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Biomedical subjects

D J Schendel

Publications and source records attributed to D J Schendel.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1993

Distribution of cell adhesion molecules (ICAM-1, VCAM-1, ELAM-1) in renal tissue during allograft rejection.

The tissue distribution of cellular adhesion molecules (CAMs) was studied in specimens from 10 normal human kidneys and in 52 biopsies from kidney allografts with cell-mediated rejection. In addition to the vascular presence of ICAM-1, a common finding in normal kidneys, expression of ICAM-1 on tubular cells was observed in 22 graft biopsies. Compared with normal kidneys, where VCAM-1 was present on Bowman's capsules and few proximal tubular cells, a markedly enhanced expression of VCAM-1 in numerous tubuli (including distal tubular segments) was observed in 51 graft biopsies. In 41 graft specimens VCAM-1 appeared also in variable numbers of peritubular capillaries. Infiltrating leukocytes carrying VCAM-1 were observed in 7 grafts. ELAM-1 could not be found in normal kidneys but was restricted to some peritubular capillaries in 29 grafts. Comparable results were obtained with cultured renal tubular cells when stimulated by TNF-alpha. That the induced appearance of adhesion molecules was in fact related to actual cellular synthesis was demonstrated by Northern blot analysis. Thus, little ICAM-1 specific mRNA of 3.4-kb length could be detected in unstimulated cultured renal tubular cells, but hybridization was markedly increased after stimulation with TNF-alpha. A substantial amount of VCAM-1 specific mRNA of 3.2-kb length was present already in unstimulated renal tubular cells. Likewise, synthesis of VCAM-1 mRNA was enhanced by stimulation with TNF-alpha. TNF-stimulated endothelial cells also showed weak synthesis of VCAM-1 mRNA. The results provide further evidence that constitutive and inducible expression of cell adhesion molecules contributes to the process of allograft rejection.

Antibodies, Monoclonal↗
PubMed1993

The multifactorial nature of MHC-linked susceptibility to insulin-dependent diabetes.

Several lines of evidence suggest that major histocompatibility complex (MHC)-linked susceptibility to insulin-dependent diabetes mellitus (IDDM) is not restricted to the presence or absence of any single gene product. The existence of population-specific haplotypes associated with IDDM supports the concept that distinct combinations of MHC alleles interact synergistically to induce disease when other environmental and genetic factors are present. MHC-controlled peptide transport and binding to MHC molecules as well as the levels of MHC class I and class II expression in the thymus and pancreatic beta cells may also play significant roles in the outbreak of IDDM. These intrinsic factors shape the T-cell receptor (TCR) repertoire during T-cell ontogeny in the thymus and later influence the efficiency of potentially autoreactive T cells in the periphery. Several extrinsic factors, such as viruses or dietary proteins, may be directly involved in the TCR/MHC interaction at the cell surface; furthermore viruses can alter the regulatory mechanisms of peptide/MHC interaction and expression. We propose that these intrinsic and extrinsic factors need not be mutually exclusive and might even be interdependent: a given virus may act deleteriously only when certain autoreactive T cells and combinations of MHC alleles are present in the individual. IDDM would develop if pathogenic T-cells are activated and an appropriate target MHC/peptide is expressed in pancreatic beta cells. Future knowledge of the host-virus relationships influenced by the MHC genes, the function of their encoded proteins and the polymorphic gene structure of well-established susceptibility MHC haplotypes will help delineate an overall picture of this issue.

Animals↗
PubMed1992

Cytotoxic T lymphocytes show HLA-C-restricted recognition of EBV-bearing cells and allorecognition of HLA class I molecules presenting self-peptides.

Human CTL have been isolated that show self-restricted recognition of autologous lymphoblastoid cell lines and allorecognition. The lymphoblastoid cell line ligand most likely used a peptide that is expressed in EBV-bearing cells when the virus enters the lytic cycle. This peptide is presented to CD8+ CTL by HLA-Cw7 molecules. The allogeneic ligand recognized on non-EBV-infected cells is composed of a class I glycoprotein and a naturally selected self-peptide. In previous studies we demonstrated that this ligand is determined by two MHC-linked genes: one gene encodes the allogeneic class I molecule whereas the other controls the self-peptide. Despite the use of different peptides and different class I molecules, seemingly equivalent structures are formed that enable these two ligands to function as antigenic mimics of each other. CTL with the same patterns of dual specificity could be isolated from four unrelated donors, indicating that HLA-Cw7 is frequently involved in self-restricted recognition of EBV-harboring cells. Such CTL could help not only to contain lytic virus during a primary infection but also may be maintained life-long to eliminate cells in which reactivated virus appears.

Burkitt Lymphoma↗
PubMed1992

The HLA-A,B "negative" mutant cell line C1R expresses a novel HLA-B35 allele, which also has a point mutation in the translation initiation codon.

The HLA-A,B negative mutant cell line C1R is widely used as a transfection recipient in functional studies of class I MHC genes. It was derived from a normal B cell line, Hmy2, by three rounds of mutagenesis and immunoselection with anti-HLA mAb. Serology characterizes C1R to be negative for the HLA-A2, A3, B35, Bw62, and Cw3 Ag of the parental cell line while retaining expression of HLA-Cw4. We find, however, that CTL specific for HLA-B35 lyse C1R cells, suggesting that expression of HLA-B35 is also retained. To resolve this paradox we examined the expression of HLA-A,B,C genes and proteins in C1R cells. The results are consistent with deletion of the HLA-A3, Bw62, Cw3 haplotype and retention of the HLA-A2, B35, Cw4 haplotype in C1R. Although present, the HLA-A2 gene appears not to be transcribed. As expected, the HLA-Cw4 gene is transcribed and the protein expressed at normal levels. Transcription of the HLA-B35 gene is also normal and comparable to that of HLA-Cw4. However, expression of the HLA-B35 protein is reduced to a few percent of the parental level. Comparison of the nucleotide sequence of B35 alleles from C1R and Hmy2 revealed that reduced translation in C1R is caused by a point mutation (ATG to TTG) in the translation initiation codon. The HLA-B35 allele from C1R and Hmy2 represents a novel subtype, B*3503, differing from B*3501 by replacement of serine by phenylalanine at the peptide binding position 116. This study shows cell surface levels of a class I molecule which are insensitive to lysis by antibody and complement can be readily recognized by alloreactive T cells, further illustrating the relative sensitivity of Ag recognition by T cells.

Alleles↗