Biomedical subjects

C Perreault

Publications and source records attributed to C Perreault.

At least 37 records · Page 2Linked to original sources

PubMed1998

Development of a highly polymorphic STR marker for identity testing purposes at the human androgen receptor gene (HUMARA).

We developed a non-isotopic method which improves the technical quality of the X-linked HUMARA locus typing process. The use of formamide and a low concentration of acrylamide increased resolution and sharpness of HUMARA alleles in silver-stained polyacrylamide gels. In addition, the construction of an allelic ladder containing amplified sequence of 9 alleles (even-numbered alleles) of the HUMARA locus, allows confident, rapid and precise assignment of discretely defined alleles. Allele and genotype frequencies for the HUMARA locus were determined in a French Canadian population sample. Observed genotype frequencies in females conformed to Hardy-Weinberg expectations. Furthermore, the HUMARA locus is highly polymorphic with 18 observed alleles and an heterozygosity value of 89.3%. Also, this locus has average powers of discrimination of 97.8% and 88.7% for testing samples of female and male origin, respectively. In the French Canadian population, the average probability of excluding a random man as the father in paternity analysis when both mother and daughter are tested for this locus is 88.0%. Together, the results indicate that the HUMARA locus provides a highly discriminatory system that is appropriate for the purposes of forensic identification and paternity testing involving a female child.

Alleles↗

PubMed1997

On the mechanisms of immunodominance in cytotoxic T lymphocyte responses to minor histocompatibility antigens.

Although there are numerous minor histocompatibility antigens (MiHA), T cell responses leading to graft-versus-host (GVH) and graft-versus-tumor effects involve only a small number of immunodominant MiHA. The goal of the present study was to analyze at the cellular and molecular levels the mechanisms responsible for MiHA immunodominance. Cytotoxic T lymphocytes (CTL) generated in eight combinations of H2b strains of mice were tested against syngeneic targets sensitized with HPLC-fractionated peptides eluted from immunizing cells. The number of dominant MiHA was found to range from as little as two up to ten depending on the strain combination used. The nature of dominant MiHA was influenced by both the antigen profile of the antigen-presenting cells (APC) and the repertoire of responding CTL. When C57BL/6 dominant MiHA (B6dom) and H-Y were presented on separate APC, they showed similar immunogenicity. In contrast, when they were presented on the same APC, B6dom MiHA totally dominated H-Y. B6dom MiHA did not suppress anti-H-Y responses by acting as T cell receptor antagonists for anti-H-Y CTL, nor were anti-B6dom CTL precursors more abundant than anti-H-Y CTL precursors. Dominance resulted from competition for the APC surface between anti-B6dom and anti-H-Y CTL; the crucial difference between the dominant and the dominated MiHA appears to depend on the differential avidity of their respective CTL for APC. The only B6dom epitope thus far identified is the nonapeptide AAPDNRETF presented by H2-D(b). We found that compared with other known D(b)-binding peptides, AAPDNRETF is expressed at very high levels on the cell surface, binds to the D(b) molecule with very high affinity, and dissociates very slowly from its presenting class I molecule. These data indicate that one cannot predict which MiHA will be dominant or dominated based simply on their respective immunogenicity when presented on separate APC. Indeed, the avidity of T cell/APC interactions appears to determine which antigen(s) will trigger T cell responses when numerous epitopes are presented by the same APC.

Animals↗

PubMed1997

Thymic and extrathymic differentiation and expansion of T lymphocytes following bone marrow transplantation in irradiated recipients.

Thymic function is severely impaired in most marrow transplant recipients. To evaluate the impact of thymic hypoplasia on T cell reconstitution following marrow transplantation, we compared the phenotype and function of T lymphocytes in thymectomized recipients with those of euthymic hosts. Irradiated C57BL/6 mice (Thy1.2+, Ly5.1+) received 10(7) T cell-depleted B6.Ly5.2 bone marrow cells (Thy1.2+, Ly5.2+), with or without 3 x 10(5) B6.PL lymph node cells (Thy1.1+, Ly5.1+) as a source of T lymphocytes. Multiparameter flow cytometry analysis showed that in euthymic mice (group 1), T cell reconstitution was carried out by donor hematopoietic stem cells that differentiated in the host's thymus, whereas the production of chimeric T cells in athymic recipients depended on the presence or absence of T cells in the graft. When T lymphocytes were present in the graft (group 2), their progeny constituted the vast majority of splenic T cells on day 100 posttransplant. When the graft did not contain T lymphocytes (group 3), T cell reconstitution resulted from extrathymic maturation of donor hematopoietic progenitors; T cells differentiating along this pathway expressed lower levels of T cell receptor and a large proportion of the CD8+ subset expressed CD8alpha alpha homodimers. The T cell receptor Vbeta profile of all chimeras was similar to that of normal C57BL/6 mice. Compared with T cells found in euthymic recipients, those in mice from groups 2 and 3 were less abundant (particularly with respect to the CD4+ subset), displayed the CD44/CD45 phenotype of activated memory cells, and expressed high levels of IL-2 receptor beta chain. These results show that both the presence or absence of the thymus and the composition of the grafted inoculum determine the source and extent of posttransplant T cell reconstitution. Because they determine the nature of the differentiation pathway taken during T cell development in the host, these two factors can exert a critical influence on the appearance of graft vs. host disease and the level of host immunocompetence.

Animals↗

PubMed1996

Elimination of neuroblastoma and small-cell lung cancer cells with an anti-neural cell adhesion molecule immunotoxin.

BACKGROUND: The development of immunotoxins has been hampered by difficulties, particularly in solid tumors, of finding appropriate target antigens and of linking sufficiently potent toxins. PURPOSE: We evaluated the tissue specificity of an immunotoxin, N901-blocked ricin (N901-bR), and assessed its potential for eliminating neural cell adhesion molecule (NCAM)-positive tumor cells in conditions appropriate for in vitro purging, prior to autologous stem cell transplantation, and its potential for myelosuppression. N901-bR consists of a monoclonal antibody (MAb), N901, directed against CD56, an antigen of the family of NCAMs, covalently linked to blocked ricin as the cytotoxic effector moiety. METHODS: The tissue specificity of the N901 MAb and the N901-bR immunotoxin was tested against a wide array of human tumor tissues and normal human tissues by immunohistochemical staining. The cytotoxic activity of N901-bR was tested against both small-cell lung cancer (SCLC) cells and neuroblastoma cells, either alone or among normal bone marrow mononuclear cells, and the efficacy of this treatment to specifically eliminate these cells was evaluated in a limiting dilution assay. In addition, normal bone marrow mononuclear cells were incubated with N901-bR, and the toxic effects of the immunotoxin on normal hematopoietic progenitors was evaluated. RESULTS: N901 and N901-bR exhibited specificity for several neoplasms of neuroectodermal origin, including SCLC and neuroblastoma. Staining of normal tissues was essentially limited to various neuroendocrine cells, cardiac muscle cells, and cells in peripheral nerve tissue. We observed a time- and dose-dependent elimination of tumor cells in vitro, with three logs (i.e., > 99.9%) of malignant cells being killed following only 5 hours of exposure to 10 nM N901-bR. Unconjugated N901 MAb specifically blocked the elimination of NCAM-positive cells by N901-bR, whereas neither an isotype-matched control MAb nor galactose (the ligand of native ricin) had any effect on the activity of the immunotoxin, confirming the specificity of its cytotoxic activity. Importantly, N901-bR used under optimal conditions for in vitro tumor cell depletion was not toxic to hematopoietic precursors. CONCLUSIONS: N901-bR has the properties required to target CD56, an antigen present not only on cells from a large number of cancers of neuroendocrine origin, but also on some important normal tissues. In addition, treatment with this immunotoxin results in the highly effective and specific elimination of neuroblastoma and SCLC cells and does not affect normal hematopoietic progenitors. IMPLICATIONS: N901-bR may have clinical utility for purging of neuroblastoma cells and SCLC cells before autologous stem cell transplantation. Further toxicology studies are warranted to assess the potential of N901-bR for in vivo administration.

Antibodies, Monoclonal↗

PubMed1996

Identification of an immunodominant mouse minor histocompatibility antigen (MiHA). T cell response to a single dominant MiHA causes graft-versus-host disease.

T cell responses to non-MHC antigens are targeted to a restricted number of immunodominant minor histocompatibility antigens whose identity remains elusive. Here we report isolation and sequencing of a novel immunodominant minor histocompatibility antigen presented by H-2Db on the surface of C57BL/6 mouse cells. This nonapeptide (AAPDNRETF) shows strong biologic activity in cytotoxic T lymphocyte sensitization assays at concentrations as low as 10 pM. C3H.SW mice primed with AAPDNRETF in incomplete Freund's adjuvant generated a potent anti-C57BL/6 T cell-mediated cytotoxic activity, and T lymphocytes from AAPDNRETF-primed mice caused graft-versus-host disease when transplanted in irradiated C57BL/6 recipients. These results (a) provide molecular characterization of a mouse dominant minor histocompatibility antigen, (b) identify this peptide as a potential target of graft-versus-host disease and, (c) more importantly, demonstrate that a single dominant minor antigen can cause graft-versus-host disease. These findings open new avenues for the prevention of graft-versus-host disease and should further our understanding of the mechanisms of immunodominance in T cell responses to minor histocompatibility antigens.

Amino Acid Sequence↗

PubMed1996

Involvement of nitric oxide in target-cell lysis and DNA fragmentation induced by murine natural killer cells.

Although it has been recognized for sometime that target cells destroyed by natural killer (NK) cells die largely by apoptosis, the underlying mechanisms are not fully understood. The aim of the present study was to examine the role of nitric oxide (NO) in mediating murine NK-cell-induced killing of YAC-1 lymphoma cells. NK calls induced extensive release of 125I-DNA and 51Cr from YAC-1 cells. The target killing ability of NK cells was associated with an increased production of NO as measured by concentrations of nitrite in the culture medium. That YAC-1 killing resulted, in part, from the production of NO was confirmed by the significant protection of cell lysis in L-arginine-depleted medium and by approximately 30 % attenuation of cell lysis and DNA fragmentation by an inhibitor of NO synthase, NG-nitro-L-arginine methyl ester (L-NAME) in a culture medium containing 1 mmol/L L-arginine. Fluorescence microscopic examination of YAC-1 cells showed the presence of changes in nuclear morphology characteristic for apoptosis. The percentage of apoptotic cells was markedly decreased by L-NAME. Further evidence for apoptosis is provided by the specific pattern of internucleosomal DNA fragmentation both in the absence and presence of L-NAME. During target-cell killing, an increased oxidation of intracellularly trapped dichlorofluorescein was observed in cells labeled with an antimouse NK-cell monoclonal antibody, as measured by flow cytometry. These increases were effectively prevented by L-NAME, but not W-13, an inhibitor of calmodulin. The ability of NO to induce cell lysis and DNA fragmentation in YAC-1 cells was further demonstrated by exposing tumor cells to chemically generated NO. Taken together, these observations suggest a role for NO as one of the mediators of NK-cell-mediated DNA fragmentation and cell lysis.

Animals↗

PubMed1996

The COI mitochondrial gene encodes a minor histocompatibility antigen presented by H2-M3.

We found that (LP x C57BL/6)F1 mice could raise a CTL response against parental C57BL/6 cells. These CTLs recognized a maternally transmitted, H2-M3wt-restricted, minor histocompatibility Ag (MiHA) that is widely distributed among many strains of mice and encoded by the COI mitochondrial gene. The wild-type MiHA is the COI N-terminal hexapeptide. Sequencing the 5' end of the COI gene in LP and C57BL/6 mice showed that the LP allele arose by a T-->C transition in the third codon, which caused substitution of threonine for isoleucine. Molecular characterization of this MiHA and the demonstration that it is presented exclusively by H2-M3: 1) support the concept that differential expression of MiHA in MHC-identical animals is caused by polymorphism of the MiHA gene proper; 2) expand our knowledge of the repertoire of self-peptides naturally presented by H2-M3 and show that this MHC class I molecule can present short endogenous peptide ligands; and 3) suggest that mitochondrial DNA mutations that modify the repertoire of H2-M3-associated mitochondrial peptides are representative of mitochondrial DNA mutations in general.

Amino Acid Sequence↗

PubMed1996

Deficient cellular cyclic AMP may cause both cardiac and skeletal muscle dysfunction in heart failure.

Deficient myocardial cyclic AMP concentrations contribute to abnormal Ca2+ handling and systolic and diastolic dysfunction in chronic heart failure (CHF). We tested the hypothesis that decreased cyclic AMP in skeletal muscle of animals with failure may contribute to the weakness and easy fatiguability also common in patients with CHF. We compared intracellular Ca2+ signaling and contractility in skeletal muscle preparations from rats 6 weeks after myocardial infarction-induced CHF versus sham-operated controls. Bundles of 100 to 200 cells were dissected from the extensor digitorum longus (EDL) muscle of control and CHF rats. Muscles from CHF rats exhibited depressed tension development compared with control muscles during twitches. Treatment with 2mM dibutyryl cyclic AMP returned tension and Ca2+ towards normal levels. There was no evidence of cellular atrophy in the CHF rats. In conclusion, EDL skeletal muscle from rats with CHF had intrinsic abnormalities in excitation-contraction coupling that could be reversed with cyclic AMP supplementation as previously reported for the heart. This suggests that deficient cyclic AMP levels may contribute to both cardiac and skeletal muscle dysfunction in CHF.

Animals↗

PubMed1996

Phenotypic and functional characterization of peripheral blood and bone marrow natural killer cells prior to autologous transplantation.

NK cells can exert potent anti-leukemia activity after either autologous or allogeneic BMT. However, in autologous blood or marrow transplant patients, NK cell number and/or function could be reduced, and also may vary according to the sampling site. In order to evaluate the hypothesis that blood or marrow grafts from autologous transplant patients exhibit impaired NK cell activity that could contribute to disease recurrence, we evaluated the immunologic characteristics of NK cells in the bone marrow (BM) and peripheral blood (PB) from 27 patients undergoing autologous BMT, and also from 20 normal donors. We measured baseline and interleukin-2 (IL-2)-activated NK cell cytotoxicity, as well as expression of IL-2 receptors (IL-2R) (alpha-chain (p55) and beta-chain (p75)), and adhesion molecules. The cytotoxic activity of PB NK cells was significantly lower in autologous transplant patients than in normal donors (P < 0.0005) and this difference was not mitigated following IL-2 activation. In contrast, BM from autologous patients showed normal NK cell cytotoxicity, but contained higher numbers of NK cells (P < 0.025), with more intense CD56 expression (P < 0.05). Expression of p75 was lower on BM than on PB NK cells in both patients and normal donors. In addition, induction of p55 by IL-2 was abrogated in autologous PB NK cells. Therefore, depending on the site of harvest and the nature of donor cells (pre-BMT vs normal), our results show significant differences in NK cell number, function, and IL-2 receptor expression. This may affect relapse rates following autologous transplants performed with either PB or BM grafts.

Adolescent↗

PubMed1996

Sequential analysis of early hematopoietic reconstitution following allogeneic bone marrow transplantation with fluorescence in situ hybridization (FISH).

Using in situ hybridization with an X and Y chromosome probe mixture, we have sequentially studied peripheral blood samples from 10 patients (four males/six females) in an HLA-matched allogeneic setting in order to monitor the kinetics of early hematopoietic reconstitution. Interphase cells from smears consisting of purified granulocytic and lymphocytic populations respectively were studied in three patients at 24, 48, 72 and 96 h post-transplant. This period was arbitrarily defined as the immediate post-transplant period. These three patients plus seven others were studied sequentially at days 5, 10, 15, 20, 25 and 50 post-transplant, defined as the intermediate post-transplant period. The X and Y probes were indirectly labelled with rhodamine and fluoresceine isothiocyanate, respectively. Donor neutrophils were detected as early as 24 h post marrow infusion followed by a significant expansion at 48 h. At 96 h post-transplant, the median percentage of donor neutrophils was > 90%. In the immediate post-transplant period, most of the lymphocytes were of recipient origin. However, we have documented a significant expansion in donor lymphocytes, starting at day 5 post-transplant in most patients. Almost complete chimerism for the myeloid and lymphoid lineages was established at days 10 and 25 post-transplant, respectively. All patients engrafted normally according to standard clinical criteria. Follow-up data for those surviving > or = 100 days (eight patients), showed persistence of this pattern of hematopoietic reconstitution in all but one patient. Molecular monitoring of early engraftment has enabled us to unravel a distinct biphasic pattern of myeloid and lymphoid engraftment.

Bone Marrow Transplantation↗

PubMed1995

Oligoclonal expansion of CTLs directed against a restricted number of dominant minor histocompatibility antigens in hemopoietic chimeras.

To understand how T cells respond to allogeneic minor histocompatibility Ags (MiHAs), we studied the fate of Thy-1.1+ lymphocytes, as well as their TCR usage and functional activity, in irradiated LP (Thy-1.2+) recipients transplanted with a mixture of C57BL/6 (Thy-1.2+) hemopoietic progenitors supplemented with either low or high numbers of B6.PL lymphocytes (Thy-1.1+). Mice transplanted with low numbers of T cells experienced a dramatic expansion (> or = 10(5)-fold) of donor Thy-1.1+/CD8+ cells during the first 15 days post-transplant. Flow-cytometric analysis and sequencing of junctional nucleotide sequences showed that the nature of this expansion was oligoclonal and involved primarily one or a few clones using V beta 5.1 or V beta 8.1 TCR elements. Expanded T lymphocyte populations displayed MHC-restricted cytotoxicity for a restricted number of MiHAs, that were found in only two peaks following fractionation of LP MiHAs by reverse-phase HPLC. Expansion of donor T cells was limited to the spleen, and short-lived in these recipients that became healthy long-term chimeras without any signs of graft-vs-host disease (GVHD). In contrast, mice transplanted with high numbers of T cells (GVHD+) showed proliferation of Thy-1.1+ donor cells not only in the spleen, but also in the thymus, and recipients died rapidly of GVHD. These results show that: 1) in the MiHA-incompatible transplantation setting, lack of GVHD cannot be explained simply by the absence of antihost T cell responses, 2) GVHD+ recipients present a massive thymic infiltration by donor mature T lymphocytes, and 3) antihost T cell responses are oligoclonal in nature and targeted to only a few MiHAs. These findings shed new light on the pathogenetic mechanisms involved in GVHD and on the selection of the T cell repertoire involved in response to immunodominant MiHAs.

Amino Acid Sequence↗

PubMed1995

Elimination of B-lineage leukemia and lymphoma cells from bone marrow grafts using anti-B4-blocked-ricin immunotoxin.

Bone marrow is the primary site of disease in patients with acute lymphoblastic leukemia (ALL) and is frequently involved in patients with non-Hodgkin's lymphoma (NHL). At the time of autologous bone marrow transplantation, marrow grafts from patients with leukemia and lymphoma are often still contaminated by malignant cells, even when such patients achieve complete clinical remission. In this study, we evaluated the potential of anti-B4-blocked-ricin (anti-B4-bR) immunotoxin to eliminate residual ALL and NHL cells from bone marrow. Anti-B4-bR binds to the CD19 antigen, which is B-lineage specific, and, at concentrations of 5 x 10(-9) M or greater, could eliminate more than 3 logs of CD19+ Nalm-6 or Namalwa cells in a 20-fold excess of normal irradiated bone marrow after only 5 hr of incubation. This activity was abrogated by the addition of anti-B4 but not by the presence of galactose, which is the natural ligand for native ricin. Also, when used at these high concentrations, anti-B4-bR showed little nonspecific toxicity against normal hematopoietic progenitors. In conclusion, a single short exposure to anti-B4-bR is capable of inducing high levels of depletion of CD19+ leukemia and lymphoma cells without significant nonspecific toxicity against normal marrow progenitors. Therefore, anti-B4-bR offers an interesting approach to the elimination of B-lineage malignant cells prior to autologous bone marrow transplantation.

Antibodies, Monoclonal↗

PubMed1995

Immunodominant minor histocompatibility antigens expressed by mouse leukemic cells can serve as effective targets for T cell immunotherapy.

Numerous minor histocompatibility antigens (MiHAs) show tissue-specific expression and can induce vigorous T cell responses. They therefore represent attractive targets for leukemia immunotherapy mediated by adoptive transfer of T cells. The main objective of this work was to determine whether MiHAs expressed by normal hematopoietic cells were present on leukemic cells and whether they could trigger lysis by cytotoxic T lymphocytes (CTLs). CTL assays showed that mouse leukemic cells of both lymphoid and myeloid lineages were sensitive to CTLs targeted toward some but not all MiHAs. In four out of four strain combinations in which we primed CTLs against immunodominant MiHAs, effectors killed leukemic blasts, whereas no cytotoxicity was observed when CTLs were targeted toward four immunorecessive MiHAs. Testing of HPLC fractions obtained from normal and leukemic cells provided molecular evidence that leukemic blasts expressed only some of the MiHAs found on normal mouse hematopoietic cells. Decreased density of H-2 class I molecules at the surface of leukemic cells suggests that down-regulation of genes encoding either class I molecules or proteins involved in antigen processing played a role in the aberrant expression of MiHAs. In vivo resistance to the leukemic cells by various strains of mice correlated with in vitro CTL activity. These results show that leukemic cells express only some (immunodominant) MiHAs and suggest that this subset of MiHAs represent prime targets for adoptive immunotherapy.

Animals↗

PubMed1995

Distinct patterns of minimal residual disease associated with graft-versus-host disease after allogeneic bone marrow transplantation for chronic myelogenous leukemia.

PURPOSE: Allogeneic bone marrow transplantation (BMT) has been shown to provide effective therapy for chronic myelogenous leukemia (CML), but previous reports have also demonstrated the persistence of bcr-abl-positive cells for months to years after BMT in the majority of patients. To evaluate the biologic significance of persistent bcr-abl-positive cells, we examined the relationship between clinical parameters known to affect the risk of relapse and the ability to detect bcr-abl-positive cells post-BMT. PATIENTS AND METHODS: We analyzed 480 samples from 92 patients at two transplant centers for the presence of bcr-abl-positive cells by polymerase chain reaction (PCR). Two different BMT preparative regimens and protocols for prevention of graft-versus-host disease (GVHD) were used. One center used cyclophosphamide plus total-body irradiation (CY/TBI) and T-cell-depleted marrow; the second center used busulfan plus cyclophosphamide (Bu/CY) and untreated marrow with cyclosporine and methotrexate (Csp/MTX) as GVHD prophylaxis. RESULTS: We first determined the percent of patients at each center with > or = one PCR-positive (PCR+) result at defined intervals post-BMT. Between 0 and 6 months post-BMT, the majority of patients (80% to 83%) in both populations had PCR-detectable bcr-abl-positive cells. Between 6 and 24 months post-BMT, 80% to 88% of patients who received T-cell-depleted marrow remained PCR+, as compared with 26% to 30% of patients who received unmodified marrow. After 24 months post-BMT, the percentage of PCR+ patients was not significantly different in the two populations. This pattern of detection of bcr-abl-positive cells post-BMT followed the development of chronic GVHD in patients who received unmodified marrow. All patients were also divided into three groups based on post-BMT PCR results as follows: (1) persistent PCR+ (n = 29), (2) intermittent PCR-negative ([PCR-] n = 40), and (3) persistent PCR- (n = 23). These three groups were found to have a low, intermediate, and high probability of maintaining remission and disease-free survival, respectively (P = .0001). Intermittent or persistent PCR- results, which reflect levels of minimal residual disease < or = the limit of detection by PCR, were clearly associated with both acute (P = .004) and chronic (P = .000005) GVHD. Nevertheless, 44% of patients without GVHD also had intermittent or persistent PCR- assays. CONCLUSION: The persistence of PCR-detectable bcr-abl-positive cells early post-BMT in more than 80% of patients suggests that neither BMT preparative regimen effectively eradicates CML cells in most patients. Subsequently, acute and/or chronic GVHD are associated with a decreased ability to detect residual bcr-abl-positive cells, which suggests that immunologic mechanisms mediated by donor cells are important for inducing long-term remissions after BMT. The demonstration that 44% of patients without GVHD had either low or undetectable levels of residual leukemia suggests the presence of mechanisms capable of suppression or eradication of CML independent of GVHD.

Acute Disease↗

PubMed1994

Graft-host tolerance in bone marrow transplant chimeras. Absence of graft-versus-host disease is associated with unresponsiveness to minor histocompatibility antigens expressed by all tissues.

Because bone marrow (BM) transplantation is used with increasing frequency, it is important to elucidate the mechanisms involved in the establishment of tolerance to host minor histocompatibility antigens (MiHA) in recipients transplanted with T-cell-undepleted marrow grafts. We have previously shown that BM chimeras transplanted across MiHA barriers showed specific unresponsiveness to MiHA expressed on recipient-type concanavalin A blasts. Because expression of many MiHA is tissue-specific, we wanted to determine if chimera T lymphocytes would be tolerant to MiHA expressed by all host tissues and organs. To investigate this issue, we measured in vivo proliferation of lymphoid cells from normal C57BL/10 (B10) mice and (B10-->LP) chimeras in tissues and organs of lethally irradiated syngeneic and allogeneic recipients. Donor B10 cells were either untreated, or depleted with anti-Thy-1.2, anti-CD4, or anti-CD8 antibodies. Transplantation of B10 cells in LP recipients triggered an important T-cell-dependent 125I-dUrd uptake in several organs that involved both CD4+ and CD8+ cells. Using Thy-1-congeneic mice we showed that in long-term chimeras practically all CD4+ and CD8+ T lymphocytes were derived from hematopoietic progenitors and not from mature T cells present in the BM graft. When (B10-->LP) BM chimera cells were injected to secondary recipients, no proliferation was observed in any organ of LP hosts whereas normal proliferation was seen in H-2k allogeneic hosts. Thus, in these BM chimeras, tolerance encompasses MiHA expressed by all organs.

Animals↗

PubMed1994

T lymphocyte responses to multiple minor histocompatibility antigens generate both self-major histocompatibility complex-restricted and cross-reactive cytotoxic T lymphocytes.

Minor histocompatibility antigens (MiHA) may represent ideal targets for cancer immunotherapy since (1) the expression of many MiHA is tissue specific and (2) they can trigger potent T lymphocyte responses. A primary objective of our research program is to characterize T cell responses to cells displaying multiple incompatible MiHA. Early in the course of this work, we observed in various stimulator/responder combinations that immunization versus multiple MiHA generated cytotoxic effectors that killed not only stimulator cells but also a large panel of MHC-identical and MHC-different targets. To characterize the cells responsible for this cytotoxic activity and their specificity, we expanded polyclonal and clonal CD3+ CD4- CD8+ LP anti-C57BL/6 effectors. LP anti-C57BL/6 polyclonal effectors (LPTc cell line) showed strong cytotoxic activity when tested against several H-2b and non-H-2b targets, but displayed, respectively, weak or absent cytotoxicity against MHC class I-deficient cells and syngeneic cells. When used as cold targets, C57BL/6 cells inhibited the lysis of all H-2b and non-H-2b cells. Some H-2b, but no H-2d or H-2k, cold targets inhibited the lysis of C57BL/6 targets. With the exception of LP and C57BL/6, all types of H-2b cells (A.BY, D1.LP and C3H.SW) showed complete reciprocal inhibition of lysis. The same observation was made for non-H-2b targets. The cytotoxicity profile of 12/14 LP anti-C57BL/6 clones was identical to that of the LPTc cell line, while 2/14 clones recognized only H-2b cells. Cytotoxicity was inhibited by incubation of effector cells with anti-CD3 or anti-CD8 antibodies and by incubation of target cells with specific anti-MHC class I antibodies. These results show that immunization against multiple MiHA in the context of self-MHC generates 2 types of CTL: some are strictly self-MHC restricted while others are strongly cross-reactive and recognized MHC-peptide complexes on allogeneic MHC-different targets. This observation has significant implications concerning the use of anti-MiHA T cells in cancer immunotherapy.

Animals↗

PubMed1994

The role of MHC-associated self-peptides in transplantation and immunosurveillance.

The antigen-binding site of most major histocompatibility complex (MHC) class I and II antigens is occupied by self-peptides that are derived from the proteolysis of endogenous proteins following instructions provided by the molecules of the MHC themselves. Together with MHC proteins, self-peptides define our immunological self and shape the repertoire of both T cells that recognize "nonself," and NK cells that may recognize "no self." Endogenous proteins of all cell compartments (nucleus, cytosol, organelles, surface membrane) can yield self peptides whose expression may be either ubiquitous or lineage-specific. Their expression allows the binary recognition mechanism of T and NK cells to check the integrity of the cell genome. A better understanding of the molecular bases of the distinction between self and nonself permits us to anticipate the possibility of modifying their expression and/or their recognition in order to: (i) make the nonself acceptable as self, thereby establishing specific transplantation tolerance, (ii) reestablish tolerance of the self lost in autoimmune diseases, and (iii) induce the rejection as nonself of neoplastic cells. These objectives are particularly pertinent to the area of bone marrow transplantation, where the ultimate goal is aimed at modulating host cell allorecognition in such a way as to both potentiate the graft-versus-leukemia reaction and prevent GVHD.

Animals↗

PubMed1993

Acute graft-versus-host disease prophylaxis with methotrexate and cyclosporine after busulfan and cyclophosphamide in patients with hematologic malignancies.

The combination of two powerful immunosuppressive agents, methotrexate (MTX) and cyclosporine (CSP), has resulted in a significant decrease in the morbidity and mortality after allogeneic bone marrow transplantation (BMT). However, the additive toxicities from ablative preparative regimens may lead to suboptimal use of this combined immunoprophylaxis. We evaluated the efficacy and feasibility of administering MTX/CSP with busulfan (4 mg/kg/d for 4 days) and cyclophosphamide (50 mg/kg/d for 4 days) (BuCy4) in 101 consecutive patients with hematologic malignancies categorized into high- and low-risk groups receiving HLA-matched marrow grafts. Postgrafting immunosuppression consisted of MTX short course (15 mg/m2 on day 1 and 10 mg/m2 on days 3, 6, and 11) and intravenous CSP (1.5 mg/kg every 12 hours). Eighty-three patients (82.1%) received 100% of MTX calculated dose and 87 (86.1%) achieved a CSP therapeutic level (250 to 600 ng/mL) within a median of 16 days. Seventy-three patients (72.2%) received optimal immunosuppressive therapy comprising a full MTX course and achieving CSP therapeutic concentrations. The Kaplan-Meier estimated incidence of grade II to IV acute graft-versus-host disease (GVHD) was 9.2% for all patients and 5.5% in patients receiving optimal GVHD prophylaxis. Eighty-nine patients (88.2%) survived > or = 100 days posttransplant and 43 (48.3%) developed chronic GVHD, the majority of which were de novo (31 of 43). The estimated incidence of relapse was 28.9% for all patients and 14.8% in the low-risk group, with a median follow-up of 24.5 months. High-risk features and the absence of chronic GVHD were significantly associated with relapse (P = .002 and .036, respectively) in multivariate analyses. Projected disease-free survival at 2 years was 52.3% for all patients and 65.2% in low-risk patients. Disease-free survival was significantly improved in optimally treated patients (P = .03) due to a lower incidence of early deaths from acute GVHD and infectious episodes. In conclusion, optimal delivery of MTX/CSP in association with BuCy4 resulted in a near complete abrogation of acute GVHD in HLA-matched transplants and a significantly improved disease-free survival.

Adult↗