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ITM2A is induced during thymocyte selection and T cell activation and causes downregulation of CD8 when overexpressed in CD4(+)CD8(+) double positive thymocytes.

Kirchner J, Bevan MJ - J. Exp. Med. (1999)

Bottom Line: We report here our studies of one gene, ITM2A, whose expression is dramatically higher in T cells in the selecting thymus.In addition, ITM2A expression is higher in the thymus than in either the spleen or lymph nodes, but can be upregulated in peripheral T cells upon activation.Expression on the surface of EL4 cells increases with activation by phorbol myristate acetate (PMA) and ionomycin.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute and the Department of Immunology, University of Washington, Seattle, Washington 98195, USA.

ABSTRACT
To identify novel genes that are involved in positive selection of thymocytes, we performed polymerase chain reaction (PCR)-based subtractive hybridization between selecting and nonselecting thymi. OT-1 T cell receptor (TCR) transgenic thymocytes on a recombination activating gene (RAG) background are efficiently selected into the CD8 lineage in H-2(b) mice (RAG-2(-/-)OT-1, selecting thymi), but are not selected on a transporter associated with antigen processing (TAP) background (RAG-2(-/-)TAP-1(-/-)OT-1, nonselecting thymi). We report here our studies of one gene, ITM2A, whose expression is dramatically higher in T cells in the selecting thymus. The expression pattern of ITM2A in thymocyte subsets correlates with upregulation during positive selection. In addition, ITM2A expression is higher in the thymus than in either the spleen or lymph nodes, but can be upregulated in peripheral T cells upon activation. ITM2A expression was also induced in RAG-2(-/-) thymocytes in vivo upon CD3 cross-linking. We demonstrate that ITM2A is a type II membrane glycoprotein that exists as two species with apparent M(r) of 45 and 43 kD and appears to localize primarily to large cytoplasmic vesicles and the Golgi apparatus, but is also expressed on the cell surface. Expression on the surface of EL4 cells increases with activation by phorbol myristate acetate (PMA) and ionomycin. Finally, overexpression of ITM2A under control of the lck proximal promoter in mice results in partial downregulation of CD8 in CD4(+)CD8(+) double positive (DP) thymocytes, and a corresponding increase in the number of CD4(+)CD8(lo) thymocytes. Possible roles for this novel activation marker in thymocyte development are discussed.

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Northern blot analysis of activated splenocytes. (A) C57BL/6 splenocytes were cultured in medium alone (medium) or supplemented with 5 μg/ml ConA (+ConA). (B) P14 splenocytes were cultured alone (−p33) or in the presence of irradiated EL4 cells coated with p33 peptide (+p33). Total RNA was extracted from cells harvested at various times during activation and from an untreated C57BL/6 thymus (Thy). The blots in A and B were hybridized with probes for ITM2A, EF1α, and CD69 (shown for A only).
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Figure 4: Northern blot analysis of activated splenocytes. (A) C57BL/6 splenocytes were cultured in medium alone (medium) or supplemented with 5 μg/ml ConA (+ConA). (B) P14 splenocytes were cultured alone (−p33) or in the presence of irradiated EL4 cells coated with p33 peptide (+p33). Total RNA was extracted from cells harvested at various times during activation and from an untreated C57BL/6 thymus (Thy). The blots in A and B were hybridized with probes for ITM2A, EF1α, and CD69 (shown for A only).

Mentions: To determine if ITM2A expression was upregulated in peripheral T cells upon activation, we examined expression in splenocytes harvested at various time points after activation. C57BL/6 splenocytes were cultured in the presence of the T cell mitogen, ConA (Fig. 4 A). Splenocytes from P14 mice, which express an MHC class I–restricted transgenic TCR, were activated by antigen presentation of the cognate peptide (Fig. 4 B). The blots were probed for ITM2A, CD69, a T cell activation marker 33343536, and EF1α, a housekeeping gene. In both experiments, the amount of ITM2A transcript started to increase within 30 min of activation and peaked at 6 h, reaching a level similar to that in an untreated C57BL/6 thymus. Expression was greatly reduced by 24 h. The kinetics of ITM2A induction in splenocytes were similar to those of CD69 (Fig. 4 A, and data not shown), but ITM2A and CD69 transcripts were present at quite different steady state levels in the thymus. Although these experiments do not exclude the possibility that some amount of the ITM2A transcript was contributed by non-T cells, expression of ITM2A, like CD69, was upregulated as a direct result of signals transmitted through the TCR upon binding of ConA or the more physiological MHC-peptide ligand.


ITM2A is induced during thymocyte selection and T cell activation and causes downregulation of CD8 when overexpressed in CD4(+)CD8(+) double positive thymocytes.

Kirchner J, Bevan MJ - J. Exp. Med. (1999)

Northern blot analysis of activated splenocytes. (A) C57BL/6 splenocytes were cultured in medium alone (medium) or supplemented with 5 μg/ml ConA (+ConA). (B) P14 splenocytes were cultured alone (−p33) or in the presence of irradiated EL4 cells coated with p33 peptide (+p33). Total RNA was extracted from cells harvested at various times during activation and from an untreated C57BL/6 thymus (Thy). The blots in A and B were hybridized with probes for ITM2A, EF1α, and CD69 (shown for A only).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2195576&req=5

Figure 4: Northern blot analysis of activated splenocytes. (A) C57BL/6 splenocytes were cultured in medium alone (medium) or supplemented with 5 μg/ml ConA (+ConA). (B) P14 splenocytes were cultured alone (−p33) or in the presence of irradiated EL4 cells coated with p33 peptide (+p33). Total RNA was extracted from cells harvested at various times during activation and from an untreated C57BL/6 thymus (Thy). The blots in A and B were hybridized with probes for ITM2A, EF1α, and CD69 (shown for A only).
Mentions: To determine if ITM2A expression was upregulated in peripheral T cells upon activation, we examined expression in splenocytes harvested at various time points after activation. C57BL/6 splenocytes were cultured in the presence of the T cell mitogen, ConA (Fig. 4 A). Splenocytes from P14 mice, which express an MHC class I–restricted transgenic TCR, were activated by antigen presentation of the cognate peptide (Fig. 4 B). The blots were probed for ITM2A, CD69, a T cell activation marker 33343536, and EF1α, a housekeeping gene. In both experiments, the amount of ITM2A transcript started to increase within 30 min of activation and peaked at 6 h, reaching a level similar to that in an untreated C57BL/6 thymus. Expression was greatly reduced by 24 h. The kinetics of ITM2A induction in splenocytes were similar to those of CD69 (Fig. 4 A, and data not shown), but ITM2A and CD69 transcripts were present at quite different steady state levels in the thymus. Although these experiments do not exclude the possibility that some amount of the ITM2A transcript was contributed by non-T cells, expression of ITM2A, like CD69, was upregulated as a direct result of signals transmitted through the TCR upon binding of ConA or the more physiological MHC-peptide ligand.

Bottom Line: We report here our studies of one gene, ITM2A, whose expression is dramatically higher in T cells in the selecting thymus.In addition, ITM2A expression is higher in the thymus than in either the spleen or lymph nodes, but can be upregulated in peripheral T cells upon activation.Expression on the surface of EL4 cells increases with activation by phorbol myristate acetate (PMA) and ionomycin.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute and the Department of Immunology, University of Washington, Seattle, Washington 98195, USA.

ABSTRACT
To identify novel genes that are involved in positive selection of thymocytes, we performed polymerase chain reaction (PCR)-based subtractive hybridization between selecting and nonselecting thymi. OT-1 T cell receptor (TCR) transgenic thymocytes on a recombination activating gene (RAG) background are efficiently selected into the CD8 lineage in H-2(b) mice (RAG-2(-/-)OT-1, selecting thymi), but are not selected on a transporter associated with antigen processing (TAP) background (RAG-2(-/-)TAP-1(-/-)OT-1, nonselecting thymi). We report here our studies of one gene, ITM2A, whose expression is dramatically higher in T cells in the selecting thymus. The expression pattern of ITM2A in thymocyte subsets correlates with upregulation during positive selection. In addition, ITM2A expression is higher in the thymus than in either the spleen or lymph nodes, but can be upregulated in peripheral T cells upon activation. ITM2A expression was also induced in RAG-2(-/-) thymocytes in vivo upon CD3 cross-linking. We demonstrate that ITM2A is a type II membrane glycoprotein that exists as two species with apparent M(r) of 45 and 43 kD and appears to localize primarily to large cytoplasmic vesicles and the Golgi apparatus, but is also expressed on the cell surface. Expression on the surface of EL4 cells increases with activation by phorbol myristate acetate (PMA) and ionomycin. Finally, overexpression of ITM2A under control of the lck proximal promoter in mice results in partial downregulation of CD8 in CD4(+)CD8(+) double positive (DP) thymocytes, and a corresponding increase in the number of CD4(+)CD8(lo) thymocytes. Possible roles for this novel activation marker in thymocyte development are discussed.

Show MeSH
Related in: MedlinePlus