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Direct ex vivo analysis of activated, Fas-sensitive autoreactive T cells in human autoimmune disease.

Bieganowska KD, Ausubel LJ, Modabber Y, Slovik E, Messersmith W, Hafler DA - J. Exp. Med. (1997)

Bottom Line: T cells expressing mRNA transcripts encoding T cell receptor (TCR)-alpha and -beta chains found in T cell clones previously isolated from these subjects recognizing the MBPp85-99 epitope were examined.MBPp85-99 TCR transcripts were present in IL-2 receptor alpha-positive T cells which were induced to undergo Fas-mediated cell death upon antigen stimulation.These data demonstrate that at least a subpopulation of patients with MS can have a very high frequency of activated autoreactive T cells.

View Article: PubMed Central - PubMed

Affiliation: Center for Neurologic Diseases, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.

ABSTRACT
The frequency of clonally expanded and persistent T cells recognizing the immunodominant autoantigenic peptide of myelin basic protein (MBP)p85-99 was directly measured ex vivo in subjects with typical relapsing remitting multiple sclerosis (MS). T cells expressing mRNA transcripts encoding T cell receptor (TCR)-alpha and -beta chains found in T cell clones previously isolated from these subjects recognizing the MBPp85-99 epitope were examined. In contrast to frequencies of 1 in 10(5)-10(6) as measured by limiting dilution analysis, estimates of the T cell frequencies expressing MBPp85-99-associated TCR chain transcripts were as high as 1 in 300. These high frequencies were confirmed by performing PCR on single T cells isolated by flow cytometry. MBPp85-99 TCR transcripts were present in IL-2 receptor alpha-positive T cells which were induced to undergo Fas-mediated cell death upon antigen stimulation. These data demonstrate that at least a subpopulation of patients with MS can have a very high frequency of activated autoreactive T cells.

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Frequency of TCR-Vα3.1 transformants expressing the  CDR3 region sequence present in an MBPp85-99–reactive T cell clone  in MS patient Ob. (A) A representative experiment is shown, using mRNA  from peripheral blood lymphocytes. cDNA was synthesized and amplified  with Vα3.1- and Cα-specific primers. PCR products were ligated into  pCRII vectors and competent Escherichia coli were transformed with ligation products. Transformants were grown in 96-well plates and were transferred to nitrocellulose paper in duplicates. Blots were hybridized with either  Vα3.1 probe (bottom) or a specific Ob-TDA probe recognizing TCRVα3.1 junctional region sequence expressed in a previously isolated  MBP-reactive T cell clone (top). (B) The same peripheral blood lymphocytes from subject Ob were stimulated with MBPp85-99 for 7 d, followed by restimulation with antigen-pulsed WMNC and, on day 9, the  addition of IL-2. On day 14, mRNA was extracted from the antigenstimulated T cells and the proportion of transformants hybridizing with the  Ob-TDA–specific probe after Vα3.1 chain amplification was measured.
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Figure 1: Frequency of TCR-Vα3.1 transformants expressing the CDR3 region sequence present in an MBPp85-99–reactive T cell clone in MS patient Ob. (A) A representative experiment is shown, using mRNA from peripheral blood lymphocytes. cDNA was synthesized and amplified with Vα3.1- and Cα-specific primers. PCR products were ligated into pCRII vectors and competent Escherichia coli were transformed with ligation products. Transformants were grown in 96-well plates and were transferred to nitrocellulose paper in duplicates. Blots were hybridized with either Vα3.1 probe (bottom) or a specific Ob-TDA probe recognizing TCRVα3.1 junctional region sequence expressed in a previously isolated MBP-reactive T cell clone (top). (B) The same peripheral blood lymphocytes from subject Ob were stimulated with MBPp85-99 for 7 d, followed by restimulation with antigen-pulsed WMNC and, on day 9, the addition of IL-2. On day 14, mRNA was extracted from the antigenstimulated T cells and the proportion of transformants hybridizing with the Ob-TDA–specific probe after Vα3.1 chain amplification was measured.

Mentions: Using this approach, we could identify TCR-Vα chains expressed in MBPp85-99–reactive T cells in MS patients (Table 1, Fig. 1 A). Specifically, the percentage of Vα3.1positive transformants hybridizing with the Ob-TDA probe was 0.8% of Vα3.1 chains expressed in patient Ob; the percentages were 1.6% for probe Hy-TDA and 2.4% for probe Hy-TDT of Vα3.1 chains expressed in patient Hy (Table 1). Repeated experiments measuring the percentage of transformants hybridizing with either probe over a two-yr time interval yielded similar frequencies (Table 1). As expected, there was no crosshybridization of Hy probes with Ob transformants or of Ob probes with Hy transformants. The sequencing of 20 transformants expressing a TCR-α chain that hybridized to the Ob-TDA probe in patient Ob and 25 transformants that hybridized to either the Hy-TDA or Hy-TDT probes in patient Hy, demonstrated the same TCR-α sequence as that expressed in the original MBPreactive T cell clones. As expected, DNA from 20 random transformants that did not hybridize to the CDR3 probes contained different TCR-α junctional region sequences. In control subjects, after screening TCR-α transformants with Jl-SSI and Jl-SGS probes for Jl and Nb-ASI probe for Nb, we were unable to detect any sequences associated with recognition of MBPp85-99 in peripheral blood T cells (Table 1).


Direct ex vivo analysis of activated, Fas-sensitive autoreactive T cells in human autoimmune disease.

Bieganowska KD, Ausubel LJ, Modabber Y, Slovik E, Messersmith W, Hafler DA - J. Exp. Med. (1997)

Frequency of TCR-Vα3.1 transformants expressing the  CDR3 region sequence present in an MBPp85-99–reactive T cell clone  in MS patient Ob. (A) A representative experiment is shown, using mRNA  from peripheral blood lymphocytes. cDNA was synthesized and amplified  with Vα3.1- and Cα-specific primers. PCR products were ligated into  pCRII vectors and competent Escherichia coli were transformed with ligation products. Transformants were grown in 96-well plates and were transferred to nitrocellulose paper in duplicates. Blots were hybridized with either  Vα3.1 probe (bottom) or a specific Ob-TDA probe recognizing TCRVα3.1 junctional region sequence expressed in a previously isolated  MBP-reactive T cell clone (top). (B) The same peripheral blood lymphocytes from subject Ob were stimulated with MBPp85-99 for 7 d, followed by restimulation with antigen-pulsed WMNC and, on day 9, the  addition of IL-2. On day 14, mRNA was extracted from the antigenstimulated T cells and the proportion of transformants hybridizing with the  Ob-TDA–specific probe after Vα3.1 chain amplification was measured.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196290&req=5

Figure 1: Frequency of TCR-Vα3.1 transformants expressing the CDR3 region sequence present in an MBPp85-99–reactive T cell clone in MS patient Ob. (A) A representative experiment is shown, using mRNA from peripheral blood lymphocytes. cDNA was synthesized and amplified with Vα3.1- and Cα-specific primers. PCR products were ligated into pCRII vectors and competent Escherichia coli were transformed with ligation products. Transformants were grown in 96-well plates and were transferred to nitrocellulose paper in duplicates. Blots were hybridized with either Vα3.1 probe (bottom) or a specific Ob-TDA probe recognizing TCRVα3.1 junctional region sequence expressed in a previously isolated MBP-reactive T cell clone (top). (B) The same peripheral blood lymphocytes from subject Ob were stimulated with MBPp85-99 for 7 d, followed by restimulation with antigen-pulsed WMNC and, on day 9, the addition of IL-2. On day 14, mRNA was extracted from the antigenstimulated T cells and the proportion of transformants hybridizing with the Ob-TDA–specific probe after Vα3.1 chain amplification was measured.
Mentions: Using this approach, we could identify TCR-Vα chains expressed in MBPp85-99–reactive T cells in MS patients (Table 1, Fig. 1 A). Specifically, the percentage of Vα3.1positive transformants hybridizing with the Ob-TDA probe was 0.8% of Vα3.1 chains expressed in patient Ob; the percentages were 1.6% for probe Hy-TDA and 2.4% for probe Hy-TDT of Vα3.1 chains expressed in patient Hy (Table 1). Repeated experiments measuring the percentage of transformants hybridizing with either probe over a two-yr time interval yielded similar frequencies (Table 1). As expected, there was no crosshybridization of Hy probes with Ob transformants or of Ob probes with Hy transformants. The sequencing of 20 transformants expressing a TCR-α chain that hybridized to the Ob-TDA probe in patient Ob and 25 transformants that hybridized to either the Hy-TDA or Hy-TDT probes in patient Hy, demonstrated the same TCR-α sequence as that expressed in the original MBPreactive T cell clones. As expected, DNA from 20 random transformants that did not hybridize to the CDR3 probes contained different TCR-α junctional region sequences. In control subjects, after screening TCR-α transformants with Jl-SSI and Jl-SGS probes for Jl and Nb-ASI probe for Nb, we were unable to detect any sequences associated with recognition of MBPp85-99 in peripheral blood T cells (Table 1).

Bottom Line: T cells expressing mRNA transcripts encoding T cell receptor (TCR)-alpha and -beta chains found in T cell clones previously isolated from these subjects recognizing the MBPp85-99 epitope were examined.MBPp85-99 TCR transcripts were present in IL-2 receptor alpha-positive T cells which were induced to undergo Fas-mediated cell death upon antigen stimulation.These data demonstrate that at least a subpopulation of patients with MS can have a very high frequency of activated autoreactive T cells.

View Article: PubMed Central - PubMed

Affiliation: Center for Neurologic Diseases, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.

ABSTRACT
The frequency of clonally expanded and persistent T cells recognizing the immunodominant autoantigenic peptide of myelin basic protein (MBP)p85-99 was directly measured ex vivo in subjects with typical relapsing remitting multiple sclerosis (MS). T cells expressing mRNA transcripts encoding T cell receptor (TCR)-alpha and -beta chains found in T cell clones previously isolated from these subjects recognizing the MBPp85-99 epitope were examined. In contrast to frequencies of 1 in 10(5)-10(6) as measured by limiting dilution analysis, estimates of the T cell frequencies expressing MBPp85-99-associated TCR chain transcripts were as high as 1 in 300. These high frequencies were confirmed by performing PCR on single T cells isolated by flow cytometry. MBPp85-99 TCR transcripts were present in IL-2 receptor alpha-positive T cells which were induced to undergo Fas-mediated cell death upon antigen stimulation. These data demonstrate that at least a subpopulation of patients with MS can have a very high frequency of activated autoreactive T cells.

Show MeSH
Related in: MedlinePlus