Heterologous immunity provides a potent barrier to transplantation tolerance

Abstract

Many strategies have been proposed to induce tolerance to transplanted tissue in rodents; however, few if any have shown equal efficacy when tested in nonhuman primate transplant models. We hypothesized that a critical distinction between specific pathogen-free mice and nonhuman primates or human patients is their acquired immune history. Here, we show that a heterologous immune response--specifically, virally induced alloreactive memory--is a potent barrier to tolerance induction. A critical threshold of memory T cells is needed to promote rejection, and CD8(+) "central" memory T cells are primarily responsible. Finally, treatment with deoxyspergualin, an inhibitor of NF-kappa B translocation, together with costimulation blockade, synergistically impairs memory T cell activation and promotes antigen-specific tolerance of memory. These data offer a potential explanation for the difficulty encountered when inducing tolerance in nonhuman primates and human patients and provide insight into the signaling pathways essential for memory T cell activation and function.

Figure 1

Analysis of viral- and allo-specific responses in vivo. B6 mice were infected with either LCMV, VV, or VSV. The results are displayed for the peak of the response as well as during the memory phase (more than 6 weeks). For comparison, the response to a skin allograft is also shown. (a) Representative dot plots displaying the number of CD8⁺ IFN-γ⁺ T cells representing the antiviral response (LCMV-NP396-404, VV-infected stimulators, VSV-N-peptide; left panels) and the corresponding heterologous allo-specific responses (right panels). (b) The number of virus-specific (NP396-404, filled squares) and virally induced, alloreactive T cells (H-2^d stimulated, filled triangles) after LCMV infection are shown over time. The allo-specific response after skin graft is shown for comparison (H-2^d stimulated, open squares; CD8⁺, upper panel; CD4⁺, lower panel). Each time point represents the average for three animals. Experiments were repeated three times with similar results.

Figure 2

A critical number of CD8⁺ central memory T cells are needed to resist tolerance. Naive B6 mice (CD45.2) received T cells from sensitized congenic B6 mice (CD45.1). (a) Titration of memory cell numbers. Naive recipients received varying doses of T cells (calculated number of memory cells transferred in brackets) from sensitized hosts (2 × 10⁷ [400,000], filled squares; 10⁷ [200,000], filled diamonds; 5 × 10⁶ [100,000], filled triangles; 2 × 10⁶ [40,000], open diamonds; 10⁶ [20,000], open triangles; 2 × 10⁷ naive cells [none], open squares). (b) CD8⁺ memory T cells represent a potent barrier to tolerance induction. Purified CD4⁺, CD8⁺, or whole T cells were isolated from sensitized mice and transferred to naive congenic mice. Mice receiving either naive selected cells (data not shown) or CD4⁺ T cells (filled diamonds) from sensitized donors developed high-level donor cell chimerism and accepted donor-type skin grafts indefinitely. The transfer of CD8⁺ T cells (filled triangles) from sensitized animals prevented tolerance induction in a similar fashion to transferred whole T cells (filled squares) (MST, 21 days). (c) CD8⁺ central memory T cells promote rejection more efficiently than “effector” memory T cells. Equal numbers of CD8⁺ (open squares), CD8⁺ CD62L^hi (filled squares), or CD8⁺ CD62L^lo (open triangles) antigen-specific memory T cells were transferred to naive congenic hosts, which were then subjected to the tolerance protocol (control naive CD8⁺ T cells, filled triangles). Both unseparated CD8⁺ memory T cells and central CD8⁺ memory T cells efficiently rejected allogeneic skin grafts in less than 3 weeks (MST, 21 and 18 days, respectively). In contrast, effector memory T cells of the traditional CD62L^lo memory phenotype were less effective at promoting rejection (MST, more than 70 days; P < 0.01).

Figure 3

Virally induced, alloreactive memory cells prevent tolerance induction. Naive, monoimmune (LCMV or VV), or polyimmune mice (LCMV→VV or LCMV→VV→VSV) received the mixed chimerism, costimulation blockade–based tolerance protocol. (a and b) Skin graft survival and donor chimerism levels after tolerance induction. Naive mice universally accept donor-type skin allografts (left panel, filled squares) and become high-level mixed chimeras (10 of 10, right panel). The majority of mice immune to a single pathogen become tolerant, although not as consistently as naive mice (9 of 12, open triangles). Mice immune to multiple pathogens are refractory to tolerance induction (3 of 12; MST, 24; P = 0.002 when compared with naive mice; filled diamonds). Inf, infection(s).

Figure 4

DSG and costimulation blockade synergistically inhibit memory cells. Both alloreactive and virus-specific memory cell responses were analyzed. (a) Skin graft survival of mice that had previously (1 week earlier) received T cells from sensitized congenic mice and were treated with either the tolerance induction protocol alone or in combination with various agents. The combination of DSG and costimulatory blockade promoted tolerance of donor-specific memory cells (n = 5; MST, 100 days; filled squares). The costimulation blockade tolerance regimen given alone (n = 5, open squares) or in combination with other agents, including rapamycin (n = 5, filled triangles), anti-CD25 (n = 5, open triangles), and anti-γ_c (n = 5, filled diamonds) failed to inhibit memory-dependent rejection. Two additional experiments demonstrated similar results. (b) LCMV-immune mice were rechallenged with LCMV clone 13 and treated with costimulation blockade (costim), DSG, or the combination of costimulation blockade and DSG. Five days after rechallenge, CD8⁺ (class I restricted NP396-404 and GP276-286) and CD4⁺ (class II restricted P13 GP60-80) responses were analyzed. The combination of costimulatory blockade and DSG synergistically inhibited LCMV-specific CD8⁺ memory T cells (*P < 0.01). No tx, no treatment; immune no rechall, immune mouse without viral rechallenge.

Figure 5

Antigen-specific tolerance of memory cells. (a) Congenic mice were either infected with LCMV (Thy 1.1) or received a skin allograft (CD45.1). Eight weeks after challenge, T cells were isolated to obtain both allo- and virus-specific memory cells and then transferred into naive B6 mice (CD45.2/Thy1.2). Mice then received the tolerance protocol or the tolerance protocol with DSG. sg, skin graft. (b) When the tolerance protocol alone was administered, both alloreactive and virus-specific memory cell populations were preserved, resulting in allograft rejection. When given in combination with DSG, donor-reactive cells were specifically deleted, and viral memory remained intact upon rechallenge. Unstim, unstimulated. (c) Model for heterologous immunity as a barrier to transplantation tolerance.