Regulatory functions of self-restricted MHC class II allopeptide-specific Th2 clones in vivo

Abstract

We studied T-cell clones generated from grafts of rejecting and tolerant animals and investigated the regulatory function of Th2 clones in vitro and in vivo. To prevent allograft rejection, we treated LEW strain recipient rats of WF strain kidney grafts with CTLA4Ig to block CD28-B7 costimulation. We then isolated epitope-specific T-cell clones from the engrafted tissue, using a donor-derived immunodominant class II MHC allopeptide presented by recipient antigen-presenting cells. Acutely rejected tissue from untreated animals yielded self-restricted, allopeptide-specific T-cell clones that produced IFN-gamma, whereas clones from tolerant animals produced IL-4 and IL-10. Adoptive transfer into naive recipients of Th1 clones, but not Th2 clones, induced alloantigen-specific delayed-type hypersensitivity (DTH) responses. In addition, Th2 clones suppressed DTH responses mediated by Th1 clones in vivo and blocked Th1 cell proliferation and IFN-gamma production in vitro. A pilot human study showed that HLA-DR allopeptide-specific T-cell clones generated from patients with chronic rejection secrete Th1 cytokines, whereas those from patients with stable graft function produce Th2 cytokines in response to donor-specific HLA-DR allopeptides. We suggest that self-restricted alloantigen-specific Th2 clones may regulate the alloimmune responses and promote long-term allograft survival and tolerance.

Figure 1

Proliferative responses of T-cell lines from rejecting and tolerant animals to specific donor-derived MHC class II allopeptides. ^AP < 0.0001 for proliferation in T-cell lines from rejecting versus tolerant animals. No significant proliferation over base line was observed against the nonspecific (control) allopeptide for both T-cell lines. Data are presented as mean ± SEM (n = 4 experiments).

Figure 2

T-cell lines cytokine profile of rejecting and tolerant animals after restimulation with the RT1.D^uβ20-44 allopeptide. Data are presented as mean ± SEM (n = 4 experiments).

Figure 3

T-cell clone cytokine profile patterns of rejecting and tolerant animals after restimulation with the RT1.D^uβ20-44 allopeptide. Data are presented as mean ± SEM (n = 5 clones per group, 3 experiments).

Figure 4

DTH responses in LEW rats were elicited after intraperitoneal injection of the Th1 or Th2 TCR Vβ9 cell clones (20 × 10⁶). Five days later, the animals were challenged by subcutaneous injection in the ear with RT1.D^uβ20-44 or whole-irradiated WF splenocytes. Changes in ear thickness were then measured 48 hours later. Bars represent the mean ± SD Δ ear thickness (inches × 10^–2). Naive LEW rats that are not injected with the clones or immunized with the allopeptides do not mount DTH responses (n = 8 animals/group, ^AP < 0.0001).

Figure 5

Regulatory function of Th2 clones in vitro. (a) Proliferation of TCR Vβ9 Th1 clone from the rejecting animal, irradiated TCR Vβ9 Th2 clone from the tolerant animal, and irradiated naive LEW T cells. 1, Th1 clone specific for RT1.D^uβ20-44 + peptide RT1.D^uβ20-44. 2, irradiated naive LEW T cells + peptide RT1.D^uβ20-44. 3, irradiated Th2 clone specific for RT1.D^uβ20-44 + peptide RT1.D^uβ20-44. 4, Th1 clone specific for RT1.D^uβ20-44 + irradiated naive LEW T cells + peptide RT1.D^uβ20-44. 5, Th1 clone + irradiated Th2 clone both specific for RT1.D^uβ20-44 + peptide RT1.D^uβ20-44. (b) Cytokine profile of TCR Vβ9 Th1 clone from the rejecting animal and irradiated TCR Vβ9 Th2 clone from the tolerant animal after restimulation with the RT1.D^uβ20-44 allopeptide. 6, Th1 clone specific for RT1.D^uβ20-44 + peptide RT1.D^uβ20-44. 7, Th2 clone specific for RT1.D^uβ20-44 + peptide RT1.D^uβ20-44. 8, irradiated Th2 clone specific for RT1.D^uβ20-44 + peptide RT1.D^uβ20-44. 9, Th1 clone + irradiated Th2 clone both specific for RT1.D^uβ20-44 + peptide RT1.D^uβ20-44. Data are presented as mean ± SEM (n = 4, ^AP < 0.0001).

Figure 6

Regulatory function of the Th2 clones in vivo. The DTH response in LEW rats was elicited in animals injected with Th1 TCR Vβ9, irradiated Th2 TCR Vβ9 cell clones and irradiated naive LEW T cells in a 1:1 ratio. Bars represent the mean ± SD Δ ear thickness (inches × 10^–2, n = 8 animals/group, ^AP < 0.0001).

Figure 7

T-cell line proliferation to specific donor-derived HLA-DR allopeptides in renal transplant recipients with chronic rejection (CR, two patients) and those with stable renal function (Stable, three patients). ^AP ≤ 0.001 for proliferation in CR group versus Stable group (a). T-cell line cytokine profile patterns after restimulation with allopeptide between CR group and Stable group (b). No significant proliferation or cytokine production over base line was observed to the nonspecific (control) allopeptide for T-cell lines from either patient group. Data are presented as mean ± SEM (n = 3).