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Biomedical subjects

V A Boussiotis

Publications and source records attributed to V A Boussiotis.

At least 19 recordsLinked to original sources
PubMed1998

The BB1 monoclonal antibody recognizes both cell surface CD74 (MHC class II-associated invariant chain) as well as B7-1 (CD80), resolving the question regarding a third CD28/CTLA-4 counterreceptor.

The identification of all CD28/CTLA-4 counterreceptors is critical to our understanding of this pivotal pathway of T cell activation. Clouding our understanding has been the reported discrepancies in expression and function of the B7-1 (CD80) molecule based upon the use of the BB1 vs other anti-B7-1 mAbs. To resolve this issue, we have cloned a BB1-binding molecule from the BB1+B7-1(-) NALM-6 pre-B cell line. Here, we demonstrate that this BB1-binding molecule is identical to the cell surface form of CD74 (MHC class II-associated invariant chain). CD74-transfected cells bound the BB1 mAb but not other anti-CD80 mAbs, CD28-Ig, or CTLA4Ig. Absorption and blocking experiments confirmed the reactivity of BB1 mAb with CD74. A region of weak homology was identified between CD74 and the region of B7-1 encoding the BB1 epitope. Therefore, the BB1 mAb binds to a protein distinct from B7-1, and this epitope is also present on the B7-1 protein. Many of the puzzling observations in the literature concerning the expression of human B7-1 are resolved by an understanding that BB1 staining is the summation of CD74 plus B7-1 expression. This observation requires the field to reconsider studies using BB1 mAb in the analysis of CD80 expression and function.

3T3 Cells↗
PubMed1998

Unbalanced expression of bcl-2 family proteins in follicular lymphoma: contribution of CD40 signaling in promoting survival.

Although highly responsive, advanced stage follicular lymphoma (FL) is not curable with conventional treatment. This relative resistance is thought to be due to the t(14;18) that results in the constitutive overexpression of the death-inhibiting protein bcl-2. However, the observation that FL cells are sensitive to treatment in vivo and prone to apoptosis on in vitro culture questions whether bcl-2 alone is responsible for the pathogenesis and clinical behavior of this disease. Therefore, multiple genes are likely to be involved in both the lymphomagenesis and the clinical course of FL. We examined whether expression of other bcl-2 family genes might also be operative. Here, we show that FL cells display a different pattern of expression of bcl-2 family proteins from normal germinal center (GC) B cells that are thought to be their normal counterpart. FL cells express the death-suppressor proteins bcl-2, bcl-xL, and mcl-1; whereas GC B cells express bcl-xL and mcl-1 but also the proapoptotic proteins bax-alpha and bad. Although maintaining constitutive levels of bcl-2 and mcl-1, FL cells are not protected from apoptosis when cultured in vitro. Their propensity to undergo apoptosis is temporally associated with downregulation of bcl-xL. More importantly, activation of FL cells via CD40 not only prevents downregulation but increases the level of bcl-xL expression and results in promotion of survival. These results support the hypothesis that the overexpression of bcl-2 is not the only antiapoptotic mechanism responsible for the pathogenesis of FL. Survival of FL cells is determined by a number of death-inhibiting proteins, among which bcl-xL appears to have the most critical role. Moreover, these findings are consistent with the hypothesis that, although FL cells are malignant, they respond to microenvironmental signals such as CD40L that appear to contribute to their survival through the upregulation of death-inhibiting proteins.

Apoptosis↗
PubMed1997

Maintenance of human T cell anergy: blocking of IL-2 gene transcription by activated Rap1.

In the absence of costimulation, T cells activated through their antigen receptor become unresponsive (anergic) and do not transcribe the gene encoding interleukin-2 (IL-2) when restimulated with antigen. Anergic alloantigen-specific human T cells contained phosphorylated Cbl that coimmunoprecipitated with Fyn. The adapter protein CrkL was associated with both phosphorylated Cbl and the guanidine nucleotide-releasing factor C3G, which catalyzes guanosine triphosphate (GTP) exchange on Rap1. Active Rap1 (GTP-bound form) was present in anergic cells. Forced expression of low amounts of Rap1-GTP in Jurkat T cells recapitulated the anergic defect and blocked T cell antigen receptor (TCR)- and CD28-mediated IL-2 gene transcription. Therefore, Rap1 functions as a negative regulator of TCR-mediated IL-2 gene transcription and may be responsible for the specific defect in IL-2 production in T cell anergy.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Induction of T cell clonal anergy results in resistance, whereas CD28-mediated costimulation primes for susceptibility to Fas- and Bax-mediated programmed cell death.

Since TCR-mediated stimulation induces T cells to become sensitive to Fas-mediated activation-induced cell death (Fas-AICD), we examined whether anergized and CD28-costimulated T cell clones were equally sensitive to Fas-AICD. Here, we show that TCR signal in the presence or absence of CD28 costimulation induced equivalent expression of Fas and Fas ligand. Although anergized cells expressed Fas and Fas ligand, they were resistant to Fas-AICD. Induction of anergy resulted in up-regulation and persistent expression of moderate amounts of bcl-xL and bax and absence of induction of bad. In contrast, CD28-costimulated cells that also expressed Fas and Fas ligand were initially resistant to Fas-AICD but became susceptible after 72 h of culture. Although Fas-mediated apoptosis was the major mechanism of AICD, the IL-1beta-converting enzyme-like protease inhibitor zVAD-FMK totally abrogated DNA fragmentation but not cell death, suggesting that additional Fas-independent apoptotic mechanisms were also operative. Resistance to apoptotic cell death was temporally associated with a dramatic increase of bcl-xL and the presence of bcl-xL:bax heterodimers. Subsequent sensitivity to AICD was associated with down-regulation of bcl-xL, induction of bad, and the displacement of bax from bcl-xL:bax heterodimers. Although induced following CD28 costimulation, bcl-2 did not protect against AICD. Therefore, besides its role in promotion of viability, prevention of anergy, and clonal expansion, CD28 costimulation also has a central role in the induction of subsequent AICD by up-regulating apoptotic mediators.

3T3 Cells↗
PubMed1997

Ex vivo generation of human anti-pre-B leukemia-specific autologous cytolytic T cells.

In contrast to other neoplasms, antigen-specific autologous cytolytic T cells have not been detected in patients with human pre-B-cell leukemias. The absence of efficient B7 family (B7-1/CD80; B7-2/CD86) -mediated costimulation has been shown to be a major defect in tumor cells' capacity to function as antigen-presenting cells. We show here the generation of autologous anti-pre-B-cell leukemia-specific cytolytic T-cell lines from the marrows of 10 of 15 patients with pre-B-cell malignancies. T-cell costimulation via CD28 is an absolute requirement for the generation of these autologous cytolytic T cells (CTL). Although costimulation could be delivered by either bystander B7 transfectants or professional antigen-presenting cells (indirect costimulation), optimal priming and CTL expansion required that the costimulatory signal was expressed by the tumor cell (direct costimulation). These anti-pre-B-cell leukemia-specific CTL lysed both unstimulated and CD40-stimulated tumor cells from each patient studied but did not lyse either K562 or CD40-stimulated allogeneic B cells. Cytolysis was mediated by the induction of tumor cell apoptosis by CD8+ T cells via the perforin-granzyme pathway. Although we were able to generate anti-leukemia-specific CTL from the bone marrow, we were unable to generate such CTL from the peripheral blood of these patients. These studies show that antigen-specific CTL can be generated from the bone marrow of patients with pre-B-cell leukemias and these findings should facilitate the design of adoptive T-cell-mediated immunotherapy trials for the treatment of patients with B-cell precursor malignancies.

Animals↗
PubMed1996

Human CD100, a novel leukocyte semaphorin that promotes B-cell aggregation and differentiation.

Herein we describe the molecular characterization of the human leukocyte activation antigen CD100 and identify it as the first semaphorin, to our knowledge, in the immune system. Semaphorins have recently been described as neuronal chemorepellants that direct pioneering neurons during nervous system development. In this study we demonstrate that CD100 induces B cells to aggregate and improves their viability in vitro. We show that CD100 modifies CD40-CD40L B-cell signaling by augmenting B-cell aggregation and survival and down-regulating CD23 expression. Thus, these results suggest that semaphorins as exemplified by CD100 also play a functional role in the immune system.

Amino Acid Sequence↗
PubMed1996

Differential association of protein tyrosine kinases with the T cell receptor is linked to the induction of anergy and its prevention by B7 family-mediated costimulation.

When stimulated through their antigen receptor, without costimulation, T cells enter a state of antigen-specific unresponsiveness, termed anergy. B7-mediated costimulation, signaling via CD28, is sufficient to prevent the induction of anergy. Here we show that ligation of T cell receptor (TCR) by alloantigen alone, which results in anergy, activates tyrosine phosphorylation of TCR zeta and its association with fyn. In contrast, TCR ligation in the presence of B7 costimulation, which results in productive immunity, activates tyrosine phosphorylation of TCR zeta and CD3 chains, which associate with activated lck and zeta-associated protein (ZAP) 70. Under these conditions, CD28 associates with activated lck and TCR zeta. These data suggest that the induction of anergy is an active signaling process characterized by the association of TCR zeta and fyn. In addition, CD28-mediated costimulation may prevent the induction of anergy by facilitating the effective association of TCR zeta and CD3 epsilon with the critical protein tyrosine kinase lck, and the subsequent recruitment of ZAP-70. Strategies to inhibit or activate TCR-associated, specific protein tyrosine kinase-mediated pathways may provide a basis for drug development with potential applications in the fields of transplantation, autoimmunity, and tumor immunity.

3T3 Cells↗
PubMed1996

Pre-B acute lymphoblastic leukemia cells may induce T-cell anergy to alloantigen.

Even if neoplastic cells express tumor associated antigens they still may fail to function as antigen presenting cells (APC) if they lack expression of one or more molecules critical for the induction of productive immunity. These cellular defects can be repaired by physiologic activation, transfection, or fusion of tumor cells with professional APC. Although such defects can be repaired, antitumor specific T cells may still fail to respond in vivo if they may have been tolerized. Here, human pre-B cell acute lymphoblastic leukemia (pre-B ALL) was used as a model to determine if primary human tumor cells can function as alloantigen presenting cells (alloAPC) or alternatively whether they induce anergy. In the present report, we show that pre-B cell ALL express alloantigen and adhesion molecules but uniformly lack B7-1 (CD80) and only a subset express B7-2 (CD86). Pre-B ALL cells are inefficient or ineffective alloAPC and those cases that lack expression of B7-1 and B7-2 also induce alloantigen specific T-cell unresponsiveness. Under these circumstances, T-cell unresponsiveness could be prevented by physiologic activation of tumor cells via CD40, cross-linking CD28, or signaling through the common gamma chain of the interleukin-2 receptor on T cells. Taken together, these results suggest that pre-B ALL may be incapable of inducing clinically significant T-cell-mediated antileukemia responses. This defect may be not only due to their inability to function as APC, but also due to their potential to induce tolerance. Attempts to induce clinically significant antitumor immune responses may then require not only mechanisms to repair the antigen presenting capacity of the tumor cells, but also reversal of tolerance.

Antigen Presentation↗
PubMed1996

Complete blockade of B7 family-mediated costimulation is necessary to induce human alloantigen-specific anergy: a method to ameliorate graft-versus-host disease and extend the donor pool.

Graft-versus-host disease (GVHD) is initiated by adoptively transferred donor T cells that recognize host alloantigens. Whereas the absence of donor T-cell proliferation to host alloantigens in a mixed-leukocyte reaction does not predict freedom from GVHD, the frequency of alloreactive precursor helper T lymphocytes (pHTL) is predictive. Complete blockade of 87 family-mediated costimulation, but not of major histocompatibility complex recognition or adhesion, induces host alloantigenic-specific energy by reducing cytokine production below threshold levels necessary for common gamma chain signaling. The associated reduction of alloreactive pHTL frequency below that predictive for GVHD, without depletion of either nonallospecific T cells or hematopoietic progenitors, has led us to embark upon human clinical trials of haplomismatched allogeneic bone marrow transplantation.

Abatacept↗
PubMed1996

R24 anti-GD3 ganglioside antibody can induce costimulation and prevent the induction of alloantigen-specific T cell clonal anergy.

R24 is a monoclonal antibody directed against the cell surface ganglioside GD3. It can detect GD3 on the surface of a subset of T lymphocytes and can stimulate proliferation and secretion of cytokines in vitro. In the present report, we examined the effects of the R24 antibody upon antigen-specific T cell response, employing an HLA-DR7-specific T cell clonal model. As previously shown, primary stimulation of HLA-DR7-specific alloreactive T cell clones by transfectants expressing HLA-DR7 alone (t-DR7) in the absence of B7 co-stimulation resulted in anergy. Binding of cell surface GD3 on HLA-DR7-specific alloreactive T cell clones with R24 under these anergizing conditions resulted in interleukin-2 (IL-2) accumulation and prevented the induction of alloantigen-specific T cell clonal anergy. Binding of GD3 by R24 also prevented anergy under conditions where B7:CD28 interactions were blocked by CTLA4-Ig. The effect of R24 was abrogated in the presence of a combination of monoclonal antibodies for the alpha and beta chains of the IL-2 receptor (IL-2R) or a neutralizing anti-IL-2 antibody. R24 does not appear to interact directly with the IL-2R since incubation of T cell clones with R24 did not induce early activation of IL-2R associated Jak kinases, Jak1 and Jak3, as was induced following incubation with IL-2. In contrast, incubation of HLA-DR7-specific clones with t-DR7 in the presence of R24 did result in phosphorylation of IL-2R related Jak kinases after 24 h. Our data indicate that the membrane ganglioside GD3 structure recognized by R24 may play an important role in antigen-specific T cell clonal response.

3T3 Cells↗
PubMed1996

Breast cancer-associated antigen, DF3/MUC1, induces apoptosis of activated human T cells.

Given the plethora of well-documented breast carcinoma-associated antigens in humans including MAGE-1, -2 and -3, mutated p53, p21ras, HER-2/neu and DF3/MUC-1, coupled with evidence that humoral and cytotoxic T-cell responses against these antigens exist, the central dilemma facing tumor immunologists is why the host immune response is so inefficient. One possibility is that tumor cells themselves are either inefficient or ineffective antigen-presenting cells (APCs). The failure of tumor cells to function as APCs may be due to their inability to process and present the antigen, the absence or insufficient numbers of adhesion and costimulatory molecules or, potentially, the secretion of inhibitory cytokines. Therefore, we sought to determine whether human breast cancer cell lines could function as APCs and, if not, to identify mechanism(s) responsible for this defect. Here, we show that human breast cancer cell lines fail to present alloantigen. This defect does not reside in their inherent capacity to present antigen but rather is due to apoptosis of activated T cells induced by exposure to the breast carcinoma-associated mucin antigen, DF3/MUC1. These results support the hypothesis that DF3/MUC1 may contribute to the paucity of clinically significant anticarcinoma-specific immune responses.

3T3 Cells↗
PubMed1996

Comparative study of the formation and repair of O6-methylguanine in humans and rodents treated with dacarbazine.

The mutagenic, carcinogenic and cytotoxic activity of dacarbazine, a drug employed in cancer chemotherapy, may be related to the induction in DNA of O6-methylguanine (O6-meG), a quantitatively minor but biologically important lesion. In the present study the kinetics of O6-meG formation and repair in blood leukocyte DNA were examined in 20 Hodgkins lymphoma patients treated i.v. with 180 +/- 13 (mean +/- SD) mg/m2 dacarbazine and compared with those observed in various tissues of rodents treated with different doses of the drug. In Hodgkin's lymphoma patients adduct levels reached a value of 0.27 +/- 0.14 fmol/microgram DNA 2 h after dacarbazine administration, while the rate of subsequent loss suggested an adduct half-life of < or = 30 h. Measurement of adduct levels in the same individuals after successive courses of treatment spaced 3 weeks apart (up to 10 treatment courses) demonstrated a consistent individual response and statistical analysis of variance confirmed that intra-individual variation in adduct accumulation after a given dose of dacarbazine accounted for only 5% of the total variance observed. In contrast, inter-individual variation accounted for 70% of the observed variance, with adduct levels 2 h after drug treatment varying approximately 7.5-fold among adduct-positive individuals. No significant depletion of lymphocyte O6-alkylguanine-DNA alkyltransferase (AGT) occurred after patient treatment with dacarbazine. No significant relationship between adduct levels and clinical response to treatment was observed. In rats treated with single or multiple doses of dacarbazine causing varying degrees of AGT depletion the highest levels of O6-meG were seen in the liver, followed by the lymph nodes, bone marrow and blood leukocytes, which showed up to approximately 2-fold lower levels. A similar tissue distribution was also observed in mice and in a single rabbit. These observations suggest that O6-meG levels assayed in blood leukocytes of therapeutically treated humans reflect those present in the -lymph nodes (target tissue for chemotherapy) and the bone marrow (target tissue for leukaemogenesis) and may be utilized as a measure of the drug dose reaching these tissues. The quantitative data reported in this study show that under conditions of no depletion of AGT O6-meG accumulates in blood leukocyte DNA of humans at a rate similar to that observed in rats, suggesting that human susceptibility to any O6-meG-mediated genotoxic effects of dacarbazine may be comparable with that of the rat.

Animals↗
PubMed1996

Fludarabine monophosphate in refractory B-chronic lymphocytic leukemia: maintenance may be significant to sustain response.

In the present study we report our results on the efficacy of Fludarabine monophosphate in 20 B-chronic lymphocytic leukemia (CLL) patients, refractory to conventional chemotherapy. Of the 20 patients 14 were males and 6 females with a median age of 58 years (44-70). Eight had Binet stage B and 12 stage C. They were previously treated with chlorambucil, prednisone, mini-CHOP or irradiation. Their disease duration prior to fludarabine administration was 49 months (7-180). Fludarabine was given at a dose of 25 mg/m2 daily, for five consecutive days, monthly for six months and if responding for six additional months. Treatment was administered on an outpatient basis. Complete response (CR) was observed in 7 patients (33%) and partial remission (PR) in 5 (25%). Of the complete responders 5 were males and 2 females with a median age of 60 years (range 55-68); three of them had stage B and 4 stage C disease; the median number of fludarabine courses for achieving CR was 3 (range 2-5). In all CR patients a residual monoclonal CD5/CD19 positive lymphocyte population was found in the peripheral blood. All CRs relapsed shortly after discontinuation of therapy within 12 months. The main toxicity observed was upper respiratory tract and/or pulmonary infections in 8 patients, requiring hospitalization. Among the CRs one patient died during the administration of the third course of therapy, due to a severe hypersensitivity reaction with Stevens-Johnson syndrome. The importance of maintenance therapy is also stressed as PR was sustained in some patients using 3 day cycles every 2-4 months. One patient was maintained in this fashion for 60 + months. This study showed that fludarabine is effective in CLL patients refractory to conventional chemotherapy thus it may be given as the treatment of choice if such patients still require treatment.

Adult↗
PubMed1995

CTLA4 mediates antigen-specific apoptosis of human T cells.

The regulation of T cell-mediated immune responses requires a balance between amplification and generation of effector function and subsequent selective termination by clonal deletion. Although apoptosis of previously activated T cells can be induced by signaling of the tumor necrosis factor receptor family, these molecules do not appear to regulate T-cell clonal deletion in an antigen-specific fashion. We demonstrate that cross-linking of the inducible T-cell surface molecule CTLA4 can mediate apoptosis of previously activated human T lymphocytes. This function appears to be antigen-restricted, since a concomitant signal T-cell receptor signal is required. Regulation of this pathway may provide a novel therapeutic strategy to delete antigen-specific activated T cells.

Abatacept↗
PubMed1995

B7-1 and B7-2 do not deliver identical costimulatory signals, since B7-2 but not B7-1 preferentially costimulates the initial production of IL-4.

The functional necessity for two CD28 counterreceptors (B7-1 and B7-2) is presently unknown. B7-1 and B7-2 equivalently costimulate IL-2 and interferon-gamma (IFN gamma) production and IL-2 receptor alpha and gamma chain expression. B7-2 induces significantly more IL-4 production than B7-1, with the greatest difference seen in naive T cells. Repetitive costimulation of CD4+ CD45RA+ T cells with B7-2 results in moderate levels of both IL-4 and IL-2, whereas repetitive costimulation with B7-1 results in high levels of IL-2 and low levels of IL-4. Therefore, B7-1 and B7-2 costimulation mediate distinct outcomes, since B7-2 provides an initial signal to induce naive T cells to become IL-4 producers, thereby directing the immune response more towards Th0/Th2, whereas B7-1 is a more neutral differentiative signal.

3T3 Cells↗
PubMed1994

Pivotal role of the B7:CD28 pathway in transplantation tolerance and tumor immunity.

The above story illustrates the translation of basic scientific discoveries to the clinic. In vitro and preclinical in vivo experimentation suggests that modulation of the B7:CD28 pathway will result in either amplification or suppression of the immune response. Considering the frequency with which diseases characterized by either inadequate or dysregulated immune function present to the practicing hematologist or oncologist, it is not difficult to envisage clinical applications for reagents that modulate this pathway. However, we still have much to learn about the function and clinical potential of this and other potentially redundant costimulatory pathways and therefore we suspect that this story will become considerably more complex over the next few years.

Abatacept↗