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Sensory adaptation in naive peripheral CD4 T cells.

Smith K, Seddon B, Purbhoo MA, Zamoyska R, Fisher AG, Merkenschlager M - J. Exp. Med. (2001)

Bottom Line: Here we show that pMHC contact modulates the expression of CD5 by naive CD4 T cells in a process that requires the continued expression of p56(lck).Reduced CD5 levels in T cells deprived of pMHC contact are predictive of elevated Ca(2)+ responses to subsequent TCR engagement by anti-CD3 or nominal antigen.Adaptation to peripheral pMHC contact may be important for regulating naive CD4 T cell responsiveness.

View Article: PubMed Central - PubMed

Affiliation: Lymphocyte Development Group, MRC Clinical Sciences Centre, ICSM Hammersmith Hospital, London W12 0NN, UK.

ABSTRACT
T cell receptor interactions with peptide/major histocompatibility complex (pMHC) ligands control the selection of T cells in the thymus as well as their homeostasis in peripheral lymphoid organs. Here we show that pMHC contact modulates the expression of CD5 by naive CD4 T cells in a process that requires the continued expression of p56(lck). Reduced CD5 levels in T cells deprived of pMHC contact are predictive of elevated Ca(2)+ responses to subsequent TCR engagement by anti-CD3 or nominal antigen. Adaptation to peripheral pMHC contact may be important for regulating naive CD4 T cell responsiveness.

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Modulation of CD5 levels by environmental MHC expression and haplotype. APC-depleted Rag1o/o H2bxk AND TCR transgenic LN cells were placed in 3-D reaggregate culture with a combination of thymic stroma and BM-derived APCs (reference 39) prepared from MHCo mice (thin line) or from mice expressing one of two selecting MHC haplotypes, H2b (C57BL/10, bold line), or H2k (B10.BR, dashed line). 3 d later cell recovery was between 60 and 80% of input for all MHC haplotypes (data not shown) and cells were analyzed (A) for forward scatter, CD4, CD45RB, CD69, and CD5 expression by flow cytometry (histogram overlays are gated on CD4 cells; the brighter CD5 staining in this and subsequent figures is due to the use of PE-conjugated mAb instead of the FITC conjugates used in previous figures) and (B) for tyrosine phosphorylation status by Western blot analysis of total cell lysates with anti-phosphotyrosine mAb. The blot was stripped and reprobed for CD3 ζ protein.
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fig4: Modulation of CD5 levels by environmental MHC expression and haplotype. APC-depleted Rag1o/o H2bxk AND TCR transgenic LN cells were placed in 3-D reaggregate culture with a combination of thymic stroma and BM-derived APCs (reference 39) prepared from MHCo mice (thin line) or from mice expressing one of two selecting MHC haplotypes, H2b (C57BL/10, bold line), or H2k (B10.BR, dashed line). 3 d later cell recovery was between 60 and 80% of input for all MHC haplotypes (data not shown) and cells were analyzed (A) for forward scatter, CD4, CD45RB, CD69, and CD5 expression by flow cytometry (histogram overlays are gated on CD4 cells; the brighter CD5 staining in this and subsequent figures is due to the use of PE-conjugated mAb instead of the FITC conjugates used in previous figures) and (B) for tyrosine phosphorylation status by Western blot analysis of total cell lysates with anti-phosphotyrosine mAb. The blot was stripped and reprobed for CD3 ζ protein.

Mentions: The data so far demonstrate that MHC contact and lck are required to sustain CD5 levels on naive peripheral CD4 T cells but they do not distinguish between TCR and coreceptor involvement in the regulation of CD5 expression. To approach this issue we made use of the fact that AND TCR transgenic T cells are positively selected on Ab as well as Ek, the latter being considered to provide ‘stronger’ positive selection (41). We adopted an in vitro 3-D reaggregate culture system (49) which allowed us to maintain naive AND CD4 T cells in an environment manipulated to either lack MHC expression or to express either of the two selecting MHC haplotypes, H-2b or H-2k. Rag1o/o H2bxk AND TCR transgenic naive CD4 LN cells were placed in reaggregate culture with a combination of thymic stroma cells (depleted of endogenous lymphoid cells by treatment with deoxyguanosine) and BM-derived APCs (39) prepared from MHCo/o, H2b (C57BL/10), or H2k (B10.BR) mice. 3 d later, although most cells maintained their initial phenotypic characteristics, CD5 levels varied according to MHC exposure. The lowest levels were evident on CD4 cells deprived of MHC interactions, intermediate levels on cells maintained in an H-2b expressing environment, and the highest levels on cells maintained in the presence of H-2k (Fig. 4) (neither H2b thymic stroma nor H2b BM-derived APCs on their own were able to sustain CD5 expression; data not shown). Basal ζ chain phosphorylation was lost in the absence of MHC, but maintained by either of the selecting MHC haplotypes (Fig. 4). These data show that CD5 expression by naive peripheral T cells is subject to haplotype-specific regulation. As described previously for the regulation of CD5 levels during thymocyte development (19, 20), this result suggests an involvement of specific TCR recognition over and above coreceptor engagement in the regulation of CD5 expression by peripheral T cells.


Sensory adaptation in naive peripheral CD4 T cells.

Smith K, Seddon B, Purbhoo MA, Zamoyska R, Fisher AG, Merkenschlager M - J. Exp. Med. (2001)

Modulation of CD5 levels by environmental MHC expression and haplotype. APC-depleted Rag1o/o H2bxk AND TCR transgenic LN cells were placed in 3-D reaggregate culture with a combination of thymic stroma and BM-derived APCs (reference 39) prepared from MHCo mice (thin line) or from mice expressing one of two selecting MHC haplotypes, H2b (C57BL/10, bold line), or H2k (B10.BR, dashed line). 3 d later cell recovery was between 60 and 80% of input for all MHC haplotypes (data not shown) and cells were analyzed (A) for forward scatter, CD4, CD45RB, CD69, and CD5 expression by flow cytometry (histogram overlays are gated on CD4 cells; the brighter CD5 staining in this and subsequent figures is due to the use of PE-conjugated mAb instead of the FITC conjugates used in previous figures) and (B) for tyrosine phosphorylation status by Western blot analysis of total cell lysates with anti-phosphotyrosine mAb. The blot was stripped and reprobed for CD3 ζ protein.
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Related In: Results  -  Collection

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fig4: Modulation of CD5 levels by environmental MHC expression and haplotype. APC-depleted Rag1o/o H2bxk AND TCR transgenic LN cells were placed in 3-D reaggregate culture with a combination of thymic stroma and BM-derived APCs (reference 39) prepared from MHCo mice (thin line) or from mice expressing one of two selecting MHC haplotypes, H2b (C57BL/10, bold line), or H2k (B10.BR, dashed line). 3 d later cell recovery was between 60 and 80% of input for all MHC haplotypes (data not shown) and cells were analyzed (A) for forward scatter, CD4, CD45RB, CD69, and CD5 expression by flow cytometry (histogram overlays are gated on CD4 cells; the brighter CD5 staining in this and subsequent figures is due to the use of PE-conjugated mAb instead of the FITC conjugates used in previous figures) and (B) for tyrosine phosphorylation status by Western blot analysis of total cell lysates with anti-phosphotyrosine mAb. The blot was stripped and reprobed for CD3 ζ protein.
Mentions: The data so far demonstrate that MHC contact and lck are required to sustain CD5 levels on naive peripheral CD4 T cells but they do not distinguish between TCR and coreceptor involvement in the regulation of CD5 expression. To approach this issue we made use of the fact that AND TCR transgenic T cells are positively selected on Ab as well as Ek, the latter being considered to provide ‘stronger’ positive selection (41). We adopted an in vitro 3-D reaggregate culture system (49) which allowed us to maintain naive AND CD4 T cells in an environment manipulated to either lack MHC expression or to express either of the two selecting MHC haplotypes, H-2b or H-2k. Rag1o/o H2bxk AND TCR transgenic naive CD4 LN cells were placed in reaggregate culture with a combination of thymic stroma cells (depleted of endogenous lymphoid cells by treatment with deoxyguanosine) and BM-derived APCs (39) prepared from MHCo/o, H2b (C57BL/10), or H2k (B10.BR) mice. 3 d later, although most cells maintained their initial phenotypic characteristics, CD5 levels varied according to MHC exposure. The lowest levels were evident on CD4 cells deprived of MHC interactions, intermediate levels on cells maintained in an H-2b expressing environment, and the highest levels on cells maintained in the presence of H-2k (Fig. 4) (neither H2b thymic stroma nor H2b BM-derived APCs on their own were able to sustain CD5 expression; data not shown). Basal ζ chain phosphorylation was lost in the absence of MHC, but maintained by either of the selecting MHC haplotypes (Fig. 4). These data show that CD5 expression by naive peripheral T cells is subject to haplotype-specific regulation. As described previously for the regulation of CD5 levels during thymocyte development (19, 20), this result suggests an involvement of specific TCR recognition over and above coreceptor engagement in the regulation of CD5 expression by peripheral T cells.

Bottom Line: Here we show that pMHC contact modulates the expression of CD5 by naive CD4 T cells in a process that requires the continued expression of p56(lck).Reduced CD5 levels in T cells deprived of pMHC contact are predictive of elevated Ca(2)+ responses to subsequent TCR engagement by anti-CD3 or nominal antigen.Adaptation to peripheral pMHC contact may be important for regulating naive CD4 T cell responsiveness.

View Article: PubMed Central - PubMed

Affiliation: Lymphocyte Development Group, MRC Clinical Sciences Centre, ICSM Hammersmith Hospital, London W12 0NN, UK.

ABSTRACT
T cell receptor interactions with peptide/major histocompatibility complex (pMHC) ligands control the selection of T cells in the thymus as well as their homeostasis in peripheral lymphoid organs. Here we show that pMHC contact modulates the expression of CD5 by naive CD4 T cells in a process that requires the continued expression of p56(lck). Reduced CD5 levels in T cells deprived of pMHC contact are predictive of elevated Ca(2)+ responses to subsequent TCR engagement by anti-CD3 or nominal antigen. Adaptation to peripheral pMHC contact may be important for regulating naive CD4 T cell responsiveness.

Show MeSH