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J R Parnes

Publications and source records attributed to J R Parnes.

At least 37 records · Page 2Linked to original sources
PubMed1995

Human CD6 possesses a large, alternatively spliced cytoplasmic domain.

Human CD6 is a monomeric 105/130-kDa T cell surface glycoprotein that is involved in T cell activation. The apparent discrepancy between the size of the cytoplasmic domain in human (44 amino acids) and mouse (243 amino acids) CD6, led us to use reverse transcriptase-polymerase chain reaction of human peripheral blood lymphocyte mRNA to isolate cDNA clones that include the carboxyl-terminal coding region of human CD6. The nucleotide sequence of the longest human cDNA clone, CD6-PB1, predicts a protein of 668 amino acids with a 244-amino acid cytoplasmic domain similar in size to and possessing 71.5% amino acid sequence identity with the cytoplasmic domain of mouse CD6. This previously unrecognized 244-amino acid cytoplasmic domain does not have significant homology to any other known protein (except mouse CD6), but does possess two proline-rich motifs containing the SH3 domain-binding consensus sequence, a serine-threonine-rich motif repeated three times, three protein kinase C phosphorylation-site motifs, and 10 casein kinase-2 phosphorylation-site motifs. These sequences are likely to play a role in the ability of CD6-specific monoclonal antibodies to stimulate T cell proliferation. Full-length CD6 cDNA containing this cytoplasmic domain sequence encodes a monomeric 105/130-kDa protein that can be immunoprecipitated from the surface of transfected cells and comigrates upon SDS-PAGE with wild-type CD6 immunoprecipitated from PBL. We also isolated two alternatively spliced forms of human CD6 cDNA lacking sequences encoding membrane-proximal regions of the cytoplasmic domain which maintain the same reading frame as CD6-PB1. The short cytoplasmic domain of the previously reported human CD6-15 cDNA clone results from a deletion of a 20-bp segment through use of an alternative 3' splice site, resulting in a frame shift and premature termination of translation relative to the clones we have isolated. These data demonstrate that human CD6 possesses a large cytoplasmic domain containing sequence motifs that are likely to be involved in signal transduction upon stimulation of T cells through CD6 ligation.

Amino Acid Sequence↗
PubMed1994

CD4 and CD8 in T cell lineage commitment: alterations induced by expression of a CD8/CD4 chimeric transgene.

The mechanism by which thymocytes expressing both the CD4 and CD8-coreceptor proteins differentiate into mature T cells expressing either CD4 or CD8 is not well understood. We have shown that expression of a chimeric CD8/CD4 transgene can alter T cell lineage commitment such that cells expressing a transgenic class I major histocompatibility complex-restricted T cell receptor can differentiate to the CD4 rather than those the CD8 lineage. The implications of these findings and those of others for existing instructive and stochastic models of thymocyte lineage commitment are discussed, and an alternative model is considered.

Animals↗
PubMed1994

Isolation of P1 bacteriophage clones containing large contiguous segments of the human and mouse loci for the T-cell coreceptor molecule CD8.

The T-lymphocyte coreceptor molecules CD8 (composed of CD8 alpha and CD8 beta chains) and CD4 undergo a complex pattern of regulated expression during T-cell maturation. In the thymus, the most immature cells progress from expressing neither molecule (the double-negative [DN] stage) to an intermediate stage at which both are coexpressed (the double-positive [DP] stage). As a result of thymic selection and further differentiation, DP cells give rise to the most mature thymic cells and peripheral T cells that express either CD8 or CD4 (the single-positive [SP] stage). Our previous studies of the transcriptional regulatory mechanisms controlling CD8 alpha expression during the DN-->DP and DP-->SP transitions suggest the existence of important cis-acting elements located a considerable distance from the CD8 alpha gene and that these elements might serve to regulate both CD8 alpha and CD8 beta. While both genes and intergenic DNA span approximately 60 kb in the mouse, the relevant cis elements could lie either within or beyond this region. As a result, we sought to isolate large contiguous segments of DNA in P1 bacteriophage that covered at least this region from the mouse and human CD8 locus. Our initial physical characterization of these clones demonstrates the value of the P1 system as all isolated clones were found to contain single contiguous 85- to 95-kb segments of DNA that are faithful replicas of the chromosomal locus. The presence of abundant native flanking DNA both upstream and downstream of the intact coding regions will make these clones extremely useful for identifying physiological CD8 cis-active regulatory elements by virtue of their ability to direct appropriate lineage- and stage-specific expression in transfected and transgenic T cells.

Animals↗
PubMed1993

Biochemical identity of the mouse Ly-19.2 and Ly-32.2 alloantigens with the B cell differentiation antigen Lyb-2/CD72.

Lyb-2/CD72 is a 45-kDa mouse B cell surface protein that binds CD5 (Ly-1) and has been shown to induce B cell proliferation upon mAb binding. The serologically defined Ly-19.2 and Ly-32.2 lymphocyte alloantigens have mouse strain distribution patterns similar to that of the Lyb-2/CD72 alleles and map to the same region on chromosome 4 as Lyb-2/CD72. Our recent isolation of the Lyb-2a, -2b, and -2c cDNA has enabled us in this report to examine the relationship between Ly-19, Ly-32, and Lyb-2/CD72. A rat T cell line transfected with a mouse Lyb-2a cDNA is recognized by Ly-19.2-specific mAb, whereas transfectants expressing the Lyb-2b cDNA are recognized by both Ly-19.2 and Ly-32.2-specific mAb. Cell surface iodination immunoprecipitation analysis from Lyb-2a cDNA transfectants using Lyb-2a- and Ly-19.2-specific mAb as well as from Lyb-2b cDNA transfectants using Lyb-2b-, Ly-19.2-, and Ly-32.2-specific Ab, produced immunoprecipitates containing comigrating 45-kDa polypeptides. Preclearing studies with these transfectants indicate that the immunoprecipitated proteins represent the same polypeptide chain. These results demonstrate that the mouse Ly-19.2 and Ly-32.2 alloantigens are in fact the B cell differentiation Ag Lyb-2/CD72.

Animals↗
PubMed1993

CD2-CD4-CD8- lymph node T lymphocytes in MRL lpr/lpr mice are derived from a CD2+CD4+CD8+ thymic precursor.

MRL lpr/lpr (lymphoproliferative, lpr) mice demonstrate an age-dependent lymphoproliferation and development of autoimmunity. Characteristic of the lymphoproliferation in these mice is the accumulation of large numbers of CD4-CD8-(CD4-8-),CD3+ T lymphocytes in their lymph nodes. The development of the CD4-8- cells, which also aberrantly express B220 and CD44 (Pgp-1) but are CD2-, has been shown to be thymus dependent. An unusual feature of lpr CD4-8-T lymphocytes is that although they appear unresponsive to stimulation, as defined by proliferation and IL-2 production, they have undergone thymic negative selection. As thymic deletion normally occurs at the CD4+CD8+ (CD4+8+) stage, this raises the dilemma that lpr CD4-8- T lymphocytes have either previously been CD4+8+, or they are able to undergo thymic selection as CD4-8- cells. We have addressed this question by examining the methylation status of the CD8 gene in MRL lpr CD4-8- lymph node cells. Demethylation of the CD8 gene has been shown to be an indicator of previous CD8 expression. We find that the CD8 gene in lpr CD4-8- lymph node cells, as well as in the abnormal B220+ CD4-8- lpr thymocytes, is demethylated, suggesting that these cells have previously expressed CD8. In addition, we find that the lpr CD4+8+ thymocyte population contains an increased percentage of atypical B220+, CD44+ cells that are virtually all CD2+. Taken together, these data are consistent with the lpr CD2-CD4-8- population of LNC having arisen from a CD2+ CD4+8+ thymic stage of differentiation.

Animals↗
PubMed1993

The CD8 alpha locus is located on the telomere side of the immunoglobulin kappa locus at a distance of 2 Mb.

The immunoglobulin kappa and CD8 alpha loci had been mapped by in situ hybridization to the short arm of chromosome 2 at 2cen-p11.2 and 2p1, respectively, but probes derived from the two loci did not hybridize to the same large PFGE fragments. We have now succeeded in linking the two loci on a 3.0-Mb fragment from a partial NruI digest. A new hybridization probe located between the two loci provided additional linking fragments and allowed the distance to be determined as 2.0-2.2 Mb.

CD8 Antigens↗
PubMed1993

Positive-negative selection gene targeting with the diphtheria toxin A-chain gene in mouse embryonic stem cells.

The diphtheria toxin A-chain gene was used in a positive-negative selection gene targeting vector to alter the CD4 gene which is transcriptionally silent in mouse embryonic stem cells. Expression of the toxin gene was driven by a constitutively active enhancer, yet the targeting construct exhibited only minimal transient toxicity while enriching for targeted clones 9- to 29-fold. Germline transmission of the stem cell-derived genome was obtained. These data suggest the usefulness of this diphtheria toxin A-chain cassette in replacement-type positive-negative selection vectors. Its potential for novel applications, particularly in the enrichment for 'hit-and-run' insertion-type vectors, is discussed.

Animals↗
PubMed1992

Extensive polymorphism in the extracellular domain of the mouse B cell differentiation antigen Lyb-2/CD72.

Lyb-2/CD72 is a 45-kDa mouse B cell surface protein that binds CD5 and has been shown to play a role in B cell proliferation and differentiation. Using the polymerase chain reaction we have isolated and sequenced cDNA clones encoding the serologically defined mouse Lyb-2a, Lyb-2b, and Lyb-2c alleles. We confirmed that our full length cDNA clones encode the Lyb-2a, -2b, and -2c alleles, respectively, by transfecting the isolated Lyb-2/CD72 cDNA clones into L cells and demonstrating that the transfectants bind only the appropriate allele specific anti-Lyb-2/CD72 antibodies. Sequence comparisons demonstrate that the Lyb-2/CD72 allels are highly conserved in their cytoplasmic and transmembrane domains but exhibit a high degree of polymorphism in their extracellular domains. This polymorphism in the extracellular region involves amino acid substitutions at a minimum of 20 residues and is concentrated primarily in the membrane distal region. cDNA sequence comparisons also demonstrate two distinct seven amino acid insertion/deletions among these allelic variants. A form of Lyb-2b cDNA lacking the sequence encoding the transmembrane region was isolated from a C57B1/6 mouse and a CH12.LX subline. The Lyb-2/CD72 PCR products from mRNA of mice expressing Lyb-2a and Lyb-2c contain a DNA fragment that corresponds in size to the transmembraneless form, suggesting that these mouse strains also express this mRNA.

Animals↗
PubMed1992

An immunological role for the CD8 beta-chain.

Mature T cells can be functionally divided into two categories distinguished by surface expression of either CD4 or CD8, which in turn corresponds to restriction by and binding to class II or class I major histocompatibility complex proteins, respectively. CD8 can be expressed as a homodimer of the alpha-chain, or as a heterodimer of alpha- and beta-chains on human and mouse T cells, although most peripheral T cells seem to express CD8 alpha beta heterodimers exclusively (reviewed in ref. 9). Functional characterization of CD8 has focused primarily on the effect of the alpha-chain, which enhances or reconstitutes T-cell responses in homodimeric form and may play a specific role in thymic selection. In contrast, no role has been ascribed to CD8 beta or alpha beta heterodimers specifically. Here we show that CD8 alpha beta transfectants produce more interleukin-2 than CD8 alpha transfectants in response to specific stimuli. Increased interleukin-2 is also observed in cells expressing hybrid CD8 beta-alpha molecules (extracellular CD8 beta plus CD8 alpha transmembrane and cytoplasmic regions) on their surface. These results indicate that external portions of CD8 beta could be critical and that they may act independently of CD8 alpha in mediating their augmentation effect.

Animals↗
PubMed1992

Signal for T-cell differentiation to a CD4 cell lineage is delivered by CD4 transmembrane region and/or cytoplasmic tail.

Mature T cells express either CD4 or CD8 on their surface. Most helper T cells express CD4, which binds to class II major histocompatibility complex (MHC) proteins, and most cytotoxic T cells express CD8, which binds to class I MHC proteins. In the thymus, mature CD4+CD8- and CD4-CD8+ T cells expressing alpha beta T-cell antigen receptors (TCR) develop from immature thymocytes through CD4+CD8+ alpha beta TCR+ intermediates. Experiments using mice transgenic for alpha beta TCR suggest that the specificity of the TCR determines the CD4/CD8 phenotype of mature T cells. These results, however, do not indicate how a T cell differentiates into the CD4 or CD8 lineage. Here we show that the CD4 transmembrane region and/or cytoplasmic tail mediates the delivery of a specific signal that directs differentiation of T cells to a CD4 lineage. We generated transgenic mice expressing a hybrid molecule composed of the CD8 alpha extracellular domains linked to the CD4 transmembrane region and cytoplasmic tail. We predicted that this hybrid molecule would bind to class I MHC proteins through the extracellular domains but deliver the intracellular signals characteristic of CD4. By crossing our transgenic mice with mice expressing a transgenic alpha beta TCR specific for a particular antigen plus class I MHC protein, we were able to express the hybrid molecule in developing thymocytes expressing the class I MHC-restricted TCR. Our results show that the signal transduced by the hybrid molecule results in the differentiation of immature thymocytes expressing a class I-restricted TCR into mature T cells expressing CD4.

Animals↗
PubMed1992

Ly-1 (CD5), a membrane glycoprotein of mouse T lymphocytes and a subset of B cells, is a natural ligand of the B cell surface protein Lyb-2 (CD72).

CD5 is a 67-kDa glycoprotein expressed on the cell surface membrane of all T lymphocytes and on a small proportion of B lymphocytes. The physiologic role of this Ag is still unknown. Structural and functional studies of CD5 suggest that it might act as a receptor for a positive signal. CD5-specific mAb augment CD3- or mitogen-induced T cell proliferation, IL-2 secretion, and IL-2R expression and induce a rise in intracellular [Ca2+]. In this report, we describe the purification of mouse CD5 protein (mCD5) and its use as a probe to search for the ligand of CD5. We demonstrate that mCD5 specifically interacts with the mouse B cell differentiation Ag CD72/Lyb-2. Three serologically defined allelic forms of mouse CD72/Lyb-2 can all interact with mCD5. We further show that mCD5 can interact with human CD72/Lyb-2, and similarly, that human CD5 can interact with mouse CD72/Lyb-2. These studies may have major implications for the mechanisms of T-B cell communication.

Animals↗
PubMed1992

Human Lyb-2 homolog CD72 is a marker for progenitor B-cell leukemias.

S-HCL 2 is the prototype antibody of the recently defined CD72 cluster (human Lyb-2). Under nonreducing conditions, S-HCL 2 monoclonal antibody (mAb) precipitates a glycoprotein of 80-86 kDa. Under reducing conditions, a dimer of 43 and 39 kDa, with core proteins of 40 and 36 kDa, is precipitated. CD72 expression in normal and malignant tissues is different from expression of all other previously described human B-cell antigens. In peripheral blood and bone marrow, the antigen appears to be present on all B lymphocytes, with the exception of plasma cells. In tissue, immunohistochemical staining revealed positivity for all known B-cell compartments; however, pulpa macrophages of the spleen and von Kupffer cells exhibited distinct positivity for CD72 also. Among 83 malignant non-Hodgkin's lymphomas examined by immunohistochemistry (alkaline phosphatase anti-alkaline phosphatase technique), all 54 B-cell lymphomas, including precursor B-cell lymphomas, Burkitt's lymphomas, germinal center lymphomas, chronic lymphocytic leukemias, and hairy cell leukemias, were CD72 positive, but no T-cell lymphomas were. Flow cytometry study of more than 80 mainly acute leukemias (52 B-cell leukemias) showed reactivity with S-HCL 2 mAb over the full range of B-cell differentiation. In particular, very early B cells in cytoplasmic Ig (cIg)-negative, CD19-positive pre-pre-B-cell leukemias and hybrid leukemias (mixed myeloid and B-cell type) were consistently positive for CD72 on the cell surface. Therefore, CD72 may become an important marker for progenitor B-cell leukemias.

Antibodies, Monoclonal↗
PubMed1991

The B-cell surface protein CD72/Lyb-2 is the ligand for CD5.

The glycoprotein CD5 is expressed on the surface membrane of all mature T cells and a small proportion of B lymphocytes. Its exact role in immune interactions is still unknown. Studies indicate that CD5 functions both in mice and humans as a receptor, delivering co-stimulatory signals to T cells in a manner similar to CD2 (ref. 11) and CD28 (ref. 12). Anti-CD5 antibodies stimulate both T-cell proliferation mediated by CD3 in association with the T-cell receptor and secretion of interleukin-2 and expression of its receptor, as well as inducing an increase in intracellular Ca2+ concentration (refs 5-10). To identify the ligand for CD5 we purified the human CD5 protein, labelled it with biotin and used it as a probe. Here we report that CD5 specifically interacts with the cell-surface protein CD72 exclusive to B cells. This interaction is blocked by anti-CD72 antibodies, but not by any other anti-B-cell antibodies. Moreover, non-B cells (mouse L-cell fibroblasts and human Jurkat T cells) expressing a transfected human CD72 complementary DNA could bind to the CD5-biotin conjugate. The results demonstrate that the B-cell surface protein CD72 (Lyb-2 in mice) is the ligand for CD5.

Antigens, CD↗
PubMed1991

Adhesion versus coreceptor function of CD4 and CD8: role of the cytoplasmic tail in coreceptor activity.

CD4 and CD8 play an important role in T-cell recognition and activation; however, their mechanisms of action are not well understood. We compare the effects of expressing CD4 and CD8 alpha either individually or together in a class II-restricted T-cell hybridoma. We also compare the effects of expressing truncated forms of CD4 or CD8 alpha that do not have a cytoplasmic tail and thus do not associate with the T-cell-specific tyrosine kinase p56lck, which has been implicated in T-cell activation. We demonstrate that, although CD4 and CD8 alpha can specifically enhance interleukin 2 secretion, maximal potentiation occurs with expression of CD4, which, unlike CD8, can bind to the same major histocompatibility complex protein as the T-cell receptor. Our data further indicate that the cytoplasmic tail and/or the associated p56lck are primarily significant for interleukin 2 secretion by the hybridomas we have examined when CD4 or CD8 can bind to the same major histocompatibility complex ligand as the T-cell receptor.

Animals↗