Dominant role of the α-chain in rejection of tumor cells bearing a specific alloantigen in TCRα transgenic mice and in in vitro experiments

Abstract

Both TCRα and TCRβ types of T-cell receptors contribute to antigen recognition. However, some TCRs have chain centricity, which means that either the α-chain or the β-chain dictates the peptide-MHC complex specificity. Most earlier reports investigated the role of well-studied β-chains in antigen recognition by TCRαβ. In a previous study, we identified TCRs specific to the H-2K^b molecule. In the present work, we generated transgenic mice carrying the α-chain of this TCR. We found that these transgenic mice rejected EL-4 tumor cells bearing alloantigen H-2K^b more effectively than wild-type mice and similarly to mice with established specific memory T cells. Moreover, we found that T cells transduced with this TCRα can inhibit EL-4 cell growth in vitro and in vivo. We also found that transgenic mice recruit fewer CD8 T cells into the peritoneal cavity at the peak of the immune response and had a significantly higher number of central memory CD8 T cells in the spleen of intact transgenic mice compared to intact wild-type control. These results indicate the ability of a single transgenic α-chain of the H-2K^b-specific TCR to determine specific recognition of the H-2K^b molecule by a repertoire of T lymphocytes and to rapidly reject H-2K^b-bearing lymphoma cells.

Keywords: TCRα; alloantigen; transgenic mice; tumor.

Figure 1. EL-4 cells were cultured with activated lymphocytes transduced with 1D1α and 1D1α-gfp constructions.

Analysis of the number of EL-4 cells (A) and splenic T-cell phenotype (B) were performed 24h after co-culturing. (A) The percentages of Kb positive cells were determined by flow cytometry in three groups of mixed cultures in vitro – control (R101 + EL-4) and two experimental (1D1α + EL-4 and 1D1α-gfp + EL-4). (B) The bar graph represents the ratio of CD4Vα11+, CD4Vα11–, CD8Vα11+, and CD8Vα11– in the culture of T cells expressing 1D1α-gfp without EL-4 relative to the culture of T cells expressing 1D1α-gfp along with EL-4. We define 1D1α-gfp positive cells as Vα11+ because GFP matches the cells expressing α-chain 1D1– a member of the Vα11 protein family. The data represent the mean ± sd (n = 4–6).

Figure 2. Flow-cytometric analysis of lymphocyte subpopulations in thymus of WT and Tg 1D1α mice.

(A) Dot plots show expression of CD8 vs CD4 on thymocytes of WT (left) and Tg (right) mice. (B) DN, CD4 SP, and CD8 SP and DP subpopulations in thymus of R101 and 1D1α mice. (C) The histogram visualizes the expression of the CD3 marker on DN thymocytes. (D) The percentages of CD3+ DN, CD3+ CD4 SP, CD3+ CD8 SP, and CD3+ DP thymocytes are shown. (E) Co-expression of CD44 and CD25 on DN-gated thymocytes. (F) The box plot shows the distribution of thymocytes over different stages of DN development defined by CD44 and CD25 surface expression. SP – single positive, DN – double negative, DP – double positive. (A), (C), (E) Data from one representative staining are shown.

Figure 3

(A) Flow-cytometric analysis of CD8 and CD4 expression on CD3-gated lymphocytes from the spleen of wild-type (left) and Tg (right) mice. Data from one representative staining are shown. (B) The box plot represents the number of CD3, CD4, CD8, and DN cells in the spleen of R101 and 1D1α mice. (C) The ratio of CD4 to CD8 T cells in the spleen of R101 and transgenic (1D1α) mice is shown. (D) Relative expression of Tg T-cell receptor α-chain 1D1 in lymphoid organs of Tg mice. Four transgenes were used for the qPCR analysis. The spleen of one Tg mouse was used as a reference sample. Tbp and ppia were used as reference genes for normalization of gene expression. The samples were run in triplicate. The data represent the mean and sd. (E) Analysis of expression of 14 distinct Vβ gene families in peripheral blood T cells of Tg and WT mice. Six transgenes and 5 WT mice were used in the experiment. The data represent the mean ± sd. (F) Proliferative response of splenocytes to specific and third-party alloantigens. Cells from spleens of 3-month-old Tg and WT mice were used as responders. Splenocytes from B10.D2(R101) (syngeneic), C57BL/6 (specific alloantigen), and FVB (third-party alloantigen) were used as stimulators. All stimulators were treated with mitomycin C. The background proliferation (i.e. the proliferative response to syngeneic stimulators) was subtracted from values obtained in response to the specific and third-party alloantigens. The excess of proliferative response of splenocytes to C57BL/6 and FVB stimulators of R101 MEMO (BL/6 R101 MEMO and FVB R101 MEMO, respectively, and Tg (BL/6 1D1α and FVB 1D1α, respectively) over R101 splenocytes is shown.

Figure 4. Estimation of tumor cell number in the peritoneal cavity on days 0, 3-4, 5-6, and 12 after immunization.

(A, B) Flow-cytometric analysis of EL-4 tumor cells in the lavage of WT (A) and Tg (B) mice. Arrows on the upper panels indicate the presence of EL-4 cells in samples as determined by forward scatter (FSC) and side scatter (SSC). The lower panels represent the number of Kb positive cells in the peritoneal cavity. We distinguished two populations of Kb+ cells – with high (Kb^high) or decreased (Kb^int, intermediate) expression of Kb. Data from one representative staining are shown. (C, D) Box plots show the number of Kb^high (left) and Kb^int (right) EL-4 cells in the lavage of WT (C) and Tg (D) mice on the days 0, 3-4, 5-6, and 12 after immunization.

Figure 5. Flow-cytometric analysis of CD3/CD8 expression on lymphocytes from the peritoneal cavity of WT (A, C) and Tg (B, D) mice on days 0, 3-4, 5-6, and 12 after immunization.

(A, B) Data from one representative staining are shown. (C, D) Box plots are used to visualize the distribution of a dataset.

Figure 6

(A–C) Flow-cytometric analysis of expression of CD8 and activation markers CD44 and CD62L on splenic T lymphocytes on days 0, 3-4, 5-6, and 12 after immunization. The expression of CD8- on CD3-gated lymphocytes was defined in the spleen of WT (A, C) and Tg (B, C) mice. (A, B) Data from one representative staining are shown. (C) The box plot shows the relative number of CD8+ T cells in the spleen of R101 (left) and 1D1α (right) mice. (D–H) Flow-cytometric analysis of co-expression of CD44 and CD62L markers on the surface of the CD8 subset of T lymphocytes in the spleen of WT (D, F, G, H) and Tg (E–H) mice. Data from one representative staining are shown. The box plots show the distribution of cells with naive (CD44-CD62L+) (F), effector memory (CD44+CD62L–) (G), and central memory (CD44+CD62L+) (H) phenotypes.