Judith Gan

Tolerance induction by megadose hematopoietic transplants : Donor-type human cd34 stem cells induce potent specific reduction of host anti-donor cytotoxic T lymphocyte precursors in mixed lymphocyte culture'

BACKGROUND Recently, the use of megadoses of CD34+ hematopoietic progenitors has been reported to abrogate resistance to engraftment, thus overcoming major histocompatibility barriers in bone marrow transplantation in leukemia patients. METHODS The ability of human CD34+ cells to possess potent tolerizing activity was studied by limiting dilution analysis of cytotoxic T lymphocyte (CTL) precursors (CTL-p) in human peripheral blood lymphocytes after addition of purified CD34+ cells. RESULTS The addition of purified human CD34+ cells to primary mixed lymphocyte culture led to a marked reduction of antiallogeneic CTL-p frequency against stimulator cells of the same origin, compared with the response against cells of third-party origin. The CD34+ cells caused a marked inhibition of the CTL activity, when added at an equal number with the responder T cells, and they were still present after the mixed lymphocyte culture, which suggests that no significant killing of CD34+ cells had occurred. The tolerizing activity is abrogated by irradiation and requires cell contact. This pattern of tolerization most closely resembles what has been ascribed to veto cells in other systems. Phenotypic analysis of the purified CD34+ cells showed that they express MHC class I and class II antigens, but do not express costimulatory molecules of the B7 family. CONCLUSIONS It is possible, that CD34+ cells in the megadose transplants-perhaps by their inability to provide costimulatory molecules-are actively reducing the frequency of CTL-p directed against their antigens, and thereby help to overcome allogeneic rejection, and enhance their own engraftment.

Hsp90 Recognizes a Common Surface on Client Kinases

Hsp90 is a highly abundant chaperone whose clientele includes hundreds of cellular proteins, many of which are central players in key signal transduction pathways and the majority of which are protein kinases. In light of the variety of Hsp90 clientele, the mechanism of selectivity of the chaperone toward its client proteins is a major open question. Focusing on human kinases, we have demonstrated that the chaperone recognizes a common surface in the amino-terminal lobe of kinases from diverse families, including two newly identified clients, NFκB-inducing kinase and death-associated protein kinase, and the oncoprotein HER2/ErbB-2. Surface electrostatics determine the interaction with the Hsp90 chaperone complex such that introduction of a negative charge within this region disrupts recognition. Compiling information on the Hsp90 dependence of 105 protein kinases, including 16 kinases whose relationship to Hsp90 is first examined in this study, reveals that surface features, rather than a contiguous amino acid sequence, define the capacity of the Hsp90 chaperone machine to recognize client kinases. Analyzing Hsp90 regulation of two major signaling cascades, the mitogen-activated protein kinase and phosphatidylinositol 3-kinase, leads us to propose that the selectivity of the chaperone to specific kinases is functional, namely that Hsp90 controls kinases that function as hubs integrating multiple inputs. These lessons bear significance to pharmacological attempts to target the chaperone in human pathologies, such as cancer.

Hsp90 restrains ErbB-2/HER2 signalling by limiting heterodimer formation

ErbB‐2/HER2 is an oncogenic tyrosine kinase that regulates a signalling network by forming ligand‐induced heterodimers with several growth factor receptors of the ErbB family. Hsp90 and co‐chaperones regulate degradation of ErbB‐2 but not other ErbB members. Here, we report that the role of Hsp90 in modulating the ErbB network extends beyond regulation of protein stability. The capacity of ErbB‐2 to recruit ligand‐bound receptors into active heterodimers is limited by Hsp90, which is dissociated from ErbB‐2 following ligand‐induced heterodimerization. We show that Hsp90 binds a specific loop within the kinase domain of ErbB‐2, thereby restraining heterodimer formation and catalytic function. These results define a role for Hsp90 as a molecular switch regulating the ErbB signalling network by limiting formation of ErbB‐2‐centred receptor complexes.

Defective ubiquitinylation of EGFR mutants of lung cancer confers prolonged signaling.

Several distinct mutations within the kinase domain of the epidermal growth factor receptor (EGFR) are associated with non-small cell lung cancer, but mechanisms underlying their oncogenic potential are incompletely understood. Although normally ligand-induced kinase activation targets EGFR to Cbl-mediated receptor ubiquitinylation and subsequent degradation in lysosomes, we report that certain EGFR mutants escape this regulation. Defective endocytosis characterizes a deletion mutant of EGFR, as well as a point mutant (L858R-EGFR), whose association with c-Cbl and ubiquitinylation are impaired. Our data raise the possibility that refractoriness of L858R-EGFR to downregulation is due to enhanced heterodimerization with the oncogene product HER2, which leads to persistent stimulation.

Tolerance Induction by Veto CTLs in the TCR Transgenic 2C Mouse Model. II. Deletion of Effector Cells by Fas-Fas Ligand Apoptosis

The direct assay of veto CTLs in the 2C mouse model enables monitoring, by FACS, the fate of the TCR transgenic effector CD8+ T cells, the transgene of which can be stained with clonotypic Ab 1B2. After the addition of veto cells, CD8+1B2+ effector cells increasingly express annexin V, and maximal apoptosis is attained 72 h after initiation of MLR. This veto activity can be partially blocked by anti-CD8 Abs directed against the allele expressed by the veto CTLs, but not by the effector cells. When effector CD8+ T cells were from 2C mice, which lack Fas expression ((2CX lpr)F2), deletion of effector cells was not exhibited by veto cells. The protein levels of the apoptosis inhibitors FLIP and Bcl2 in purified CD8+1B2+ effector cells at different time points after MLR showed an initial up-regulation of these inhibitors, with marked reduction of FLIP, but not of Bcl2, by 48 h after initiation of culture. Taken together, these results are in accordance with a Fas-FasL-based mechanism in which prolonged binding between the effector cell and the veto cell might be required to allow FLIP to be down-regulated. Such prolonged interaction might be afforded through the interaction of CD8 molecules on the veto cell with the α3 domain of H2 class 1 on the effector cell.

Tolerance Induction by Veto CTLs in the TCR Transgenic 2C Mouse Model. I. Relative Reactivity of Different Veto Cells

Several bone marrow cells and lymphocyte subpopulations, known as veto cells, were shown to induce transplantation tolerance across major histocompatibility Ags. Due to the low frequency of the effector T cells against which the veto cells inhibitory activity is aimed, the fate of the effector cells was traditionally followed indirectly by functional limiting dilution assays, which are cumbersome and depend on numerous parameters. In the present study the fate of the effector T cells was monitored directly by FACS, using TCR transgenic mouse CD8+ T cells in which the transgene is directed against H-2d (the 2C model). This assay is validated by demonstrating the potency, selectivity, radiation sensitivity, and contact dependency of anti-third-party CTLs previously demonstrated by the limiting dilution assay. In contrast to veto CTLs, nonactivated CD8+ T cells lack veto activity. Comparison by FACS in the 2C model revealed a hierarchy of veto cells, in the order of veto CTLs activated NK cells, activated CD4+ T cells, and activated B cells. The latter cells as well as nonactivated CD4+ or NK cells were shown to be completely devoid of veto activity.

Donor-type chimerism determination by competitive polymerase chain reaction (PCR) in a primate model for bone marrow transplantation.

BACKGROUND Evaluation of the outcome of successful bone marrow transplantation (BMT) and in-depth studies of transplantation biology rely increasingly on accurate detection of donor origin cells in the transplanted recipients. This study describes a quantitative competitive polymerase chain reaction (PCR) assay for accurate evaluation of chimerism after allogeneic BMT in a cynomologous primate model, based on detection of monkey Y-specific DNA. METHODS A competitor standard was generated via PCR using a mutagenic primer that makes the competitor DNA 22 bp less than the wild type monkey Y-specific DNA. The mutated form can still be amplified by the primer pair for the detection of monkey Y-specific DNA. A fixed amount of sample subjected to chimerism detection was co-amplified with a range of competitor DNA using a touch down program and hot start PCR technique. The PCR products were analyzed by computing densitometry. The ratio of competitor/target (Y-specific) DNA for each sample pair was calculated. RESULTS Using DNAs prepared from an artificial mixture of male and female cells, a set of standard curves has been obtained and the sensitivity of the established quantitative PCR was found to be 25 pg of male DNA, which corresponds approximately to 0.005 fg competitor DNA. A DNA sample taken from a female monkey, transplanted with purified CD34+ stem cells from a male monkey donor 26 days after BMT, was subjected to the competitive PCR with 10% male DNA as a control; the level of male DNA in this sample was calculated to be around 50%. CONCLUSIONS This quantitative PCR assay offers both a high degree of specificity as well as a very accurate and sensitive evaluation of chimerism in a sex-mismatched monkey BMT model.