For the analysis of mucosal immunity to HIV-1, we have recently established a line of transgenic (Tg) mice expressing the TCRalpha and TCRP genes of the murine CTL clone RT1 specific for P18-I10 (RGPGRAFVTI), an immunodominant gp160 envelope-derived epitope of IIIB isolate, restricted by the H-2D(d) MHC-I molecule. Here we examine those cells bearing specific TCR among the intraepithelial lymphocytes (IELs), with flow cytometric analysis using H-2D(d)/P18-I10 tetramers. We observed three distinct CD3(+), tetramer positive populations among the lELs: extra-thymic CD8alphaalpha(+), alphabetaTCR T-cells; CD8alphaalpha(+), gammadeltaTCR T-cells; and thymus-derived CD8alphabeta(+), alphabetaTCR T-cells. Challenge of these Tg mice with P18-I10 encoded by a vaccinia virus vector, either intrarectally (i.r.) or intraperitoneally (i.p.), revealed that the intraepithelial compartment seems to be a major site for prevention of the spread of viral infection. Such immunity appears due to the thymus-derived, CD8alphabeta(+) antigen-specific CTLs together with CD8alphaalpha(+) gammadelta cells, which regulate virus spread. This model system for studying CTL based immunity at mucosal sites should prove helpful in developing rational approaches for HIV control. (C) 2004 Elsevier Inc. All rights reserved.