Cyclic nucleotide phosphodiesterases from purified human CD4+ and CD8+ T lymphocytes

Background CD4⁺ and CD8⁺ T-lymphocytes are suggested to differentially affect airway inflammation in asthma. Agents which increase intracellular cAMP levels, such as PDE inhibitors, have been shown to diminish lymphocyte growth and differentiation, and to affect cytokine expression. Differences in the PDE isoenzyme profile between CD4⁺ and CD8⁺ cells might form a basis to differentially modify their functions by PDE inhibitors. Objective The study investigates and compares the PDE isoenzyme activity profiles of human peripheral blood CD4⁺ and CD8⁺ T-lymphocytes. Methods CD4⁺ and CD8⁺ T-lymphocytes were purified (>98%) from peripheral blood mononuclear cells by negative selection. PDE isoenzyme activity profiles were investigated using PDE isoenzyme selective inhibitors and activators. Results In CD4⁺ and CD8+ T-lymphocyte homogenates, PDE IV and PDE III activities were the predominant PDE isoenzyme activities at 0.5μM cyclic nucleotide substrate concentrations. PDE IV was localized in the soluble fraction whereas PDE III was membrane bound. Low PDE I, II and V activities were detected. About 20% of total eAMP hydrolysing capacity at 0.5 μM cAMP was insensitive to PDE isoenzyme selective inhibitors and activators and therefore could not be assigned to PDE I-IV. The PDE isoenzyme pattern was not different between CD4⁺ and CDS⁺ T-lymphocytes. Moreover, representative inhibitors of PDE HI and IV activity inhibited cAMP hydrolysis in soluble fractions of both T-lymphocyte subsets with similar potency. Enzyme kinetic analysis similarly did not reveal differences between CD4^h and CD8⁺ T-lymphocytes. Conclusion Normal CD4⁺ and CD8⁺ T-lymphocytes are likely to be equally sensitive targets for the effects of PDE inhibitors.     

Increased frequencies of the CD29 and CD57 markers and decreased frequency of CD45RA within CD4+ and CD8+ subsets after allogeneic bone marrow transplantation in man.

Two monoclonal antibodies, anti-CD45RA and anti-CD29, reciprocally divide the CD4+ and CD8+ lymphocytes into CD4+ CD45RA+, CD4+ CD29+, CD8+ CD45RA+ and CD8+ CD29+ subsets. The CD4+ CD45RA+, CD4+ CD29+ and CD8+ CD45RA+ possess suppressor-inducer, helper-inducer and suppressor-effector functions respectively. Since the role of these subsets has not been established after allogenic bone marrow transplantation we studied lymphocyte subpopulations in 12 patients 45-227 days after the procedure. The fraction of CD4+ lymphocytes was significantly (P = 0.0005) decreased to 20 +/- 9% versus 43 +/- 3% in controls. Within the CD4+ compartment, we found an increase in the fraction of CD4+ cells that co-expressed CD29 (CD29+/CD4+) to 92 +/- 10% versus 48 +/- 15% (P = 0.008) in controls and a concomitant decrease in CD45RA+/CD4+ to 16 +/- 12% versus 56 +/- 25% (P = 0.008). Patients were also noted to have an increase in the percentage of CD8+ lymphocytes to 41 +/- 5% compared to 23 +/- 4% in controls (P = 0.0004). Examination of the CD8+ subsets revealed a significant increase in the CD29+/CD8+ fraction to 97 +/- 3% versus 64 +/- 2% in controls (P = 0.008) and a decrease in the CD45RA+/CD8+ fraction to 36 +/- 11% versus 70 +/- 21% (P = 0.008). The number of cells co-expressing CD57 were also determined within the CD4+ and CD8+ subsets. In patients CD57+/CD4+ were increased to 29 +/- 7% versus 1 +/- 1% in controls (P = 0.04), and CD57+/CD8+ to 49 +/- 12% versus 23 +/- 9% (P = 0.02). Since CD29+ and CD57+ cells have a poor capability for IL-2 production and proliferation this shift in subset distribution may account for some of the defects in cellular immunity seen within the first year after allogeneic bone marrow transplantation.     

Apoptosis among CD45RA−/low CD3+ Progeny Accompanies Differentiation of Human Multinegative Thymocytes

Apoptotic cell death is widely believed to be the fate of negatively selected cells in the thymus. In this work we have used multiparameter flow cytometry to analyse reductions in DNA content that occur among differentiating human CD3^?4^?8^? multinegative (MN) thymocytes as they acquire CD3/TCR during in vitro culture. DNA content was measured as the intensity of the DNA-binding dye DAPI. The position of the diploid peak was identified by comparison to chicken red blood cells and to unfractionated uncultured thymocytes which have a sharply defined diploid peak. Apoptosis, was defined as a reduction in DNA content. Apoptotic cell death occurred continuously throughout the 7 day culture period and at the latest stages of culture DNA fragmentation was apparent on gels. Although both CD3^? and CD3⁺ progeny became apoptotic, it was more frequent among the CD3⁺progeny of the MN thymocytes. Apoptotic progeny included 60–70% CD3⁺ cells, while progeny remaining diploid were 8–36% CD3⁺. Fifty five per cent of CD3⁺ TCRδl⁺ progeny had less than 75% of the diploid DNA content, while for CD3⁺ TCRδ1^? progeny only 28% were in this category. CD3⁺ TCRδ1⁺ progeny also comprised 66% of the cycling cells at days 6–7 of culture, suggesting a pattern of rapid cell division followed by apoptotic cell death for this subset. A lack of positive selection in culture may trigger apoptosis among the TCRδ1 progeny. In contrast, TCRαβ progeny arising in culture appear to be less susceptible to apoptosis, perhaps due to their lack of CD4 and CD8. The expression of CD45RA and CD45R0 isoforms were assessed on the progeny of MN thymocytes based on their DNA content. Although 30% of apoptotic progeny expressed CD45RA, it was present at relatively low density compared to that on diploid or cycling cells, 55% of which were CD45RA^hi. The majority of apoptotic cells expressed neither CD45RA or CD45R0, but were CD45⁺, indicating the presence of an isoform not detected by our monoclonal antibodies (MoAbs). This is consistent with speculations that apoptotic cell death among thymocytes is preceded by a transition in CD45 isoform expression. These conditions may model early selective events resulting from high avidity TCR engagement that is independent of CD4 and/or CD8.     

A Functional Study of Purified CD4+ and CD8+ Cells Isolated from Synovial Fluid of Patients with Rheumatoid Arthritis and Other Arthritides

The purpose of this investigation was to study purified synovial fluid (SF) CD4⁺ and CD8⁺ cells from patients with rheumatoid arthritis (RA) and other inflammatory joint diseases (non-RA) with respect to the proliferative response to mitogens and recombinant interleukin 2 (rIL-2). Highly purified cell subsets were isolated by an immunomagnetic technique, and spontaneous proliferation as well as proliferative responses to rIL-2 and a combination of phytohaemagglutinin (PHA) and phorbol myristate acetate (PMA) (to substitute for accessory cells) were measured. Some patients had SF CD4⁺ and/or CD8⁺ cells with moderately increased spontaneous proliferation, but only the CD4⁺ cells of the two patient groups differed significantly from the peripheral blood (PB) T-cell subsets of healthy individuals who served as controls. The response to rIL-2 was variable but generally low, although about 50% of the CD4⁺ and 20% of the SF CD8⁺ cells of both patient groups expressed the Tac antigen. The response to PHA/PMA was significantly lower for RA SF CD4⁺ cells than for non-RA SF CD4⁺ cells, which again was lower than for normal PB CD4⁺ cells. SF CD8⁺ response to PMA/PHA by both groups of patients was somewhat decreased, but not significantly lower than in the controls. Thus, the CD4⁺ cells seemed functionally more deviant than the CD8⁺ cells in both patient groups, but the abnormality was most pronounced in the RA group. The results demonstrate that the previously reported diminished response to mitogens by SF mononuclear cells is present even when SF CD4⁺ cells are cultured alone. This indicates that these T cells have a reduced response, probably because of prior activation.     

CD4+ and CD8+ Distribution Profile in Individuals Infected by Schistosoma mansoni

Rationale: Patients with chronic Schistosoma mansoni infection show lower anti‐soluble egg antigen (SEA) proliferation responses and higher responses to soluble worm antigen preparation (SWAP). Objective: To compare the activation status and proliferation response of peripheral blood mononuclear cells (PBMC) of infected (XTO) and egg‐negative individuals (NI) living in the same endemic area. Methods: XTO (n = 51) and NI individuals from the same geographical area (n = 37) and healthy blood donors (n = 22) were evaluated before and after stimulation with SEA and SWAP. The expression of activation markers (CD4⁺ HLADR⁺, CD8^high+HLA‐DR⁺ and CD8⁺ CD28⁺) and proliferation assay was assessed by flow cytometry. Findings: PBMC from infected patients showed lower frequency of CD4⁺ but no change in CD8⁺ T cells when compared with the healthy donor group. The ratio CD4⁺/CD8⁺ was 1.3, 0.6 and 0.5 in healthy donors, infected and non‐infected individuals, respectively. The HLA‐DR⁺ expression on CD8⁺ was higher in PBMC from infected and non‐infected individuals than from healthy donors, but similar in both total lymphocytes and CD4⁺ populations. No intergroup proliferation response differences were observed in CD4⁺ and CD8⁺ PBMC unstimulated and stimulated with SEA and SWAP. The SEA but not SWAP‐stimulated cells showed a decrease in the expression of phosphorylated extracellular signal‐regulated kinase (ERK1/2). Conclusions: XTO and NI individuals living in the same area presented a smaller per cent of CD4⁺ and a higher per cent of CD8⁺ cells. The activation by either CD8^high+HLA‐DR⁺ or CD8^high+HLA‐DR⁺/CD8⁺ was enhanced and decreased in XTO and NI by CD8⁺ CD28⁺ and CD8⁺ CD28⁺/CD8⁺ when compared with healthy donor. ERK phosphorylation was attenuated in XTO and NI individuals when stimulated with SEA but not SWAP.     

Linomide Enhances Apoptosis in CD4+CD8+ Thymocytes

Linomide, a quinoline-3-carboxamide, has a pleiotropic immune modulating capacity and inhibits development as well as progression of disease in animal models of autoimmunity. Linomide treatment of mice resulted in a dramatic, dose-dependent decrease of the thymic cell number shortly after the start of administration. Flow cytometric analysis revealed that the major thymocyte subset, the early immature type CD4⁺CD8⁺ thymocytes, were reduced in number by 75%, mature CD4⁺CD8^? or CD4^?CD8⁺ thymocytes were less sensitive to treatment. The polyclonal T cell activator Con A (Concanavalin A) was used together with IL-2 to evaluate the potential proliferative responsiveness of ex vivo thymocytes. Thymocytes from mice treated with Linomide exhibited a more vigorous proliferation than control cultures. An effect shown to not only be due to the enrichment of mature thymocytes in the cultures from Linomide treated animals, but also when purified, mature thymocytes (CD4⁺CD8^? and CD4^?CD8⁺) were cultured with Con A and IL-2, these cells responded with a significantly enhanced proliferation. In vivo Linomide treatment did not result in increased plasma concentrations of corticosterone and treatment of adrenalectomized mice resulted in a reduction of thymocytes which was comparable to the effect in intact mice, indicating that glucocorticoids (GC) are not major mediators of Linomide-induced thymocyte deletion. In addition to this, and supporting a glucocorticoid independent mode of action, Linomide treatment of thymocytes in vitro resulted in a significant increase in the number of apoptotic cells, specifically in the CD4⁺CD8⁺ subset, implicating apopotosis as one component in the course of thymocyte reduction. In addition to this, in vivo treatment with Linomide resulted in an identical pattern to that seen in vitro in that there was significantly increased apoptosis only in the CD4⁺CD8⁺. These data indicate that Linomide modifies thymocyte development using a glucocorticoid independent pathway and results in the increased apoptosis of the CD4⁺CD8⁺ subset.     

Comparison of lymphokine secretion and mRNA expression in the CD45RA+ and CD45RO+ subsets of human peripheral blood CD4+ and CD8+ lymphocytes

Flow cytometric analysis of human peripheral blood T lymphocytes demonstrated that the majority of the CD4⁺ cells were CD29⁺ or CD45RO⁺ “mature” cells while the CD8⁺ cells were primarily CD45RA⁺ “naive” cells. After an initial separation into CD4⁺ and CD8⁺ cells and a secondary separation into CD45 subsets, lymphokine secretion was assessed after phorbol 12-myristate 13-acetate and ionomycin or fixed anti-CD3 stimulation. Within the respective CD45 subsets, CD4⁺ cells produced more interleukin (IL)-2, IL-4, and IL-6; but the CD8⁺ cells secreted more interferon-γ and granulocyte/macrophage-colony-stimulating factor. Tumor necrosis factor-α secretion was similar in the matched CD45 subsets. Northern analysis revealed a parallel pattern of lymphokine mRNA expression in the four lymphocyte subsets. These results suggest that human CD8⁺ peripheral blood lymphocytes have a significant capacity to secrete lymphokines, and that the low lymphokine production observed in unseparated CD8⁺ cells reflects the higher percentage of less functional CD45RA⁺ cells.     

The effect of disodium cromoglycate (DSCG) on in vitro proliferation of CD4+, CD8+, and CD19+ cell populations derived from allergic and healthy donors

The effect of disodium cromoglycate (DSCG) on in vitro proliferation of CD4⁺ and CD8⁺ T cells and CD19⁺ B cells, positively selected by immunomagnetic separation, was investigated. The cells were obtained from allergic patients with moderate serum IgE levels and mild to moderate atopic dermatitis, and healthy controls. The different cell subfractions were stimulated with mitogens or specific allergens, as well as cell supernatants from the lymphoblastoid B- (RPMI 8866) and T-hybridoma (166 A₂) cell lines. Proliferative responses of T- and B-cell subsets stimulated with mitogens together with recombinant interleukin-2 (rIL-2) or accessory cells (AC) could be inhibited by DSCG. In allergic individuals, significant allergen-specific stimulation could be observed in the CD8-depleted peripheral blood mononuclear cell (PBMC) fractions. Isolated CD4⁺ T cells, without AC or IL-2, could also be stimulated with specific allergen, but the responses were rather low. DSCG inhibited, concentration dependently, all allergen-induced responses. Interestingly, only atopic derived CD4⁺ and CD8⁺ T cells were stimulated by soluble low-affinity IgE receptor (Fc?RII/sCD23) and IgE binding factor (IgEBF), including IgE enhancing factor, present in culture supernatants from RPMI 8866 and 166 A₂, respectively. These responses were also inhibited by DSCG. This was in contrast to the amplifying effect of DSCG on spontaneously proliferating RPMI 8866 and 166 A₂ cells, cultured in fresh cRPMI 1640 medium without sCD23 and IgE enhancing factor. Our results show that DSCG delivers an inhibitory signal or signals to PBMC subpopulations expressing Fc?RII/sCD23, either upregulated by phytohemagglutinin in normal and atopic cells, or by allergens or sCD23 in atopic cells. The findings suggest that sCD23 in supernatants or in serum may reverse the general inhibitory mode of DSCG.     

1142292807Expression of CD4+CD25+FOXP3+ regulatory T cells in women with idiopathic recurrent spontaneous abortion and implantation failure: 3-color flow cytometric analysis

Introduction: A population of CD4⁺CD25⁺ regulatory T (Treg) cells is thought to regulate alloreactive T cells in many autoimmune diseases. Lack of Treg cells resulted in abortions in mice and transfer of them prevented miscarriage. FOXP3 is now considered the most specific marker for Treg cells. In this study, we investigated whether levels of peripheral blood Treg cells in women with recurrent spontaneous abortion (RSA) of unknown etiology or with repeated implantation failures (IF) are different from those of normal fertile women. Materials and Methods: Non‐pregnant women with a history of idiopathic RSA or repeated IF were enrolled in the study group (n = 15) and non‐pregnant fertile females served as controls (n = 7). A flow cytometry assay was used. Peripheral blood mononuclear cells (PBMCs) were isolated and stained with appropriate monoclonal antibodies that identify Treg cells: for surface markers, such as anti‐CD4 and anti‐CD25, and for intracellular marker, anti‐FOXP3. Results: The proportions of CD4⁺CD25⁺FOXP3⁺ Treg cells were significantly different between the patients and controls, 1.5% versus 2.4%, respectively (P < 0.05). In addition, an alteration in the expression of surface CD25 was noted after permeabilization of PBMC which allows antibodies to enter the cells and bind to FOXP3. The mean percentage of CD25+ cells before permeabilization, 9.9 + 4.8%, was decreased to 5.3+3.5% after permeabilization (P < 0.01). The ratios of CD4⁺CD25⁺/CD4⁺ between the surface and the intracellular staining also decreased by 46% (P < 0.001). Conclusion: CD4⁺CD25⁺FOXP3⁺ cells were significantly lower in the patients with idiopathic RSA or multiple IF than in the controls. Permeabilization for intracellular staining induces a decrease in expression of surface markers.     

CD4+ memory T cells: functional differentiation and homeostasis

Summary: CD4⁺ T cells are central regulators of both humoral and cellular immune responses. There are many subsets of CD4⁺ T cells, the most prominent being T‐helper 1 (Th1), Th2, Th‐17, and regulatory T cells, specialized in regulating different aspects of immunity. Without participation by these CD4⁺ T‐cell subsets, B cells cannot undergo isotype switching to generate high‐affinity antibodies, the microbicidal activity of macrophages is reduced, the efficiency of CD8⁺ T‐cell responses and CD8⁺ T‐cell memory are compromised, and downregulation of effector responses is impaired. It therefore stands to reason that memory CD4⁺ T cells are likely to fulfill an important facilitator role in the maintenance and control of protective immune responses. This review discusses some issues of importance for the generation of memory CD4⁺ T cells and focuses in particular on their heterogeneity and plasticity, with respect to both phenotypic characteristics and function. Finally, we discuss a number of factors that affect long‐term maintenance of memory CD4⁺ T cells.     

CD4+CD25+ T cell depletion impairs tolerance induction in a murine model of asthma

Background Regulatory T cells (Tregs) are key players in controlling the development of airway inflammation. However, their role in the mechanisms leading to tolerance in established allergic asthma is unclear. Objective To examine the role of Tregs in tolerance induction in a murine model of asthma. Methods Ovalbumin (OVA) sensitized asthmatic mice were depleted or not of CD25⁺ T cells by anti‐CD25 PC61 monoclonal antibody (mAb) before intranasal treatment (INT) with OVA, then challenged with OVA aerosol. To further evaluate the respective regulatory activity of CD4⁺CD25⁺ and CD4⁺CD25^? T cells, both T cell subsets were transferred from tolerized or non‐tolerized animals to asthmatic recipients. Bronchoalveolar lavage fluid (BALF), T cell proliferation and cytokine secretion were examined. Results Intranasal treatment with OVA led to increased levels of IL‐10, TGF‐β and IL‐17 in lung homogenates, inhibition of eosinophil recruitment into the BALF and antigen specific T cell hyporesponsiveness. CD4⁺CD25⁺Foxp3⁺ T cells were markedly upregulated in lungs and suppressed in vitro and in vivo OVA‐specific T cell responses. Depletion of CD25⁺ cells before OVA INT severely hampered tolerance induction as indicated by a strong recruitment of eosinophils into BALF and a vigorous T cell response to OVA upon challenge. However, the transfer of CD4⁺CD25^? T cells not only suppressed antigen specific T cell responsiveness but also significantly reduced eosinophil recruitment as opposed to CD4⁺CD25⁺ T cells. As compared with control mice, a significantly higher proportion of CD4⁺CD25^? T cells from OVA treated mice expressed mTGF‐β. Conclusion Both CD4⁺CD25⁺ and CD4⁺CD25^? T cells appear to be essential to tolerance induction. The relationship between both subsets and the mechanisms of their regulatory activity will have to be further analyzed.     

Expression of naive/memory (CD45RA/CD45RO) markers by peripheral blood CD4+ and CD8+ T cells in children with asthma

Introduction: The role of CD4⁺ T cells in the immunopathogenesis of asthma is well documented. Little is known about the role of CD8⁺ T cells. The aim of this study was to assess peripheral blood subsets of CD4⁺ and CD8⁺ T cells expressing naive/memory markers (CD45RA⁺/RO⁺) and the activation marker (CD25⁺) in children with allergic asthma. Materials and Methods: Peripheral blood mononuclear cells were isolated from children with allergic asthma and healthy children. T cell subsets were analyzed by flow cytometry for the expressions of CD45RA, CD45RO, and CD25. In this study, some differences in the memory compartment of peripheral blood T cells between asthmatic children and healthy controls were detected. Results: The absolute number of CD8⁺ T cells expressing CD45RO was significantly elevated and the percentages of CD3⁺ T cells expressing activation marker CD25 and of CD4⁺ T cells expressing memory marker CD45RO were significantly lower in children with asthma compared with controls. No correlation was found between severity of asthma and peripheral blood lymphocyte subsets. Conclusions: There were some differences in the memory compartment of peripheral blood T cells between asthmatic children and healthy controls. The increase in the number of CD8⁺ T cells expressing the memory marker (CD45RO) in children with allergic asthma may indicate that CD8⁺ T cells play a role in the pathogenesis of asthma.     

CD8+ T‐cell memory in tumor immunology and immunotherapy

Summary: The cellular and molecular mechanisms underlying the formation of distinct central, effector, and exhausted CD8⁺ T‐cell memory subsets were first described in the setting of acute and chronic viral diseases. The role of these T‐cell memory subsets are now being illuminated as relevant to the tumor‐bearing state. The generation and persistence of productive CD8⁺ T‐cell memory subsets is determined, in part, by antigen clearance, costimulation, responsiveness to homeostatic cytokines, and CD4⁺ T‐helper cells. By contrast, chronic exposure to antigen, negative costimulation, and immunomodulation by CD4⁺ T regulatory cells corrupt productive CD8⁺ T memory formation. It has become clear from human and mouse studies that the mere generation of CD8⁺ T‐cell memory is not a ‘surrogate marker’ for cancer vaccine efficacy. Some current cancer vaccine strategies may fail because they amplify, rather than correct or reset, the corrupted CD8⁺ memory population. Thus, much of the present effort in the development of vaccines for cancer and chronic infectious diseases is aimed at creating effective memory responses. Therapeutic vaccines for cancer and chronic infectious diseases may achieve consistent efficacy by ablation of the dysfunctional immune state and the provision of newly generated, non‐corrupted memory cells by adoptive cell transfer.     

Age-related accumulation of LFA-1high cells in a CD8+CD45RAhigh T cell population

The differential expression of CD45 isoforms has been suggested as a marker for stages of post-thymic T cell development, that is, CD45RA+CD45R0^? T cells and CD45RA^?CD45R0⁺ T cells are supposed to be virgin and memory cells respectively. Recently, several adhesion molecules have been shown to be up-regulated on the cell surface of memory T cells, and have been suggested to serve as a memory marker. In this study, we investigated the levels of LFA-1 expression on T cells in various subpopulations defined by CD45 isoform expression in donors of various ages. In CD4⁺ T cells, the proportion of LFA-1^high cells among CD45RA^highCD45R0-T cells remained low in all age groups and did not show significant accumulation with age. CD4⁺CD45RA^?CD45R0^highTcells expressed LFA-1 at a higher level than CD4⁺CD45RA^highCD45R0^? T cells. Thus, the currently prevailing view that CD45RA and CD45R0 can be markers for virgin and primed cells was consistent with LFA-1 expression in CD4⁺ T cell population. In CD8⁺ T cells, however, CD45RA^highCD45R0^? T cells consisted of two distinct subpopulations, LFA-1^low and LFA-1^high cells, whereas CD45RA^?CD45R0^high T cells were almost exclusively LFA-1^high When CD29 expression was examined in place of LFA-1 expression, similar results were obtained; CD45RA^high CD45R0^? T cells consisted of two distinct subpopulations, CD29-^{to low} and CD^29high cells, while CD45RA-CD45R0^high T cells were mostly CD29^high. The proportion of LFA-1^high cells in the CD8+CD45RA^high T cell subpopulation increased significantly as a function of age (r = 0.9, p < 0.001). It ranged from 8% in a 14-year-old donor to 94% in a 79-year-old donor. Furthermore, the proportion of CD8⁺CD45RA^highLFA-1^high cells in the CD8⁺ T cell population increased significantly as a function of age (r = 0.85, p < 0.001). On the other hand, the proportion of LEA-1^high cells in CD8⁺CD45RA^? T cell subpopulation exceeded 90% in most donors irrespective of age. These results indicate that the CD8⁺CD45RA^highCD45R0^? T cell subpopulation contains a considerable number of LFA-1^high cells and CD29^high cells, phenotypically similar to previously activated cells. Thus, in terms of LFA-1 and CD29 expressions, the simple scheme that CD45RA is a marker of virgin cells is not applicable to the CD8⁺ T cell population.     

CD57+ T lymphocytes are derived from CD57− precursors by differentiation occurring in late immune responses

CD3⁺ T cells expressing the 110-kDa CD57 antigen are found in survivors of renal, cardiac and bone marrow transplants, in patients with acquired immune deficiency syndrome and in patients with rheumatoid arthritis. They are also present in normal individuals and expand upon ageing. They do not grow in culture and their role in the immune response is poorly understood. The expression of the various isoforms of the leukocyte common antigen (CD45) identifies a spectrum of differentiation in CD4⁺ and CD8⁺ T cells ranging from naive (CD45RA⁺CD45RB^brightCD45RO^?) through early primed cells (CD45RA^?RB^brightRO^dull) to highly differentiated memory cells which are CD45RA^?RB^dullRO^bright. CD45 isoforms expressed by CD57⁺ T cells showed distinct differences between CD4⁺ and CD8⁺ populations, but in each case indicated an advanced state of differentiation. The expression of T cell receptor Vβ families was highly variable between individuals, but both CD57⁺ and CD57^? cells show a full range of the specificities tested. Vβ expression was more closely related within either the CD4⁺ or the CD8⁺ subsets, irrespective of CD57 expression, than between these subsets, suggesting a relationship between CD57⁺ and CD57^? cells within the same T cell pool. This possibility was supported by experiments showing that CD3⁺CD57⁺ lymphocytes were similar to CD3⁺CD57^? T cells in terms of the production of basic T cell cytokines [interleukin (IL)-2, IL-4, and interferon-γ]. Furthermore, in vitro stimulation of CD3⁺CD57^? T cells in secondary mixed leukocyte reaction or by co-culture with IL-2 and IL-4 induced the appearance of CD3⁺CD57⁺ cells with phenotypic and functional similarities to in vivo CD3⁺CD57⁺ cells. These data strongly suggest that the expression of CD57 is a differentiation event which occurs on CD57^? T cells late in the immune response.     

A subset of CD8+ T cells from allergic patients produce IL-4 and stimulate IgE production in vitro

Background and Objective A subset of IL-4 producing CD8⁺ T cells was recently identified in HIV patients. Based on these findings we examined whether IL-4 producing CD8⁺ T cells would also be present in allergic patients and what would be the functional relevance of this T-cell population. Methods We investigated the role of CD8⁺ T cells in IgE production of allergic diseases by analysing the cytokine profile of individual CD4⁺ and CD8⁺ T cells. Results In allergic patients about twice as many CD4⁺ T cells and six times as many CD8⁺ T cells produced IL-4 as in non-allergic controls. In contrast the frequency of IFNγ⁺ T-cell subsets did not significantly differ between the allergic and non-allergic individuals. The frequency of 1L4⁺CD8⁺ T cells correlated with the level of serum IgE. Coculture experiments with T cells or purified CD8⁺ T cells together with autologous B cells indicated that CD8⁺ T cells enhanced IgE in vitro, but not IgM production, even when they were physically separated from B cells. This effect could be partially blocked by addition of an IL-4 binding protein, a soluble IL-4 receptor indicating that lL-4 is involved in CD8⁺ T-cell mediated IgE production. Conclusions These data indicate a positive role of IL-4 secreting CD8⁺ T cells in IgE regulation in allergic patients.     

Programmed death (PD)‐1 molecule and its ligand PD‐L1 distribution among memory CD4 and CD8 T cell subsets in human immunodeficiency virus‐1‐infected individuals

Human immunodeficiency virus (HIV)‐1 causes T cell anergy and affects T cell maturation. Various mechanisms are responsible for impaired anti‐HIV‐1‐specific responses: programmed death (PD)‐1 molecule and its ligand PD‐L1 are negative regulators of T cell activity and their expression is increased during HIV‐1 infection. This study examines correlations between T cell maturation, expression of PD‐1 and PD‐L1, and the effects of their blockade. Peripheral blood mononuclear cells (PBMC) from 24 HIV‐1⁺ and 17 uninfected individuals were phenotyped for PD‐1 and PD‐L1 expression on CD4⁺ and CD8⁺ T cell subsets. The effect of PD‐1 and PD‐L1 blockade on proliferation and interferon (IFN)‐γ production was tested on eight HIV‐1⁺ patients. Naive (CCR7⁺CD45RA⁺) CD8⁺ T cells were reduced in HIV‐1 aviraemic (P = 0·0065) and viraemic patients (P = 0·0130); CD8 T effector memory subsets [CCR7^‐CD45RA^–(T_EM)] were increased in HIV‐1⁺ aviraemic (P = 0·0122) and viraemic (P = 0·0023) individuals versus controls. PD‐1 expression was increased in CD4 naive (P = 0·0496), central memory [CCR7⁺CD45RA^– (T_CM); P = 0·0116], T_EM (P = 0·0037) and CD8 naive T cells (P = 0·0133) of aviraemic HIV‐1⁺versus controls. PD‐L1 was increased in CD4 T_EMRA (CCR7^‐CD45RA⁺, P = 0·0119), CD8 T_EM (P = 0·0494) and CD8 T_EMRA (P = 0·0282) of aviraemic HIV‐1⁺versus controls. PD‐1 blockade increased HIV‐1‐specific proliferative responses in one of eight patients, whereas PD‐L1 blockade restored responses in four of eight patients, but did not increase IFN‐γ‐production. Alteration of T cell subsets, accompanied by increased PD‐1 and PD‐L1 expression in HIV‐1 infection contributes to anergy and impaired anti‐HIV‐1‐specific responses which are not rescued when PD‐1 is blocked, in contrast to when PD‐L1 is blocked, due possibly to an ability to bind to receptors other than PD‐1.     

CD3 antigen-mediated calcium signals and protein kinase C activation are higher in CD45R0+ than in CD45RA+ human T lymphocyte subsets

T lymphocytes may be separated into subsets according to their expression of CD45 isoforms. The CD45R0⁺ T cell subset has been reported to proliferate in response to recall antigen and to mitogenic mAb to a much greater extent than the CD45RA⁺ subset. This difference could be due to more efficient coupling of the T cell antigen receptor complex to mitogenic signaling pathways. To investigate this possibility, CD3 antigen-induced calcium signals, diacylglycerol (DAG) production and protein kinase C (PKC) activation levels were compared in CD45RA⁺ and CD45R0⁺ human T lymphocyte subsets derived from peripheral blood. The mean CD3-induced rise in intracellular calcium was 80% greater in CD45R0⁺ than in CD45RA⁺ cells. Basal DAG levels in CD45R0⁺ cells were found to be, on average, 60% higher than in CD45RA⁺ cells (p = 0.002), but the CD3-induced production of DAG over background was not different in the two subsets (p = 0.4). Basal PKC activity, and CD3-induced PKC activation levels over background, were found to be 50% and 140% higher, respectively, in CD45R0⁺ cells than in CD45RA⁺ cells (p = 0.015 and 0.023). The CD45R0⁺ subset contained a higher proportion of cells expressing activation markers, such as CD25, CD71 and major histocompatibility complex class II, when compared to the CD45RA⁺ subset. Our results suggest that the elevated basal DAG levels observed in the CD45R0⁺ subset may reflect the recent activation of these cells. Both the higher basal DAG and CD3-induced elevation in intracellular calcium observed in the CD45R0⁺ cells may contribute to the greater PKC activation signals triggered by CD3 mAb in this subset. These findings elucidate the greater response of CD45R0⁺ T cells to mitogenic stimuli compared to CD45RA⁺ cells.     

Role of TCR specificity in CD4+CD25+ regulatory T-cell selection

Summary: CD4⁺CD25⁺ regulatory T cells play a crucial role in preventing autoimmune disease and can also modulate immune responses in settings such as transplantation and infection. We have developed a transgenic mouse system in which the role that T‐cell receptor (TCR) specificity for self‐peptides plays in the formation of CD4⁺CD25⁺ regulatory T cells can be examined. We have shown that interactions with a single self‐peptide can induce thymocytes bearing an autoreactive TCR to undergo selection to become CD4⁺CD25⁺ regulatory T cells and that thymocytes bearing TCRs with low affinity for the selecting peptide do not appear to undergo selection into this pathway. In addition, thymocytes with identical specificity for the selecting self‐peptide can undergo overt deletion versus abundant selection to become CD4⁺CD25⁺ regulatory T cells in response to variations in expression of the selecting peptide in different lineages of transgenic mice. Finally, we have shown that CD4⁺CD25⁺ T cells proliferate in response to their selecting self‐peptide in the periphery, but these cells do not proliferate in response to lymphopenia in the absence of the selecting self‐peptide. These studies are determining how the specificity of the TCR for self‐peptides directs the thymic selection and peripheral expansion of CD4⁺CD25⁺ regulatory T cells.