Asialo GM1(+) CD8(+) T cells play a critical role in costimulation blockade-resistant allograft rejection

Abstract

Simultaneous blockade of the CD40 and CD28 costimulatory pathways is an effective treatment strategy to promote allograft acceptance but does not lead to indefinite allograft survival. The immune mechanisms responsible for costimulation-independent rejection are not defined. Here we have studied the rejection responses of murine C57BL/6 recipients, which we show to be relatively resistant to inhibition by combined CD40/CD28 blockade. We demonstrate that asialo GM1(+) CD8(+) cells play a critical role in this costimulation blockade-resistant rejection. These results provide new insights into the costimulatory requirements for T-cell subsets and demonstrate for the first time that combined blockade of the CD40 and CD28 pathways does not adequately inhibit CD8-mediated skin allograft rejection. Furthermore, we provide evidence that asialo GM1 is a potentially important therapeutic target for CD8-dependent immune responses.

Figure 1

Skin allograft rejection in B6 mice is relatively resistant to combined blockade of the CD40 and CD28 pathways. B6 recipients of Balb/c skin allografts treated with anti-CD40L (500 μg) and CTLA4-Ig (500 μg) on days 0, 2, 4, and 6 (MST 20 days; n = 7) had minimally prolonged survival compared with a control group that received no treatment (MST 10 days; n = 7; P < 0.01). Recipients treated with chronic costimulation blockade therapy (days 0, 2, 4, and 6 and weekly until rejection) (MST 17 days; n = 7) did not have prolonged survival compared with our standard CD40/CD28 blockade protocol.

Figure 2

Anti–asialo GM1 antibodies prolong skin allograft survival in B6 recipients treated with combined CD40/CD28 blockade. B6 recipients of Balb/c skin allografts treated with anti-CD40L, CTLA4-Ig (500 μg on days 0, 2, 4, and 6) and anti–asialo GM1 (50 μL on days 0, 4, 8, and 12) (MST 86 days; n = 7) had significantly prolonged graft survival compared with recipients treated with costimulation blockade alone (MST 20 days; n = 7; P < 0.01). Recipients treated with anti–asialo GM1 alone (MST 11 days; n = 7) did not have prolonged graft survival compared with control-treated (Rabbit IgG-treated) recipients (MST 10 days; n = 7).

Figure 3

Asialo GM1 is expressed on NK cells and subsets of γδ T cells and αβ T cells in B6 mice. Nylon wool nonadherent splenocytes from B6 mice were analyzed for NK1.1, γδ TCR, αβ TCR, CD4, CD8, and asialo GM1 in the lymphoid compartment using 2-color flow cytometric analysis. All axes represent fluorescence intensity on a logarithmic scale. Asialo GM1 is expressed on approximately 93% of NK1.1⁺ cells, 53% of γδ T cells, 13% of αβ T cells, less than 1% of CD4⁺ T cells, and 20% of CD8⁺ T cells.

Figure 4

CD8⁺ T cells mediate costimulation blockade–resistant rejection. B6 recipients were depleted of CD4 or CD8 cells with GK1.5 or TIB105, respectively (100 μg on days –3, –2, and –1 and weekly until rejection) or left undepleted. Recipient mice were transplanted with Balb/c skin allografts on day 0 and treated with costimulation blockade alone (CTLA4-Ig, anti-CD40L, 500 μg on days 0, 2, 4, and 6) or left untreated. (a) Costimulation blockade has no effect on CD8-mediated rejection. Untreated recipients depleted of CD4 cells (MST 16 days; n = 7) had minimally prolonged allograft survival compared with undepleted mice without treatment (MST 9 days; n = 6). Treatment with costimulation blockade did not prolong allograft survival in recipients depleted of CD4 cells (MST 18 days; n = 7) compared with costimulation blockade treatment of undepleted mice (MST 20.5 days; n = 6). (b) Costimulation blockade significantly inhibits CD4-mediated allograft rejection. In the same experiment as a, depicted on a separate graph for clarity, treatment of CD8-depleted recipients with costimulation blockade (MST 67 days; n = 5) significantly prolonged allograft survival compared with undepleted recipients treated with costimulation blockade (MST 20.5 days; n = 6; P < 0.01) or recipients treated with anti-CD8 alone (MST 11 days; n = 7).

Figure 5

Anti–asialo GM1 delays skin allograft rejection mediated by CD8⁺ T cells. (a and b) B6 Rag1^–/– recipients were reconstituted with 10⁷ B6 CD4⁺ or CD8⁺ T cells and transplanted with Balb/c skin allografts 2 days later. (a) Recipients reconstituted with B6 CD4⁺ T cells promptly rejected Balb/c skin allografts when treated with rabbit Ig (MST 12 days; n = 5) or anti–asialo GM1 (MST 13 days; n = 5). (b) Recipients reconstituted with CD8⁺ T cells treated with rabbit Ig (MST 11 days; n = 5) promptly rejected Balb/c skin allografts. Recipients reconstituted with CD8 cells treated with anti–asialo GM1 had significantly prolonged allograft survival (MST 37 days; n = 4; P < 0.02). (c) B6 B₂-microglobulin^–/– promptly rejected Balb/c skin allografts when treated with rabbit IgG (MST 10 days; n = 5) or anti–asialo GM1 (MST 10 days; n = 7). (d) Balb/c skin graft survival was significantly prolonged in B6 class II^–/– recipients treated with anti–asialo GM1 (MST 26 days; n = 7) compared with recipients treated with rabbit Ig (MST 12 days; n = 5; P < 0.01). Treatment of B6 class II^–/– recipients with anti-NK1.1 did not significantly prolong graft survival (MST 13 days; n = 7).

Figure 6

Anti–asialo GM1 prevents CD8⁺ T cell expansion. CFSE-labeled B6 T cells were adoptively transferred into irradiated (18 Gy) Balb/c recipients treated with rabbit IgG or anti–asialo GM1. (a) Flow cytometric analysis of cells harvested from rabbit IgG–treated mice 66 hours after transfer demonstrates expansion in the CD8 gate, including cells that have undergone more than 6 divisions (left of line). (b) Cells harvested from mice treated with anti–asialo GM1 show considerably less CD8 expansion and an almost complete absence of cells that have undergone more than 6 divisions. Histograms of CFSE fluorescence are gated on CD8 cells and depict data from 1 mouse representative of 5.