression of PAR-1 correlated with the expression of CD57. Here we show, by three-colour immunofluorescence, that the LGLs from a patient with a rare CD4⁺ CD57⁺ monoclonal expansion also expressed PAR-1. Northern blot analysis confirmed the presence of high levels of mRNA for PAR-1 in these LGLs.     

CD34+ cell immunoselection from G-CSF-alone-primed peripheral blood in children with low body mass

Summary. We report the data of CD34⁺ cell immunoselection from peripheral blood after G-CSF-alone mobilization (lOμg/kg/d s.c.) in nine children with neuroblastoma (median age 4 5 years (2-8), median body weight 16kg (10-20). Leukaphereses were carried out on a Cobe Spectra separator and two consecutive harvests (4 blood volumes processed) were used for immunoselection on a Ceprate(tm) column. The yield of CD34⁺ cells in the purified fraction was 50% (23-80), with a median number of 2.8xl0⁶ CD34+ cells/kg (1-9-4). All patients were reinfused with selected CD34⁺ cells after busulfan 600 mg/m²+ melphalan 180mg/m² and achieved successful haemopoietic recovery.     

CD4+ T lymphocytes contribute to protective immunity induced in sheep and goats by Haemonchus contortus gut antigens

Immunization with parasite antigens derived from the gut of adult Haemonchus contortus induces significant levels of protection against the parasite in sheep and goats. However, the mechanisms of immunity involved in this protection are not clear. Here, we investigate the requirement for CD4⁺ T lymphocytes in gut antigen-induced immunity against H. contortus. Gut antigen immunized animals were depleted (>98%) of their CD4⁺ T lymphocytes in peripheral blood by intravenous injection of an anti-CD4 MoAb. Depletion in peripheral blood persisted for at least eight days, after which there was gradual recovery of CD4⁺ T lymphocytes. Serum antibody levels in gut antigen-immunized animals correlated significantly with worm parameters, suggesting a contribution by antibody to the immunity observed. By covariate analysis, using ELISA OD as the covariate, CD4⁺ T lymphocyte depletion was shown to partially abrogate immunity induced by gut antigen immunization, against challenge infection with H. contortus. The greatest effect of CD4⁺ T lymphocyte depletion was observed at 14 days post-infection, with differences between CD4⁺ T lymphocyte depleted and intact animals less apparent between days 21 and 25. Collectively, our data indicate that CD4⁺ T lymphocytes contribute to immunity induced by gut antigens. Our results also suggest that antibody works synergistically with CD4⁺ T lymphocytes to confer this immunity.     

Increased number of peripheral blood CD34+ cells in lithium-treated patients

Eight adult patients with bipolar disorder were prospectively examined to find whether lithium carbonate increased their peripheral blood CD34⁺ haemopoietic stem cells. Following lithium therapy for 3–4 weeks their neutrophil counts increased by a mean of 88% (from 4625 ± 1350 × 10⁹/l, mean ± SD pretreatment, to a peak of 8300 ± 3910 × 10⁹/l). Concommitantly, there was a significant increment in their CD34⁺ cells (from 0.11 ± 0.01% to a peak of 0.18 ± 0.08%). There was a significant correlation between the rise in neutrophil count and that of the CD34⁺ cells ( r  = 0.795, P  = 0.019). Lithium therapy may be used to mobilize peripheral blood CD34⁺ cells for marrow transplantation.     

Chronic myelogenous leukaemia CD34+ cells exit G0/G1 phases of cell cycle more rapidly than normal marrow CD34+ cells

To investigate the mechanisms behind the leukaemic expansion of chronic myelogenous leukaemia (CML), we examined the cell cycle status and activation kinetics of purified subpopulations of CD34⁺ cells from normal and CML bone marrow (BM). Propidium iodide staining was used to assess cell cycle status of fresh cells or those stimulated with cytokines. Although the cell cycle status of fresh low-density cells from CML and normal BM was similar, a larger percentage of CML CD34⁺ cells were cycling than those from normal BM. The HLA-DR⁻ compartment of CML CD34⁺ cells, a fraction enriched for normal, non-leukaemic progenitors, contained a higher percentage of quiescent cells than the CD34⁺ HLA-DR⁺ fraction. When the activation of CD34⁺ cells was examined in response to SCF or IL-3 alone, or SCF+IL-3+IL-6, CML CD34⁺ cells exited G₀/G₁ more rapidly than normal CD34⁺ cells. Interestingly, although normal BM CD34⁺ cells failed to cycle in response to IL-6 alone, or in the absence of exogenous cytokines, 30% of CML cells cycled under these conditions. No differences in the degree of apoptosis were documented among CML and normal CD34⁺ cells in these cultures. These data suggest that enhanced cell cycle activation of CML CD34⁺ cells, by either autocrine stimuli or via enhanced sensitivity to exogenous stimuli, may be partially responsible for the pronounced cellular expansion characteristic of CML.     

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.     

Alloreactive CD4+ T lymphocytes can exert cytotoxicity to chronic myeloid leukaemia cells processing and presenting exogenous antigen

Existing evidence supports that CD4⁺ T lymphocytes play a role in the graft-versus-leukaemia (GVL) reaction after allogeneic bone marrow transplantation (BMT) for chronic myeloid leukaemia (CML), not only as initiators of the immune response but also as effectors of GVL. In BMT between HLA-identical pairs this CD4-mediated GVL would require CML cells to process and present antigens through MHC class II molecules. To investigate whether CML cells are capable of processing and presenting antigens, and suitable targets for CD4⁺ T-cell-mediated cytotoxicity, we generated HLA-DR1-restricted CD4⁺ cytotoxic T-cell clones that specifically recognized tuberculous purified protein derivative (PPD). We have shown that CML cells and B lymphoblastoid cell line (B-LCL) cells but not PHA-blasts from patients with CML processed exogenous antigen, PPD, and induced proliferative and cytotoxic CD4⁺ T-cell responses. Antigen presentation was blocked by antibodies to HLA-DR but not to MHC class I and by treatment with chloroquine and brefeldin. This indicates that CML cells use a classic MHC class II antigen processing pathway to present PPD antigens to CD4⁺ T cells. Cytotoxicity to CML was shown by antibody blocking studies to be mediated mainly through fas antigen. These findings indicate that donor CD4⁺ T cells alone are sufficient to mediate GVL effects following allogeneic BMT for CML.