Quantitation of T‐cell receptor Vβ chain expression on lymphocytes from blood, brain, and spinal fluid in patients with multiple sclerosis and other neurological diseases

Abstract

Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the central nervous system in which large numbers of T cells enter the brain and cerebrospinal fluid (CSF). To determine whether these cells represent restricted populations, we studied expression of T-cell receptor V beta chains on paired samples from the central nervous system and blood of patients with MS or other neurological diseases (OND) using a quantitative polymerase chain reaction. The distribution of V beta chain expression in blood was skewed, with a significant preponderance of message from V beta genes 1 through 8 (p = 0.0001). Such skewing was not present in samples from the CSF and brain. Patterns of V beta gene expression were different among paired samples from spinal fluid and blood and were relatively heterogeneous. Blood and CSF samples from a patient with acute meningitis were studied on two separate occasions. The patterns of V beta expression changed over 72 hours in both the blood and the CSF. With one exception, no oligoclonal populations of T cells were observed nor were there disease-specific patterns of V beta gene expression in the blood or CSF. Samples from 2 MS brains and 1 OND brain expressed patterns of V beta genes that were different and less heterogeneous than those in paired blood. In addition, expression of V beta 12 was remarkably increased in the 2 MS brains, suggesting a selective recruitment or expansion of T cells expressing this gene. These data demonstrate that populations of T cells from blood, spinal fluid, and brain differ from one another and can fluctuate during periods of acute inflammation.(ABSTRACT TRUNCATED AT 250 WORDS)

Developments in Quantitative PCR

“…This is particularly relevant when Q-PCR is applied to the measurement of temporal and functional variations of mRNA molecules (20)(21)(22)(23)(24). The measurement of gene expression in cultured cells is a typical experimental condition in which the absolute evaluation of the number of mRNA molecules can be of limited interest.…”

T cell receptor Vb5 and Vb17 clonal diversity in cerebrospinal fluid and peripheral blood lymphocytes of multiple sclerosis patients

“…30 Vb12 gene segment was found to be also frequently used (albeit to a lesser extent than Vb17) by MBP-speci®c T cell clones derived from PBL of HLA-DR2 MS patients and expansion of oligoclonal Vb12 + CSF T cells was found in three patients with chronic progressive MS. 27 Vb12 transcripts were also detected in brain lesions of half the HLA-DR2 MS patients in one study 22 and is predominantly expressed in two MS brains studied in an independent work. 28 Usuku et al, 29 have shown that cytokine expansion of CSF T cells led to the selective enrichment of a limited number of Vb families: Vb2, Vb6, Vb15 and Vb17 being the most frequently expressed. This heterogeneous Vb gene usage between patients at (or in close proximity to) the site of the lesions makes it unlikely that a given superantigen is involved in MS pathophysiology.…”

Quantitative PCR: An Overview

“…This type of PCR format has been used to analyze temporal and differential expression of mRNA (Rappolee et aI., 1988;Arrigo et aI., 1989Arrigo et aI., , 1990Choi et aI., 1989;Chelly et aI., 1988Chelly et aI., , 1990aSinger-Sam et aI., 1990b;Makino et aI., 1990;Murphy et aI., 1990;Park and Mayo, 1991;Golay et aI., 1991;Birnbaum and Van Ness, 1992;Mohler and Butler, 1991;Abe et aI., 1992;Wood et aI., 1992;Horikoshi et aI., 1992;Hall and Finn, 1992), to study alternative splicing (Neve et aI., 1990;Mochizuki et aI., 1992), to evaluate the amount of allele-specific transcripts differing by a single nucleotide (Singer-Sam et aI., 1992), or to anatomize the reverse transcription process of HIV-l in quiescent cells (Zack et aI., 1990(Zack et aI., , 1992.…”

“…In the majority of these quantitative methods the relative levels of mRNAs are determined by (1) coamplifying the target RNA with an internal control, and (2) comparing the ratio target RNA: control RNA in which the internal control is either an endogenous (Singer-Sam et aI. , 1992;Golay et aI., 1991;Park and Mayo, 1991;Neve et aI., 1990;Wood et aI., 1992;Birnbaum and Van Ness, 1992;Abe et aI., 1992;Noonan et aI., 1990) or an exogenous mRNA (Chelly et aI., 1990;Chapter 8, this volume;Rappolee et aI., 1988;Wood et aI., 1992;Ferre et aI., 1992bFerre et aI., , 1994.…”

“…RNA species connected through function or structure to the actual target have also been used. For example, for the estimation of relative level of the different T-cell receptor Vi3 chain mRNA, the Ca chain mRNA can be coamplified to normalize the expression of the V i3 chains (Birnbaum and Van Ness, 1992;Abe et aI., 1992). The Ca mRNA is better suited to play the role of an internal control than the i32-microglobulin or ribosomal RNA because its level of expression is closer to that of the target's.…”