CD157 undergoes ligand-independent dimerization and colocalizes with caveolin in CHO and MCA102 fibroblasts

CD157, a glycosylphosphatidylinositol (GPI)-anchored glycoprotein, has recently been shown to induce protein tyrosine phosphorylation in monocytes differentiated from HL-60 cells (mHL-60) in a ligand-dependent manner, but in a ligand-independent manner in stable CD157-transfected CHO (CHO/CD157) and MCA102 (MCA/CD157) fibroblasts [Cell Signal. 11 (1999) 891-897.]. Many GPI-anchored proteins need to be clustered by their ligands or antibodies to induce redistribution to caveolae and a concomitant activation of the associated signal-transducing proteins [Nature 387 (1997) 569-572.]. Here, we demonstrate that CD157, independent of antibody crosslinking, undergoes dimerization with disulfide bond formation and localization in caveolae in CHO/CD157 and MCA/CD157 fibroblasts. However, the native CD157 induced in mHL-60 cells remains a monomer form. The structural integrity of caveolae is required for the association of CD157 with caveolin and CD157-mediated tyrosine kinase signalling in the fibroblasts. We propose that an overexpression of CD157 could lead to its dimerization and relocation to caveolae and to further result in the initiation of signalling processes.     

Signalling of GPI-anchored CD157 via focal adhesion kinase in MCA102 fibroblasts

CD157, a glycosylphosphatidylinositol-anchored protein, has previously been shown to mediate tyrosine phosphorylation of a 130 kDa protein (p130) in several cell lines. In this study, we have identified the p130 protein to be focal adhesion kinase (FAK or pp125(FAK)). FAK undergoes phosphorylation at Tyr-397 and Tyr-861 in intact MCA102 cells stably transfected with CD157 (MCA/CD157). MCA/CD157 cells, which displayed a rounded and compact cell morphology, exhibited a dispersed distribution, in contrast to a more closely associated and elongated spindle cell shape in the vector-transfected cells. MCA/CD157 cells proliferated at a rate 20-25% slower than the control cells. Our results demonstrate, for the first time, that FAK is a downstream signalling molecule of CD157.     

Regulation of neuronal nitric-oxide synthase activity by somatostatin analogs following SST5 somatostatin receptor activation

Somatostatin receptor SST5 is an inhibitory G protein-coupled receptor that exerts a strong cytostatic effect on various cell types. We reported previously that the SST5 anti-proliferative effect results in the inhibition of mitogen-induced increases in intracellular cGMP levels and MAPK activity. This study was conducted to define the early molecular events accountable for the SST5-mediated anti-proliferative effect. Here, we demonstrate that, in Chinese hamster ovary cells expressing SST5 (CHO/SST5 cells), somatostatin inhibited cell proliferation induced by nitric oxide donors and overexpression of the neuronal nitric-oxide synthase (nNOS) protein isoform. Accordingly, nNOS activity and dimerization were strongly inhibited following SST5 activation by the somatostatin analog RC-160. In CHO/SST5 cells, nNOS was dynamically recruited by the SST5 receptor and phosphorylated at tyrosyl residues following RC-160 treatment. RC-160 induced SST5-p60(src) kinase complex formation and subsequent p60(src) kinase activation. Coexpression of an inactive p60(src) kinase mutant with SST5 blocked RC-160-induced nNOS phosphorylation and inactivation and prevented the SST5-mediated anti-proliferative effect. In CHO/SST5 cells, p60(src) kinase associated with nNOS to induce its inactivation by phosphorylation at tyrosyl residues following RC-160 treatment. Using recombinant proteins, we demonstrated that such phosphorylation prevented nNOS homodimerization. Next, surface plasmon resonance and mutation analysis revealed that p60(src) directly associated with nNOS phosphorylated Tyr604. SST5-mediated inhibition of nNOS activity was demonstrated to be essential to the RC-160 anti-proliferative effect on pancreatic endocrine tumor-derived cells. We therefore identified nNOS as a new p60(src) kinase substrate essential for SST5-mediated anti-proliferative action.     

Cytoplasmic relocalization and inhibition of the cyclin-dependent kinase inhibitor p27(Kip1) by PKB/Akt-mediated phosphorylation in breast cancer

The cyclin-dependent kinase inhibitor p27(kip1) is a putative tumor suppressor for human cancer. The mechanism underlying p27(kip1) deregulation in human cancer is, however, poorly understood. We demonstrate that the serine/threonine kinase Akt regulates cell proliferation in breast cancer cells by preventing p27(kip1)-mediated growth arrest. Threonine 157 (T157), which maps within the nuclear localization signal of p27(kip1), is a predicted Akt-phosphorylation site. Akt-induced T157 phosphorylation causes retention of p27(kip1) in the cytoplasm, precluding p27(kip1)-induced G1 arrest. Conversely, the p27(kip1)-T157A mutant accumulates in cell nuclei and Akt does not affect p27(kip1)-T157A-mediated cell cycle arrest. Lastly, T157-phosphorylated p27(kip1) accumulates in the cytoplasm of primary human breast cancer cells coincident with Akt activation. Thus, cytoplasmic relocalization of p27(kip1), secondary to Akt-mediated phosphorylation, is a novel mechanism whereby the growth inhibitory properties of p27(kip1) are functionally inactivated and the proliferation of breast cancer cells is sustained.     

Effector phenotype and immunologic specificity of T-cell-mediated adoptive therapy for a murine tumor that lacks intrinsic immunogenicity

The MCA 102 sarcoma has been defined by a variety of immunologic studies as a tumor lacking intrinsic immunogenicity. Nevertheless, we have recently demonstrated the feasibility of generating therapeutically effective lymphocytes for adoptive immunotherapy of this tumor. Procedures to achieve this required in vivo priming of syngeneic mice to elicit preeffector cells followed by in vitro sensitization (IVS) with tumor cells in the presence of IL-2. By selective depletion of T cell subsets in vivo, we identified the involvement of both CD4+ (L3T4+) and CD8+ (Lyt-2+) T cells in mediating tumor regression. The CD4+ cells exerted their helper function via the secretion of IL-2 because antitumor effects abrogated by depletion of CD4+ cells could be reconstituted by exogenous IL-2. In order to elicit preeffector cells with reactivity against the MCA 102 tumor, we found that in vivo sensitization could be accomplished with either the MCA 102 or MCA 106 tumor but not with the MCA 101 or MCA 105 tumor. Analysis of specificity of tumor stimulation during IVS of MCA 102 tumor-primed preeffector cells demonstrated cross-reactivity between not only the MCA 102 and MCA 106 tumors but also the MCA 105 tumor whereas the MCA 101 tumor was ineffective. In adoptive immunotherapy, transfer of IVS cells generated from MCA 102 tumor-primed and stimulated lymph node cells was able to mediate reductions of pulmonary metastases established from the MCA 102, MCA 105, and MCA 106 tumors but not from the MCA 101 tumor. We conclude that regression of the MCA 102 tumor is probably mediated through T cell recognition of a set of common tumor-associated Ag shared by several other syngeneic tumors. Immunologically, the tumor-associated Ag are characteristically different from classical tumor-specific transplantation Ag (TSTA) because immunity to TSTA on the MCA 105 or MCA 106 tumor does not cross-react with the MCA 102 tumor. Thus, this study demonstrates that Ag other than TSTA on chemically induced tumors can serve as target molecules for T cell-mediated adoptive immunotherapy.     

Identification of p130Cas as a Mediator of Focal Adhesion Kinase–promoted Cell Migration

Previously we have demonstrated that focal adhesion kinase (FAK)-promoted migration on fibronectin (FN) by its overexpression in CHO cells is dependent on FAK autophosphorylation at Y397 and subsequent binding of Src to this site. In this report, we have examined the role of FAK association with Grb2 and p130^Cas, two downstream events of the FAK/Src complex that could mediate integrin-stimulated activation of extracellular signal-regulated kinases (Erks). We show that a Y925F FAK mutant was able to promote cell migration as efficiently as FAK and that the transfected FAK demonstrated no detectable association with Grb2 in CHO cells. In contrast, cells expressing a FAK P712/715A mutant demonstrated a level of migration comparable to that of control cells. This mutation did not affect FAK kinase activity, autophosphorylation, or Src association but did significantly reduce p130^Cas association with FAK. Furthermore, FAK expression in CHO cells increased tyrosine phosphorylation of p130^Cas and its subsequent binding to several SH2 domains, which depended on both the p130^Cas binding site and the Src binding site. However, we did not detect increased activation of Erks in cells expressing FAK, and the MEK inhibitor PD98059 did not decrease FAK-promoted cell migration. Finally, we show that coexpression of p130^Cas further increased cell migration on FN and coexpression of the p130^Cas SH3 domain alone functioned as a dominant negative mutant and decreased cell migration. Together, these results demonstrate that p130^Cas, but not Grb2, is a mediator of FAK-promoted cell migration and suggest that FAK/ p130^Cas complex targets downstream pathways other than Erks in mediating FAK-promoted cell migration.     

Lack of effector cell function and altered tetramer binding of tumor-infiltrating lymphocytes

Tumor-specific CD8 T cell responses to MCA102 fibrosarcoma cells expressing the cytotoxic T cell epitope gp33 from lymphocytic choriomeningitis virus were studied. MCA102(gp33) tumors grew progressively in C57BL/6 mice, despite induction of peripheral gp33-tetramer(+) T cells that were capable of mediating antiviral protection, specific cell rejection, and concomitant tumor immunity. MCA102(gp33) tumors were infiltrated with a high number ( approximately 20%) of CD11b(+)CD11c(-) macrophage-phenotype cells that were able to cross-present the gp33 epitope to T cells. Tumor-infiltrating CD8 T cells exhibited a highly activated phenotype but lacked effector cell function. Strikingly, a significant portion of tumor-infiltrating lymphocytes expressed TCRs specific for gp33 but bound MHC tetramers only after cell purification and a 24-h resting period in vitro. The phenomenon of "tetramer-negative T cells" was not restricted to tumor-infiltrating lymphocytes from MCA102(gp33) tumors, but was also observed when Ag-specific T cells derived from an environment with high Ag load were analyzed ex vivo. Thus, using a novel tumor model, allowing us to trace tumor-specific T cells at the single cell level in vivo, we demonstrate that the tumor microenvironment is able to alter the functional activity of T cells infiltrating the tumor mass.     

Tetraspanin CD9 regulates beta 1 integrin activation and enhances cell motility to fibronectin via a PI-3 kinase-dependent pathway

Tetraspanin CD9 regulates cell motility and other adhesive processes in a variety of tissue types. Using transfected Chinese Hamster Ovary cells as our model system, we examined the cellular pathways critical for CD9 promoted cell migration. alpha 5 beta 1 integrin was directly involved as CD9 enhanced migration was abolished by the alpha 5 beta 1 blocking antibody PB1. Furthermore, the ligand mimetic peptide RGDS, significantly upregulated the expression of a beta1 ligand induced binding site (LIBS) demonstrating for the first time that CD9 expression potentiates beta1 integrin high affinity conformation states. CD9 promoted cell motility was significantly blocked by phosphatidylinositol-3 kinase (PI-3K) inhibitors, wortmannin and LY294002, whereas inhibitors targeting protein kinase C or mitogen-activated protein kinase had no effect. PI-3K dominant/negative cDNA transfections confirmed that PI-3K was an essential component. CD9 enhanced the phosphorylation of the PI-3K substrate, Akt, in response to cell adhesion on FN. CD9 expression also upregulated p130Cas phosphorylation and total protein levels; however, p130Cas siRNA knockdown did not alter the motile phenotype. CD9 enhanced migration was also unaffected by serum deprivation suggesting that growth factors were not critical. Our studies demonstrate that CD9 upregulates beta1 LIBS, and in concert with alpha 5 beta 1, enhances cell motility to FN via a PI-3K dependent mechanism.     

The protein kinase inhibitor SB203580 uncouples PMA-induced differentiation of HL-60 cells from phosphorylation of Hsp27

HL-60 cells are an attractive model for studies of human myeloid cell differentiation. Among the well-examined parameters correlated to differentiation of HL-60 cells are the expression and phosphorylation of the small heat shock protein Hsp27. Here we demonstrate that PMA treatment of HL-60 cells stimulates different MAP kinase cascades, leading to significant activation of ERK2 and p38 reactivating kinase (p38RK). Using the protein kinase inhibitor SB 203580, we specifically inhibited p38RK and, thereby, activation of its target MAP kinase-activated protein kinase 2(MAPKAP kinase 2), which is the major enzyme responsible for small Hsp phosphorylation. As a result, PMA-induced Hsp27 phosphorylation is inhibited in SB 203580-treated HL-60 cells indicating that p38RK and MAPKAP kinase 2 are components of the PMA-induced signal transduction pathway leading to Hsp27 phosphorylation. We further demonstrate that, although PMA-induced phosphorylation is inhibited, SB 203580-treated HL-60 cells are still able to differentiate to the macrophage-like phenotype as judged by decrease in cell proliferation, induction of expression of the cell surface antigen CD11b and changes in cell morphology. These results indicate that, although correlated, Hsp27 phosphorylation is not required for HL-60 cell differentiation. However, the results do not exclude that increased Hsp27 expression is involved in HL-60 cell differentiation.     

CD40 ligand promotes priming of fully potent antitumor CD4(+) T cells in draining lymph nodes in the presence of apoptotic tumor cells.

The presence or absence of CD4(+) T cell help can determine the direction of adaptive immune responses toward either cross-priming or cross-tolerance. It has been demonstrated that interactions of CD40-CD40 ligand can replace CD4(+) T cell help and enable dendritic cells to prime cytotoxic T cells. Here, we demonstrate that antitumor reactivity induced in regional lymph nodes (LNs) by s.c. injection of CD40 ligand (CD40L)-transduced tumor (MCA205 CD40L) showed far superior therapeutic efficacy against established brain tumors of a weakly immunogenic fibrosarcoma, MCA205, when adoptively transferred. Coinjection of apoptotic, but not necrotic parental tumor cells with CD40L-expressing tumor cells caused a strong synergistic induction of antitumor reactivity in tumor-draining LNs. Freshly isolated T cells from LNs immunized with apoptotic parental tumor cells and MCA205 CD40L were capable of mediating regression of the parental tumor in vivo. In contrast, T cells derived from LNs immunized without MCA205 CD40L required ex vivo anti-CD3/IL-2 activation to elicit therapeutic activity. On anti-CD3/IL-2 activation, cells from LNs immunized with MCA205 CD40L exhibited superior per cell antitumor reactivity. An in vitro depletion study revealed that either CD4(+) or CD8(+) T cells could mediate therapeutic efficacy but that the antitumor efficacy mediated by CD4(+) T cells was far superior. Cytosolic flow cytometric analyses indicated that priming of CD4(+) cells in LNs draining CD40L-expressing tumors was polarized to the Th1 type. This is the first report that fully potent antitumor CD4(+) T cell priming was promoted by s.c. injection of CD40L-transduced tumor in the presence of apoptotic tumor cells.     

XB130 Mediates Cancer Cell Proliferation and Survival through Multiple Signaling Events Downstream of Akt

XB130, a novel adaptor protein, mediates RET/PTC chromosome rearrangement-related thyroid cancer cell proliferation and survival through phosphatidyl-inositol-3-kinase (PI3K)/Akt pathway. Recently, XB130 was found in different cancer cells in the absence of RET/PTC. To determine whether RET/PTC is required of XB130-related cancer cell proliferation and survival, WRO thyroid cancer cells (with RET/PTC mutation) and A549 lung cancer cells (without RET/PTC) were treated with XB130 siRNA, and multiple Akt down-stream signals were examined. Knocking-down of XB130 inhibited G(1)-S phase progression, and induced spontaneous apoptosis and enhanced intrinsic and extrinsic apoptotic stimulus-induced cell death. Knocking-down of XB130 reduced phosphorylation of p21Cip1/WAF1, p27Kip1, FOXO3a and GSK3β, increased p21Cip1/WAF1protein levels and cleavages of caspase-8 and-9. However, the phosphorylation of FOXO1 and the protein levels of p53 were not affected by XB130 siRNA. We also found XB130 can be phosphorylated by multiple protein tyrosine kinases. These results indicate that XB130 is a substrate of multiple protein tyrosine kinases, and it can regulate cell proliferation and survival through modulating selected down-stream signals of PI3K/Akt pathway. XB130 could be involved in growth and survival of different cancer cells.     

VE-PTP and VE-cadherin ectodomains interact to facilitate regulation of phosphorylation and cell contacts

VE-cadherin is the essential adhesion molecule in endothelial adherens junctions, and the regulation of protein tyrosine phosphorylation is thought to be important for the control of adherens junction integrity. We show here that VE-PTP (vascular endothelial protein tyrosine phosphatase), an endothelial receptor-type phosphatase, co-precipitates with VE-cadherin, but not with beta-catenin, from cell lysates of transfected COS-7 cells and of endothelial cells. Co-precipitation of VE-cadherin and VE-PTP required the most membrane-proximal extracellular domains of each protein. Expression of VE-PTP in triple-transfected COS-7 cells and in CHO cells reversed the tyrosine phosphorylation of VE-cadherin elicited by vascular endothelial growth factor receptor 2 (VEGFR-2). Expression of VE-PTP under an inducible promotor in CHO cells transfected with VE-cadherin and VEGFR-2 increased the VE-cadherin-mediated barrier integrity of a cellular monolayer. Surprisingly, a catalytically inactive mutant form of VE-PTP had the same effect on VE-cadherin phosphorylation and cell layer permeability. Thus, VE-PTP is a transmembrane binding partner of VE-cadherin that associates through an extracellular domain and reduces the tyrosine phosphorylation of VE-cadherin and cell layer permeability independently of its enzymatic activity.     

The SH2 and SH3 domains of pp60src direct stable association with tyrosine phosphorylated proteins p130 and p110.

Transformation of chicken embryo cells with the tyrosine kinase oncogene src results in the tyrosine phosphorylation of numerous cellular proteins. We have recently generated monoclonal antibodies to individual tyrosine phosphorylated cellular src substrates, several of which are directed to the phosphotyrosine-containing proteins p130 and p110. These proteins form stable complexes with activated variants of pp60src. Mutagenesis of the src homology domains (SH2 and SH3) of activated pp60src resulted in src variants with altered association with p130 and p110. Analysis of these variants showed that the SH3 domain was required for association of p110, while the SH2 domain contained residues necessary for the formation of the ternary complex involving p130, p110 and pp60src. Both the tyrosine phosphorylation status and pp60src association of p130 and p110 appeared to correlate, in part, with the extent of cell transformation. Biochemical analysis demonstrated that p130 and p110 were substrates of both serine/threonine and tyrosine kinases. In addition, p130 was redistributed from the nucleus to cellular membranes upon src transformation, whereas p110, which normally colocalized with cytoskeletal elements, was observed in adhesion plaques (podosomes) in src transformed cells. These data indicate that tyrosine phosphorylation of two different phosphoproteins may play a role during src transformation either by directing their interaction with pp60src, by redirecting subcellular distribution or both.     

Involving of the cytoplasmic region of leukemia inhibitory factor receptor α subunit,IL-6 related signal transducer-gp130 or Fas death domain for MAPK p42/44 activation in HL-60 cell with LIF or anti-Fas IgG

The chimeric receptors were prepared by exchanging the cytoplasmic region between leukemia inhibitory factor (LIF) receptor subunit (gp190) and the other subunit-gp130 (190/130,130/190) and separately transduced into leukemia line HL-60 (to have the wild type subunit). The purpose is to investigate which subunit for activating MAPK p42/44 in leukemia cell while the cytoplasmic region homodimerization (190cyt-190cyt, 130cyt-130cyt) was induced by LIF. The results showed that MAPK p42/44 expression level after LIF stimulation 5 h was lower in the transformants with pED 130/190 (190cyt-190cyt) (p < 0.01) and higher in the transformants with pED 190/130 (130cyt-130cyt) (p < 0.05) than those in the parent cells. Meanwhile, MAPK p42/44 phosphorylation (Thr202/Tyr204) was ascended and the highest at 10 min in the 190/130 and descended in the 130/190. It suggests that gp130 activate MAPK p42/44 and gp190 indirectly regulate its expression and function. In order to analyses the relation of the subunit oligomerization and MAPK p42/44 we also prepared the recombination of the extracellular and transmembrane region of Fas and the cytoplasmic region of each LIFR subunit (Fas/190, Fas/130). After transduction into HL-60 with lipofection and induction by anti-Fas IgG, we found that MAPK p42/44 expression levels were lower in the Fas/190 than in the Fas/130 and parent cells (p < 0.01) and no difference between the Fas/130 and the wild type receptor. However, phospho-MAPK p42/44 were increased in the Fas/130 than the parent cells. It suggests that the oligomerization of the cytoplasmic regions of gp130 be potential to normally initiate MAPK p42/44 for the signal of HL-60 proliferation. We also determine that the separated oligomerization FasDD (no dimerization) can initiate the corresponding signal molecules, then regulate MAPK p42/44 expression and phosphorylation in leukemia cells.     

Characterization of endogenous Chinese hamster ovary cell surface molecules that mediate T cell costimulation

Chinese hamster ovary (CHO) cells are commonly used in the generation of transfectants for use in in vitro costimulation assays. However, we have noted that nontransfected CHO cells can themselves provide a low-level B7/CD28 independent costimulatory signal for CD3-mediated murine T cell activation and IL-2 production. This study set out to identify those molecules that contribute to this CHO-dependent costimulatory activity. We describe a CHO subline capable of delivering potent CD28-independent costimulation to murine T cells and the generation of monoclonal antibodies against these CHO cells that inhibited this costimulatory activity. These blocking antibodies do not affect CHO cell-independent costimulation or bind mouse cells, suggesting an effect mediated by their target molecules on the costimulatory competent CHO cells. Immunoprecipitation and expression cloning revealed that these antibodies bound the hamster homologues of Crry (CD21/35), CD44, CD54 (ICAM-1), CD63, CD87, CD147, and an 80- to 90-kDa protein which could not be cloned. Expression of these hamster genes on COS cells demonstrated that hamster CD54 was able to costimulate both CD3-mediated IL-2 secretion and T cell proliferation by naive murine T cells independent of the other molecules identified.     

Synergy between CD40 ligation and IL-4 on fibroblast proliferation involves IL-4 receptor signaling.

Fibrosis can be an undesired consequence of activated cellular immune responses. The purpose of this work was to determine whether CD40 ligation and the pro-fibrotic cytokine IL-4 interact in regulating fibroblast proliferation and collagen production, and, if so, the mechanisms used. This study found that the combination of IL-4 and ligation of CD40 on the fibroblast cell surface had synergistic effects in stimulating fibroblast proliferation. In contrast, CD40 ligation negated the inhibitory effects of IFN-gamma on fibroblast proliferation. Western blotting analyses of fibroblast crude lysates revealed that a potential mechanism of the synergy between CD40 ligation and IL-4 was the phosphorylation of proteins at 130 kDa and, to a lesser degree, at 95, 85, and 75 kDa. Immunoprecipitation-Western blotting experiments showed that phosphorylation levels of IL-4Ralpha, Janus kinase 1, insulin receptor substrate 1, and insulin receptor substrate 2, factors with molecular mass close to the observed 130 kDa major phosphorylation band, increased in response to the combined CD40 ligation and IL-4 action. In contrast, there was no evidence that synergy was mediated by an increased expression of IL-4Ralpha chain, CD40, or the autocrine profibrotic cytokines IL-6 and TGF-beta. These findings suggest that CD40-CD40 ligand contacts between fibroblasts and cells secreting IL-4 may promote the profibrotic effects of IL-4 by affecting signal transduction and reducing the anti-fibrotic effects of IFN-gamma.     

Carbohydrate supplementation and exercise-induced changes in T-lymphocyte function.

Carbohydrate (CHO) ingestion during exercise has been shown to reduce perturbations in immune cell numbers and function, possibly through a reduction in the cortisol response to exercise. We have previously observed that exercise decreases T-lymphocyte responses to mitogen via an increase in cell death of both CD4 and CD8 T lymphocytes (Green KJ and Rowbottom DG. J Appl Physiol. 95: 57-63, 2003). This study tested the hypothesis that CHO ingestion rather than placebo (Pl) would result in an attenuation of the cortisol response to exercise and a reduction of the exercise-associated alterations in cell death. Six well-trained cyclists completed two exercise trials consisting of 2.5 h of cycling at 85% of individual ventilatory threshold. In a random order, trials were completed under either CHO (6% CHO solution, 3.2 g CHO/kg body wt total) or Pl conditions. Blood samples were collected before exercise, midexercise (after 60 min of exercise), immediately after exercise, and after 60 min of recovery. T-lymphocyte responses to mitogen were determined by using carboxyfluorescein diacetate succinimidyl ester fluorescent cell division tracking and expansion rates, and cell death rates were calculated for each sample as well as mitosis rates for each cell generation. Cellular expansion of T lymphocytes was decreased after exercise in Pl only. The reduction in cellular expansion was related to an increase in cell death of both CD4 and CD8 cells in culture rather than a decrease in the ability of cells to undergo mitosis. CHO ingestion compared with Pl was associated with no reductions in cellular expansion or increases in cell death. CHO ingestion during exercise acted to reduce the impairment of T-lymphocyte function by decreasing cell death within mitogen-stimulated cell cultures; however, the mechanism of action appears to be independent of cortisol.