γδ T-Cell Precursor-Derived CD4− CD8−αβ T Cells Retain γδ Cell Function

We have previously shown that some of the DNαβ⁺ T cells arising in TcRα-chain transgenic mice are of γδ T cell origin, based on phenotypic data and on their status of TcR gene rearrangements. In the present report we investigated the impact of αβ TcR expression on the functional programme of the mature γδ precursor-derived DNαβ⁺ T cells. Our results demonstrate that both their proliferative capacity and their cytokine production profile are similar to that of γδ T cells. Furthermore, both transgenic DNαβ⁺ T cells and DNγδ⁺ T cells up-regulate CD8α expression after activation, but, in contrast to CD4⁺αβ T cells, are unable to induce proliferation of naive B cells. Thus, our results suggest that the effector functions of mature T cells are determined independently of the TcR isotype, probably at an early stage of differentiation, and thereby have important implications for current models of T-cell lineage commitment.     

Primary Stimulation of CD4+ Cells in the Presence of IL-4 or IFN-γ Alters the Frequencies of Cytokine-Producing Cells at Restimulation

The induction of specific effector functions in naive T cells may be directed by accessory signals during activation. These could be elicited through binding to cell surface molecules or through factors secreted by antigen-presenting cells or other simultaneously activated cells. We have investigated the influence of CD8⁺ cells and of exogenousiy added cytokines (interleukin (IL)-2, IL-4 and interferon (IFN)-γ) on the cylokine production in splenic CD4⁺ T cells. IL-2, IL-4, IL-5 and IFN-γ production in CD4⁺ cells was measured at the single cell level during primary mitogen stimulation in vitro in the presence or absence of factors or CD8⁺ cells. On day 5 the cells were restimulated with mitogen alone and analysed to evaluate the short-term development of cytokine-producing cells in such cultures. Preactivation in the presence of either exogenous IL-4 or IFN-γ led to an increased production of IL-4 and IFN-γ respectively at restimtilation, and the effects of both IL-4 and IFN-γ were augmented by IL-2. After preactivation in the presence of IL-2 and IL-4, every third CD4⁺ cell could be induced to produce IL-4. Exogenous IL-4 or IFN-γ further decreased each other's production. Depletion of CD8⁺ cells before activation resulted in a slight increase of IL-4-producing cells, indicating that simultaneous activation of CD8⁺ cells will influence lymphokine production in CD4⁺ cells. The results suggest that the pattern of lymphokines induced in naive cells may be influenced by factors secreted by preactivated CD4⁺ and CD8⁺ cells, and that naive cells are preferentially 'recruited' to produce similar cytokines.     

Immunophenotypic and Functional Characteristics of Haemopoietic Cells from Human Cord Blood

Cord blood (CB) as a new source for bone marrow transplantation represents advantageous features concerning stem cell and leucocyte compartments and function. We attempted to get more information about the phenotypes and function of CB cells by investigating their cell surface markers and also the production of IL-2, IFN-γ and IL-6 by mitogen and alloantigen stimulation. The CB cells were characterized by a low proportion of CD3⁺ T cells, CD4⁺ T subpopulation, activated T cells and CD3⁺ CD16/CD56⁺ cytotoxic cells, suggesting reduced graft versus host potential. The significant increase of CD19/CD3 double positive cells and decrease of CD19/HLA-DR double positive mature B cells reflect that immature B cells exist in CB. In the functional studies, a 27- and 5-fold reduction was observed in the production of IFN-γ by CB cells stimulated with PHA and allogeneic cells, respectively. The production of IL-2 in PHA-stimulated CB cells also showed a 50% determination. Decrease in the production of these cytokines by CB cells is supported by the decline of the proportion of CD3⁺ T cells. However, an increase was observed in the production of IL-6 by CB cells stimulated with allogeneic cells as compared with the controls. These results suggest a difference in the functional activity of the T helper cell subsets between the CB and peripheral blood and/or differences in the functional maturity of T helper cell subsets and B cells in these compartments.     

Contrary Roles of IL-4 and IL-12 on IL-10 Production and Proliferation of Human Tumour Reactive T Cells

The cytokine profile of tumour reactive T cells is likely to play a central role in their function. However, little is known about how cytokine patterns of tumour reactive T cells can be regulated. Here, the authors investigated the influence of exogenous regulatory cytokines in addition to interleukin-2 (IL-2) on cytokine patterns and the proliferation of T cells recognizing an autologous sarcoma cell line. In this system, IL-4 and IL-12 showed the most polarizing influences on tumour reactive T cells. Exogenous IL-4 induced a predominant production of IL-4 while decreasing the interferon-γ (IFN-γ) and IL-10 production by tumour reactive T cells. It also stimulated the growth of tumour reactive CD4⁺ T cell clones. In contrast, IL-12 substantially increased the production of IL-10 and IFN-γ. This was accompanied by a growth inhibition of tumour reactive T cells. The growth of CD4⁺ tumour reactive T cells was also suppressed by exogenous IL-10. This study shows that cytokine patterns and proliferation tumour reactive T cells can be significantly influenced by exogenous cytokines and confirms the hypothesis of a negative feedback loop of IL-12 by the induction of IL-10 in the context of human tumour reactive T cells.     

Infective Dose Modulates the Balance between Th1- and Th2-Regulated Immune Responses during Blood-Stage Malaria Infection

Plasmodium chabaudi infection of mice provides an excellent model for examining acquired immunity to the blood-borne stage of malaria infection. CD4⁺ T-cell receptor (TCR) αβ-bearing T lymphocytes play a critical role in mediating protection, ascribed to both T helper (Th) 1 and Th2 subsets. One factor that may influence the Th1/Th2 cell balance is infective dose. In this study, we found that the size of the infective dose of P. chabaudi , and thus the level of antigen presented to the immune system, correlated with the balance of responder CD4⁺ T-cell phenotypes. Increasing the infective dose in a resistant mouse strain enhanced the Th1 cytokine (interferon-γ; IFN-γ) response and reduced the Th2 cytokine (interleukin-4; IL-4) response. In contrast, increasing the infective dose in a susceptible mouse strain led to a prominent and accelerated up-regulation of IL-4 production. These data show that the dose of antigen can significantly affect the balance between Th1- and Th2-mediated immune functions during infection of the mammalian host with blood-stage malaria parasites. This demonstration that parasite numbers may modulate CD4⁺ T-cell regulation has novel implications for the successful implementation of antimalarial vaccination and chemotherapeutic strategies.     

Interleukin-4 and Interferon-Gamma Production by Leishmania Stimulated Peripheral Blood Mononuclear Cells from Nonexposed Individuals

Interferon-gamma (IFN-γ) and interleukin-4 (IL-4) production by Leishmania reactive peripheral blood mononuclear cells (PBMC) from non-exposed individuals was investigated. IFN-γ was measured in culture supernatants after antigen stimulation. For the measurement of IL-4, antigen stimulated cells were pulsed with PMA and ionomycin before IL-4 release was measured. L. donovani and L. major antigens induced IL-4 production (105–1748pg/ml) in 13 and seven cultures, and IFN-γ production (1.7- > 66IU/ml) in 14 and 11 of 20 cultures, respectively. IL-4 production rose steeply after 6 days of antigen stimulation suggesting a response due to antigen recognition. Both IL-4 and IFN-γ production was abrogated by depletion of CD2⁺ or CD4⁺ but not CD8⁺ cells. CD2⁺ or CD4⁺ but not CD8⁺ enriched cultures produced cytokines as unseparated PBMC. Thus, in non-exposed individuals circulating Leishmania reactive CD4⁺ T cells could be demonstrated. The cells from different individuals showed different patterns of IFN-γ and/or IL-4 production upon antigenic stimulation. In experimental leishmaniasis the early balance between IFN-γ and IL-4 is important for the clinical outcome. Our findings call for studies of the importance of cytokine production by cross-reactive T cells for the outcome of L. donovani infections in humans and show that the method for IL-4 detection is useful for this purpose.     

Vδ1+ Gamma/Delta T Lymphocytes Infiltrating Human Lung Cancer Express the CD8α/α Homodimer

Murine γ/δ T lymphocytes localize to different epithelial tissues and are phenotypically distinct from peripheral γ/δ T cell-populations in that they show limited TCR diversity, express the CD8 α/α homodimer and lack the CD8β chain. In humans, a compartmentalization of γ/δ cells sharing similar phenotypic features has been documented to date only in the case of intestinal epithelium. In the present study we show that about half of Vδ1⁺ (as well as Vδ1⁻Vδ2⁻) γ/δ lymphocytes, which can be selectively expanded from human lung cancers, coexpress the CD8α/α homodimer. The accumulation of intraepithelial CD8⁺γ/δ⁺ lymphocytes might then be a more general phenomenon, possibly as a result of common mechanisms operating at those sites.     

CD8+ Cells are the Main Producers of IL10 and IFNγ after Superantigen Stimulation

Purified CD8⁺ T cells were recently shown to produce TH1 as well as TH2 types of cytokines upon restimulation, indicating an important role for these cells in regulation of immune responses. However, it is not known if the CD8⁺ cells would contribute to cytokine production in the presence of cytokine secreting CD4⁺ cells. In the present study the authors have investigated the proportion of cytokine-producing CD4⁺ and CD8⁺ cells in the spleen after in vitro or in vivo stimulation. They found that stimulation of spleen cells with the superantigen Staphylococcal Enterotoxin B (SEB) in the presence of IL4 promoted production of IL10 and IFNγ predominately by CD8⁺ cells. In contrast, the production of IL4 was almost exclusively confined to the CD4⁺ subset. When priming with SEB in vivo before subsequent restimulation in vitro , a protocol previously shown to induce anergy, up to 80% of the IL10 and IFNγ positive cell expressed the CD8 marker. Taken together, these results emphasize the important role of cytokine-producing CD8⁺ cells and indicate that CD4⁺ and CD8⁺ T cells may, in a given situation, produce distinct cytokines.     

Different Percentages of Peripheral Blood γδ+ T Cells in Healthy Individuals from Different Areas of the World

The frequency of γδ⁺ T cells in the peripheral blood of 26 Turkish, 24 Swedish, 35 Japanese and 14 'Asian' (non-Japanese) healthy blood donors and healthy volunteers were investigated by flow cytometry. In the Turkish group, 9.3% (median value) of the CD3⁺ peripheral blood T cells expressed the γδ T cell receptor. A similar level of γδ⁺ T cells was found in the non-Japanese 'Asian' healthy volunteers (9.2%), while significantly lower values were detected in the Swedish (4.2%) and Japanese (4.5%) groups. These dramatic differences in normally occurring γδ⁺ T cells in different groups of healthy individuals were further reflected by a low incidence of >10% γδ⁺ T cells in the Swedish (0/24) and Japanese (6/35) groups compared to the Turkish (12/26) and 'Asian' (5/14) groups. The described γδ⁺ T cell differences between distinct ethnic groups are thus likely to be a consequence of environmental factors, but additional genetic influences cannot be ruled out. The present study demonstrates the potential importance of the ethnic origin and environmental history of subjects examined in studies of γδ⁺ T cells–disease relations.     

γδ and αβ T Cells are Equally Susceptible to Apoptosis

Gamma/delta TCR bearing T lymphocytes represent a T-cell subset whose functional relevance remains unclear. Nevertheless these T cells may play a role in the early immune reponse against bacteria. Until now the regulatory mechanisms on this response have not been investigated. The study described here evaluated the immunoregulatory effects of Interleukin-10 on γ/δ and α/β TCR-positive T-cell clones and freshly isolated peripheral-blood mononuclear cells (PBMC). IL-10 has been shown previously to inhibit lectin and antigen-induced proliferation and cytokine production by α/β T cells. The results outlined below show that rhIL-10 strongly inhibits lectin-induced production of IFN-γ, TNF-α. IL-2, and to a lesser degree proliferation and IL-4 production of both T-cell subsets. As IL-10 did not inhibit proliferation but at the same time strongly suppressed cytokine production in various experiments, the hypothesis that it could function as a growth factor for human T cells as has been described for murine thymoeytes was tested. The data demonstrate that, although the γ/δ T-cell clones tested do not produce IL-10 they can use it as a growth factor in combination with IL-2, IL-4 or alone. Furthermore, IL-10 has the same properties on human α/β T-cell clones and PBMC. In summary, it is shown that IL-10 has pleiotropic effects on γ/δ and α/β TCR⁺ T cells by inhibiting lectin-induced cytokine production and by acting as a growth factor for these cells alone or in combination with IL-2 or IL-4.     

GD2 Ganglioside-Specific Monoclonal Antibody Reacts with Murine Cytotoxic T Lymphocytes Reactive with FBL-3N Erythroleukaemia

Ganglioside expression on cytotoxic T lymphocytes induced against the mouse erythroleukaemia line FBL-3N, was analysed, compared with that of naive T lymphocytes in the thymus, lymph nodes and spleen. Although normal uncultured and cultured T lymphocytes expressed no GD2, GD3 or GM2 gangliosides, cytotoxic T cells with CD4⁺ CD8⁻, CD4⁻CD8⁺, and CD4⁻CD8⁻ phenotypes reacted with anti-GD2 monoclonal antibody with various intensities. The reactivity with anti-GD2 was associated with the intensity of cytotoxic activity as analysed using cytotoxic T lymphocyte (CTL) clones established from the bulk CTLs of each phenotype. An increase of the mRNA expression of GM2/GD2 synthase gene was demonstrated by Northern blot hybridization using RNA extracted from thymocytes, spleen cells, Con A-treated spleen cells and various types of CTL cells. These results indicated that GD2 ganglioside expression might be associated with the functional differentiation of murine T lymphocytes.     

Control of T-cell activation by CD4+ CD25+ suppressor T cells

Summary: Depletion of the minor (∼10%) subpopulation of CD4⁺ T cells that co-expresses CD25 (interleukin (IL)-2 receptor α-chain) by thymectomy of neonates on the third day of life or by treatment of adult CD4⁺ T cells with anti-CD25 and complement results in the development of organ-specific autoimmunity. Autoimmune disease can be prevented by reconstitution of the animals with CD4⁺ CD25⁺ cells. CD4⁺ CD25⁺-mediated protection of autoimmune gastritis does not require the suppressor cytokines IL-4, IL-10, or transforming growth factor (TGF)-β. Mice that express a transgenic T-cell receptor (TCR) derived from a thymectomized newborn that recognizes the gastric parietal cell antigen H/K ATPase all develop severe autoimmune gastritis very early in life. CD4⁺ CD25⁺ T cells are also powerful suppressors of the activation of both CD4⁺ and CD8⁺ T cells in vitro . Suppression is mediated by a cell contact-dependent, cytokine-independent T–T interaction. Activation of CD4⁺ CD25⁺ via their TCR generates suppressor effector cells that are capable of non-specifically suppressing the activation of any CD4⁺ or CD8⁺ T cell. Activation of suppressor effector function is independent of co-stimulation mediated by CD28/CTLA-4 interactions with CD80/CD86. We propose that CD4⁺ CD25⁺ T cells recognize organ-specific antigens, are recruited to sites of autoimmune damage where they are activated by their target antigen, and then physically interact with autoreactive CD4⁺ or CD8⁺ effector cells to suppress the development of autoimmune disease.     

Natural CD4+ CD25+ regulatory T cells. Their role in the control of superantigen responses

Summary: CD4 regulatory T cells have a major role in controlling the immune response to self and foreign antigens. Natural CD4⁺ CD25⁺ T cells are a major component of the regulatory subset. Their absence is associated with the development of autoimmune and inflammatory diseases and with abnormal peripheral T-cell homeostasis. Two main characteristics discriminate natural CD4⁺ CD25⁺ T cells from their CD4⁺ CD25⁻ counterparts, namely their cytokine production profile and their behavior during tolerance induction. Natural CD4⁺ CD25⁺ T cells produce interleukin (IL)-10, a cytokine that contributes to their regulatory role. They do not produce IL-2 and are dependent on exogenous IL-2 for proliferation in vitro and in vivo . Studies of their response to superantigen administration in vivo show that they are resistant to clonal deletion but can be tolerized by anergy. Their resistance to apoptosis may contribute to their continuous regulatory function, as it allows them to maintain permanent control over effector T cells.     

Priming of T-Cell Responses in Mice by Porins of Salmonella typhimurium

An imbalance in signals delivered to T cells via T-cell receptor and accessory molecules can lead to anergy, apoptosis, or both. In the present study we have demonstrated that Salmonella typhimurium infection in mice leads to a progressive loss of CD4⁺ T helper (Th) cell population, abnormal T-cell death by apoptosis and loss of accessory molecules (B7 and intracellular adhesion molecule-1) on macrophages. Quantification of interleukin-2 (IL-2), interleukin-4 (IL-4) and interferon-γ (IFN-γ) secretion revealed a Th2-type of response in lymphocytes isolated from spleen. However, preimmunization of mice with porins resulted in an increased CD4⁺ Th cell population and accessory molecules on the surface of macrophages. Quantification of cytokines revealed a Th1-type of response. We conclude that preimmunization of mice with porins provides a microenvironment in which a well-balanced accessory molecule and cytokine network is established, which results in the prevention of cell death by apoptosis.     

Lack of Polarized Type 1 or Type 2 Cytokine Profile in Asymptomatic HIV-1-Infected Patients During a Two-Year Bimonthly Follow-Up

The production of type 1 (interferon or IFN-γ) and type 2 (interleukin or IL-4 and IL-10) cytokines by mitogen-stimulated peripheral blood mononuclear cells (PBMCs) obtained from asymptomatic human immunodeficiency virus-seropositive (HIV⁺) patients untreated with any antiviral, antibacterial or antimycotic drugs, and from healthy individuals, was evaluated by quantitative ELISA. Patients who were HIV⁺ were characterized by the absence of abnormal cytokine production. The level of each cytokine differed among individuals in the same group with intersubject variations greater for HIV⁺ patients than for healthy individuals. The longitudinal evaluation of IFN-γ, IL-4 and IL-10 production showed intrasubject variations which were particularly marked in HIV⁺ patients. Accordingly, HIV⁺ patients and, to a lesser extent, healthy individuals were characterized by a wide spectrum of possible profiles, which were confined to type 0 phenotype. In HIV⁺ patients no correlation was found between each cytokine level and the number of CD4⁺ T cells, not even in those with a falling CD4⁺ T-cell count and clinical symptoms.     

Peripheral Immune Response in Pulmonary Tuberculosis

Cellular immune response and delayed-type hypersensitivity reactions are considered to play a major role in the immunopathogenesis of pulmonary tuberculosis (PTB). But the exact mechanism is still to be clarified. Th1 cells are mainly involved in cellular immune responses in PTB and provide a normal healing process with minimal or no sequela whereas Th2 cell and CD8⁺ T lymphocyte responses may lead to more severe type of disease. In this study, we investigated the peripheral blood immune responses in PTB. The study group consisted of acid fast positive young male soldiers with PTB and a negative HIV serology. The control group included healthy young volunteer male soldiers without a history of PTB. Intracytoplasmic cytokine content of CD8⁺ T cells and lymphocytes, including IL-2, IL-4, IL-5, IL-10 and IFN-γ were determined by flow cytometry, and IL-2, IL-4, IL-5, IL-10, IFN-γ and TNF-α serum levels were measured by cytometric bead array (CBA). No difference was observed between the percentages of T, B, NK cells and HLA-DR expression in both groups, however, the number of CD3⁺HLA-DR⁺ activated T cell percentages was higher in PTB group as compared to healthy subjects. IL-2, IL-4, IL-5, IL-10 contents of lymphocytes and IFN-γ⁺CD8⁺ T cells were found to be significantly lower in PTB patients when compared with healthy subjects, and in parallel, serum IL-2, IL-4, IL-5 and TNF-α levels were also significantly lower in PTB patients. In conclusion we suggest that, CD8⁺ T cells producing both Th1 and Th2 type cytokines, may play important role in the peripheral immune response to mycobacteria.     

T Helper-Independent Activation of Human CD8+ Cells: the Role of CD28 Costimulation

The concept that activation of MHC class I-restricted CD8⁺ cells entirely depends on help from MHC class II-restricted CD4⁺ T cells has recently been supplemented with an alternative model in which CD8⁺ cells can directly be activated by MHC class I-expressing professional antigen-presenting cells (APC), which are able to deliver an accessory signal. The authors analysed the role of CD28-mediated costimulation for T helper cell-independent activation of purified human CD8⁺ T cells in two different in vitro models. Freshly isolated CD8⁺ cells could be activated (proliferation, IL-2 production and cytotoxic activity) by anti-CD3-presenting FcγR⁺ mouse cells transfected with the human CD28 ligand, CD80, as the only accessory signal. On the other hand, activation of CD8⁺ cells by allogeneic MHC class I on EBV-transformed B cells, which express two different CD28 ligands, CD80 and CD86, also proceeded very efficiently (proliferation, cytotoxic activity and CD25 expression), but was either not, or only partially, blocked by anti-CD80 and anti-CD86 MoAb or CTLA-4Ig. This indicates that other costimulatory signals are also effective, and that CD28 triggering is not absolutely required for initial T-cell activation. CsA and CD80/CD86-blocking agents were synergistic in completely inhibiting activation of CD8⁺ cells in the MLR with allogeneic B-cell lines. This combination also induced non-responsiveness of CD8⁺ cells upon restimulation in the absence of blocking agents. Therefore, although professional APC can apparently provide multiple costimulatory signals for direct activation of CD8⁺ T cells, the signal derived from CD80/CD86 is unique in providing CsA-resistance.