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Biomedical subjects

P K Gregersen

Publications and source records attributed to P K Gregersen.

At least 55 records · Page 3Linked to original sources
PubMed1996

Shortened telomeres in clonally expanded CD28-CD8+ T cells imply a replicative history that is distinct from their CD28+CD8+ counterparts.

Long term in vitro culture of clonally expanded CD8+T cells, generally found within the CD57+ or CD28-subset, has generally been unsuccessful, suggesting that these cells may have a limited replicative potential. Telomeric shortening may reflect the action of a "mitotic clock" regulating the number of divisions a cell can undergo. In this study, we have compared the telomeric lengths of CD28-CD8+ and CD28+CD8+ T cells in 10 normal individuals to assess their replicative history. Overall, the telomeric lengths were found to be significantly shorter in the CD28-CD8+ T cell subset compared with the CD28+CD8+ subset. Furthermore, clonally expanded TCRBV11+CD8+ T cells from an individual exhibited telomeric lengths that were 2.9 kb shorter than those found in the polyclonal CD28+CD8+ T cell subset. These findings indicate that clonally expanded CD28-CD8+ T cells have undergone many more rounds of replication than CD28+CD8+ T cells, and consistent with the loss of CD28 expression, they may have reached a state of replicative senescence.

Adult↗
PubMed1996

Evidence for progenitors of chronic lymphocytic leukemia B cells that undergo intraclonal differentiation and diversification.

Peripheral blood mononuclear cells from five patients with IgG+ B-type chronic lymphocytic leukemia (B-CLL) were analyzed for the presence of clone-specific Ig H chain variable region gene mRNA transcripts linked to C mu and/or C alpha. This was assessed by (1) comparing the lengths of portions of the VHDJH of the IgG+ CLL clones with those of the mu and alpha isotype-expressing B cells, (2) performing clone-specific endonuclease digestion studies, and (3) determining the DNA sequences of the mu and alpha isotype-expressing cDNA. Thus, when B-cell mRNA from these five patients were reverse transcribed with C gamma-specific primers and then amplified by polymerase chain reaction, dominant cDNA were found with lengths corresponding to those of the IgG+ CLL B cell. In addition, in four cases, cDNA of lengths identical to those of the CLL B cell were detected when mRNA was reverse transcribed and amplified using c mu- and/or C alpha-specific primers, strongly suggesting clonal relatedness. These CLL-related mu- and alpha-expressing cDNA were present in greater amounts that unrelated (non-CLL) mu- and alpha-expressing cDNA from normal B cells that used genes of the same VH family. When the sequences of these CLL-related C mu- and C alpha-expressing cDNA were compared with those of the IgG+ CLL clones, it was clear that they were derived from the same ancestral gene as the IgG-expressing CLL B cell, thus documenting their common origin. Finally, nucleotide point mutations were observed in the mu- and alpha-expressing cDNA of certain patients, indicating divergence with the CLL. These data suggest that IgM+ B cells, which are precursors of the leukemic B cells, exist in increased numbers in the blood of most patients with IgG+ B-CELL and that these cells may differentiate, accumulate V genes mutations, and undergo isotype switching in vivo. In addition, the data are consistent with a sequential-hit model for the evolution of CLL.

Base Sequence↗
PubMed1996

Oligoclonality of V beta 3 TCR chains in the CD8+ T cell population of rheumatoid arthritis patients.

It has been established that oligoclonal expansion is a common feature of the CD8+ T cell population, particularly within the CD8+ CD57+ lymphocyte subset. In addition, clonal malignancies involving CD8+ CD57+ T cells (large granulocytic lymphocytic leukemias) are often accompanied by rheumatoid arthritis, Felty's syndrome, or both. Therefore, to identify disease-related alterations in the CD8+ T cell repertoire, we have compared the patterns of oligoclonality in the CD8+ T cells of rheumatoid arthritis patients (n = 32) with those of age-matched controls (n = 25). By using a multiplex PCR assay for the CDR3 length of TCR beta-chains, we have found a striking increase in the frequency of CD8+ oligoclonality involving V beta 3 TCR: 50% of the rheumatoid arthritis patients had evidence of oligoclonality in this TCR family compared with 4% of controls (p < 0.0002). In addition, two unrelated RA patients had clonally dominant CD8+ T cell beta receptors that were identical in amino acid sequence, suggesting selection by a common Ag. An analysis of a subset of RA patients with mAbs specific for V beta 3 TCR revealed the presence of clonal expansion in a minority of patients usually, but not exclusively, involving the CD57+ subset. These data define a phenotype of the T cell repertoire that is strongly associated with rheumatoid arthritis; the mechanisms and genetic and environmental factors that explain this phenomenon remain to be defined.

Adult↗
PubMed1996

Unexpected HLA haplotype sharing in dizygotic twin pairs discordant for rheumatoid arthritis.

Dizygotic twins are generally believed to be no more genetically similar than sibs born from separate pregnancies. In the present study, a panel of 93 dizygotic twin pairs discordant for rheumatoid arthritis were typed for HLA-A, -B, -Cw, and -DR antigens. HLA haplotype sharing identical by descent between the twins showed a trend towards increased sharing of both HLA haplotypes; this increased sharing was statistically significant when the female/female twin pairs were considered separately. In contrast, the pattern of HLA haplotype sharing in sib pairs (n = 128) was consistent with a 1:2:1 ratio of 2, 1, or 0 haplotypes shared. An analysis of 16 normal dizygotic twin pairs was consistent with these results raising the possibility that dizygotic twins in general are genetically more similar at the HLA complex than sibs born from separate pregnancies.

Arthritis, Rheumatoid↗
PubMed1996

Oligoclonality of CD8+ T cells in multiple sclerosis.

We have shown earlier that CD8+ T cell oligoclonality occurs frequently in normal individuals and general exhibits a very diverse repertoire. In order to investigate the role of CD8+ T cells in MS, we analysed CD8 oligoclonality in 125 patients with MS in varying stages of disease. A multiplex PCR assay for CDR3 length variation was employed to detect oligoclonality in 25 TCRBV segments/families. CD8 clonal dominance was found to be frequent in MS. Comparison of the CD8 T cell repertoire in MS with that in normal controls revealed an increased frequency of oligoclonality involving the TCRBV9, -18 and -23 families. Sequence analysis of the TCRs from these clonally dominant CD8+ cells revealed a high degree of diversity overall. However, we observed one instance of identical TCRBV18 sequences in CD8 cells from two unrelated MS patients. In addition, several TCRs with motifs homologous to those found in MS brain and MBP specific T cell clones in EAE and MS were also detected. Future characterization of the function and specificity of these clonally expanded populations may provide insight into the nature of immune dysregulation in this autoimmune disorder.

Adult↗
PubMed1995

Oligoclonal CD8+ T cells are preferentially expanded in the CD57+ subset.

A number of recent reports have established that oligoclonality and/or clonal expansion is a common feature of the CD8+ T cell population. Oligoclonal expansion has also been observed in bone marrow transplant recipients and rheumatoid arthritis patients, disease states in which CD57+CD8+ T cells are occasionally elevated. In this study we have compared the TCR repertoire of the CD57+ and CD57- subsets of CD8+ T cells in normal persons by using three-color FACS analysis with a panel of 16 mAbs specific for TCR V segments. The CD57 surface marker was highly variable in frequency but generally present on a minority of CD8+CD3+ T cells (mean 16.3%, SD 12.7) in a group of 41 normal volunteers. Dramatic oligoclonal expansion was present in the CD57+CD8+ T cell population in 15 of 41 (37%) of our study population and thus is a characteristic feature of the normal immune system. No such prominent oligoclonal expansions were observed in the CD57-CD8+ subset, although preliminary experiments suggest that oligoclonality per se is occasionally present at a lower frequency in CD57- cells. The reasons for this persistent accumulation of oligoclonal CD8+CD57+ T cells and their function in immune homeostasis are unclear.

Adult↗
PubMed1995

Alternative splicing of CD79a (Ig-alpha/mb-1) and CD79b (Ig-beta/B29) RNA transcripts in human B cells.

The CD79a (Ig-alpha/mb-1) and CD79b (Ig-beta/B29) molecules form a membrane heterodimer that is non-covalently associated with surface membrane immunoglobulin and is the major signaling component of the B cell antigen receptor complex. We have defined variant RNA transcripts for both CD79a (Ig-alpha/mb-1) and CD79b (Ig-beta/B29) which appear to arise by alternative splicing. These splice variants are predicted to encode truncated forms of these molecules that result in the deletion of the entire extracellular Ig-like domain of CD79b and of a major portion of the extracellular domain of CD79a. The presence of these short transcripts in a variety of human B cells and B cell lines was established by an RNAse protection assay. The definition of these variant transcripts provides a basis for a continuing effort to define variant protein products of CD79a and CD79b and examine their role in B cell physiology.

Alternative Splicing↗
PubMed1995

Oligoclonality in the human CD8+ T cell repertoire in normal subjects and monozygotic twins: implications for studies of infectious and autoimmune diseases.

BACKGROUND: We have previously demonstrated CD8+ T cell clonal dominance using a PCR assay for the CDR3 length of T cell receptors belonging to a limited number of TCRBV segments/families. In this study, we have modified this approach in order to analyze more comprehensively the frequency of oligoclonality in the CD8+ T cell subset in 25 known TCRBV segments/families. In order to assess the relative roles of genes and environment in the shaping of a clonally restricted CD8+ T cell repertoire, we have analyzed clonal dominance in the CD8+ T cell population of monozygotic twins, related siblings, and adoptees. MATERIALS AND METHODS: Oligoclonality was assessed in the CD8+ T cell subsets using a multiplex PCR approach to assay for CDR3 length variation across 25 different TCRBV segments/families. Specific criteria for oligoclonality were established, and confirmed by direct sequence analysis of the PCR products. This assay was used to investigate the CD8+ T cell repertoire of 56 normal subjects, as well as six sets of monozygotic (MZ) twins. RESULTS: Seventy-two percent of normal subjects (n = 56) had evidence of oligoclonality in the CD8+ T cell subset, using well-defined criteria. Although MZ twins frequently displayed CD8+ T cell clonal dominance, the overall pattern of oligoclonality was very diverse within each twin pair. However, we occasionally observed dominant CD8+ T cell clones that were highly similar in sequence in both members of some twin pairs. Not a single example of such similarity was observed in normal controls or siblings. CONCLUSIONS: Oligoclonality of circulating CD8+ T cells is a characteristic feature of the human immune system; both host genetic factors and environment shape the pattern of oligoclonality in this T cell subset. The high frequency of this phenomenon in normal subjects provides a background with which to evaluate CD8+ T cell oligoclonality in the setting of infection or autoimmune disease. Further phenotypic and functional characterization of these clonally expanded T cells should provide insight into normal immune homeostasis.

Adolescent↗
PubMed1994

The complete sequence of the human CD79b (Ig beta/B29) gene: identification of a conserved exon/intron organization, immunoglobulin-like regulatory regions, and allelic polymorphism.

We determined the complete genomic sequence of the human CD79b (Ig beta/B29) gene. The CD79b gene product is associated with the membrane immunoglobulin signaling complex which is composed of immunoglobulin (Ig) itself, associated in a noncovalent fashion with CD79b and a second polypeptide chain, CD79a (Ig alpha/mb1). The sequence and exon/intron organization of the human and mouse CD79b genes are highly similar. The gene organization suggests that some variant forms of CD79b may arise by virtue of alternative splicing of mRNA. In addition, a number of conserved regulatory sequences commonly found in Ig genes are present in sequences which flank the human CD79b gene. Some of these sequences are distinct from those found in the CD79a promoter. These differences may explain why transcription of CD79b, but not CD79a, is observed in plasma cells. A new Taq 1 restriction fragment length polymorphism is described that is not associated with any structural polymorphisms of the expressed CD79b polypeptide.

Alleles↗
PubMed1994

Chromosomal localization, genomic structure, and allelic polymorphism of the human CD79 alpha (Ig-alpha/mb-1) gene.

The germline DNA sequence of the human CD79 alpha (Ig-alpha/mb-1) gene was determined by polymerase chain reaction sequencing of a cosmid clone derived from an arrayed human chromosome 19 library. The CD79 alpha gene was localized to chromosome 19q13.2; this localization places the gene within the CEA-like gene cluster with the following gene order: -CEA-CGM1-CD79 alpha-RPS11-ATP1A3-BGP-CGM9-. The genomic organization of the human CD79 alpha gene resembles the mouse counterpart with five exons interrupted by four introns. Computer analyses suggest the presence of transcription regulatory elements known to be important in the regulation of mouse CD79 alpha (AP-1, EBF, AP-2, MUF2, and SP-1 sites), as well as elements not found in the mouse gene (an NK-kappa B binding site and a series of E-box motifs). Similar to the mouse gene, the 5' flanking region of human CD79 alpha lacks a TATA box; however, unlike mouse CD79 alpha, a classical octamer motif could not be identified in the human gene. Finally, a new Rsa I restriction fragment length polymorphism was defined in the non-coding regions of the human gene.

Amino Acid Sequence↗
PubMed1994

X chromosome inactivation patterns correlate with fetal-placental anatomy in monozygotic twin pairs: implications for immune relatedness and concordance for autoimmunity.

BACKGROUND: Monozygotic (MZ) twinning is a poorly understood phenomenon that may result in subtle biologic differences between twins, despite their identical inheritance. These differences may in part account for discordant expression of disease in MZ twin pairs. Due to their stochastic nature, differences in X chromosome inactivation patterns are one source of such variation in female MZ twins. MATERIALS AND METHODS: We investigated X chromosome inactivation patterns in the blood of 41 MZ twin pairs based on methylation of the androgen receptor gene using a Hpa II-PCR assay. Twenty-six female MZ twin pairs with autoimmune disease (rheumatoid arthritis or multiple sclerosis) were studied. In addition, we studied 15 newborn female MZ twin pairs who were characterized at birth with respect to the anatomy of chorionic membranes (dichorionic versus monochorionic). RESULTS: We found a strong correlation between dichorionic fetal anatomy and differences in X chromosome inactivation patterns between members of an MZ twin pair. In contrast, all monochorionic twin pairs had closely correlated patterns of X chromosome inactivation. X chromosome inactivation patterns did not distinguish between MZ twin pairs who were concordant or discordant for autoimmune disease. CONCLUSIONS: The highly similar patterns of X chromosome inactivation among monochorionic twin pairs may result from their shared placental blood supply during intrauterine life. Alternatively, these patterns may indicate that X chromosome inactivation occurs before the twinning event in this anatomic subgroup of MZ twins. The data further suggest that these factors do not make a major contribution to the high discordance rates for autoimmune disease in MZ twin pairs.

Autoimmune Diseases↗
PubMed1993

Clonal predominance of T cell receptors within the CD8+ CD45RO+ subset in normal human subjects.

Structural models for the TCR alpha/beta predict that the CDR1, CDR2, and CDR3 loops of both the alpha- and beta-chains contribute to specific interactions with the Ag/MHC complex. The CDR3 loops are constructed by joining events involving the V-(D)-J segments, and thus may vary in both sequence and length. We have developed a polymerase chain reaction assay to assess the length variation of the CDR3 loop in TCR derived from seven V beta segment families (V beta 2, V beta 3, V beta 4, V beta 9, V beta 14, V beta 16, and V beta 17). Peripheral blood T cells from 10 normal adults as well as five cord blood samples were studied. CD4+ and CD8+ T cells were analyzed separately. We observed extreme predominance of particular CDR3 lengths in half of the normal adults. These TCR were shown to be clonal by direct sequence analysis. This clonal dominance was found in the CD8+, CD45RO+ T cell population, and was observed in various V segment families. These patterns of TCR clonality were persistent over many months of observation in some individuals. In one subject, the new appearance of a predominant clone was associated with a booster vaccination for hepatitis B. These studies reveal a surprising degree of oligoclonality in the CD8+ cells of normal subjects which may be due to both environmental and genetic factors; the functional significance of persistent clonal dominance in the CD8 compartment remains to be determined.

Adult↗
PubMed1993

The human Ig-beta cDNA sequence, a homologue of murine B29, is identical in B cell and plasma cell lines producing all the human Ig isotypes.

The B cell Ag receptor complex consists of at least two disulfide-linked, heterodimeric structures: the clonally restricted membrane Ig (mIg) molecule and the nonpolymorphic Ig-alpha:Ig-beta protein dimer. The latter molecule is encoded by two separate genes, mb-1 and B29. The DNA sequences of murine and human mb-1 and murine B29 have been determined previously. This study describes the sequence of the full-length human cDNA homologue of the murine Ig-beta/B29 message. The human sequence codes for a protein that displays the typical subunit features of a transmembrane member of the Ig superfamily. The transmembrane and intracytoplasmic domains exhibit striking nucleotide and amino acid sequence similarity between the two species. These regions show almost complete conservation of areas presumed to be involved in noncovalent interactions with other members of the receptor complex and with intracellular kinases and cytoskeletal components. The only sequence dissimilarity seen in these presumed critical areas involves the Y-E-G-L-N motif, a potential target for tyrosine phosphorylation. In contrast, the extracellular portion is much more divergent. Inasmuch as similar patterns of species diversity have been reported for Ig-alpha, the Ig-alpha and Ig-beta molecules may have coevolved to maintain species-specific extracellular interactions between one another and with mIg. Similar to the Ig-alpha molecule, the Ig-beta sequence is identical in B lineage cells expressing all five Ig isotypes. However, in contrast to the Ig-alpha molecule, the Ig-beta sequence is expressed at apparently similar levels in terminally differentiated, mIg- plasma cells as well as in mIg+, mature B cells. These data suggest that Ig-beta has functions in addition to those associated with surface mIg expression.

Amino Acid Sequence↗