Human Major Histocompatibility Class Research Articles

Adeno-Associated Virus D-Sequence-Mediated Suppression of Expression of a Human Major Histocompatibility Class II Gene: Implications in the Development of Adeno-Associated Virus Vectors for Modulating Humoral Immune Response.

A 20-nt long sequence, termed the D-sequence, in the adeno-associated virus (AAV) inverted terminal repeat was observed to share a partial sequence homology with the X-box in the regulatory region of the human leukocyte antigen DRA (HLA-DRA) promoter of the human major histocompatibility complex class II (MHC-II) genes. The D-sequence was also shown to specifically interact with the regulatory factor binding to the X-box (RFX), binding of which to the X-box is a critical step in the MHC-II gene expression, suggesting that D-sequence might compete for RFX transcription factor binding, thereby suppressing expression from the MHC-II promoter. In DNA-mediated transfection experiments, using a reporter gene under the control of the HLA-DRA promoter, D-sequence oligonucleotides were found to inhibit expression of the reporter gene expression in HeLa and 293 cells by ∼93% and 96%, respectively. No inhibition was observed when nonspecific synthetic oligonucleotides were used. D-sequence oligonucleotides had no effect on expression from the cytomegalovirus immediate-early gene promoter. Interferon-γ-mediated activation of MHC-II gene expression was also inhibited by D-sequence oligonucleotides as well as after infection with either the wild-type AAV or transduction with recombinant AAV vectors. These studies suggest that the D-sequence-mediated downregulation of the MHC-II gene expression may be exploited toward the development of novel AAV vectors capable of dampening the host humoral response, which has important implication in the optimal use of these vectors in human gene therapy.

Alu elements within the human major histocompatibility class I region in the Comoros Islands: genetic variation and population relationships

Background: Alu elements are attractive markers for population genetics, disease, forensics and paternity analyses, due to their particular characteristics. Five polymorphic Alu insertions within the MHC class I region have been little examined in human populations.Aim: The analysis of the genetic diversity of autochthonous Comorians from the three major islands of the archipelago by these polymorphic MHC Alus and to assess their relationships together and with other populations.Subjects and methods: Two hundred and fifty-seven unrelated participants from the Comoros archipelago, Grande Comore (86), Anjouan (93) and Moheli (78), were examined for five MHC Alu insertions. The data were analysed for intra- and inter-population genetic variation.Results: All MHC Alu were polymorphic in the three samples and only one significant differentiation was observed between Anjouan and Moheli. According to the MDS and AMOVA results, the populations included in the inter-population analyses were grouped in three major clusters according to their genetic ancestry. The haplotype diversity showed by the Comorians is higher than in previously studied African populations and occupies an intermediate position between African and Asian clusters.Conclusion: MHC Alu insertions are useful markers to study micro-geographical genetic variations. Using these polymorphisms, new insights have been obtained about the biological history and evolution of the Comoros.

Epitopes rationally selected through computational analyses induce T-cell proliferation in mice and are recognized by serum from individuals infected with Schistosoma mansoni.

Schistosomiasis is the second leading cause of death due to parasitic diseases in the world. Seeking an alternative for the control of disease, the World Health Organization funded the genome sequencing of the major species related to schistosomiasis to identify potential vaccines and therapeutic targets. Therefore, the aim of this work was to select T and B-cell epitopes from Schistosoma mansoni through computational analyses and evaluate the immunological potential of epitopes in vitro. Extracellular regions of membrane proteins from the Schistosoma mansoni were used to predict promiscuous epitopes with affinity to different human Major Histocompatibility Class II (MHCII) molecules by bioinformatics analysis. The three-dimensional structure of selected epitopes was constructed and used in molecular docking to verify the interaction with murine MHCII H2-IAb . In this process, four epitopes were selected and synthesized to assess their ability to stimulate proliferation of CD4+ T lymphocytes in mice splenocyte cultures. The results showed that Sm041370 and Sm168240 epitopes induced significant cell proliferation. Additionally, the four epitopes were used as antigens in the Indirect Enzyme-Linked Immunosorbent Assay (ELISA) to assess the recognition by serum from individuals infected with Schistosoma mansoni. Sm140560, Sm168240, and Sm041370 epitopes were recognized by infected individuals IgG antibodies. Therefore, Sm041370 and Sm168240 epitopes that stood out in in silico and in vitro analyses could be promising antigens in schistosomiasis vaccine development or diagnostic kits. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:804-814, 2017.

Abstract 4899: The regulation by kinases of the expression of human major histocompatibility class I molecules

Abstract The major histocompatability complex (MHC) is a central receptor in the adaptive immune response and is the underlying target of several effective therapies for cancer. Druggable kinases may provide the opportunity to modulate the immune response toward MHC. However, the regulation of MHC-I by kinases is largely unstudied, even though many patients with cancer are receiving therapeutic kinase inhibitors. The entire human kinome was screened using a pooled shRNA interference-based approach in a human mesothelioma cell line to uncover kinase regulators of MHC-I. Negative and positive regulators of cell surface HLA levels were discovered. A subset of highly scoring positive and negative kinase hits were subsequently validated by additional RNAi, and pharmacologic inhibitors when available. MAP2K1 (MEK), EGFR, and RET were validated as negative regulators of HLA expression in multiple cancer types. We mapped the pathways responsible for increased HLA upon kinase inhibition. Interestingly, inhibition of the MAP Kinase pathway broadly influenced expression of other components of the antigen presentation machinery. Moreover, DDR2 and MINK1 were shown to positively regulate HLA-A*02:01. This had therapeutic relevance, as shown with a therapeutic TCR mimic antibody to a MHC/peptide complex. Druggable kinases may thus serve as immediately applicable targets for modulating immunotherapy for many diseases. Citation Format: Elliott J. Brea, Claire Oh, Eusebio Manchado, Ron Gejman, George Mo, Patrizia Mondello, Ralph Garippa, Neal Rosen, David A. Scheinberg. The regulation by kinases of the expression of human major histocompatibility class I molecules. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4899.

Regulation of interlocking gene regulatory network subcircuits by a small molecule inhibitor of retinoblastoma protein (RB) phosphorylation: Cancer cell expression of HLA-DR

The induction of the major histocompatibility (MHC), antigen-presenting class II molecules by interferon-gamma, in solid tumor cells, requires the retinoblastoma tumor suppressor protein (Rb). In the absence of Rb, a repressosome blocks the access of positive-acting, promoter binding proteins to the MHC class II promoter. However, a complete molecular linkage between Rb expression and the disassembly of the MHC class II repressosome has been lacking. By treating A549 lung carcinoma cells with a novel small molecule that prevents phosphorylation-mediated, Rb inactivation, we demonstrate that Rb represses the synthesis of an MHC class II repressosome component, YY1. The reduction in YY1 synthesis correlates with the advent of MHC class II inducibility; with loss of YY1 binding to the promoter of the HLA–DRA gene, the canonical human MHC class II gene; and with increased Rb binding to the YY1 promoter. These results support the concept that the Rb gene regulatory network (GRN) subcircuit that regulates cell proliferation is linked to a GRN subcircuit regulating a tumor cell immune function.

Human Umbilical Cord Blood–Derived Endothelial Cells Reendothelialize Vein Grafts and Prevent Thrombosis

To accelerate vein graft reendothelialization and reduce vein graft thrombosis by infusing human umbilical cord blood-derived endothelial cells (hCB-ECs) because loss of endothelium contributes to vein graft thrombosis and neointimal hyperplasia. Under steady flow conditions in vitro, hCB-ECs adhered to smooth muscle cells 2.5 to 13 times more than ECs derived from peripheral blood or human aorta (P<0.05). Compared with peripheral blood and human aorta ECs, hCB-ECs had 1.4-fold more cell surface α(5)β(1) integrin heterodimers per cell (P<0.05) and proliferated on fibronectin 4- to 10-fold more rapidly (P<0.05). Therefore, we used hCB-ECs to enhance reendothelialization of carotid interposition vein grafts implanted in NOD.CB17-Prkdc(scid)/J mice. Two weeks postoperatively, vein grafts from hCB-EC-treated mice demonstrated approximately 55% reendothelialization and no luminal thrombosis. In contrast, vein grafts from sham-treated mice demonstrated luminal thrombosis in 75% of specimens (P<0.05) and only approximately 14% reendothelialization. In vein grafts from hCB-EC-treated mice, 33±10% of the endothelium was of human origin, as judged by human major histocompatibility class I expression. The hCB-ECs adhere to smooth muscle cells under flow conditions in vitro, accelerate vein graft reendothelialization in vivo, and prevent vein graft thrombosis. Thus, hCB-ECs offer novel therapeutic possibilities for vein graft disease.

Transgenic mice with a diverse human T cell antigen receptor repertoire

Because of tolerance mechanisms, it has been hard to identify the T cell receptors (TCRs) of high-avidity T cells against self (for example, tumor) antigens. TCRs that are specific for foreign human antigens from the nontolerant T cell repertoire can be identified in mice. Moreover, if mice are constructed to express the human TCR repertoire, they can be used to analyze the unskewed repertoire against human self antigens. Here we generated transgenic mice with the entire human TCRalphabeta gene loci (1.1 and 0.7 Mb), whose T cells express a diverse human TCR repertoire that compensates for mouse TCR deficiency. A human major histocompatibility class I transgene increases the generation of CD8+ T cells with human compared to mouse TCRs. Functional CD8+ T cells against several human tumor antigens were induced, and those against the Melan-A melanoma antigen used similar TCRs to those that have been detected in T cell clones from individuals with autoimmune vitiligo or melanoma. These mice will allow researchers to identify pathogenic and therapeutic human TCRs.

Influence of intermolecular disulfide bonds on assembly of mouse MHC class I molecules (130.25)

Abstract Within the major histocompatibility (MHC) class I peptide-loading complex, tapasin and ERp57 are joined by a stable disulfide bond which is necessary for the efficient assembly of MHC class I molecules. In our investigation of murine tapasin, we identified a large covalently bound complex involving wild type mouse tapasin and ERp57. This complex, which migrates at ~150 kDa, is noticeably larger than the 110 kDa tapasin-ERp57 complex previously identified in human cells. Additional data suggest that the mouse MHC class I molecule Kd may be a part of this ~150 kDa complex. In order to investigate the importance of tapasin-ERp57 disulfide bonding for the assembly of murine MHC class I molecules, we introduced a tapasin C95S mutant into tapasin knockout mouse fibroblasts. Although this tapasin mutant is unable to disulfide bond with ERp57, mouse tapasin C95S was found to maintain a non-covalent association with ERp57. Mutation of tapasin C95 impaired the maturation and stability of MHC class I Kd molecules to a greater extent than was previously observed for the human MHC class I molecule, B*4402. Furthermore, the maturation and stability of mouse MHC class I Kb molecules was also impaired in cells expressing mouse tapasin C95S. In total, these results highlight inter-species differences in the MHC class I assembly pathway and emphasize the importance of studying diverse MHC class I allotypes.

Expression of Bisecting Type and Lewisx/Lewisy Terminated N-Glycans on Human Sperm

Human sperm lack major histocompatibility class I molecules, making them susceptible to lysis by natural killer (NK) cells. Major histocompatibility class I negative tumor cells block NK cell lysis by expressing sufficient amounts of bisecting type N-glycans on their surfaces. Therefore, sperm could employ the same strategy to evade NK cell lysis. The total N-glycans derived from sperm were sequenced using ultrasensitive mass spectrometric and conventional approaches. Three major classes of N-glycans were detected, (i) high mannose, (ii) biantennary bisecting type, and (iii) biantennary, triantennary, and tetraantennary oligosaccharides terminated with Lewisx and Lewisy sequences. Immunostaining of normal sperm showed that glycoproteins bearing Lewisy sequences are localized to the acrosome and not the plasma membrane. In contrast, defective sperm showed distinct surface labeling with anti-Lewisy antibody. The substantial expression of high mannose and complex type N-glycans terminated with Lewisx and Lewisy sequences suggests that sperm glycoproteins are highly decorated with ligands for DC-SIGN. Based on previous studies, the addition of such carbohydrate signals should inhibit antigen-specific responses directed against sperm glycoproteins in both the male and female reproductive systems. Thus, the major N-glycans of human sperm are associated with the inhibition of both innate and adaptive immune responses. These results provide more support for the eutherian fetoembryonic defense system hypothesis that links the expression of carbohydrate functional groups to the protection of gametes and the developing human in utero. This study also highlights the usefulness of glycomic profiling for revealing potential physiological functions of glycans expressed in specific cell types.

Expression, purification and preliminary X-ray crystallographic analysis of the human major histocompatibility antigen HLA-B*1402 in complex with a viral peptide and with a self-peptide.

The product of the human major histocompatibility (HLA) class I allele HLA-B*1402 only differs from that of allele HLA-B*1403 at amino-acid position 156 of the heavy chain (Leu in HLA-B*1402 and Arg in HLA-B*1403). However, both subtypes are known to be differentially associated with the inflammatory rheumatic disease ankylosing spondylitis (AS) in black populations in Cameroon and Togo. HLA-B*1402 is not associated with AS, in contrast to HLA-B*1403, which is associated with this disease in the Togolese population. The products of these alleles can present peptides with Arg at position 2, a feature shared by a small group of other HLA-B antigens, including HLA-B*2705, the prototypical AS-associated subtype. Complexes of HLA-B*1402 with a viral peptide (RRRWRRLTV, termed pLMP2) and a self-peptide (IRAAPPPLF, termed pCatA) were prepared and were crystallized using polyethylene glycol as precipitant. The complexes crystallized in space groups P2(1) (pLMP2) and P2(1)2(1)2(1) (pCatA) and diffracted synchrotron radiation to 2.55 and 1.86 A resolution, respectively. Unambiguous solutions for both data sets were obtained by molecular replacement using a peptide-complexed HLA-B*2705 molecule (PDB code 1jge) as a search model.

Multivalent recombinant protein vaccine against coccidioidomycosis.

Coccidioidomycosis is a human respiratory disease that is endemic to the southwestern United States and is caused by inhalation of the spores of a desert soilborne fungus. Efforts to develop a vaccine against this disease have focused on identification of T-cell-reactive antigens derived from the parasitic cell wall which can stimulate protective immunity against Coccidioides posadasii infection in mice. We previously described a productive immunoproteomic/bioinformatic approach to the discovery of vaccine candidates which makes use of the translated genome of C. posadasii and a computer-based method of scanning deduced sequences of seroreactive proteins for epitopes that are predicted to bind to human major histocompatibility (MHC) class II-restricted molecules. In this study we identified a set of putative cell wall proteins predicted to contain multiple, promiscuous MHC II binding epitopes. Three of these were expressed by Escherichia coli, combined in a vaccine, and tested for protective efficacy in C57BL/6 mice. Approximately 90% of the mice survived beyond 90 days after intranasal challenge, and the majority cleared the pathogen. We suggest that the multicomponent vaccine stimulates a broader range of T-cell clones than the single recombinant protein vaccines and thereby may be capable of inducing protection in an immunologically heterogeneous human population.

Paucity of class I MHC gene heterogeneity between individuals in the endangered Hawaiian monk seal population

The Hawaiian monk seal population has experienced precipitous declines in the last 50 years. In this study, we provide evidence that individuals from remaining endangered population exhibit alarming uniformity in class I major histocompatibility (MHC) genes. The peripheral blood leukocyte-derived mRNA of six captive animals rescued from a stranding incident on the French frigate shoals in the Hawaiian archipelago was used to characterize genes in the monk seal class I MHC gene family, from which techniques for genotyping the broader population were designed using degenerate primers designed for the three major established human MHC class I loci (HLA-A, HLA-B, and HLA-C), and by sequencing multiple clones, six unique full-length classical MHC class I gene transcripts were identified among the six animals, three of which were only found in single individuals. Since The low degree of sequence variation between these transcripts and the similarity of genotype between individuals provided preliminary evidence for low class I MHC variability in the population. The sequence information from the class I transcripts from these six animals was used to design several primer sets for examining the extent of MHC variability in the remaining population using a combination of polymerase chain reaction and denaturing gradient gel electrophoresis (DGGE). Several DGGE assays, each one amplifying subtly different class I MHC gene combinations, were designed to compare exons encoding the highly polymorphic domains of the putative peptide-binding region of MHC class I. In combination, these assays failed to show interindividual variability at any of the class I MHC gene loci examined in either the six captive seals or in 80 free-ranging animals ( approximately 6.7% of the estimated population) representing all six major subpopulations of Hawaiian monk seal.

Oct-1 DNA binding activity unresponsive to retinoblastoma protein expression prevents MHC class II induction in a non-small cell lung carcinoma cell line

Numerous human tumor lines fail to induce major histocompatibility (MHC) class II expression following interferon-γ (IFN-γ) treatment, a response that is considered to be a normal function for almost all human parenchymal and connective tissue cell-types. The effect of MHC class II non-inducibility on solid tumor growth is controversial, but an extensive body of literature indicates that tumor cell MHC class II expression can lead to an antitumor response or tumor tolerance, depending on a number of variables. Thus, understanding the molecular basis for MHC class II induction failures in solid tumor cells will likely lead to ideas for manipulating the antitumor immune response. To date, a handful of tumor associated molecular anomalies have accounted for all the known failures of MHC class II inducibility. In particular, lack of the retinoblastoma tumor suppressor protein (Rb) has been shown in both human and mouse cells to be strongly associated with failure to induce MHC class II. The basis for this relationship is traceable to, among other things, high level Oct-1 DNA binding activity in Rb-defective cells, which represses the prototypical human MHC class II gene, HLA-DRA. Ordinarily, re-establishment of Rb expression leads to elimination of, or substantially reduced Oct-1 DNA binding activity and to rescue of HLA-DRA inducibility. However, in the case of one non-small cell lung carcinoma line (NSCLC), Rb re-expression failed to rescue HLA-DRA inducibility despite successful re-establishment of Rb-function. We now report that this failure is traceable to the failure of Rb to rescue normal Oct-1 function. Furthermore, histone deacetylase inhibitor treatment allows a bypass of the Rb requirement and facilitates the MHC class II induction in this NSCLC line.

US2, a Human Cytomegalovirus-encoded Type I Membrane Protein, Contains a Non-cleavable Amino-terminal Signal Peptide

The human cytomegalovirus US2 gene product targets major histocompatibility class I molecules for degradation in a proteasome-dependent fashion. Degradation requires interaction between the endoplasmic reticulum (ER) lumenal domains of US2 and class I. While ER insertion of US2 is essential for US2 function, US2 lacks a cleavable signal peptide. Radiosequence analysis of glycosylated US2 confirms the presence of the NH(2) terminus predicted on the basis of the amino acid sequence, with no evidence for processing by signal peptidase. Despite the absence of cleavage, the US2 NH(2)-terminal segment constitutes its signal peptide and is sufficient to drive ER translocation of chimeric reporter proteins, again without further cleavage. The putative US2 signal peptide c-region is responsible for the absence of cleavage, despite the presence of a suitable -3,-1 amino acid motif for signal peptidase recognition. In addition, the US2 signal peptide affects the early processing events of the nascent polypeptide, altering the efficiency of ER insertion and subsequent N-linked glycosylation. To our knowledge, US2 is the first example of a membrane protein that does not contain a cleavable signal peptide, yet otherwise behaves like a type I membrane glycoprotein.

Identification of CCAAT Displacement Protein (CDP/cut) as a Locus-specific Repressor of Major Histocompatibility Complex Gene Expression in Human Tumor Cells

Human major histocompatibility (MHC) class I antigen expression is important in controlling the metastatic growth of malignant tumors. Locus-specific down-regulation of MHC class I gene expression is frequently observed in human tumors, leading to decreased susceptibility to cytotoxic T-cell-mediated lysis. The mechanism of this down-regulation is incompletely understood. Here, we describe the identification of human CCAAT displacement protein (CDP/cut) as a locus-specific repressor of HLA-B and C gene expression. Transient and stable transfections in HeLa and K562 cells demonstrated the presence of a repressor element 650 base pairs upstream of the first exon of HLA-B7. A specific binding complex with the HLA-B7 and Cw2 repressor elements was demonstrated by EMSA. Formation of the EMSA complex was inhibited specifically with polyclonal antiserum to human CDP/cut, demonstrating that CDP/cut binds the HLA-B7 repressor element. The corresponding region of the HLA-A2 promoter neither repressed HLA-A2 gene expression nor bound CDP/cut. Overexpression of CDP/cut in cell lines deficient in CDP/cut resulted in a nearly 4-fold repression of reporter constructs containing the HLA-B7 repressor element but not the corresponding region of the HLA-A2 promoter. Repression of HLA-B and C gene expression by CDP/cut does not involve displacement of NF-Y, nor is CDP/cut associated with the histone deacetylase HDAC1 when bound to the HLA-B7 repressor element. To our knowledge, these results identify CDP/cut as the first example of a locus-specific repressor of MHC class I gene transcription in human tumor cells.

Hla-DR2-restricted responses to proteolipid protein 95-116 peptide cause autoimmune encephalitis in transgenic mice.

In multiple sclerosis (MS) patients who carry the Class II major histocompatibility (MHC) type HLA-DR2, T cells specific for amino acids 95-116 in the proteolipid protein (PLP) are activated and clonally expanded. However, it remains unclear whether these autoreactive T cells play a pathogenic role or, rather, protect against the central nervous system (CNS) damage. We have addressed this issue, using mice transgenic for the human MHC class II region carrying the HLA-DR2 (DRB1* 1502) haplotype. After stimulating cultured lymph node cells repeatedly with PLP95-116, we generated 2 HLA-DR2-restricted, PLP95-116-specific T-cell lines (TCLs) from the transgenic mice immunized with this portion of PLP. The TCLs were CD4+ and produced T-helper 1 (Th1) cytokines in response to the peptide. These TCLs were adoptively transferred into RAG-2/2 mice expressing HLA-DR2 (DRG1* 1502) molecules. Mice receiving 1 of the TCLs developed a neurological disorder manifested ataxic movement without apparent paresis on day 3, 4, or 5 after cell transfer. Histological examination revealed inflammatory foci primarily restricted to the cerebrum and cerebellum, in association with scattered demyelinating lesions in the deep cerebral cortex. These results support a pathogenic role for PLP95-116-specific T cells in HLA-DR2+ MS patients, and shed light on the possible correlation between autoimmune target epitope and disease phenotype in human CNS autoimmune diseases.

Differential expression of human major histocompatibility class I loci: HLA-A, -B, and -C

Expression of the human class I MHC loci, HLA-A, -B, and -C, was examined by reverse transcription and competitive PCR with locus-specific primers. This approach allows unambiguous identification of target sequences by analysis of the amplified products. JY and Pala lymphoblastoid B cells express more HLA-A than HLA-B mRNAs and little HLA-C mRNA. Raji Burkitt lymphoma and HeLa carcinoma cells express approximately equal amounts of HLA-A and HLA-C mRNAs but less HLA-B mRNA. Jar trophoblast cells express no HLA class I mRNAs. Surprisingly, K562 leukemia cells express significant amounts of HLA-C mRNA. However, K562 cells contain no detectable HLA-A or -B mRNAs, suggesting that these loci are regulated independently. Furthermore, cultured endothelial cells and smooth muscle cells express low, approximately equal amounts of HLA-A, -B, and -C mRNAs, whereas donor-matched, EBV transformed B cells express much more HLA-B mRNA, suggesting that cell type dependent regulation underlies differential locus expression. Finally, expression of HLA class I molecules on the cell surface correlates with total HLA mRNAs but not with mRNAs encoded by any one locus. Differential expression of these HLA class I loci may contribute to cell-type dependent immune reactions by preferentially presenting distinct peptides to T cells.

Structure of the Human Palmitoyl-Protein Thioesterase-2 Gene (PPT2) in the Major Histocompatibility Complex on Chromosome 6p21.3

Palmitoyl-protein thioesterase-2 (PPT2) is a homolog of PPT1, the enzyme that is deficient in the lysosomal storage disorder, infantile neuronal ceroid lipofuscinosis (NCL). As a first step toward determining whether mutations in the gene encoding PPT2 (PPT2) are associated with any of the molecularly uncharacterized forms of NCL, we report here the structure and chromosomal localization of humanPPT2. PPT2spans about 10 kb and is composed of nine exons. One major (2.0 kb) and two minor (7.0 and 2.8 kb) mRNAs are transcribed from the gene, and the larger transcripts appear to be messenger RNAs in whichPPT2exons are spliced into a downstream gene encoding a homolog of human latent transforming growth factor-β binding protein (human LTBP).PPT2is located in the human major histocompatibility class III locus on chromosome 6p21.3, a position that rules outPPT2as the causative gene in any of the NCLs at defined chromosomal loci. No mutations were detected by SSCP analysis in a preliminary analysis of 12 subjects referred with a suspected diagnosis of infantile NCL who had normal PPT activity. However, five single nucleotide polymorphisms were found in unrelated normal individuals. These polymorphisms (and a microsatellite discovered withinPPT2) will aid in the further delineation of the possible role ofPPT2in lysosomal storage disorders of unknown etiology.

Isolation of Tumor-Specific Cytotoxic CD4+ and CD4+CD8dim+ T-Cell Clones Infiltrating a Cutaneous T-Cell Lymphoma

We have isolated several T-cell clones from lymphocytes infiltrating a human major histocompatibility class (MHC) II negative cutaneous T-cell lymphoma (CTCL). We describe here two of these clones, TC5 and TC7, with, respectively, a CD4+CD8dim+ and CD4+CD8− phenotype. Both clones mediated a specific MHC class I–restricted cytotoxic activity toward the fresh autologous tumor cells, and autologous tumor cell lines previously established with interleukin-2 (IL-2) and IL-7 from the skin and from the blood. Analysis of the T-cell receptor (TCR) Vβ gene expression showed that the tumor cells, which were shown to have a trisomy 7 by fluorescent in situ hybridization, expressed Vβ7/Jβ2.3, Vβ13/Jβ2.5, and Vβ22/Jβ2.5 rearrangements. Phenotypic analysis using specific anti-Vβ monoclonal antibodies indicated that only Vβ13 could be detected on the cell membrane of the tumor cells. Analysis of the TCR Vβ gene expression of the clones showed that TC5 and TC7 expressed a unique TCR-Vβ transcript, corresponding, respectively, to Vβ5/Jβ2.3 and Vβ17/Jβ2.7 gene segments. To determine whether these reactive T lymphocytes were present in vivo, we used specific primers corresponding to TC5- and TC7-Vβ TCR transcripts. The results showed that both cytotoxic T-cell clones were present at the lesional skin site and amplified in vitro. TC7 was found in the patient peripheral blood invaded by tumoral cells, whereas TC5 was not, indicating that the repertoire of the reactional lymphocytes differs in the blood and at the tumor site. These results show for the first time the presence of reactive T lymphocytes with CD4 or double-positive phenotype infiltrating a CTCL. These findings raise the question of the role of these antitumoral effector T cells in the tumor growth.

Isolation of Tumor-Specific Cytotoxic CD4+ and CD4+CD8dim+ T-Cell Clones Infiltrating a Cutaneous T-Cell Lymphoma

AbstractWe have isolated several T-cell clones from lymphocytes infiltrating a human major histocompatibility class (MHC) II negative cutaneous T-cell lymphoma (CTCL). We describe here two of these clones, TC5 and TC7, with, respectively, a CD4+CD8dim+ and CD4+CD8− phenotype. Both clones mediated a specific MHC class I–restricted cytotoxic activity toward the fresh autologous tumor cells, and autologous tumor cell lines previously established with interleukin-2 (IL-2) and IL-7 from the skin and from the blood. Analysis of the T-cell receptor (TCR) Vβ gene expression showed that the tumor cells, which were shown to have a trisomy 7 by fluorescent in situ hybridization, expressed Vβ7/Jβ2.3, Vβ13/Jβ2.5, and Vβ22/Jβ2.5 rearrangements. Phenotypic analysis using specific anti-Vβ monoclonal antibodies indicated that only Vβ13 could be detected on the cell membrane of the tumor cells. Analysis of the TCR Vβ gene expression of the clones showed that TC5 and TC7 expressed a unique TCR-Vβ transcript, corresponding, respectively, to Vβ5/Jβ2.3 and Vβ17/Jβ2.7 gene segments. To determine whether these reactive T lymphocytes were present in vivo, we used specific primers corresponding to TC5- and TC7-Vβ TCR transcripts. The results showed that both cytotoxic T-cell clones were present at the lesional skin site and amplified in vitro. TC7 was found in the patient peripheral blood invaded by tumoral cells, whereas TC5 was not, indicating that the repertoire of the reactional lymphocytes differs in the blood and at the tumor site. These results show for the first time the presence of reactive T lymphocytes with CD4 or double-positive phenotype infiltrating a CTCL. These findings raise the question of the role of these antitumoral effector T cells in the tumor growth.