Cutting edge: Highly alloreactive dual TCR T cells play a dominant role in graft-versus-host disease

Abstract

Alloreactivity is the response of T cells to MHC molecules not encountered during thymic development. A small population (1-8%) of peripheral T cells in mice and humans express two TCRs due to incomplete allelic exclusion of TCRalpha, and we hypothesized they are highly alloreactive. FACS analysis of mouse T cell MLR revealed increased dual TCR T cells among alloreactive cells. Quantitative assessment of the alloreactive repertoire demonstrated a nearly 50% reduction in alloreactive T cell frequency among T cells incapable of expressing a secondary TCR. We directly demonstrated expansion of the alloreactive T cell repertoire at the single cell level by identifying a dual TCR T cell with distinct alloreactivities for each TCR. The importance of dual TCR T cells is clearly demonstrated in a parent-into-F(1) model of graft-vs-host disease, where dual TCR T cells comprised up to 60% of peripheral activated T cells, demonstrating a disproportionate contribution to disease.

FIGURE 1

Dual TCR T cells are increased among alloreactive T cells. CFSE-labeled T cells from B6 (H-2^b) mice cultured in MLR with irradiated splenocytes from either CBA (H-2^k) or BALB/c (H-2^d) mice or anti-CD3 and anti-CD28 mAbs for 4 d were analyzed by flow cytometry for the presence of dual TCR T cells. Cells were gated on CD4⁺ and CD8⁺ T cells, and alloreactive T cells were distinguished by gating on cells with diluted CFSE. A, Dual TCR T cells were consistently observed in higher frequency among T cells that had divided in response to allogeneic stimulation as compared to undivided controls. FACS data of dual TCRα T cells from a representative experiment with allogeneic stimulation by B6.K splenocytes show the increased dual TCRα T cells among T cells that had divided in response to allogeneic stimulation as measured by CFSE dilution. B, To compare the frequency of T cells expressing dual TCRα between populations and 5 independent experiments, T cell numbers were normalized to Vα2⁺ cells, demonstrating a consistent and significant (mean ± s.d. * p < 0.05 ** p < 0.01) increase in dual TCR T cells among alloreactive T cells. C, Increased dual TCR T cells were also consistently and significantly (mean ± s.d. * p < 0.05 ** p < 0.01) observed among alloreactive CD4⁺Vα2⁺ T cells. Stimulation with anti-CD3 and anti-CD28 mAbs did not result in significantly increased dual TCRα T cells (p = 0.18–0.10).

FIGURE 2

Secondary TCRα contribute nearly half of the naive alloreactive T cell repertoire. A, Alloreactive T cell frequency of CD4⁺ T cells from B6 mice and from B6.Cα^+/−mice incapable of expressing secondary TCRα were measured by ELISPOT analysis for IFN-γ production following 48 h MLR with irradiated B6.K splenocytes. Data from a representative experiment illustrates the consistent 40–60% reduction in alloreactive T cell frequency among CD4⁺ T cells from B6.Cα^+/−mice. B, Integration of multiple cell concentrations from 6 independent experiments by normalizing the frequency of CD4⁺ T cell alloreactivity as a percentage of the alloreactivity of CD4⁺ T cells from B6 mice demonstrates the consistent and significant (mean ± s.d. p = 0.001) reduction in alloreactivity among CD4⁺ T cells from B6.Cα^+/− mice.

FIGURE 3

2.102 secondary TCRα generates a non-thymically-selected TCR with specific and distinct alloreactivity. A secondary Vα2, Vβ1 TCR was identified from the Vα4, Vβ1 expressing 2.102 T cell clone. A, The Vα2, Vβ1 secondary TCR is not thymically selected, as RAG^−/− mice transgenic for this receptor demonstrate thymocytes arrested at the double positive stage of development, and no peripheral αβ T cells (data not shown). B, Double positive thymocytes express the transgenic Vα2. C, The secondary Vα2, Vβ1 TCR demonstrates specific alloreactivity against H-2^d, distinct from the alloreactivity against H-2E^p demonstrated by the primary Vα4, Vβ1 2.102 TCR. T cell hybrids expressing the secondary Vα2, Vβ1 TCR were tested for reactivity against a panel of allogeneic irradiated splenocytes, and T cell hybrid reactivity was assessed by measurement of IL-2 production by ELISA. T cell hybrids expressing the Vα2 secondary TCR were specifically reactive to H-2^d APC, but not to H-2^p APC, demonstrating a distinct alloreactivity (mean ± s.d. p = 0.05).

FIGURE 4

Dual TCR T cells are profoundly increased among T cells mediating GvHD. B6 Ly5.1⁺ bone marrow cells and splenocytes were transferred into lethally irradiated (B6 × CBA)F₁ or control irradiated B6 mice. Lymphocytes were collected from spleens and lymph nodes and analyzed by flow cytometry using the 4 available Vα mAbs. A, Mice with GvHD demonstrated dramatically increased numbers of dual TCR T cells by FACS analysis compared to control B6 mice. FACS analysis of representative mice illustrate markedly increased dual TCR T cell frequency in mice with GvHD. Dual TCR T cells from mice with GvHD also exhibited down-regulation of their TCR, consistent with T cell activation. B, Dual TCR T cells from mice with GvHD are larger as measured by forward scatter, consistent with activation. C, Normalization of Vα2⁺ T cells expressing a second TCRα quantifies the dramatic increase in dual TCR T cells in mice with GvHD. Significantly more Vα2⁺ T cells from mice with GvHD expressed a second TCRα (mean ± s.d * p < 0.05 ** p < 0.01). D, The increase in dual TCR T cells was also observed among both alloreactive CD4⁺Vα2⁺ T cells and E, CD8⁺Vα2⁺ T cells.