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Biomedical subjects

D J Schendel

Publications and source records attributed to D J Schendel.

At least 19 recordsLinked to original sources
PubMed1998

Human renal cell carcinoma antigen-specific CTLs: antigen-driven selection and long-term persistence in vivo.

Renal cell carcinomas (RCCs) are thought to be immunogenic, because cytokine-induced and even spontaneous tumor regression has been observed in a significant number of patients. However, little is known about the nature of immune responses that might lead to tumor regression. We studied naturally arising human T-cell responses against RCC by combining molecular analyses of T-cell receptor (TCR) usage in primary tumors in situ with functional analyses of tumor-infiltrating lymphocytes (TILs) in vitro. TILs of patient 26 that were cultured in vitro showed a human leukocyte antigen (HLA-A*0201)-restricted cytotoxic activity specific for autologous tumor cells. These tumor-derived lymphocytes were dominated by a family of T cells expressing V alpha20- and V beta22-positive TCRs. Their specificity-conferring third complementarity-determining regions were highly homologous with respect to the loop length and selection of particular amino acids in both TCR chains. These characteristics are similar to those reported for antigen-selected murine T cells recognizing immunodominant epitopes of non-self proteins. To evaluate the biological significance of these CTLs in vivo, we analyzed the corresponding TCR transcripts in the cryopreserved tumor material of patient 26 and in a second HLA-A*0201-positive RCC patient whose tumor cells were also lysed by TIL-26. The in situ TIL populations of both patients used related families of highly homologous TCRs, supporting the contention that immunodominant responses directed against a shared tumor-associated antigen occurred in both individuals in vivo. Furthermore, in the absence of overt metastatic disease, the tumor antigen-specific CTLs of patient 26 were shown to persist in the periphery 4 years after removal of the primary tumor. These results demonstrate that antigen-driven T-cell responses specific for spontaneously arising carcinomas developed in these patients and showed long-term persistence, even in the absence of immunotherapy.

Amino Acid Sequence↗
PubMed1998

Double suicide gene (cytosine deaminase and herpes simplex virus thymidine kinase) but not single gene transfer allows reliable elimination of tumor cells in vivo.

Suicide genes such as cytosine deaminase (CD) and herpes simplex virus thymidine kinase (TK) encode products that convert nontoxic substances (prodrugs) into toxic metabolites. Suicide gene transfer is currently being used in cancer therapy or can be used as a safety modality. To analyze the reliability of suicide genes as a safety modality for a vaccination study with viable cytokine/B7 gene-modified tumor cells, the individual and combined efficacy of the two suicide genes was compared for in vitro and in vivo cell killing of a murine mammary adenocarcinoma cell line (TS/A). To adapt the system to an in vivo gene delivery situation, bulk cultures cotransfected with the CD and TK gene were used instead of selected clones. In vitro, both CD and TK conferred sensitivity to the respective prodrug but the combined cytotoxic effects of both gene products were always superior. For in vivo analysis BALB/c mice were injected subcutaneously with CD- and TK-modified TS/A cells, treated with prodrugs, and tumor size was evaluated for a period of 100 days. In the in vivo situation the combination of both enzyme/prodrug systems was again most effective. The highest single concentration of 5-FC (500 mg/kg) or GCV (100 mg/kg) was not able to fully protect the animals from developing tumors, whereas a combination of 5-FC (250 mg/kg) and GCV (50 mg/kg) resulted in complete tumor eradication. In nude mice treated in the same way, most CD/TK tumors could not be eliminated. Furthermore, BALB/c mice cured of TS/A-CD/TK tumors developed a systemic tumor immunity against challenge with parental TS/A cells. These findings indicate that reliable tumor elimination by the suicide genes depends on T cells. The cooperative effect of both suicide genes was confirmed in vitro with the human renal cell carcinoma line RCC26. We conclude that TK and CD together, but neither gene alone, act as a safety mechanism for the elimination of tumor cells in a reliable fashion and suggest that a rapid and quantitative antigen release by effective TK- and CD-mediated tumor destruction is necessary for T cell immunity to develop.

Adenocarcinoma↗
PubMed1997

Identification of naturally processed T cell epitopes from glutamic acid decarboxylase presented in the context of HLA-DR alleles by T lymphocytes of recent onset IDDM patients.

Glutamic acid decarboxylase (GAD) has been defined as a major target antigen in insulin-dependent diabetes mellitus (IDDM). To identify the molecular ligands triggering a T cell response to GAD, a panel of human GAD65-specific T lymphocyte lines was generated from peripheral blood of three recent onset IDDM patients. All lines derived from a patient expressing the high-risk-conferring HLA-DR*0301/ *0401 haplotypes recognized a single epitope localized between amino acid positions 270 and 283 of GAD65, a stretch that is located in close proximity to the homology region shared with Coxsackie virus P2-C protein. All lines with this specificity were restricted to the DRA, B1*0401 product of the DR4 haplotype. Analysis of the GAD-specific T cell response in a second patient homozygous for DR4 haplotypes demonstrated that the same DRA, B1*0401 allele selected T cells specific for a different determinant. The T cell response profile in a third patient showed that DR*1501/ *1601-encoding haplotypes could present at least three different epitopes to GAD65-specific T lymphocytes. One of these epitopes was presented by a DR allele associated with the resistance-conferring DRB1*1501 haplotype. GAD-specific T cell lines could not be isolated from HLA class II-matched normal individuals. Our data reveal that (a) the T cell response to GAD65 is quite heterogenous in recent onset IDDM patients; (b) HLA-DR, not DQ, seems to be the principal restriction element used by T cells present at the onset of the disease; and (c) T cells responding to epitopes containing identical sequences to Coxsackie virus P2-C protein were not detected.

Alleles↗
PubMed1997

HLA-C revisited. Ten years of change.

During the past 10 years knowledge about the interactions between major histocompatibility complex (MHC) class I molecules and the T-cell receptor (TCR) complex of cytotoxic T-cells (CTL) has developed dramatically. But the primary interest, both with respect to structure as well as function, has concentrated on HLA-A and -B molecules because of their high sequence polymorphism and their dominating presence at the cell surface. In contrast, HLA-C molecules seemed to be of only minor importance in the cascade of immune reactions owing to their more limited polymorphism and reduced levels of surface expression. The inability to define a number of antigen specificities had the result that HLA-C molecules were often neglected in studies of immune response, transplantation, and disease association. More recently a new function has been identified for HLA class I molecules where they act as inhibitors of the lytic capacity of natural killer (NK) cells and non-MHC-restricted T-cells. Moreover, the understanding of this novel mode of negative regulation of cytotoxicity was remarkably influenced by HLA-C since these were the first HLA class I molecules found to have such inhibitory potential. With this new inhibitory function serving as an essential component of the immune system, HLA-C molecules can no longer be neglected.

Alleles↗
PubMed1997

Cellular and molecular analyses of major histocompatibility complex (MHC) restricted and non-MHC-restricted effector cells recognizing renal cell carcinomas: problems and perspectives for immunotherapy.

Renal cell carcinomas belong to the small group of tumors that are able to induce antitumor responses. Here we describe two general types of cytotoxic effector lymphocytes that can eliminate autologous tumor cells and discuss the role that major histocompatibility complex encoded molecules play in governing their specificities. Improved understanding of the cellular and molecular basis of renal cell carcinoma recognition opens new avenues of research with the potential to develop better immunotherapies for patients with metastatic disease.

Antigen-Antibody Reactions↗
PubMed1995

Expression of HLA-C molecules confers target cell resistance to some non-major histocompatibility complex-restricted T cells in a manner analogous to allospecific natural killer cells.

Specific HLA molecules have recently been shown to confer target cell resistance to lysis by some CD3- natural killer (NK) cells. For certain NK clones, resistance is governed by two specificities (NK1 and NK2) that are associated with particular HLA-C alleles: in general, target cells expressing Cw1, Cw3, Cw7, or Cw8 are susceptible to NK1 but resistant to NK2 clones, whereas target cells expressing Cw2, Cw4, Cw5, or Cw6 are susceptible to NK2 and resistant to NK1 cells. These two clusters of HLA-C alleles are distinguished by a dimorphism in the alpha 1 helical region, localized at amino acid positions 77 and 80. In this report, we show that highly enriched CD3+/CD56- cytotoxic T cell sublines and CD3-/CD56+ NK sublines derived from the same donor have identical cytolytic specificities when tested against a panel of allogeneic LCL and various HLA-B and -C transfectant cell lines. The lysis pattern of the allogeneic cells appeared to be related to the NK2 specificity for both effector cells: LCL expressing HLA-Cw2, Cw4, Cw5, or Cw6 alleles were lysed, while LCL expressing HLA-Cw1, Cw3, or Cw7 molecules were resistant. Resistance to lysis could be conferred to susceptible target cells by transfection with a Cw*0702 gene, while expression of a Cw*0602 gene did not provide protection. Similar patterns of HLA-C-mediated resistance were also found with two polyclonal T cell lines generated from the peripheral blood lymphocytes of unrelated donors. Thus, major histocompatibility complex (MHC) molecules that induced resistance to particular NK cells also regulated target cell resistance to lysis by these non-MHC-restricted effector T cells. For both types of effector cells, direct binding to HLA-C molecules was necessary to achieve inhibition since preincubation with mAb specific for class I molecules destroyed the protection from lysis of HLA-Cw7 expressing target cells. mAbs specific for CD3 and CD8 molecules had no influence on lysis or inhibition of the NK-like T cells. Formation of MHC complexes with particular peptides did not appear to be essential to confer resistance, since a cell line with defective peptide transporter genes (TAP genes), when transfected with an appropriate HLA-C allele, was as resistant to lysis as HLA-C transfectant lines with normal TAP function. These results suggest that HLA-C molecules may deliver negative regulatory signals to some non-MHC-restricted T cells in a manner similar to that described previously for particular NK cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

In vivo expansion of HLA-B35 alloreactive T cells sharing homologous T cell receptors: evidence for maintenance of an oligoclonally dominated allospecificity by persistent stimulation with an autologous MHC/peptide complex.

The nature of alloantigens seen by T lymphocytes, in particular the role of peptides in allorecognition, has been studied intensively whereas knowledge about the in vivo emergence, diversity, and the structural basis of specificity of alloreactive T cells is very limited. Here we describe human T cell clones that recognize HLA-B35 alloantigens in a peptide-dependent manner. TCR sequence analysis revealed that several of these allospecific clones utilize homologous TCR: they all express TCRAV2S3J36C1 and TCRBV4S1J2S7C2 chains with highly related CDR3 sequences. Thus peptide-specific alloreactivity is reflected in homologous CDR3 sequences in a manner similar to that described for T cells that recognize nominal peptide/self-MHC complexes. The in vivo frequency of this TCR specificity was studied in unstimulated PBL of the responding cell donor who was not sensitized against HLA-B35. The vast majority (approximately 75%) of the VA2S3J36 junctional regions obtained from two samples of PBL, isolated at a 9-yr interval, encode CDR3 identical or homologous to those of the functionally characterized HLA-B35 allospecific T cells. These data are most easily explained by a model of alloreactivity in which persistent or recurrent exposure to a foreign peptide/self-MHC complex led to the in vivo expansion and long-term maintenance of specific T cells that show fortuitous crossrecognition of an HLA-B35/peptide complex and dominate the alloresponse against HLA-B35.

Amino Acid Sequence↗
PubMed1994

HLA-DQ-restricted, islet-specific T-cell clones of a type I diabetic patient. T-cell receptor sequence similarities to insulitis-inducing T-cells of nonobese diabetic mice.

We established a T-cell line and 20 CD4+ T-cell clones from the peripheral blood lymphocytes of a type I diabetic patient using a membrane preparation of a rat insulinoma cell line (beta membrane antigen [BMA]) as a source of antigen. The T-cell line and three selected clones proliferated specifically to stimulation with BMA and to membranes prepared from human islets, rat pancreas, and NOD pancreas, but not to control antigens. Proliferation-inhibition studies using human leukocyte antigen (HLA)-specific monoclonal antibodies revealed HLA-DQw1-restricted recognition of BMA. An analysis of the T-cell receptor (TCR) repertoire of the T-cell line after 8 and 40 days of culture showed a prominent usage of the V alpha 1 and V alpha 12 gene families. Although sequencing of the TCR V alpha and V beta chains of the three clones demonstrated that each used different V alpha and V beta gene segments, structural similarities were found in complementary-determining region 3 (CDR3), the region that is postulated to interact with the peptide component of the TCR ligand. When we compared these TCR sequences with published sequences of disease-inducing T-cells of NOD mice, highly related TCR V beta families were detected. Furthermore, stretches of identical amino acids within the CDR3 region were found between two pairs of human and mouse T-cells. If one considers the species differences in TCR genes and sequence differences in the restriction elements for these cells (HLA-DQ vs. H-2 I-A nod), these sequence similarities are striking and may be useful for pinpointing T-cells of primary importance in the development of disease.

Amino Acid Sequence↗
PubMed1994

MHC class I allorecognition: the likes and dislikes of CTL and NK cells.

An allogeneic culture was established using cells of two HLA disparate individuals. No pattern of specificity could be discerned when the 6 day culture that contained a mixture of CD4+, CD8+ and CD56+ lymphocytes was tested for cytotoxicity against a panel of target cells. Three approaches were utilized to dissect this alloresponse. A CD8+ population was selected and expanded as a line. This CTL population had predominant specificity for HLA-B35 and showed differential recognition of molecularly defined B35 subtypes. A set of CD8+ T cell clones was selected that recognized B35 transfectant cells. These clones were shown to discriminate among B35 molecular subtypes and to be peptide dependent, presumably recognizing a variety of endogenous peptides expressed in human cells. A second component of the response seemed to be mediated by activated CD56+ lymphocytes. Here strong cytotoxicity was found to be directed against MHC class I mutant cell lines, such as 721.221 and C1R, as well as against some allogeneic target cells. Transfection of Cw7 DNA into the mutant cell lines led to resistance to lysis. A hierarchy in susceptibility was found to correlate with HLA-C type in the unrelated panel: cells expressing HLA-Cw7 were found to be most resistant to lysis by this effector cell population.

Animals↗
PubMed1993

Tumor-specific lysis of human renal cell carcinomas by tumor-infiltrating lymphocytes. I. HLA-A2-restricted recognition of autologous and allogeneic tumor lines.

Metastatic renal cell carcinoma (RCC), like melanoma, belongs to the small group of human tumors in which partial or complete remission has been observed in some patients after treatment with various forms of immunotherapy. In contrast to melanoma, CTL showing MHC-restricted lysis of RCC have not been easily found among tumor-infiltrating lymphocytes (TIL). This has led to the suggestion by some that responses to immunotherapy are mediated predominantly by non-MHC-restricted effector cells. We have characterized an MHC-restricted, CD8+ CTL line obtained from an uncloned TIL population of a primary RCC using a low concentration of rIL-2; in fact, these CTL represented a majority of the short-term cultured TIL population. The CTL lysed autologous tumor cells but not normal kidney cells or target cells sensitive to non-MHC-restricted effector cells. In contrast, lymphokine-activated killer (LAK) cells grown in a high concentration of rIL-2 from the patient's PBL lysed autologous tumor and normal kidney cells in addition to several allogeneic tumors. The TIL could be expanded optimally using an autologous tumor line retrovirally transduced with the human cDNA encoding IL-2. TIL were 20-fold more potent than LAK cells in eliminating the IL-2 expressing tumor cells in vitro. The cultured TIL utilized a restricted number of V alpha gene families, suggesting that they may recognize only a limited number of MHC-peptide complexes presented by autologous tumor cells. HLA-A2 was identified as an MHC restriction molecule for presentation of one tumor-derived peptide to these CTL. Only some allogeneic HLA-A2 RCC tumors were lysed. Sequencing of the second and third exons of the HLA-A2 alleles of these cells revealed that both heterogeneity in MHC and peptide availability influenced CTL recognition. These studies demonstrate that some RCC express common antigenic determinants that can be recognized by MHC-restricted CTL and open the possibility of defining the nature of RCC-derived peptides, which, combined with HLA-A2, can generate specific immune responses.

Antibodies, Monoclonal↗
PubMed1993

Tumor-specific lysis of human renal cell carcinomas by tumor-infiltrating lymphocytes: modulation of recognition through retroviral transduction of tumor cells with interleukin 2 complementary DNA and exogenous alpha interferon treatment.

Two cytotoxic effector cell populations were isolated from a patient with renal cell carcinoma. The tumor-infiltrating lymphocytes comprised a population of highly specific, major histocompatibility complex-restricted, cytotoxic T lymphocytes (CTL). An autologous non-major histocompatibility complex-restricted lymphokine-activated killer (LAK) cell population was generated by culturing the peripheral blood lymphocytes with high doses of recombinant interleukin 2 (rIL-2). The capacity of these two effector cell types to lyse cytokine-modulated autologous tumor cells was compared in vitro. A complementary DNA for rIL-2 was introduced into the tumor cells by retroviral transduction, and tumor cells secreting low doses of rIL-2 were isolated. The CTL recognition of these tumor cells was enhanced, compared to unmodified tumor cells, whereas LAK cell recognition was unchanged or slightly reduced. Pretreatment of tumor cells with exogenous alpha interferon led to an up-regulation of some major histocompatibility complex class I molecules and to slightly better recognition by the CTL; little effect on LAK cell recognition was observed. CTL were found to be 50-150-fold more effective than LAK cells in lysing autologous tumor cell lines or clones modulated with both rIL-2 and alpha interferon. The assessment of a patient's cytotoxic immune capacity directed against genetically modified autologous tumor cells in vitro provides important insight for cytokine-mediated gene therapy of cancer.

Antibodies, Monoclonal↗
PubMed1993

Soluble T cell receptor-like properties of an HLA-B35-specific monoclonal antibody (TU165).

A mouse monoclonal antibody of IgM class (TU165) was produced using Epstein-Barr virus (EBV)-infected mutant cells derived from the human BJAB-B95.8.6 cell line as immunogen. Binding studies with several HLA deletion mutant cell lines indicated that TU165 recognized the HLA-B35 molecule. In a panel of 89 EBV-transformed lymphoblastoid cell lines, all HLA-B35+ cells (n = 24) reacted with TU165 while all but two HLA-B35- lines (n = 65) were unreactive (r = 0.95). Surprisingly, peripheral blood lymphocytes of HLA-B35+ donors were unreactive; however, strong enhancement of TU165 recognition was observed with B cells of one of these individuals after transformation with EBV (B95.8 strain). Transfection of both HLA-B35 and human beta 2-microglobulin genomic DNA into mouse P815 cells led to high expression of HLA-B molecules; yet, expression of the TU165 epitope was not observed. Furthermore, the EBV-negative cell line BJAB as well as the EBV-infected (P3HR1 strain) line BJAB-HR1K were only weakly reactive, whereas the BJAB-B95.8 cell line was strongly positive. These results indicate that EBV-encoded or -controlled peptide(s) must be bound by HLA-B35 antigens to create the epitope which allows efficient binding of TU165.

Antibodies, Monoclonal↗
PubMed1993

Identification of predominant T-cell receptor rearrangements by temperature-gradient gel electrophoresis and automated DNA sequencing.

To assess the diversity of T-cell receptor (TCR) gene rearrangements in uncloned lymphocytes we used a three-stage strategy that allows the detection of a restricted TCR repertoire and the identification of the predominant, rearranged sequence(s). We have analyzed in parallel T cells obtained from a renal cell carcinoma infiltrate that specifically lyse the autologous tumor after in vitro culture and T cells from autologous peripheral blood. First, DNA amplification by the polymerase chain reaction (PCR) was performed with a number of oligonucleotide primers specific for several TCR V alpha gene families. All V alpha primers displayed specific amplification products in the peripheral blood, while a restricted TCR repertoire was present in the tumor-infiltrating lymphocytes. Subsequently, positively amplified PCR products were run in a temperature-gradient gel electrophoresis. A limited number of bands corresponding to predominant homo- and heteroduplexes was only found in the tumor-infiltrating lymphocytes. The presence of a low number of rearranged TCR sequences in these samples was confirmed by automated single-stranded DNA sequencing using a single fluorescent dye. These results support the broad application of this strategy for targeted sequencing of those PCR products carrying predominant DNA templates without previous DNA cloning.

Autoanalysis↗
PubMed1993

Microheterogeneity in HLA-B35 alleles influences peptide-dependent allorecognition by cytotoxic T cells but not binding of a peptide-restricted monoclonal antibody.

Strong peptide dependency of HLA-B*3501-specific alloreactive T-cell clones was observed in the recognition of cells bearing closely related B35 variants. The single amino acid exchange in the beta-pleated sheet of B*3503 completely abolished the responses of all clones, whereas an amino acid exchange in the alpha 2 helix of the newest B35 member (B*3508) only altered allorecognition of one T-cell clone, demonstrating the differential impact of these positions on peptide binding to B35 molecules. In contrast to T cells, a mAb (TU165) recognizing the B35 specificity in a peptide-dependent manner bound to the B35 variants irrespective of their sequence heterogeneity. However, quantitative binding differences were detected with cells bearing the same B35 alleles. This is most likely due to variations in the amount of peptide(s) that associates with B35 and forms the ligand seen by this mAb. These results reveal how naturally occurring single amino acid substitutions have led to generation of functionally distinct molecules of another multimember HLA class I cluster.

Amino Acid Sequence↗