CD160Ig fusion protein targets a novel costimulatory pathway and prolongs allograft survival

Abstract

CD160 is a cell surface molecule expressed by most NK cells and approximately 50% of CD8(+) cytotoxic T lymphocytes. Engagement of CD160 by MHC class-I directly triggers a costimulatory signal to TCR-induced proliferation, cytokine production and cytotoxic effector functions. The role of CD160 in alloimmunity is unknown. Using a newly generated CD160 fusion protein (CD160Ig) we examined the role of the novel costimulatory molecule CD160 in mediating CD4(+) or CD8(+) T cell driven allograft rejection. CD160Ig inhibits alloreactive CD8(+) T cell proliferation and IFN-γ production in vitro, in particular in the absence of CD28 costimulation. Consequently CD160Ig prolongs fully mismatched cardiac allograft survival in CD4(-/-), CD28(-/-) knockout and CTLA4Ig treated WT recipients, but not in WT or CD8(-/-) knockout recipients. The prolonged cardiac allograft survival is associated with reduced alloreactive CD8(+) T cell proliferation, effector/memory responses and alloreactive IFN-γ production. Thus, CD160 signaling is particularly important in CD28-independent effector/memory CD8(+) alloreactive T cell activation in vivo and therefore may serve as a novel target for prevention of allograft rejection.

Figure 1. CD160 is expressed on CD8 and CD4 T cells.

Naïve or stimulated (ConA) splenocytes from wildtype C57BL/6 (WT), CD4^−/−, CD8^−/− and CD28^−/− mice were stained with anti-CD160. Mean fluorescence intensity of CD160 on naïve CD8⁺ and CD4⁺ T cells or CD8⁺ and CD4⁺ T cells expressing an effector/memory phenotype (CD44^highCD62L^low) was analyzed. Upper Panel: Representative dot plots of CD160 expressing cells (shaded histograms) in WT mice. Lower Panel: The histograms demonstrate the MFI of CD160⁺ cells of the overall CD4⁺ or CD8⁺ T cell population as the mean ± SEM of 3–5 independent experiments.

Figure 2. CD160Ig inhibits allospecific T cell proliferation and Th1 cytokine generation.

Wildtype C57BL/6 (WT), CD4^−/−, CD8^−/− and CD28^−/− mice (all H-2^b) were sensitized with skins from fully missmatched BALB/c (H-2^d) donors. Recipients splenocytes were harvested and cultured with irradiated donor splenocytes in the absence and in the presence of increasing concentrations of CD160Ig or control Ig. A) Cell proliferation is expressed as percent inhibition of CD160Ig treated cells compared with control Ig treated cells of that strain. Results are presented as the mean ± SEM of 3 experiments performed in triplicates. B) Frequency of alloreactive IFN-γ producing cells was quantified by ELISPOT assay after 24 hours. Percent inhibition of IFN-γ production of CD160Ig treated cells compared with control Ig treated cells of that strain is shown. Results shown are mean ± SEM values of 3 independent experiments performed in quadruplicate wells. C.) Statistics of percent inhibition of alloreactive IFN-γ production at 100 µg/ml CD160Ig/well.

Figure 3. CD160Ig prolongs fully mismatched heart allograft survival in CD28^−/− and CD4^−/−mice.

A) C57BL/6 WT (n = 10); B) CD4^−/−, (n = 10); C) CD8^−/− (n = 10); D) CD28^−/− (n = 10) or E) C57BL/6 WT treated with CTLA4-Ig (n = 5) received Balb/c heart grafts and were treated with CD160Ig (⧫; n = 5) or control Ig (⧫; n = 5); F) C57BL/6 WT (n = 5) received bm1 skin grafts and were treated with CD160Ig (⧫, n = 5) or control-Ig (⧫; n = 5); Survival is shown by Kaplan-Meier plots.

Figure 4. CD160Ig reduces allospecific Th1 cytokine generation in CD28^−/− and CD4^−/− mice.

Heart grafts from BALB/c donors were transplanted into C57BL/6 WT, CD28^−/− or CD4^−/− recipients, which were treated with CD160Ig +/− CTLA4Ig or control Ig. Recipient splenocytes were isolated 10 days (WT recipients) or 14 days (CD28^−/− and CD4^−/− recipients, CTLA4Ig treated WT recipients) after transplantation, cultured with irradiated donor splenocytes. A) The frequency of alloreactive IFN-γ and IL-5 producing T cells was analyzed by ELISPOT. B). The concentration of TNF-α, IL-6, IFN-γ, IL-17, IL-4 and IL-5 was examined by Luminex Assay in the supernatants of the cultures with CD28^−/− responder cells. The data shown are pooled from 3–5 independent experiments performed in quadruplicate wells (Mean±SEM).

Figure 5. CD160Ig reduces effector/memory cell generation in CD28^−/− mice.

Splenocytes from C57BL/6 WT, C57BL/6 WT treated with CTLA4Ig or CD28^−/− recipients of BALB/c hearts were harvested and CD4⁺ or CD8⁺ T cells were stained for the effector/memory phenotype, characterized as CD44^highCD62L^low. Left Panel: Representative dot plots of CD8⁺CD44^highCD62L^low cells. Right Panel: The histograms demonstrate the frequency of CD44^highCD62L^low cells as a percentage of the overall CD4⁺ or CD8⁺ T cell population as the mean ± SEM of 3–5 independent experiments.

Figure 6. CD160Ig decreases in vivo proliferation of CD28 deficient CD8⁺ T cells.

CFSE-labeled splenocytes from C57BL/6 WT, CD8^−/−, CD4^−/− or CD28^−/− mice (6−8×10⁷) were injected into irradiated BALB/c hosts and treated with CD160Ig or control Ig. The precursor division frequency of CD4⁺ and CD8⁺ T cell subsets in the host spleen was analyzed 3 days later by FACS and calculated as described in Materials and Methods. Representative histograms from one of 3 experiments are shown.