As allograft rejection is a T lymphocyte–dependent event 2
, we undertook Northern blot analysis of CXCR3 expression by primary T cells and T cell clones in culture, and assessed CXCR3 involvement in allograft rejection in vivo. Freshly isolated mouse splenocytes lacked CXCR3 expression, but upon activation and culture in conditions that promote Th cell differentiation, Th1 cells expressed CXCR3 mRNA ( a). Established Th1 clones (PL17 and OF6), but not Th2 clones (CDC35 and D10), also expressed CXCR3 mRNA ( a). Heterotopic cardiac allografts in the fully MHC-mismatched BALB/C to C57BL/6 combination survive 7–8 d 17
. RNase protection assays showed that normal hearts and isografts lacked CXCR3 mRNA, whereas allografts showed progressive CXCR3 mRNA expression, peaking at day 6 after transplant, just before end-stage rejection ( b). CXCR3 mRNA expression closely followed intragraft mRNA levels of the three known CXCR3 ligands IP-10, Mig, and I-TAC, plus IFN-γ ( c). Expression of CXCR3 mRNA was localized to infiltrating leukocytes by in situ hybridization ( d), and CXCR3+
mononuclear cell infiltration was confirmed by immunohistology ( d). These results show that activated T cells, especially Th1 cells, express CXCR3 and infiltrate cardiac allografts in conjunction with graft expression of the ligands IP-10, Mig, and I-TAC 19
Analysis of CXCR3 expression by activated T cells in vitro and during graft rejection. (a) Northern blot analysis of CXCR3 expression by primary T cells activated under polarizing conditions and by Th1 cell clones. (b) RNase protection assays of CXCR3 expression in serial cardiac transplants (T) showing rejection by day 7 but not corresponding native hearts (N). (c) Northern blots showing serial allograft but not native (N) heart expression of IFN-γ–induced CXCR3 ligands IP-10, Mig, and I-TAC. (d) Graft CXCR3 mRNA (blue, left) expression is restricted to focal round cells (sense control, bottom left); immunoperoxidase at high power (brown, right) shows CXCR3 protein expression by infiltrating mononuclear leukocytes (IgM control, bottom right). Bars, 100 and 10 μm, respectively.
To establish the role of CXCR3 expression in allograft rejection, we used homologous recombination to disrupt exon 2 of the X chromosome–linked 20
CXCR3 gene ( a). Mice heterologous and homozygous for the CXCR3 mutation were normal in appearance, growth, and fertility. CXCR3 deficiency was transmitted in a Mendelian fashion, as identified by Southern blot analysis of tail DNA ( b). T cell blasts from homozygous CXCR3-deficient (CXCR3−/
−) mice lacked CXCR3 protein expression ( c), and failed to respond in chemotaxis assays to IP-10 ( d) or Mig, indicating that CXCR3 is the sole functional receptor for these chemokines. With relevance to subsequent in vivo studies, T cell blasts from CXCR3+/+
but not CXCR3−/
− mice were also labeled using the CXCR3-specific mAb ( e).
Figure 2 Targeted disruption of the murine CXCR3 gene. (a) Wild-type (WT) allele, targeted vector, and mutated allele of mouse CXCR3 gene. The wild-type gene contains exon 2 of the receptor (black rectangle) whereas in the mutant, most of exon 2 (800 bp of coding (more ...)
− mice had normal T cell–proliferative responses upon mitogen stimulation ( a), but decreased MLR responses ( b). The addition of anti-CXCR3 mAb reduced the MLR of wild-type mice ( c), but had no effect on mitogen responses (data not shown). In vivo, CXCR3−/
− mice showed profoundly decreased alloresponses ( d). Whereas CXCR3+/+
mice rejected BALB/c cardiac allografts within 1 wk, CXCR3−/
− mice accepted corresponding allografts for a mean of 58 ± 3 d (P
< 0.001); no benefit was seen when hearts from CXCR3−/
− mice were used as donor organs in BALB/c mice. Targeting of CXCR3 resulted in strong synergy with the immunosuppressive agent, CsA, consistent with data that CsA does not affect CXCR3 ligand induction 1521
. 2 wk of therapy with CsA (10 mg/kg) prolonged allograft survival by only 3 d in wild-type recipients, but induced permanent engraftment (>100 d, P
< 0.001) in CXCR3−/
− mice ( d); evaluation of the latter allografts harvested at day 100 after transplant showed normal morphology, with a lack of graft leukocyte infiltration, myocardial injury, or evidence of transplant arteriosclerosis (data not shown). Use of the CXCR3 mAb in CXCR3+/+
mice significantly prolonged allograft survival when used from the time of transplantation, and was also effective when commenced once rejection had begun, i.e., at day 4 after transplant ( e). These findings demonstrate a profound effect of targeting CXCR3 on the development of cardiac allograft rejection.
Figure 3 In vitro and in vivo effects of CXCR3 targeting. (a) CXCR3−/− knockout (KO) mice have (a) normal mitogen responses but (b) diminished alloreactivity (MLR); bars show the mean ± SD for 12 wells. Asterisks indicate significantly (more ...)
Histologic analysis of allografts harvested at day 5 after transplant showed that in contrast to control grafts, grafts in CXCR3−/− or CXCR3 mAb–treated CXCR3+/+ mice were well preserved (, a–d). Compared with control grafts, allografts in CXCR3−/− recipients had significantly reduced numbers of CD4+ and CD8+ T cells and macrophages, and no IL-2R+ (CD25+) cells indicative of immune activation ( e). Allografts in CXCR3−/− recipients contained decreased IFN-γ mRNA ( f), and decreased expression of the chemokines macrophage inflammatory protein (MIP)-1β and regulated upon activation, normal T cell expressed and secreted (RANTES; g). Consistent with the latter findings, allografts in CXCR3−/− mice showed decreased expression of the corresponding chemokine receptors CCR1, CCR2, and CCR5, produced by T cells and macrophages ( h).
Figure 4 Mechanisms underlying beneficial effects of targeting CXCR3 in allograft recipients. (a) Histology showing acute rejection with extensive mononuclear cell infiltration and myocyte necrosis in CXCR3+/+ recipients (day 7 after transplant). (b) The lack (more ...)
In conclusion, targeting of CXCR3 markedly decreases the tempo and severity of allograft rejection in vivo. This effect is associated with significantly decreased intragraft accumulation of activated T cells producing IFN-γ and consequent impairment of chemokine-dependent recruitment of multiple effector cell types, suggesting a rationale for targeting of CXCR3, in synergy with conventional immunosuppression, in clinical transplantation, and potentially in the management of acute allograft rejection.