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M L Alegre

Publications and source records attributed to M L Alegre.

At least 19 recordsLinked to original sources
PubMed2001

Cutting edge: membrane lymphotoxin regulates CD8(+) T cell-mediated intestinal allograft rejection.

Blocking the CD28/B7 and/or CD154/CD40 costimulatory pathways promotes long-term allograft survival in many transplant models where CD4(+) T cells are necessary for rejection. When CD8(+) T cells are sufficient to mediate rejection, these approaches fail, resulting in costimulation blockade-resistant rejection. To address this problem we examined the role of lymphotoxin-related molecules in CD8(+) T cell-mediated rejection of murine intestinal allografts. Targeting membrane lymphotoxin by means of a fusion protein, mAb, or genetic mutation inhibited rejection of intestinal allografts by CD8(+) T cells. This effect was associated with decreased monokine induced by IFN-gamma (Mig) and secondary lymphoid chemokine (SLC) gene expression within allografts and spleens respectively. Blocking membrane lymphotoxin did not inhibit rejection mediated by CD4(+) T cells. Combining disruption of membrane lymphotoxin and treatment with CTLA4-Ig inhibited rejection in wild-type mice. These data demonstrate that membrane lymphotoxin is an important regulatory molecule for CD8(+) T cells mediating rejection and suggest a strategy to avoid costimulation blockade-resistant rejection.

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PubMed2001

CTLA-4 is not required for induction of CD8(+) T cell anergy in vivo.

Recent studies of T cell anergy induction have produced conflicting conclusions as to the role of the negative regulatory receptor, CTLA-4. Several in vivo models of tolerance have implicated the interaction of CTLA-4 and its ligands, B7.1 and B7.2, as an essential step in induction of anergy, while results from a number of other systems have indicated that signals from the TCR/CD3 complex alone are sufficient to induce T cell unresponsiveness. One explanation for this disparity is that the requirements for anergy induction depend closely on the details of the system: in vivo vs in vitro, route of stimulus administration, naive vs memory cells, CD4(+) vs CD8(+) cells, etc. To test this possibility, we established an in vivo anergy model using mice transgenic for the 2C TCR on a recombination-activating gene-2-deficient background, that either express or lack the CTLA-4 molecule. This system provides us with a very homogeneous pool of naive Ag-specific CD8(+) T cells, allowing us to control some of the conditions mentioned above. We found that T cells from CTLA-4-deficient mice were anergized by injections of soluble antigenic peptide as efficiently as were CTLA-4-expressing cells. These results indicate that CTLA-4 is not universally required for in vivo T cell anergy induction and may point to distinctions between regulation of peripheral tolerance in CD4(+) and CD8(+) T cells.

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PubMed2001

CD8 T cell-mediated rejection of intestinal allografts is resistant to inhibition of the CD40/CD154 costimulatory pathway.

BACKGROUND: Disruption of the CD40/CD154 pathway inhibits rejection in numerous models. The importance of this pathway on intestinal allograft rejection was examined in this study. METHODS: Intestinal grafts from B6C3F1 mice transplanted into C57BL/6 recipients were assessed histologically for rejection. RESULTS: The monoclonal antibody to CD154, MR1, failed to inhibit rejection in wild-type mice. Similarly, CD154-/- recipient mice rejected intestinal allografts. MR1 did inhibit early rejection in CD8-/- mice, but had no effect in CD4-/- recipients. All MR1-treated CD8-/- recipients eventually developed rejection. No benefit was observed when blockade of the CD40/CD154 pathway by MR1 was combined with blockade of the CD28/B7 pathway by mCTLA4Ig. CONCLUSIONS: These data suggest that CD4+ T cells mediating intestinal allograft rejection may be more dependent upon the CD40/CD154 pathway than CD8+ T cells. This finding highlights the importance of identifying agents that suppress CD8+ T cell-mediated rejection.

Animals↗
PubMed2001

Absence of CTLA-4 lowers the activation threshold of primed CD8+ TCR-transgenic T cells: lack of correlation with Src homology domain 2-containing protein tyrosine phosphatase.

To examine the role of CTLA-4 in controlling Ag-specific CD8(+) T cell activation, TCR-transgenic/CTLA-4 wild-type or -deficient mice were generated in a recombination-activating gene 2-deficient background. Naive T cells from these mice responded comparably whether or not CTLA-4 was expressed. In contrast, primed T cells responded more vigorously if they lacked CTLA-4 expression. We took advantage of the difference between naive and primed T cell responses to approach the mechanism of CTLA-4 function. Single-cell analyses demonstrated that a greater fraction of CTLA-4-deficient cells responded to a fixed dose of Ag compared with CTLA-4-expressing cells, whereas the magnitude of response per cell was comparable. A shift in the dose-response curve to APCs was also observed such that fewer APCs were required to activate CTLA-4-deficient T cells to produce intracellular IFN-gamma and to proliferate. These results suggest that CTLA-4 controls the threshold of productive TCR signaling. Biochemical analysis comparing stimulated naive and primed TCR-transgenic cells revealed no obvious differences in expression of total CTLA-4, tyrosine-phosphorylated CTLA-4, and associated Src homology domain 2-containing protein tyrosine phosphatase. Thus, the biochemical mechanism explaining the differential inhibitory effect of CTLA-4 on naive and primed CD8(+) T cells remains unclear.

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PubMed2001

Different mechanisms of cardiac allograft rejection in wildtype and CD28-deficient mice.

Although CD28 blockade results in long-term cardiac allograft survival in wildtype mice, CD28-deficient mice effectively reject heart allografts. This study compared the mechanisms of allogeneic responses in wildtype and CD28-deficient mice. Adoptive transfer of purified CD28-deficient T cells into transplanted nude mice resulted in graft rejection. However, this model demonstrated that the allogeneic T cell function was severely impaired when compared with wildtype T cells, despite similar survival kinetics. Cardiac allograft rejection depended on both CD4+ and CD8+ T cell subsets in CD28-deficient mice, whereas only CD4+ T cells were necessary in wildtype recipients. These results suggested that CD8+ T cells were more important in CD28-deficient than wildtype mice. In addition to the CD8+ T cell requirement, allograft rejection in CD28-deficient mice was dependent on a sustained presence of CD4+ T cells, whereas it only required the initial presence of CD4+ T cells in wildtype mice. Taken together, these data suggest that CD4+ T cells from CD28-deficient mice have impaired responses to alloantigen in vivo, thus requiring long-lasting cooperation with CD8+ T cell responses to facilitate graft rejection. These results may help to explain the failure to promote graft tolerance in some preclinical and clinical settings.

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PubMed2001

T-cell regulation by CD28 and CTLA-4.

Activation of T lymphocytes is thought to require at least two signals, one delivered by the T-cell receptor complex after antigen recognition, and one provided on engagement of co-stimulatory receptors, such as CD28. Recent studies are providing clues as to the specific signalling roles of co-stimulatory receptors. Furthermore, superimposition of inhibitory signals, such as those delivered by cytotoxic T-lymphocyte antigen 4 (CTLA-4), leads to a complex network of positive and negative co-stimulatory signals, the integration of which modulates immune responses.

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PubMed2000

CTLA-4 gene polymorphism at position 49 in exon 1 reduces the inhibitory function of CTLA-4 and contributes to the pathogenesis of Graves' disease.

Activation of T cells requires at least two signals transduced by the Ag-specific TCR and a costimulatory ligand such as CD28. CTLA-4, expressed on activated T cells, binds to B7 present on APCs and functions as a negative regulator of T cell activation. Our laboratory previously reported the association of Graves' disease (GD) with a specific CTLA-4 gene polymorphism. In theory, reduced expression or function of CTLA-4 might augment autoimmunity. In the present study, we categorized autoimmune thyroid disease patients and normal controls (NC) by genotyping a CTLA-4 exon 1 polymorphism and investigated the function of CTLA-4 in all subjects. PBMCs and DNA were prepared from GD (n = 45), Hashimoto's thyroiditis (HT) (n = 18), and NC (n = 43). There were more GD patients with the G/G or A/G alleles (82.2% vs 65.1% in NC), and significantly fewer patients with the A/A allele (17.8% vs 34.9% in NC). In the presence of soluble blocking anti-human CTLA-4 mAb, T cell proliferation following incubation with allogeneic EBV-transformed B cells was augmented in a dose-dependent manner. Augmentation induced by CTLA-4 mAb was similar in GD and NC (GD, HT, NC = 156%, 164%, 175%, respectively). We related CTLA-4 polymorphism to mAb augmentation of T cell proliferation in each subgroup (GD, HT, NC). Although PBMC from individuals with the G/G alleles showed 132% augmentation, those with the A/A alleles showed 193% augmentation (p = 0.019). CTLA-4 polymorphism affects the inhibitory function of CTLA-4. The G allele is associated with reduced control of T cell proliferation and thus contributes to the pathogenesis of GD and presumably of other autoimmune diseases.

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PubMed2000

Role of STAT4 and STAT6 signaling in allograft rejection and CTLA4-Ig-mediated tolerance.

STAT4(-/-) mice have impaired type 1 T cell differentiation, whereas STAT6(-/-) mice fail to generate type 2 responses. The role of type 1 and type 2 T cell differentiation in acute cardiac allograft rejection and in the induction of tolerance was examined in wild-type, STAT4(-/-), and STAT6(-/-) recipients. All recipients rejected the grafts promptly. Analysis of in situ cytokine gene expression in the allografts confirmed decreased levels of IFN-gamma in STAT4(-/-) recipients and undetectable levels of IL-4 and IL-5 in STAT6(-/-) mice. Blockade of the CD28/B7 costimulatory pathway prolonged cardiac graft survival for >100 days in 100% of wild-type and STAT4(-/-) mice. However, 14% of CTLA4-Ig-treated STAT6(-/-) mice rejected their grafts between 20 and 100 days. Moreover, of those animals followed past 100 days, 60% of the STAT6(-/-) mice rejected their grafts. Splenocytes harvested on day 145 posttransplant from CTLA4-Ig-treated rejecting STAT6(-/-) recipients were transfused into syngeneic SCID mice transplanted with donor or third party cardiac allografts. Both donor and third party grafts were rejected, indicating that the initial graft loss may be due to an immunological rejection. In contrast, when splenocytes from CTLA4-Ig-treated wild-type or nonrejecting STAT6(-/-) mice were transferred into SCID recipients, donor allografts were accepted, but third party hearts were rejected. Thus, long-term prolongation of cardiac allograft survival by CTLA4-Ig is STAT4-independent but, at least in part, STAT6-dependent. These data suggest that the balance of type 1 and type 2 T lymphocyte differentiation is not critical for acute rejection but influences the robust tolerance induced by CD28/B7 blockade in this model.

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PubMed2000

Induction of T cell anergy in the absence of CTLA-4/B7 interaction.

Immunologic tolerance in T lymphocytes is maintained through both thymic and peripheral contributions. One peripheral tolerance mechanism is the induction of T cell anergy, a form of nonresponsiveness resulting from incomplete T cell activation, such as stimulation through the TCR in the absence of costimulation. Recent reports have suggested that engagement of the inhibitory receptor CTLA-4 by its B7 ligand is critical for the initiation of anergy. We tested the importance of CTLA-4 in anergy induction in primary T cells with an in vitro anergy system. Using both CTLA-4/B7-blocking agents and CTLA-4-deficient T cells, we found that T cell anergy can be established in the absence of CTLA-4 expression and/or function. Even in the absence of CTLA-4 signal transduction, T cells activated solely through TCR ligation lose the ability to proliferate as a result of autocrine IL-2 production upon subsequent receptor engagement. Thus, CTLA-4 signaling is not required for the development of T cell anergy.

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PubMed2000

In vitro characterization of five humanized OKT3 effector function variant antibodies.

Orthoclone OKT 3 (mOKT3) is a highly effective agent for the reversal of steroid-resistant renal allograft rejection. However, its wider use has been limited by the development of a human anti-mouse antibody response (HAMA) and by the "cytokine release syndrome" (CRS). CRS has been associated with T cell/monocyte activation and, secondarily, with activation of the complement cascade. These processes are mediated through Abs' Fc regions by their abilities to cross-link T cells and mononuclear cells and to activate complements. To alleviate these problems, a group of five huIgG1- and huIgG4-based OKT3 wild-type antibodies and their corresponding Fc mutants with altered residues at amino acids 234, 235, and 318, reported to be required for FcgammaRI and FcgammaRII binding and complement fixation, were constructed. Characterization of these humanized OKT3 Abs, denoted huOKT3gamma1, huOKT3gamma4, huOKT3gamma1(A(234), A(235)), huOKT3gamma4(A(234), A(235)), and huOKT3gamma1(A(318)), has demonstrated that huOKT3gamma1(A(234), A(235)) and huOKT3gamma4(A(234), A(235)), and have at least a 100-fold reduced binding to FcgammaRI and FcgammaRII. As expected, they are much less potent in the induction of T cell activation and cytokine release, yet retain in vitro immunosuppressive effects as potent as those of mOKT3. Unexpectedly, while huOKT3gamma1(A(318)) did not show any reduction in its ability to bind C1q and to fix a complement, huOKT3gamma1(A(234), A(235)) was completely inactive. The in vitro characteristics of huOKT3gamma1(A(234), A(235)) are consistent with recent in vivo studies, in which this Ab showed greatly reduced HAMA and CRS with the retention of its ability to reverse ongoing graft rejection in man.

Animals↗
PubMed1999

Downregulation of antigen-presenting cell functions after administration of mitogenic anti-CD3 monoclonal antibodies in mice.

Antibodies against CD3epsilon are widely used as immunosuppressive agents. Although it is generally assumed that these reagents exert their immunomodulatory properties by inducing T-cell deletion and/or inactivation, their precise mechanism of action remains to be elucidated. Using a murine model, we demonstrate in this report that administration of anti-CD3epsilon antibodies causes the migration and maturation of dendritic cells (DC) in vivo, as determined by immunohistochemical analysis. This maturation/migration process was followed by selective loss of splenic DC, which resulted in a selective inhibition of antigen-presenting cell (APC) functions in vitro. Spleen cells from anti-CD3epsilon-treated animals were unable to productively stimulate naive alloreactive T cells and Th1-like clones in response to antigen, while retaining the ability to present antigen to a T-cell hybridoma and Th2 clones. Anti-CD3epsilon treatment was found to induce a selective deficiency in the ability of spleen cells to produce bioactive interleukin-12 in response to CD40 stimulation. APC dysfunction was not observed when nonmitogenic forms of anti-CD3epsilon antibodies were used, suggesting that splenic DC loss was a consequence of in vivo T-cell activation. Nonmitogenic anti-CD3epsilon monoclonal antibodies were found to be less immunosuppressive in vivo, raising the possibility that APC dysfunction contributes to anti-CD3epsilon-induced immunomodulation. Collectively, these data suggest a novel mechanism by which mitogenic anti-CD3epsilon antibodies downregulate immune responses.

Animals↗
PubMed1999

Down-regulation of interleukin-2 and interferon-gamma and maintenance of interleukin-4 and interleukin-10 production after administration of an anti-CD3 monoclonal antibody in mice.

BACKGROUND: Activating anti-CD3 monoclonal antibodies (mAbs), such as OKT3, are potent immunosuppressive agents that are widely used in clinical transplantation. We investigated whether the in vivo induction of T cell unresponsiveness contributes to the immunosuppressive properties of the anti-mouse-CD3 mAb 145-2C11. METHODS: After a single in vivo administration of 145-2C11 residual T cells were restimulated in vivo and in vitro to assess cytokine production. Mice were also transplanted with allogeneic skin 9 days after 145-2C11 administration to investigate whether the immunosuppressive properties of the antibody persist after the reexpression of the T cell receptor. RESULTS: Pretreatment with anti-CD3 mAbs caused a profound deficit in both interleukin- (IL) 2 and interferon- (IFN) y secretion upon restimulation in vivo, whereas IL-4 was only partially inhibited and IL-10 production was significantly increased. Purified T cells obtained from mice injected with anti-CD3 mAb also displayed deficient IL-2 and IFN-gamma production together with persisting IL-4 and IL-10 secretion. 145-2C11 had immunosuppressive properties that per sisted after the reexpression of the T cell receptor because mice transplanted with allogeneic skin 9 days after a single anti-CD3 mAb injection still had significantly prolonged graft survival (14.1+/-0.6 days vs. 10.7+/-0.4 days in controls, P<0.02). Blocking IL-4 and IL-10 by neutralizing mAbs further prolonged skin graft survival in mice injected with 145-2C11 (18.3+/-0.7 vs. 14.8+/-0.6 days, P<0.02). CONCLUSION: The in vivo administration of the 145-2C11 anti-CD3 mAb results in the selective inhibition of Thl-type cytokine secretion upon restimulation, which correlates with a state of immunosuppression. The persistent production of Th2-type cytokines does not contribute to the anti-CD3 mAb-mediated prolonged survival of skin allografts in our experimental model.

Animals↗
PubMed1999

Regulation of cytotoxic T lymphocyte-associated molecule-4 by Src kinases.

Cytotoxic T lymphocyte-associated molecule-4 (CTLA-4) is a cell surface receptor expressed on activated T cells that can inhibit T cell responses induced by activation of the TCR and CD28. Studies with phosphorylated peptides based on the CTLA-4 intracellular domain have suggested that tyrosine phosphorylation of CTLA-4 may regulate its interactions with cytoplasmic proteins that could determine its intracellular trafficking and/or signal transduction. However, the kinase(s) that phosphorylate CTLA-4 remain uncharacterized. In this report, we show that CTLA-4 can associate with the Src kinases Fyn and Lck and that transfection of Fyn or Lck, but not the unrelated kinase ZAP70, can induce tyrosine phosphorylation of CTLA-4 on residues Y201 and Y218. A similar pattern of tyrosine phosphorylation was found in pervanadate-treated Jurkat T cells stably expressing CTLA-4. Phosphorylation of CTLA-4 Y201 in Jurkat cells correlated with cell surface accumulation of CTLA-4. CTLA-4 phosphorylation induced the association of CTLA-4 with the tyrosine phosphatase SHP-2, but not with phosphatidylinositol 3-kinase. In contrast, Lck-induced phosphorylation of CD28 resulted in the recruitment of phosphatidylinositol 3-kinase, but not SHP-2. These findings suggest that phosphorylation of CD28 and CTLA-4 by Lck activates distinct intracellular signaling pathways. The association of CTLA-4 with Src kinases and with SHP-2 results in the formation of a CTLA-4 complex with the potential to regulate T cell activation.

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PubMed1998

Expression and function of CTLA-4 in Th1 and Th2 cells.

CTLA-4 is expressed on T cells after activation and shares homology with the CD28 costimulatory receptor. In contrast to CD28, CTLA-4 is thought to be a negative regulator of T cell activation. Cross-linking of CTLA-4 during activation of peripheral T cells reduces IL-2 production and arrests T cells in G1. Much less is known about the function of CTLA-4 in differentiated T cells. We have investigated the expression and function of CTLA-4 in established Th1 and Th2 clones and in bulk populations of Th1 and Th2 cells freshly derived in vitro from TCR transgenic splenocytes. We found that CTLA-4 was induced under similar conditions and with similar kinetics following activation of both Th1 and Th2 clones. However, CTLA-4 expression was much higher in Th2 than Th1 clones and lines. This was confirmed by flow cytometry, confocal microscopy, and Northern blot analysis. The ratio of surface to intracellular expression of CTLA-4 and its rate of endocytosis were similar in Th1 and Th2 clones. Inhibition of binding of CTLA-4 to its ligands using soluble anti-CTLA-4 mAb during stimulation with Ag increased the production not only of IL-2 by Th1 clones, but also that of IL-3 and IFN-gamma by Th1 clones and of IL-3, IL-4, IL-5, and IL-10 by Th2 clones. In contrast, when anti-CTLA-4 was coimmobilized with anti-CD3 and anti-CD28 mAbs, a decrease in the production of multiple cytokines was observed. We conclude that CTLA-4 can function to suppress the production of cytokines produced by both Th1 and Th2 cells.

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PubMed1998

Impaired negative selection in CD28-deficient mice.

T cell antigen receptors (TCR) expressed on developing T cells can react with self-peptides presented by proteins encoded by the major histocompatibility complex (MHC). Depending on the relative strength of these interactions, thymocytes are either negatively selected as potentially autoreactive and deleted or positively selected to become mature T cells. Developmental selection may also be regulated by signals in addition to those mediated through the TCR. In peripheral T cells, the CD28 receptor plays an important role in enhancing the survival and expansion of T cells activated by TCR engagement. Therefore, we have investigated the role of CD28 in regulating the selection of thymocytes using CD28-deficient mice. Surprisingly, we found a 50% increase in cell number in the thymi of CD28-deficient compared to wildtype mice, suggesting that CD28 might play a role in negative selection. Negative selection of double-positive thymocytes was found to be significantly reduced in response to either antigen or antibody crosslinking of the TCR complex in CD28-deficient animals. This was not due to a generalized defect in thymocyte survival as thymocytes from CD28-deficient and wildtype mice displayed similar sensitivity to apoptosis initiated by either gamma-irradiation or dexamethasone. In contrast to its role in T cell activation and survival in the peripheral immune system, the CD28 receptor appears to participate in the intracellular signaling events that result in negative selection in the thymus.

Amino Acid Sequence↗
PubMed1998

Cytotoxic T lymphocyte antigen 4 (CTLA4) blockade accelerates the acute rejection of cardiac allografts in CD28-deficient mice: CTLA4 can function independently of CD28.

Cytotoxic T lymphocyte antigen 4 (CTLA4) appears to negatively regulate T cell activation. One mechanism by which CTLA4 might antagonize T cell function is through inhibition of CD28 signaling by competing for their shared ligands B7-1 and B7-2. In addition, CTLA4 ligation could initiate a signaling cascade that inhibits T cell activation. To address whether CTLA4 could inhibit immune responses in the absence of CD28, rejection of heart allografts was studied in CD28-deficient mice. H-2(q) hearts were transplanted into allogeneic wild-type or CD28-deficient mice (H-2(b)). Graft rejection was delayed in CD28-deficient compared with wild-type mice. Treatment of wild-type recipients with CTLA4-immunoglobulin (Ig), or with anti-B7-1 plus anti-B7-2 mAbs significantly prolonged allograft survival. In contrast, treatment of CD28-deficient mice with CTLA4-Ig, anti-B7-1 plus anti-B7-2 mAbs, or a blocking anti-CTLA4 mAb induced acceleration of allograft rejection. This increased rate of graft rejection was associated with more severe mononuclear cell infiltration and enhanced levels of IFN-gamma and IL-6 transcripts in donor hearts of untreated wild-type and CTLA4-Ig- or anti-CTLA4 mAb-treated CD28-deficient mice. Thus, the negative regulatory role of CTLA4 extends beyond its potential ability to prevent CD28 activation through ligand competition. Even in the absence of CD28, CTLA4 plays an inhibitory role in the regulation of allograft rejection.

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