Intrathymic selection of NK1.1⁺α/β T cell antigen receptor (TCR)⁺cells in transgenic mice bearing TCR specific for chicken ovalbumin and restricted to I-A^d

Abstract

A BSTR ACTGeneration and negative selection of NK1.1 ؉ ␣͞␤ T cell receptor (TCR) ؉ thymocytes were analyzed using TCR-transgenic (B10.D2 ؋ DO10)F 1 and (C57BL͞6 ؋ DO10)F 1 mice and Rag-1 ؊͞؊ ͞DO10 mice, which had been established by breeding and backcrossing between Rag-1 ؊͞؊ and DO10 mice. Almost all T cells from these mice were shown to bear V␣13͞V␤8.2 that is specific for chicken ovalbumin (cOVA) and restricted to I-A d . A normal proportion of the NK1.1 ؉ V␣13͞V␤8.2 ؉ thymocytes was generated in these mice. However, the actual cell number of both NK1.1 ؉ and NK1.1 ؊ thymocytes in I-A d͞d mice (positive selecting background) was larger than that in I-A b͞d mice (negative selecting background). Markedly low but significant proportions of NK1.1 ؉ V␣13͞V␤8.2 ؉ cells were detected in the spleens from I-A d͞d and I-A b͞d mice. It was shown that the splenic NK1.1 ؉

Immediate antigen‐specific effector functions byTCR‐transgenic CD8+ NKT cells

“…Therefore, we consider the NK1.1-positive population of T cells we found in TCR-transgenic animals as a genuine NKT cell population. In line with such an interpretation, other groups have reported the presence of NKT cells in CD8 + TCR-transgenic mouse lines H-Y [36,37], BM3.3 [38,39] and 2C [40], as well as in the CD4 + TCR-transgenic mouse lines DO11.10 [41] and 3BBM74 [42]. Here, we extend the list of mouse lines bearing tgNKT cells to include OT-I and P14 (Fig.…”

“…However, the mice analyzed in this study were unprimed and therefore have to be considered as antigen naive. Nonetheless, NK1.1 surface expression is probably not due to a general upregulation of NK1.1 on activated CD8 + T cells, as we never detected increased NK1.1 expression on transgenic CD8 + T cells after challenge with virally encoded antigen (ovalbumin-expressing adenovirus for OT-I) or soluble antigen (OVA 257-264 for OT-I and gp33 [33][34][35][36][37][38][39][40][41] for P14) in vitro or in vivo (data not shown). Schüler et al recently reported that thymic emigrants in neonates give rise to natural memory T cells with an activated phenotype [32], although the authors did not analyze the expression of NK1.1 on these cells.…”

“…Cytokine expression in tgNKT cells following anti-TCR Ab treatment has been reported for 2C [40] and DO11.10 [41] and antigen-specific cytotoxicity was shown for male H-Y T cells, but only after 8 days in culture with IL-2 (200 U/mL) [36]. None of these publications addressed early effector functions following antigen-specific stimulation.…”

“…For antigen-specific stimulation, we incubated OT-I and P14 lymphocytes in vitro with OVA 257-264 or gp33 [33][34][35][36][37][38][39][40][41] peptide, respectively. In contrast to the stimulation with anti-CD3e Ab in vivo, cytokine production (IFN-c, TNF-a and IL-2) was slower and was only seen after 1.5 h of incubation.…”

“…C57BL/6 mice were purchased from Jackson Laboratories (Bar Harbor, ME). The following utilized TCR-transgenic animals have been described previously: chicken ovalbumin-specific MHC class I (OT-I, OVA 257-264 , Va2/b5) [57] or MHC class II (OT-II, OVA 323-339 , Va2/b5 [58] and DO11.10, OVA 323-339 , Va13/ b8.2) [59,60]; LCMV-specific MHC class I (P14, gp33 [33][34][35][36][37][38][39][40][41] , Va2/b8.1 CD8 + T cells) [61], and male antigen-specific MHC class I H-Y (Va3/b8.2) [14]. To exclude endogenous TCR rearrangement, OT-I, P14 and H-Y mice were crossed onto the RAG2 -/-background.…”