Defective interleukin 2 receptor expression is associated with the T cell disfunction subsequent to bone marrow transplantation

In the present work we have used monoclonal antibodies (mAb) as probes to attempt a dissection of the mechanisms underlying the immunodeficiency subsequent to bone marrow transplantation (BMT). To this end we have studied 19 allogeneic BMT recipients, analyzing the proliferative response of peripheral blood mononuclear cells (PBMC) after activation with either phytohemagglutinin (PHA), anti-CD3 or anti-CD2 mAb. All patients presented normal proportions of CD2+ and CD3+ lymphocytes, as assessed by flow cytometry. Our results indicated that in most cases both CD2 and CD3-mediated activation pathways were inefficient to trigger normal T cell proliferation. The addition of exogenous interleukin 2 (IL2) did not restore in most cases the proliferative response, pointing out that additional defects contribute to the hyporesponsiveness. This was more evident in the group of patients studied during the first 6 months. To further dissect the T cell defect we analyzed the effect of a phorbol ester (phorbol myristate acetate, PMA), which activates protein kinase C, on the anti-CD3-induced response. Our data showed that PMA synergized with anti-CD3 similarly to exogenous IL2, and restored the proliferative response only in certain cases. The expression of IL2 receptors (CD25) as assessed by cytofluorimetry, after either PHA or anti-CD3 and PMA stimulation, was shown to be depressed, and the addition of IL2 did not restore it. Finally, we observed that the early increase of intracytoplasmic Ca2+ after anti-CD3 stimulation was comparable to that detected in normal PBMC. Altogether these results indicate that a diminished CD25 expression is associated with the T cell defect, and cannot apparently be attributed to an inability of the CD3 molecule to transduce early activation signals thus suggesting that either protein kinase C itself or an as yet undefined metabolic step preceding IL2 receptor expression is abnormal in variable proportions of T cells after BMT, and constitutes another manifestation of this complex immunodeficiency.     

CD8hi+CD57+ T lymphocytes are enriched in antigen-specific T cells capable of down-modulating cytotoxic activity

Major expansions of CD8hi+CD57+ T lymphocytes frequently occur during human immunodeficiency virus (HIV) infection and after transplantation. To investigate mechanisms of such cell expansion, we compared the activation and functional status of CD8hi+CD57+ and CD57-peripheral blood lymphocytes (PBL) from normal, bone marrow transplantation (BMT) and HIV+ donors. The CD8hi+CD57+ PBL from BMT and HIV+ donors preferentially displayed CD38 and HLA-DR activation markers without correlation between CD8hi+CD57+ percentages and HIV load, the CD45RA+ isoform in all ex vivo conditions but acquired CD45RO after in vitro expansion, CD11b and CD11c in BMT and HIV+ donors but decreased expression of CD62-L, VLA-2 and VLA-6. The CD8hi+CD57+ cells were positive for perforin and granzyme B and spontaneously mediated cytolytic activity in a CD3-redirected assay. In contrast the inhibitor of cytolytic functions (ICF) produced by CD8hi+CD57+ cells down- modulated the CD3-redirected cytolytic activity but only at low levels of CD3 cross-linking. While CD3-triggering induced a low, if any, short- term proliferation of CD8+CD57+ cells, this subset could be amplified after long-term stimulation either with mitogens or with HIV antigens, thereby enriched in HIV-specific T cells producing tumor necrosis factor-alpha. Altogether these data suggest that CD8hi+CD57+ cells represent a terminal differentiation state of activated effector cytotoxic T lymphocytes which are enriched in antigen-specific T cells and down-modulate their own cytolytic potential, thus participating in a negative control of effector cell functions during persistent viral infections or transplantations.      

CD8 lymphocytosis in primary cytomegalovirus (CMV) infection of allograft recipients: expansion of an uncommon CD8+ CD57- subset and its progressive replacement by CD8+ CD57+ T cells.

Allograft recipients undergoing cytomegalovirus infection present increased proportions of circulating CD8+ lymphocytes. A longitudinal study of 11 kidney and five liver allograft recipients with primary CMV infection but no other etiological factor of graft dysfunction revealed selective imbalances of peripheral blood CD8+ T cell subsets. Initially, CMV viraemia is associated with elevated CD8+bright T cell numbers and T cell activation. Activation markers fall to normal when viral cultures become negative (before the end of the first month). During the second to sixth month, most (12/16) patients keep up high CD8+ T cell counts (1050-2900 CD8+ cells/mm3), comprising an uncommon CD8+ T cell subset, as 45-73% of CD8+bright lymphocytes were CD3+ and TCR alpha beta+, but were not stained by anti-CD28, CD11b, CD16, CD56, and CD57 antibody. Unexpectedly, CD8+CD57+ T cells, a hallmark of CMV infection, do not appear until the second to sixth month of primary CMV infection, and their numbers increase progressively thereafter. They become the predominant CD8+ T cell subset after 6 months of infection and their persistence for several (up to 4) years is strongly correlated (r = 0.87) with expansion of CD8+ cells. By analysis with MoAbs, there was no bias towards the use of particular TCR-V beta gene families at any time of primary CMV infection. Persistence of CD8 lymphocytosis is thus directly related to the rate of expansion of an uncommon CD8+CD57- subset and its progressive replacement by CD8+CD57+ T cells that are chronically elicited by CMV.     

Induction of auto-reactive regulatory T cells by stimulation with immature autologous dendritic cells

We have shown in ex vivo studies in donor bone marrow-infused kidney transplant recipients, that chimeric cells of either donor or recipient origin taken from the recipient's bone marrow down-regulated the recipient's cellular immune responses. In the present study, we have now induced regulatory T cells from peripheral blood mononuclear cells (PBMC) of renal transplant recipients or laboratory volunteers by multi-stimulation with autologous immature dendritic cell (iDC) enriched populations derived from either bone marrow cells (BMC) of the (immunosuppressed) kidney transplant recipients or PBMC of the laboratory volunteers (i.e., ibDC and ipDC, respectively). These regulatory T cells, induced by ibDC and ipDC, were autoreactive and designated as TAb and TAp with similar phenotypes and functional profiles. They were largely CD4 + CD25high, CD45RA low and CD45RO high, and uniformly expressed intracellular CTLA-4, and message of IL-4, IL-10, Foxp3, and differentially expressed TGFbeta. Their proliferative responses to autologous mature dendritic stimulating cells (mDC) were approximately two-fold stronger than to allogeneic mDC, and to allogeneic mDC were significantly lower than those of (control) autologous TPBL, suggesting an anergic state. TAb and TAp were not cytotoxic to autologous cells expressing Epstein-Barr virus (EBV) antigens, but were able to inhibit (regulate) the effector phase of this TPBL response to both autologous and allogeneic EBV lymphoblasts. This regulation appeared to require cell-to-cell contact.     

Cytokine-induced expansion of human CD34+ stem/progenitor and CD34+CD41+ early megakaryocytic marrow cells cultured on normal osteoblasts

Thrombocytopenia remains a significant cause of morbidity in cancer patients undergoing allogeneic bone marrow transplantation (BMT), which consumes millions each year for frequent platelet transfusions. Using a novel culture system containing appropriate cytokine(s) on a layer of normal human osteoblasts, we investigated the expansion of early megakaryocytic progenitor cells while maintaining the number of CD34+ stem/progenitor marrow cells in an attempt to provide an effective solution for the problem of post-transplant thrombocytopenia. After seven days of culture, normal human osteoblasts alone without cytokines significantly increased the number of CD34+ and CD34+CD41+ marrow cells. Among the various cytokine combinations tested, both stem cell factor (SCF), interleukin 3 (IL-3)+IL-11 and SCF+IL-3+IL-11+thrombopoietin (TPO) emerged as the most effective in expanding early CD34+CD41+ megakaryocytic cells. Early CD34+CD41+ megakaryocytic cells have increased by 3.1- and 4.7-fold compared with day 7 control cultures, and by 62- and 94-fold, respectively, compared with day 0 input, respectively. Also, late CD41+ megakaryocytic cells have increased by 15.4- and 27.5-fold compared with day 7 control cultures in the presence of the same two combinations. In addition, the same cytokine combinations achieved 17.6- and 13.3-fold increases in the number of CD34+ marrow cells after the same seven days of culture on a layer of human osteoblasts. The combination (SCF+IL-3+IL-11+TPO) achieved the highest expansion of CD34+CD41+ early megakaryocytic cells from human marrow CD34+ cells reported so far in the literature. Recently, transplantation of SCF+IL-1+IL-3+TPO ex vivo expanded megakaryocytic progenitor cells as a supplement has been shown to accelerate platelet recovery by three to five days in mice. Therefore, the clinical use of the combination (SCF+IL-3+IL-11+TPO) for ex vivo expansion of CD34+ and megakaryocytic progenitor cells from a portion of the donor's marrow harvest is warranted in allogeneic BMT. Such a protocol would accelerate platelet recovery and shorten the period of hospitalization after allogeneic BMT. The present study has confirmed the role of human osteoblasts in supporting the proliferation and maintenance of human CD34+ stem/progenitor marrow cells. Given the facilitating role of osteoblasts shown previously in several allogeneic BMT studies in mice, it is possible to envisage a future role for donor osteoblasts in clinical BMT. Transplantation of the cultured donor osteoblasts together with the ex vivo expanded CD34+ marrow cells as a supplement might not only accelerate platelet recovery but also prevent acute graft-versus-host disease in allogeneic BMT. The present novel culture system should have useful clinical application in allogeneic BMT.     

IL-21R expression on CD8+ T cells promotes CD8+ T cell activation in coxsackievirus B3 induced myocarditis

IL-21 is a multi-functional cytokine which can promote survival, proliferation and activation of T and B lymphocytes including CD8 T cells. Previous studies have shown that autoimmune CD8+ T cells are the primary pathogenic effector cell in coxsackievirus B3 (CVB3) induced myocarditis in C57Bl/6 mice. To evaluate the role of IL-21 in promoting CD8+ T cell mediated cardiac injury in myocarditis, C57Bl/6 and IL-21RKO mice were infected with CVB3. IL-21RKO mice developed significantly less myocarditis than C57Bl/6 animals although cardiac virus titers were equivalent between the mouse strains. Numbers of CD8+IFNγ+ cells were decreased in IL-21RKO mice but numbers of either CD4+IFNγ+ or CD4+IL-4+ cells were not significantly different from C57Bl/6 animals indicating a selective effect of IL-21 signaling on the CD8+ T cell response. To confirm that IL-21 signaling exclusively functions at the level of the CD8+ T cell in CVB3 induced myocarditis, purified CD8+ cells were isolated from either C57Bl/6 or IL-21RKO donors and adoptively transferred into CD8KO recipients prior to CVB3 infection. CD8KO recipients given either C57Bl/6 or IL-21RKO CD8+ cells showed equivalent reconstitution of the CD8+ cells in the spleen but the recipients given C57Bl/6 CD8+ cells showed significantly greater myocarditis than recipients of IL-21RKO CD8+ cells. These data demonstrate that IL-21 signaling directly in the CD8+ cell population is required for CVB3-induced myocarditis.     

CD8high(CD57+) T cells in normal,healthy individuals specifically suppress the generation of cytotoxic T lymphocytes to Epstein-Barr virus-transformed B cell lines

We have previously identified two subsets of CD8⁺, CD57⁺ lymphocytes in normal peripheral blood: i) T cells expressing high levels [CD8^high(CD57⁺)] and ii) natural killer cells expressing low levels of surface CD8 [CD8^low(CD57⁺)]. We investigated the cytotoxic and suppressive function of CD8^high(CD57⁺) T lymphocytes from normal, healthy individuals using standard chromium-release assays and limiting dilution analysis. In normal, healthy subjects, this cell subset suppressed the generation of cytotoxic T lymphocytes (CTL) to autologous, Epstein-Barr virus (EBV)-transformed B cell lines (BCL). Depletion of CD8^high(CD57⁺) T lymphocytes from peripheral blood mononuclear cells (PBMC) resulted in a three- to sevenfold rise in CTL precursor frequency to autologous EBV-transformed BCL, but not allogeneic PBMC or BCL by LDA. Replacement of CD8^high(CD57⁺) T lymphocytes in limiting dilution cultures led to the dose-dependent suppression of EBV-specific, but not allogeneic, CTL generation. Supernatant from CD8^high(CD57⁺) T lymphocytes cultured with autologous BCL did not exhibit suppression, suggesting that soluble factors were not responsible. As CD8^high(CD57⁺) T lymphocytes did not, themselves, exhibit cytotoxicity against autologous BCL, removal of BCL stimulator cells in co-culture was not the mechanism of suppression. Furthermore, while the CD8^high(CD57⁺) T lymphocytes from healthy subjects suppressed the generation of CTL to autologous BCL, they did not suppress the cytotoxic activity of established mixed lymphocyte reactions or peptide-specific CTL clones, as has been reported in bone marrow transplant recipients and human immunodeficiency virus patients. This suggests that CD8^high(CD57⁺) T lymphocytes from healthy subjects suppress the generation of, rather than killing by, CTL in a contact-dependent manner. To our knowledge, this is the first identification of a phenotypically distinct subset of human CD8⁺ T cells that can suppress generation of antigen-specific major histocompatibility complex class I-restricted CTL.     

Impaired expression of CD28 on T cells in hairy cell leukemia

T cells from patients with active hairy cell leukemia (HCL), a chronic B cell malignancy, show poor proliferation in response to allogeneic peripheral blood mononuclear cells (PBMC). In order to study the T cell dysfunction, the expression of several adhesion and costimulatory molecules was analyzed by flow cytometry. Circulating T cells from HCL patients showed increased percentages of CD28(-) in all T cell subsets. In some patients the percentage of CD28(-) T cells within the CD4(+) subset was increased up to 80%. These CD4(+)CD28(-) T cells did not proliferate in a mixed lymphocyte culture (MLC) against allogeneic PBMC. After enrichment for CD4(+)CD28(+) T cells, the proliferative response in the MLC was recovered, but this response was still lower than the proliferative response from control T cells. In conclusion, lack of CD28 on T cells and a restricted T cell repertoire may contribute to immune deficiency in patients with HCL.     

Expression of p75 chain of IL-2 receptor in the early immunological reconstitution after allogeneic bone marrow transplantation.

Expression of p55 and p75 chains of IL-2 receptor (IL-2R) on the surface of both T and natural killer (NK) circulating lymphocytes was investigated in 14 paediatric patients given allogeneic bone marrow transplantation (BMT) from HLA-identical sibling or partially matched family donors. IL-2-induced proliferative and cytotoxic responses were also studied and all analysis was performed within 45 days from transplant. We found that, early after transplant, the percentage of p55+ and of p75+ peripheral blood lymphocytes (PBL) was not significantly different in patients who had received HLA-identical BMT (p55+ 8.04 +/- 4.87%; p75+ 28.27 +/- 18.85%) compared with healthy controls (p55+ 7.26 +/- 6.17%; p75+ 19.42 +/- 10.49%), while recipients of T cell-depleted marrow included a remarkably high percentage (76-90%) of p75+ PBL, which were mostly CD3- and co-expressed CD56 molecule. Comparable values of p55+ lymphocytes were observed in all patients and controls. However, in contrast to the other two groups, in recipients of T cell-depleted BMT the majority of these cells co-expressed p75 chain and CD56 antigen. IL-2-induced proliferation and lymphokine-activated killer (LAK) activity were detectable in all patients, and their values did not correlate with expression of p55 or p75 chains. Our data suggest that expansion of NK subsets expressing IL-2R chains after T cell-depleted BMT may be related to early reconstitution of natural immunity in the presence of allogeneic stimuli.     

Induction of Auto-reactive Regulatory T Cells by Stimulation with Immature Autologous Dendritic Cells

We have shown in ex vivo studies in donor bone marrow-infused kidney transplant recipients, that chimeric cells of either donor or recipient origin taken from the recipient's bone marrow down-regulated the recipient's cellular immune responses. In the present study, we have now induced regulatory T cells from peripheral blood mononuclear cells (PBMC) of renal transplant recipients or laboratory volunteers by multi-stimulation with autologous immature dendritic cell (iDC) enriched populations derived from either bone marrow cells (BMC) of the (immunosuppressed) kidney transplant recipients or PBMC of the laboratory volunteers (i.e., ibDC and ipDC, respectively). These regulatory T cells, induced by ibDC and ipDC, were autoreactive and designated as T_Ab and T_Ap with similar phenotypes and functional profiles. They were largely CD4 + CD25^high, CD45RA low and CD45RO high, and uniformly expressed intracellular CTLA-4, and message of IL-4, IL-10, Foxp3, and differentially expressed TGFβ. Their proliferative responses to autologous mature dendritic stimulating cells (mDC) were ～two-fold stronger than to allogeneic mDC, and to allogeneic mDC were significantly lower than those of (control) autologous T_PBL, suggesting an anergic state. T_Ab and T_Ap were not cytotoxic to autologous cells expressing Epstein-Barr virus (EBV) antigens, but were able to inhibit (regulate) the effector phase of this T_PBL response to both autologous and allogeneic EBV lymphoblasts. This regulation appeared to require cell-to-cell contact.     

Proliferation of human CD4+45R+ and CD4+45R- T helper cells is promoted by both IL-2 and IL-4 while interferon-gamma production is restricted to IL-2 activated CD4+45R- T cells

Recombinant IL-2 (rIL-2) and IL-4 (rIL-4) promote proliferation of human CD4+ T cells activated in the presence of PHA, TPA or OKT-3 monoclonal antibody (MAb), whereas the production of interferon-gamma (IFN) can be induced only by rIL-2. rIL-4 induced strong proliferative responses both in accessory cell independent assays and in the presence of autologous monocytes, but has failed to induce IFN production in any of these systems. The ability of rIL-2 to induce IFN production was strongly enhanced by the addition of monocytes, although a similar proliferative response was recorded in the absence or presence of monocytes. The MAb anti-Tac inhibited the proliferative response and the production of IFN by CD4+ T cells activated in the presence of rIL-2, whereas the proliferative response to rIL-4 was unaffected. CD4+45R+ and CD4+45R- T helper cell subsets proliferated in response to both IL-2 and IL-4. A kinetic analysis demonstrated that the production of IFN throughout a five day activation period was restricted to stimulation of CD4+45R- T cells with rIL-2. This report clearly demonstrates a dissociation of IFN production and T cell proliferation in man. While proliferation can be induced by both IL-2 and IL-4 in both the helper T cell subsets studied, IFN production was induced only in the CD4+45R- subsets and only in response to IL-2.     

Recovery of CD3+ and CD5- lymphocyte subpopulation after autologous bone marrow transplantation and chemotherapy.

Although most circulating T cells in normal subjects express both CD3 and CD5 antigens on the cell surface, a small number lack the CD5 antigen. Recipients of allogeneic bone marrow transplants develop increased numbers of CD3+ CD5- cells, particularly those who develop graft versus host disease (GVHD). This CD3+ CD5- population may rise transiently in patients who have received an autologous bone marrow transplant (BMT) and in patients following completion of intensive chemotherapy for acute myeloid leukaemia (AML). These findings suggest that these CD3+ CD5- cells are a normal component of the regenerating lymphoid system after BMT or chemotherapy.     

Analysis of the age-related decline in alloreactivity of CD4+ and CD8+ T cells in CBA/RIJ mice

CD4+ T cells from healthy old CBA/Rij mice were studied for their ability to respond to alloantigens by IL-2 production and proliferation. IL-2 production by these purified cells in response to BALB/c spleen cells was about 4 times lower than the IL-2 production (50 U/ml) by CD4+ T cells from young mice. After stimulation with concanavalin A only a two-fold difference in IL-2 production was found. The extent of proliferation by CD4+ T cells from old mice in response to allogeneic cells was at least 4 times lower than that by the cells from young mice. This difference was not influenced by the addition of IL-2 containing conditioned medium (CM). Proliferative responses by CD8+ T cells were only found after the addition of CM and then the response by cells from old mice was 2-10 times lower than the response by cells from young mice. Limiting dilution analysis of the separate T cell subpopulations showed that these low responses to alloantigens by cells from old mice were only in part due to a decline in the frequency of antigen-specific CD4+ or CD8+ T cells. As far as old CD8+ T cells were concerned, an additional explanation for the low responsiveness was found in a diminished expression of the IL-2 receptor (IL-2R) alpha-chain. However, CD4+ T cells from old mice expressed normal levels of IL-2R alpha-chain. The observation that proliferative responses by CD4+ T cells may be low, despite a normal frequency of antigen-specific cells, an apparently normal IL-2R expression and despite the presence of exogenous IL-2, indicates that CD4+ T cells from old mice are not only impaired in their ability to produce IL-2, but are also impaired in their ability to handle the IL-2 signal.     

CD25 expression distinguishes functionally distinct alloreactive CD4 CD134 (OX40) T-cell subsets in acute graft-versus-host disease.

CD134 (OX40) is expressed on activated CD4(+) donor T cells in allogeneic stem cell transplant recipients with acute graft-versus-host disease. The data presented here reveal that differential expression of CD25 by CD4(+) CD134(+) T cells allows separation of these activated cells into 2 phenotypically and functionally distinct alloreactive T-cell subsets. These subsets exhibit distinct tissue associations, with CD4(+) CD134(+) CD25(-) T cells preferentially found in lymphoid tissues and CD4(+) CD134(+) CD25(+) T cells located in lymphoid tissues and inflamed extralymphoid tissues. The CD25(-) T-cell subset exhibited potent proliferative responses to both concanavalin A and allogeneic host leukocytes. By contrast, the CD25(+) T-cell subset proliferated minimally in response to either treatment and inhibited alloantigen-induced proliferation of the CD25(-) subset. Proliferative unresponsiveness associated with the CD25(+) T-cell subset did not extend to cytokine secretion. When stimulated with alloantigen, both CD4(+) CD134(+) T-cell subsets responded by secreting interferon-gamma and interleukin (IL)-10, and neither T-cell subset produced detectable levels of IL-2 or IL-4. Three-day treatment of the CD25(+) T-cell subset with IL-2 restored the proliferative responsiveness of these cells to host alloantigens, suggesting that the proliferative unresponsiveness associated with this T-cell subset reflected a requirement for IL-2. The preferential tissue associations and distinct functional properties associated with these separable alloreactive CD4(+) CD134(+) T-cell subsets suggest that they participate differentially in clinical graft-versus-host disease.     

Peripheral Blood Mononuclear Cell Sonicates as an Alternative to Irradiated Allogeneic Cells to Stimulate a Mixed Lymphocyte Reaction and to Enumerate CD69 Alloreactive T Cells

ABSTRACT: An alloreactive reaction similar to that occurring during GvHD can be generated in a mixed lymphocyte culture. The presence of both stimulator and responder cells in these cultures makes the identification and enumeration of alloreactive cells difficult and unreliable. We describe the use of PBMC sonicates as an alternative to the standard MLC method to stimulate an allogeneic reaction. Using combinations of autologous or allogeneic PBMC sonicates, we showed that the lymphocyte proliferative response to cell sonicates was comparable to the response using irradiated cells. The proliferative response was concentration dependent and reached maximum levels at day 6. Both irradiated cells and PBMC sonicates induced significantly lower responses when the stimulating cells were partially HLA-DR matched rather than completely mismatched. Alloreactive T cells stimulated with sonicates were enumerated by the flow cytometric detection of CD69 or CD25. In HLA-mismatched cultures, approximately 7% of CD3+ T cells were CD69+ or CD25+, suggesting alloreactivity. Although there was a significant correlation between the expression of these activation markers and lymphocyte proliferative responses, significant individual variations in the results of these two assays were observed. The results in this study demonstrate the potential of using PBMC sonicates instead of irradiated lymphocytes for the study and identification of alloreactive cells at the cellular and molecular level.     

Systematic characterization of human CD8+ T cells with natural killer cell markers in comparison with natural killer cells and normal CD8+ T cells

We investigated the function of CD56+ CD8+ T cells (CD56+ T cells) and CD56- CD57+ CD8+ T cells (CD57+ T cells; natural killer (NK)-type T cells) and compared them with those of normal CD56- CD57- CD8+ T cells (CD8+ T cells) and CD56+ NK cells from healthy volunteers. After the stimulation with immobilized anti-CD3 antibodies, both NK-type T cells produced much larger amounts of interferon-gamma (IFN-gamma) than CD8+ T cells. Both NK-type T cells also acquired a more potent cytotoxicity against NK-sensitive K562 cells than CD8+ T cells while only CD56+ T cells showed a potent cytotoxicity against NK-resistant Raji cells. After the stimulation with a combination of interleukin (IL)-2, IL-12 and IL-15, the IFN-gamma amounts produced were NK cells > or = CD56+ T cells > or = CD57+ T cells > CD8+ T cells. The cytotoxicities against K562 cells were NK cells > CD56+ T cells > or = CD57+ T cells > CD8+ T cells while cytotoxicities against Raji cells were CD56+ T cells > CD57+ T cells > or = CD8+ T cells > or = NK cells. However, the CD3-stimulated proliferation of both NK-type T cells was smaller than that of CD8+ T cells partly because NK-type T cells were susceptible to apoptosis. In addition to NK cells, NK-type T cells but not CD8+ T cells stimulated with cytokines, expressed cytoplasmic perforin and granzyme B. Furthermore, CD3-stimulated IFN-gamma production from peripheral blood mononuclear cells (PBMC) correlated with the proportions of CD57+ T cells in PBMC from donors. Our findings suggest that NK-type T cells play an important role in the T helper 1 responses and the immunological changes associated with ageing.     

CD4+ CD30+ T cells perpetuate IL-5 production in Dermatophagoides pteronyssinus allergic patients

BACKGROUND: Airway allergic diseases are regulated by interleukin (IL)-5, which causes infiltration of eosinophils into the bronchial epithelium, and by IL-4 which increases serum immunoglobulin E (IgE) production and promotes CD30 expression on Th cells. CD30 generates a costimulatory signal involved in apoptosis or cell proliferation, depending on the microenvironment. Our aims were: (i) to analyze if CD4+ CD30+ T cells from allergic patients proliferate in response to Dermatophagoides pteronyssinus, and (ii) if upon stimulation this cell population produces IL-4 and IL-5. METHODS: Peripheral blood mononuclear cell (PBMC) from 17 allergic rhinitis and mild allergic asthma patients and 12 healthy nonallergic individuals were stimulated with allergen in the presence or absence of anti-IL-4, anti-IL-5 or anti-IL-4Ralpha monoclonal antibodies (mAbs). TdT-mediated dUTP nick end-labeling (TUNEL) assay, 7-aminoactinomycin-D (7-AAD) intercalation, and flow cytometry were used to determine the CD4+ CD30+ blasts percentage, cell proliferation, apoptosis, and intracellular cytokines after 7 culture days. RESULTS: Cell proliferation induced with allergen showed that 90% of the allergen-stimulated blasts were CD4+, 50% of which were CD30+. Allergen-stimulated PBMC showed a progressive increase (mean: from 7% to 23%) of CD4+ CD30+IFN-gamma+ and CD4+ CD30+IL-4+ blasts which diminished (mean: 6%) after 5 culture days. In contrast, CD4+ CD30+IL-5+ blasts showed a continuous progression (from 12% to 24%) that maintained after 7 culture days. The vast majority of CD4+ CD30+ blasts were negative to 7-AAD or TUNEL. Additionally, a significant decrease (34%) was observed in the number of CD4+ CD30+ blasts when IL-4 was neutralized. CONCLUSIONS: These data suggest that specific allergen stimulation of PBMC isolated from allergic patients generates a nonapoptotic CD4+ CD30+ blast subset that produces IL-5.     

Impairment of CD3-dependent and CD3-independent activation pathways in CD4+ and in CD8+ T cells from old CBA/RIJ mice

Stimulation of T cells from old mice with anti-CD3 antibodies resulted in a high variability of proliferative responses, which were 2- to 8-fold lower than the responses by T cells from young mice, even in the presence of exogenous rIL-2. Moreover, the CD4+ T cells from these old mice displayed a diminished capacity to produce IL-2 in response to anti-CD3. A partial explanation was found in the observation that T cells from the majority of old mice displayed a diminished expression of CD3 of variable intensity. However, after stimulation of the T cells with the combination of phorbol-12-myristate-13-acetate (PMA) and ionomycin to bypass CD3, 3 out of 6 old mice still exhibited 2-fold lower proliferative responses than T cells from young mice; IL-2 production by the CD4+ T cells was lower in all old mice tested. Comparison of CD4+ T cells and CD8+ T cells from old mice revealed a defective PMA/ionomycin response in both subsets, although this defect seemed more pronounced in CD4+ T cells when compared with the young counterparts. The diminished response of CD8+ T cells was accompanied by a diminished expression of the IL-2R alpha-chain. In contrast, old CD4+ T cells expressed rather higher levels of IL-2R alpha-chain than young CD4+ T cells. Altogether, multiple defects which are not necessarily the same in CD4+ and CD8+ T cells are responsible for defective T cell responses in old mice.     

Differential effects of CD4 and CD8 engagement on the development of cytokine profiles of murine CD4+ and CD8+ T lymphocytes

A simple culture system devoid of antigen-presenting cells was used to examine the ability of immobilized antibodies to lymphocyte function-associated antigen-1 (LFA-1) (CD11a), CD28 and CD4 or CD8 to modulate the responses of normal murine CD4+ and CD8+ lymph node T cells to immobilized anti-CD3 antibody and interleukin-2 (IL-2). All the antibodies enhanced proliferative responses to limiting anti-CD3 antibody. Both CD4+ and CD8+ cells produced substantial titres of IL-3 and interferon-gamma (IFN-gamma) in primary and secondary cultures regardless of the coactivating antibodies used for priming. By contrast, the combination of anti-CD4 with anti-CD3 antibody stimulated significantly higher titres of IL-4 than any other antibody combination in cultures of CD4+ cells. This CD4-dependent IL-4 response was induced in CD4+ T cells of naive (CD44low) phenotype and was similar in magnitude to the response induced by exogenous IL-4 but, unlike the latter, was not associated with elevated IL-3 synthesis. A comparable effect of anti-CD8 antibodies on CD8+ cells was not observed: although IL-4 production by CD8+ cells was induced by exogenous IL-4, it was not detected following coactivation with anti-CD8 or any other antibodies. We conclude that anti-CD4 antibody is a potent inducer of IL-4-secreting CD4+ T cells whose effects can be distinguished from those of anti-CD8 antibody on CD8+ T cells and from those of IL-4 on either subset.     

Characterization of a cytotoxic CD57+ T cell subset from patients with pulmonary tuberculosis

We investigated the proportion, phenotype, and cytotoxicity of CD8+CD57+ and CD57- T cells in peripheral blood from 20 tuberculosis (TB)-patients and 20 healthy tuberculin skin test-positive donors. Our results showed an increase in CD8+CD57+ T cells from TB-patients as compared with those from age-matched healthy donors (p<0.0001). CD8+CD57+ T cells from TB-patients expressed CD69, perforin, granzyme-A, and a CD28-CD62L-CD161- phenotype without recognition for the alpha-galactosylceramide-CD1d complex. This cell subset also expressed TNF-alpha and IFN-gamma, under phorbol-myristate-acetate/ionomycin stimulation. Interestingly, the cytotoxicity against autologous monocytes was higher in CD57- cells from TB-patients and donors than their CD57+ counterparts, in the presence of Mycobacterium tuberculosis H37Rv culture filtrate. However, only CD8+CD57+ T cells from TB-patients exhibited spontaneous cytotoxicity against monocytes in the absence of antigen. Our results suggest that CD8+CD57+ T cells are a subset of effector cells that could be helpful to evaluate the cell-mediated immune response to M. tuberculosis.