Tofacitinib Halts Progression of Graft Dysfunction in a Rat Model of Mixed Cellular and Humoral Rejection

Abstract

Background: The progression from acute to chronic antibody-mediated rejection in kidney transplant recipients is usually not prevented by current therapeutic options. Here, we investigated whether the use of tofacitinib (TOFA), a Janus kinase 3 inhibitor, was capable of preventing the progression of allograft dysfunction in a Fisher-to-Lewis rat model of kidney transplantation.

Methods: Rats were treated from the third week after transplantation to allow the development of rejection. Treatment was based on cyclosporin A, rapamycin or TOFA. Renal function was assessed at 1, 4, 8, and 12 weeks after transplantation, whereas rat survival, histological lesions, and infiltrating lymphocytes were analyzed at 12 weeks.

Results: Tofacitinib prolonged graft survival, preserved tubular and glomerular structures and reduced humoral damage characterized by C4d deposition. Tofacitinib was able to reduce donor-specific antibodies. In addition, T and natural killer cell graft infiltration was reduced in TOFA-treated rats. Although rapamycin-treated rats also showed prolonged graft survival, glomerular structures were more affected. Moreover, only TOFA treatment reduced the presence of T, B and natural killer cells in splenic parenchyma.

Conclusions: Tofacitinib is able to reduce the immune response generated in a rat model of kidney graft rejection, providing prolonged graft and recipient survival, better graft function, and less histological lesions.

FIGURE 1.

Effect of JAK-3 inhibition on renal function. A, BUN levels. B, Serum creatinine. C, Proteinuria. D, Urine creatinine. L-L, LEW-to-LEW; F-L, F344-to-LEW; Ø, without treatment; RAPA, rapamycin; TOFA, tofacitinib. *Significantly different when compared to F-L + Ø group at the same time of analysis (*P < 0.05; **P < 0.01;***P < 0.001).

FIGURE 2.

Effect of immunosuppression on recipient survival. *Significantly different when compared to F-L + Ø group at the same time of analysis (*P < 0.05; **P < 0.01;***P < 0.001). F-L, F344-to-LEW; Ø, without treatment.

FIGURE 3.

Detection of DSAs at 4 weeks after transplantation (A-C), 8 weeks after transplantation (D-F) and twelve weeks after transplantation (G-I). We can distinguish DSA that recognize antigens expressed by B cells, CD4 or CD8 T cells. *Significantly different when compared to F-L+ Ø group at time (*P < 0.05; **P < 0.01; ***P < 0.001). ^#Statistical difference between groups.

FIGURE 4.

Quantification of different damage entities observed in renal allograft during rejection process. Tubular atrophy (A), tubulitis (B), capillaritis (C) and fibrosis (D). E, Quantification of glomerular lesions including MC, glomerulitis, FSGS, and TG. F, Quantification of C4d deposition on PTCs. E, Percentage of glomerulus with C4d deposition. 3w and 12w, 3 and 12 weeks after transplantation.*Significantly different when compared with F-L (3w) group (*P < 0.05; **P < 0.01). MC, minimal changes.

FIGURE 5.

Effect of JAK-3 inhibition on renal damage induced by rejection process. Representative photomicrographs from different entities observed in allogenic renal graft after 12 weeks of transplantation: tubular atrophy (A); tubulitis (B); capillaritis (C); and fibrosis (D). Its quantification is presented to its right side. A representative image from different glomerular lesions was presented: glomerulitis (E), FSGS (F) and transplant glomerulopathy (G). H, Quantification of glomerular lesions in allogenic renal graft after 12 weeks of transplantation. Periodic acid Schiff stain was used in A-C and E-G whereas Sirius Red stains was used in D. Scale bar is 200 μm (D), 100 μm (A-B) or 50 μm (C, E, F, G). ^#Significantly different when compared to L-L group. *Significantly different when compared to F-L + Ø group (*P < 0.05; **P < 0.01;***P < 0.001).

FIGURE 6.

Effect of JAK-3 inhibition on C4d deposition. A-C, Representative images of C4d deposition on peritubular capillaries and glomeruli. D, Quantification of C4d deposition on PTCs. E, Percentage of glomerulous with C4d deposition. ^#Significantly different when compared to L-L group. *Significantly different when compared to F-L + Ø group (*P < 0.05; **P < 0.01; ***P < 0.001).

FIGURE 7.

Effect of JAK-3 inhibition on immune cell infiltrating renal graft. A, T cells, CD3⁺. B, T_helpers + Treg cells, CD3⁺CD4⁺. C, Treg cells, CD3⁺CD4⁺CD25⁺FOXP3⁺. D, T_cytotoxic cells, CD3⁺CD8⁺. E, NK cells, CD3⁻CD314⁺CD161⁺. ^#Significantly different when compared to L-L group (^#P < 0.05; ^##P < 0.01; ^###P < 0.001). *Significantly different when compared to F-L + Ø group (*P < 0.05; **P < 0.01;***P < 0.001).

FIGURE 8.

Effect of JAK-3 inhibition on immune cells in spleen. A, T cells, CD3⁺. B, T_helpers + Treg cells, CD3⁺CD4⁺. C, T_cytotoxic cells, CD3⁺CD8⁺. D, FO B cells, CD3⁻CD161⁻His24^HiHis57^lo. E, MZ B cells, CD3⁻CD161⁻His24^DimHis57^Hi. F, NK cells, CD3⁻CD314⁺CD161⁺. ^#Significantly different when compared to L-L group (^#P < 0.05). *Significantly different when compared with F-L + Ø group (*P < 0.05; **P < 0.01; ***P < 0.001).