OT-I T cells are deleted late in development in RIP-mOVA thymuses. (A) OT-I transitional cells and CD8 SP thymocytes are reduced in RIP-mOVA thymuses. Grafting OT-I BM into lethally irradiated B6 or RIP-mOVA recipients generated [OT-I→B6] or [OT-I→RIP-mOVA] BM-chimeric thymuses. Thymocytes from the indicated mice were analyzed for expression of CD4 and CD8 by flow cytometry. The numbers below indicate the percentages of cells in each gate. (B) Remaining CD8 SP thymocytes in RIP-mOVA mice are immature. CD24 and TCR (Vα2) expression after gating on CD8 SP thymocytes. Numbers in the quadrants indicate the percentage of mature phenotype cells. Results presented here are representative of three independent studies with two to four mice per group. (C) Vα2 expression by thymocyte populations gated as shown in A. Solid black line represents thymocytes from RIP-mOVA mice, and shaded gray histograms represent thymocytes from B6 mice. The bar indicates Vα2 levels on transitional thymocytes from B6 mice. (D) Schematic representation of OT-I thymocyte development and postulated stage of deletion in RIP-mOVA thymuses.

Deletion of OT-I/RIP-mOVA thymocytes does not require antigen presentation by BM-derived cells. Lethally irradiated RIP-mOVA mice were reconstituted with either OT-I or OT-I.MHC I^−/− BM. Results presented here are representative of three independent studies with two to four mice per group. (A) Thymocytes from the indicated mice were analyzed for CD4 and CD8 expression by flow cytometry. The numbers indicate the percentage of cells in each gate. (B) CD24 and Vα2 expression after gating on CD8 SP thymocytes. Numbers in the quadrants indicate the percentage of mature phenotype cells. (C) Vα2 expression was analyzed on DP dull, CD4, and CD8 thymocytes taken from the indicated mice. The data analyzed are from the gated populations shown in Fig. 2 A for [OT-I→B6] BM chimeras and from the gated populations shown in A for [OT-I→RIP-mOVA] and [OT-I.MHC I^−/−→RIP-mOVA] BM chimeras. Shaded gray histogram represents the DP dull population, and blue and red lines represent CD4 SP (transitional) and CD8 SP populations, respectively.

BM-derived cells in RIP-mOVA thymus can efficiently delete OT-I thymocytes. OT-I.MHC I^−/− or OT-I BM was grafted onto lethally irradiated K14-K^b.RIP-mOVA.MHC I^−/− recipients. (A) Thymocytes from the indicated mice were analyzed for CD4 and CD8 expression. The numbers below indicate the percentage of cells in each gate. (B) CD24 and Vα2 expression after gating on CD8 SP thymocytes. Numbers in the quadrants indicate the percentage of mature phenotype cells. Results presented here are representative of two studies with two mice per group.

Expression of OVA by Mtecs leads to deletion of a polyclonal repertoire of OVA-specific CD8 T cells. (A) OVA is selectively expressed in Mtecs in RIP-mOVA thymuses. Ctecs and Mtecs from RIP-mOVA thymus were enriched and expression of OVA was investigated by real-time PCR. Cathepsin L (Cat L), cathepsin S (Cat S), and insulin were monitored as controls for cell purity. Ctecs expressed high levels of cathepsin L, low levels of cathepsin S, and no insulin. Meanwhile, Mtecs expressed low levels of cathepsin L, high levels of cathepsin S, and insulin (reference 3). (B) Grafting Vβ5 BM (CD45.2) into lethally irradiated B6 or RIP-mOVA recipients generated [Vβ5→B6] or [Vβ5→RIP-mOVA] BM-chimeric thymuses. 8 wk after BM transfer, 10⁶ CD8 thymocytes were transferred into wild-type, congenically marked B6 mice (CD45.1). 1 d later, these mice were immunized with rLmOva. 7 d after immunization, splenocytes were isolated and their ability to produce IFN-γ was determined in response to a 5-h incubation with OVA peptide or medium alone. This experiment was performed twice with two mice per group. (C) IFN-γ and CD45.2 staining on gated CD8 T cells. Trasfer of thymocytes from [Vβ5→B6] is shown in the left panels. Transfer of thymocytes from [Vβ5→RIP-mOVA] is shown in the right panels. Numbers below indicate percentages of cells in each quadrant. Vβ5 transgenic donor-derived CD8 T cells are CD45.2⁺, whereas host-derived CD8 T cells are CD45.2⁻.

Deletion of OVA-specific polyclonal CD8 thymocytes does not require antigen presentation by BM-derived cells. (A) Grafting Vβ5 or Vβ5.MHC I^−/− marrow into lethally irradiated B6 or RIP-mOVA recipients generated [Vβ5→B6], [Vβ5→RIP-mOVA], [Vβ5.MHC I^−/−→B6], and [Vβ5.MHC I^−/−→RIP-mOVA] BM-chimeric thymuses. 2–4 mo after BM transfer, 0.5 × 10⁶ Vβ5 CD8 thymocytes matured in the indicated thymic environments were transferred into NK1.1-depleted (i.p. injection of 500 μg of the monoclonal antibody PK136) RAG-1^−/− mice. 1 d later, these mice were immunized with rLmOva. To eradicate remaining rLmOVA, on day 4 after immunization, mice were injected i.p. with 1 mg ampicillin and were given ampicillin in their drinking water (5 mg/ml) for the remainder of the experiment. 7 d after immunization, splenocytes were isolated and their ability to produce IFN-γ was determined in response to a 5-h incubation with OVA peptide or medium alone. (B) IFN-γ and CD8 staining on gated CD8 T cells from the indicated transfers. Numbers indicate percentages of cells within the gate.

OT-I CD8 T cells are positively selected in K14-K^b thymuses and are not negatively selected when RIP-mOVA is also expressed. Results presented here are representative of two studies with one to three mice per group. All mice are littermates. (A) Thymocytes from the indicated mice were analyzed for CD4 and CD8 expression. The numbers below indicate the percentage of cells in each gate. (B) CD24 and Vα2 expression after gating on CD8 SP thymocytes. Numbers in the quadrants indicate the percentage of cells with a mature phenotype.

Antigen presentation by BM-derived cells is required for deletion of OT-II thymocytes in RIP-mOVA thymus. Lethally irradiated B6 or RIP-mOVA mice were grafted with either OT-II or OT-II.MHC II^−/− BM. (A) Thymocytes from the indicated mice were analyzed for CD4 and CD8 expression. The numbers indicate the percentage of cells in each gate. (B) CD24 and Vα2 expression after gating on CD4 SP thymocytes. Numbers in the quadrants indicate the percentage of mature phenotype cells. (C) Graphic representation of results from three studies. Each symbol represents data from one mouse.