*Purpose: Hepatocyte transplantation represents a treatment for metabolic diseases and a bridge therapy to whole organ transplant but is limited in part by cell immunogenicity. Our prior work identified the critical role of both CD4-dependent and CD4-independent CD8⁺ T cells in mediating hepatocyte rejection. We evaluated the influence of invariant natural killer T (iNKT) cells, known to be uniquely abundant in the liver, upon CD8-mediated immune responses in the presence and absence of CD4⁺ T cells.

*Methods: C57BL/6 (wild-type; WT), iNKT deficient Jα18 KO, and RAG1 KO mice (all H-2^b) were transplanted with FVB/N (H-2^q) hepatocytes. Cohorts of each strain were depleted of CD4⁺ T cells. Some, cohorts of Jα18 KO mice received iNKT cell adoptive transfer. Recipients were evaluated for CD8-mediated in vivo and in vitro cytotoxicity, CD8⁺ T cell phenotype, and allograft rejection.

*Results: By comparing CD4-sufficient WT recipients to CD4-sufficient, iNKT cell-deficient Jα18 KO recipients, the presence of iNKT cells significantly enhanced CD8-mediated in vivo allocytotoxicity (WT= 83.8 ± 3.3%, n=15 vs. Jα18 KO 23.4 ± 3.1%, n=15, p<0.0001) but did not affect allograft median survival time (WT= 10 days, n=15 vs. Jα18 KO= 14 days, n=10; p>0.05). However, in the absence of CD4⁺ T cells, iNKT cells were critically important for CD8-mediated in vivo allocytotoxicity (WT= 31.1 ± 3.1%, n=14 vs. Jα18 KO= 1.9 ± 0.7%, n=16, p<0.0001) and allograft median survival time (WT= 14 days, n=12 vs. Jα18 KO= 56 days, n=7, p<0.0001). Adoptive transfer of iNKTs restored cytotoxicity to WT levels (not shown). iNKT cells significantly enhance the quantity of a novel CD8⁺ T cell subset (alloprimed CD44⁺IFN-γ⁺CXCR3⁺CCR4⁺CD8⁺ T cells) in the liver (2-fold) of both CD4-sufficient and CD4-deficient recipients posttransplant (p<0.035 for all comparisons). When compared to alloprimed CD8⁺ T cells that lack CCR4 expression (CXCR3⁺CCR4^–CD8⁺ T cells), CXCR3⁺CCR4⁺CD8⁺ T cells express higher amounts of IFN-γ (32.0 ± 3.2%, n=9 vs. 9.1± 1.2%, n=9; p<0.0001) and TNF-α (37.0 ± 3.0%, n=9 vs. 6.6 ± 1.3%, n=9; p<0.0001). In addition, CXCR3⁺CCR4⁺CD8⁺ T cells are more potent than CXCR3⁺CCR4^–CD8⁺ T cells since the transfer of small quantities (250 x 10³ cells) induced significantly greater in vivo cytotoxicity (13.3 ± 1.1%; n=7, vs. 5.4 ± 0.6%; n=9; p<0.0001) and rapid hepatocyte rejection following adoptive transfer into RAG1 KO recipients with stable hepatocellular allografts (MST= day 5, n=4 vs. day 21, n=3; p=0.02).

*Conclusions: iNKT cells enhance the expansion of highly cytotoxic alloprimed CXCR3⁺CCR4⁺CD8⁺ T cells that mediate rapid rejection of allogeneic hepatocytes transplanted to the host liver.

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