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     

Epinephrine test and plasma elastase as diagnostic tools in a patient with CD3+ large granular lymphocyte proliferation

Large granular lymphocytes (LGL) proliferation is characterized by expansion of cytotoxic lymphocytes and associated with neutropenia. In a case of CD3⁺ LGL-proliferation the epinephrine stimulation test (EST) induced a striking elevation of CD3⁺, CD8⁺, CD57⁺ LGL in peripheral blood from 2.7 x 10⁹/1 to 20 x 10⁹/1 and might be an additional diagnostic tool in patients with normal or low absolute numbers of circulating LGL. After treatment with steroids, plasma elastase - a marker of neutrophil destruction - decreased from 162 to 40μg/1 (normal < 47μg/1) which correlated well with a simultaneous increase in peripheral neutrophil counts from 0.14 to 1.0 x 10⁹/1. This finding supports the hypothesis that neutropenia in CD3⁺ LGL proliferation is due to neutrophil destruction, possibly mediated by LGL.     

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.     

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.     

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.     

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.     

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.     

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.     

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 .     

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.     

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.     

Immunophenotype of c-kit cells in normal human bone marrow: implications for the detection of minimal residual disease in AML

Summary. In the present study, seven normal human bone marrow samples from healthy volunteers have been analysed in order to investigate the immunophenotypic characteristics of the normal CD117⁺ cells and their utility for the detection of minimal residual disease in 71 acute myeloid leukaemia patients.
Our results show that most of normal BM CD117⁺ cells coexpress the HLADR and the myeloid associated CD33 antigen. In addition, almost half of CD117⁺ cells are CD34⁺, these cells displaying a different FSC/SSC distribution when compared to the CD117⁺/CD34⁻ cells. No CD117⁺/CD15⁺ and CD117⁺/CD10⁺ cells were detected and very few CD117⁺ cells (<1 × 10⁻³) expressing the HLADR⁻/CD34⁻, CD33⁺/HLADR⁻ and CD34⁺/HLADR⁻ phenotypes were found to be present in normal BM. In contrast, from the 71 AML patients analysed, 34 had CD117⁺/CD15⁺ blast cells and eight had the CD117⁺ phenotypes detected at low frequencies (<1 × 10⁻³) in normal BM.
In summary, the present study shows that the use of the CD117 antigen in different monoclonal antibodies combinations may be of great help for the detection of minimal residual disease in a high proportion of AML cases, especially in those patients displaying the CD117⁺/CD15⁺ phenotype, because cells coexpressing both antigens in normal BM, if present, are at very low frequencies.     

Direct and reversible inhibition of platelet factor 4 on megakaryocyte development from CD34+ cord blood cells: comparative studies with transforming growth factor β1

Mechanisms of the action of platelet factor 4 (PF4) on the growth of megakaryocyte (MK) progenitor cells in CD34⁺ cord blood (CB) cells were studied in comparison with transforming growth factor β1 (TGFβ1). Development of MK from CD34⁺ CB cells in both plasma clot culture and liquid culture was significantly inhibited by either purified human PF4 and by recombinant human TGFβ1. Inhibition of MK colony formation by PF4 was reversible, because CD34⁺ cells preincubated with PF4 could regenerate colonies after washing and replating into secondary cultures. In contrast, TGFβ1-preincubated CD34⁺ cells gave rise to few colonies following replating. Moreover, incubation of CD34⁺ cells with PF4 in liquid culture caused the increased number of both stem cell factor (SCF)-binding cells and CD34 antigen-bearing cells. In addition, PF4-preincubated CD34⁺ cells exibited a higher potential in MK colony formation in the presence of 5-fluorouracil (5FU). These results demonstrate that both PF4 and TGFβ1 inhibit MK development from CD34⁺ CB cells by different mechanisms, and suggest that PF4, unlike TGFβ1, exerts its inhibitory effect on the growth of the target cells in a reversible manner which results in a preservation of a more immature and 5FU-resistant cell population.     

Nematode infection induces Th2 cell-associated immune responses in LEC mutant rats with helper T cell immunodeficiency

Long-Evans Cinnamon (LEC) rats have maturational arrest of CD4⁺8⁻ T cells from CD4⁺8⁺ cells in the thymus. Despite this, CD4⁺8⁻ T cells are always present in peripheral lymphoid organs of LEC rats, suggesting that these CD4⁺8⁻ T cells are generated by an uncommon pathway. We investigated the role of LEC rat peripheral CD4⁺8⁻ T cells in Th2-associated responses to infection with the nematode Nippostrongylus brasiliensis . After infection, the numbers of CD4⁺8⁻ TCRαβ⁺ T cells significantly increased in mesenteric lymph nodes (MLN) and the spleen, while those in the thymus were still negligible. Infection also induced significant up-regulation of IL-4 gene expression in LEC rat MLN cells. Total serum IgE levels in LEC rats were markedly increased two weeks after infection. Mucosal mast cell responses in the gut and lungs of LEC rats were induced as prominently as in control Long-Evans Agouti (LEA) rats. Faecal egg count data indicated that LEC rats rejected nematodes faster than LEA rats. These results suggested that Th2-associated responses can be induced by nematode infection in LEC rats probably through the extrathymic recruitment and proliferation of CD4⁺8⁻ TCRαβ⁺ T cells .     

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.     

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 .     

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.     

Monitoring of CD95 and CD38 expression in peripheral blood T lymphocytes during active human cytomegalovirus infection after orthotopic liver transplantation

Aim:  The aim of the present study was to quantitatively monitor the response of CD95 molecules expressed on CD3⁺ T cells (CD95⁺CD3⁺ cells) and CD38 molecules expressed on CD8⁺ T cells (CD38⁺CD8⁺ cells) to ganciclovir treatment after orthotopic liver transplant (OLT) in recipients with active human cytomegalovirus (HCMV) infection.
Methods:  Blood samples were collected from 20 liver transplanted recipients with active HCMV infection and 24 recipients without HCMV infection. CD95⁺CD3⁺ cells and CD38⁺CD8⁺ cells were quantitatively detected with QuantiBRITE bead methods by dual-color flow cytometry analysis during the post-transplantation period.
Results:  CD95⁺CD3⁺ cells and CD38⁺CD8⁺ cells were not significantly different among different ages of healthy adults ( P  > 0.05). CD95⁺CD3⁺ cells and CD38⁺CD8⁺ cells were drastically increased in the active HCMV infection group compared with that in the stable group or in the healthy group ( P  < 0.001), and then they were gradually decreased within the next several weeks after ganciclovir treatment when compared with active HCMV infection recipients ( P  < 0.001).
Conclusions:  The present study showed that CD38⁺CD8⁺ T cells can be an appropriate immunological marker for early detection and antiviral therapeutic monitoring of HCMV infection. The evaluation of CD95 molecule levels may be used routinely in clinical practice to assess the level of immunosuppression.