Influence of direct and indirect allorecognition pathways on CD4+CD25+ regulatory T-cell function in transplantation

Abstract

While both direct and indirect allorecognition are involved in allograft rejection, evidence to date suggests that tolerance is primarily dependent on indirect pathway-triggered CD4+CD25+ T cell-mediated immunoregulation. However, the precise influence of these two pathways on CD4+CD25+ T-cell function has not been addressed. In the current study, we have utilized an adoptive transfer model to assess selectively how the absence of either direct or indirect allorecognition affects CD4+CD25+ T-cell function. The effects of the loss of the direct pathway were assessed by transplanting skin grafts from minor histocompatibility mismatched B10.D2 (H-2d) donors onto Balb/c (H-2d) recipients, or by placing bone marrow chimeric DBA/2 (H-2d/H-2b) allografts onto C57BL/6 (H-2b) hosts. The requirement for indirect allorecognition was tested by grafting DBA/2 skin allografts onto either C57BL/6- or MHC-II-deficient C57BL/6 recipients. We report here that although CD4+CD25+ regulatory T cells can suppress both directly and indirectly generated alloresponses, immunoregulation is favored when indirect presentation is the sole mechanism of allorecognition. Hence, in the absence of indirect presentation, net CD4+CD25+ T cell-dependent immunoregulation is weak, and high ratios of CD4+CD25+ to CD4+CD25 T cells are required to ensure graft survival.

Figure 1

T_Regs powerfully prevent the rejection of minor histocompatibility mismatched skin allografts. (a) 4 × 10⁵ T_Regs adoptively transferred into syngeneic Rag^−/− Balb/c hosts together with 4 × 10⁵ CD4⁺CD25⁻ T cells show a greater capacity to delay the acute rejection of minor histocompatibility mismatched (B10.D2; H-2^d) than that of MHC-mismatched (C57BL/6; H-2^b) skin allografts (P < 0.0039). (b) The transfer of 4 × 10⁵ CD4⁺CD25⁻ T cells results in more rapid acute rejection of C57BL/6 than B10.D2 allografts (P < 0.0499). MST, median survival time in days.

Figure 2

In the absence of donor APCs, the capacity of T_Regs to prevent the rejection of MHC-mismatched skin allografts is increased. Skin allografts harvested from irradiated DBA/2 mice reconstituted with either DBA/2 (DBA/2-DBA/2 chimera) or C57BL/6 (C57BL/6-DBA/ 2 chimera) BM, and placed onto Rag⁻/⁻ C57BL/6 recipients, are similarly rejected after the transfer of 4 × 10⁵ syngeneic CD4⁺CD25⁻ T cells. In contrast, while the co-transfer of 4 × 10⁵ T_Regs together with 4 × 10⁵ CD4⁺CD25⁻ T cells delays the rejection of C57BL/6-DBA/ 2 chimeric grafts, it has virtually no effect on the survival of DBA/2-DBA/2 chimeric skin allografts (DBA/2-DBA/2 versus C57BL/6-DBA/2 chimeras, P < 0.045). MST, median survival time in days.

Figure 3

T_Regs are less efficient at suppressing the cytopathic alloimmune responses in hosts lacking class II self-MHC molecules. (a) Adoptive transfer of 2 × 10⁵ C57BL/6 CD4⁺CD25⁻ T cells results in rapid rejection of DBA/2 grafts placed onto Rag^−/− C57BL/6 recipients regardless of the presence or absence of recipient MHC molecules. (b) The absence of MHC-II in Rag^−/− /MHC-II^−/− C57BL/6 recipients markedly diminishes the immunoregulatory function of 4 × 10⁵ T_Regs transferred together with 2 × 10⁵ CD4⁺CD25⁻ T cells (Rag^−/− recipients versus Rag^−/−/ MHC-II^−/− hosts, P < 0.0064).

Figure 4

Absence of class II self-MHC molecules similarly compromises the expansion and/or survival of T_Regs and CD4⁺CD25⁻ T cells. 1 × 10⁶ CD45.1⁺ T_Regs or CD45.1⁺ CD4⁺CD25⁻ T cells were transferred into CD45.2⁺ Rag^−/− syngeneic recipients bearing DBA/2 skin allografts, and 14 days later the number of CD4⁺ CD45.1⁺ cells recovered from either spleen or draining lymph nodes was quantified by flow cytometry. In Rag^−/− recipients genetically lacking MHC-II, the expansion of both T_Regs and CD4⁺CD25⁻ T cells was similarly decreased. The results shown here correspond to 1 experiment representative of 3.