Given the importance of LIGHT expression in the development of T cell activation in vivo, including in host T cell–dependent antitumor responses (5
), we undertook a serial study of LIGHT and HVEM expression during the development of cardiac allograft rejection across a full MHC disparity. Total RNA was prepared from each recipient's heterotopic transplant and own native heart after collection on days 3 and 7 after transplant, and HVEM and LIGHT mRNA expression were analyzed by RT-PCR. Negligible LIGHT or HVEM mRNA was detected in native hearts, but both LIGHT and HVEM were markedly upregulated at days 3 and 7 after transplant ( a). LIGHT mRNA expression was localized by ISH to cells with the morphology of small mononuclear cells ( b), and similar localization of HVEM mRNA, plus focal endothelial expression, was detected by ISH for HVEM (data not shown). Immunoperoxidase studies confirmed expression of LIGHT protein by infiltrating leukocytes ( c); labeled cells included lymphocytes, plus some inflammatory macrophages and tissue DCs.
Figure 1. Analysis of LIGHT and HVEM expression during allograft rejection. (a) Northern analysis of LIGHT and HVEM in serial cardiac transplants (T) rejected by day 7 versus each recipient's native heart (N). (b) ISH analysis of graft LIGHT mRNA expression at (more ...)
To determine the role of LIGHT expression in allograft rejection, we used homologous recombination to disrupt exon 1, containing the ATG initiation codon, of the LIGHT gene (). Mice heterologous and homozygous for the LIGHT mutation were normal in appearance, growth and fertility, had normal numbers of T and B cells, monocytes and granulocytes, and normal lymphoid architecture. LIGHT−/−
mice also had normal levels of plasma C3 despite the chromosomal proximity of LIGHT and C3 genes (14
); detailed characterization of these mice is underway.
Figure 2. Generation of LIGHT−/− mice. (a) Genomic organization of the murine LIGHT locus and resulting mutation induced by the targeting event; exons are filled boxes. A LIGHT gene-targeting construct was generated to replace exon 1 of (more ...)
Homozygous LIGHT−/− mice were used as recipients of fully MHC-disparate cardiac allografts. Whereas LIGHT+/+ mice rejected BALB/c cardiac allografts within 1 wk, LIGHT−/− mice maintained their grafts for an extra 3–4 d ( a), which was about as effective as CsA (10 mg/kg/d) in mice (both P < 0.05 vs. untreated LIGHT+/+ recipients). However, use of the same regimen of CsA in LIGHT−/− mice led to significantly prolonged engraftment (~30 d, P < 0.001) ( a), indicating a synergistic effect of CsA and LIGHT targeting on allograft survival. Histologic analysis showed that rejecting grafts harvested at 7 d from LIGHT−/− mice, or LIGHT+/+ recipients treated with CsA, were morphologically similar to allografts harvested form control untreated LIGHT+/+ recipients ( b), with diffuse mononuclear cell infiltrates and focal myocyte necrosis. In contrast, allografts harvested at day 7 from LIGHT−/− recipients treated with CsA showed a marked absence of leukocyte infiltration and essentially normal morphology.
Figure 3. Effects of targeting LIGHT on the survival of fully MHC-mismatched cardiac allografts (H-2d→H2b). (a) Compared with untreated LIGHT+/+ recipients, LIGHT−/− recipients, or LIGHT+/+ recipients treated with a subtherapeutic course (more ...)
Given concerns that gene-targeted mice may not always reveal the role of a given gene in the normal state because of secondary effects or compensatory responses, we investigated whether targeting of LIGHT was also beneficial in wild-type allograft recipients. We constructed an HVEM–Ig fusion protein for therapeutic blockade of the effects of endogenous LIGHT on host HVEM+ T cells. In line with the modest effects of LIGHT targeting by homologous recombination in this strong MHC disparity, we found neither HVEM-Ig nor control IgG1 had any significant effect on allograft survival in LIGHT+/+ recipients (P > 0.05), whereas HVEM-Ig, but not control IgG1, was markedly synergistic with a subtherapeutic dose of CsA in prolonging graft survival (P < 0.001) ( c).
Expression of cytokines, chemokines, and their receptors by host leukocytes vary during graft rejection. We used RNase protection assays to examine the likely mechanisms by which targeting of LIGHT, especially with concomitant CsA, induced prolonged graft survival. Use of CsA in LIGHT−/−
mice suppressed the intragraft upregulation of multiple cytokine mRNAs, including IFN-γ, IL-2, and IL-10 ( a), plus LT-β and TNF-α ( b). Consistent with these effects and the reduction in cellularity apparent histologically, expression of several IFN-γ-induced chemokines, including RANTES, MIP-1α, MIP-1β, and IP-10 was down-regulated in LIGHT−/−
mice treated with CsA ( a). Along with the decreased chemokine expression, LIGHT−/−
mice treated with CsA had modest reductions in CC-chemokine receptor expression ( b) and markedly decreased expression of the chemokine receptor for IP-10, CXCR3 ( c), which is expressed by Th1 and Tc1 lymphocytes (13
Figure 4. Mechanisms underlying beneficial effects of targeting LIGHT in allograft recipients. (a) RNase protection assay comparison of intragraft Th1- and Th2-associated cytokines in control normal heart versus allografts harvested at day 7 after transplant. Use (more ...)
Figure 5. Targeting of LIGHT is synergistic with CsA in effects at day 7 after transplant on intragraft expression of (a) the chemokines lymphotactin (LTN), RANTES, MIP-1α, MIP-1β, MCP-1, IP-10, and TCA-3; (b) the CC chemokine receptors (more ...)
Our prior studies in this model showed that donor-derived IP-10 production (19
) and concomitant infiltration by CXCR3+
) play central roles in the development of allograft rejection, such that targeting of either IP-10 production or CXCR3 expression markedly prolongs allograft survival. We also recently reported that the IP-10/CXCR3 pathway is active during development of human cardiac allograft rejection (20
). IP-10 production is regulated by NF-κB (21
), and LIGHT-induced costimulation causes NF-κB activation and translocation in T cells (9
), leading to production of IFN-γ (7
). It is likely that modulation of this pathway by inhibition of LIGHT costimulation is at least one important mechanism of action in our model, and one which is potentiated by the effects of a subtherapeutic regimen of CsA, which can also diminish NF-κB activation and IFN-γ production (22
). Nevertheless, given recent evidence of several distinct molecular forms of LIGHT, which are directed to distinct cellular compartments, including the extracellular space, the membrane, and the cytosol (14
), and findings that LIGHT overexpression by T cells promotes inflammation via activation of multiple pathways (10
), there remain many additional potential mechanisms by which targeting of LIGHT in conjunction with CsA could be beneficial.
Our studies show a modest role for LIGHT costimulation by itself in promoting allograft rejection, but in contrast to some other combinations of therapeutic agents and costimulation blockade, such as the use of CD154 mAb plus CsA (23
), the effects of targeting LIGHT-HVEM interactions are markedly synergistic with CsA. Their combined use prevents acute allograft rejection, modulates intragraft cytokine and chemokine production, and decreases the infiltration of host immunocompetent cells. These data suggest exploration of the role of LIGHT costimulation in less rigorous models as well as in combination with other therapeutic approaches. We conclude that the LIGHT-HVEM pathway is yet another of the rapidly expanding number of costimulation pathways which require attention in efforts to promote the development of safer, less toxic therapeutic protocols which may eventually facilitate development of clinical allograft tolerance.