Induction of Foxp3-Expressing Regulatory T-Cells by Donor Blood Transfusion Is Required for Tolerance to Rat Liver Allografts

BACKGROUND:Donor-specific blood transfusion (DST) prior to solid organ transplantation has been shown to induce long-term allograft survival in the absence of immunosuppressive therapy. Although the mechanisms underlying DST-induced allograft tolerance are not well defined, there is evidence to suggest DST induces one or more populations of antigen-specific regulatory cells that suppress allograft rejection. However, neither the identity nor the regulatory properties of these tolerogenic lymphocytes have been reported. Therefore, the objective of this study was to define the kinetics, phenotype and suppressive function of the regulatory cells induced by DST alone or in combination with liver allograft transplantation (LTx). METHODOLOGY/PRINCIPAL FINDINGS:Tolerance to Dark Agouti (DA; RT1(a)) rat liver allografts was induced by injection (iv) of 1 ml of heparinized DA blood to naïve Lewis (LEW; RT1(l)) rats once per week for 4 weeks prior to LTx. We found that preoperative DST alone generates CD4(+) T-cells that when transferred into naïve LEW recipients are capable of suppressing DA liver allograft rejection and promoting long-term survival of the graft and recipient. However, these DST-generated T-cells did not express the regulatory T-cell (Treg) transcription factor Foxp3 nor did they suppress alloantigen (DA)-induced activation of LEW T-cells in vitro suggesting that these lymphocytes are not fully functional regulatory Tregs. We did observe that DST+LTx (but not DST alone) induced the time-dependent formation of CD4(+)Foxp3(+) Tregs that potently suppressed alloantigen-induced activation of naïve LEW T-cells in vitro and liver allograft rejection in vivo. Finally, we present data demonstrating that virtually all of the Foxp3-expressing Tregs reside within the CD4(+)CD45RC(-) population whereas in which approximately 50% of these Tregs express CD25. CONCLUSIONS/SIGNIFICANCE:We conclude that preoperative DST, in the absence of liver allograft transplantation, induces the formation of CD4(+) T-cells that are not themselves Tregs but give rise directly or indirectly to fully functional CD4(+)CD45RC(-)Foxp3(+)Tregs when transferred into MHC mismatched recipients prior to LTx. These Tregs possess potent suppressive activity and are capable of suppressing acute liver allograft rejection. Understanding the mechanisms by which preoperative DST induces the generation of tolerogenic Tregs in the presence of alloantigens may lead to the development of novel antigen-specific immunological therapies for the treatment of solid organ rejection

Alloreactive responses of T-cells obtained from LEW rats subjected to DST alone or DSTx4+LTx <i>in vitro</i>.

<p>(A) <b>T-cells</b> (1×10⁵) from LEW rats subjected to one or more weekly DST treatments with or without LTx were incubated with irradiated DA splenocytes (2×10⁵) for 5 days to induce T-cell proliferation. The dotted line shows the proliferation of T-cells from naïve LEW rats. DSTx4 alone at 14, 37 and 107 days represent alloreactive responses of T-cells obtained from LEW rats pretreated with 4 weekly DST treatments and then harvested at 14, 37 and 107 days following the last DST treatment (no LTx). *P<0.05 compared with proliferation of T-cells from Naïve LEW rat. (B) <b>CD4⁺ T-cells</b> (1×10⁵) from LEW rats subjected to one or more weekly DST treatments with or without LTx were incubated with irradiated DA splenocytes as described above. The dotted line shows the proliferation of CD4⁺ T-cells from naïve LEW rats. *P<0.05 compared with Naïve LEW rat. ^#P<0.05 compared with DSTx4. ^¶P<0.05 compared with DSTx4 alone at 14 d. Data represent mean±SD from at least three independent experiments.</p

Cytokine mRNA Expression by Foxp3⁺CD25⁺ and Foxp3⁻CD25⁺T-cells from long term survivors.

<p>Message levels of TGFβ, IL-10 and IFNγ in CD4⁺Foxp3⁺CD25⁺ T-cells obtained from spleens of long term survivors (DSTx4+LTx rats; survival>100 days) were quantified and compared to those expressed by CD4⁺Foxp3⁻CD25⁺ T-cells obtained from these same animals. Quantitative Real-time (RT)-PCR was used to quantify cytokine mRNA expression in each population. HRPT was used as an endogenous control to normalize each sample. Data represent mean±SD. * P<0.05 compared with Foxp3⁻CD25⁺ T-cells. N = 5 for each group.</p

Allograft survival following adoptive transfer of splenocytes or indicated T-cell subsets obtained from different treatment groups.

<p>(A) Adoptive transfer of whole splenocytes from DSTx4+LTx rats induces tolerance to liver allografts in naïve LEW recipients in a time-dependent manner. Splenocytes (250×10⁶ cells) obtained from naïve (black diamonds, n = 7), DSTx4 (white diamonds,n = 9), DSTx4+LTx at 7 d post LTx (white circles, n = 4), DSTx4+LTx at 14 d post LTx (black circles, n = 5), DSTx4+LTx at 30 d post LTx (white squares, n = 4), DSTx4+LTx at 60 d post LTx (hash squares, n = 4) or DSTx4+LTx at 100 d post LTx (black squares, n = 9) were injected into lightly-irradiated (100rad) LEW rats one day before LTx. Control rats received 100 rad irradiation and received LTx but no cell transfer (black triangles, n = 14). *P<0.05 compared with no cell transfer. (B) Adoptive transfer of CD4⁺ but not CD8⁺ T-cells obtained from DST-primed LEW rats induces tolerance to liver allografts in naive LEW recipients. CD4⁺ (white squares, n = 6) or CD8⁺ (white diamonds, n = 6) T-cells (50×10⁶ cells) obtained from spleens of naïve rats and CD4⁺ (black squares, n = 6) or CD8⁺ (black diamonds, n = 6) T-cells (50×10⁶ cells) obtained from spleens of DSTx4 rats were injected into lightly-irradiated (100 rad) LEW rats one day before LTx. ^#P = 0.05 compared with naïve CD4⁺ (white squares). (C) Adoptive transfer of CD4⁺CD45RC⁻, CD4⁺CD25⁺ or CD4⁺CD25⁻ T-cells induces tolerance to liver allografts in naïve LEW recipients. CD4⁺ T-cells (50×10⁶ cells; (black squares, n = 7); CD4⁺CD45RC⁻T-cells(20×10⁶ cells; black diamonds, n = 5); CD4⁺CD45RC⁺ T-cells (20×10⁶ cells; white diamonds,n = 5); CD4⁺CD25⁻ T-cells (40×10⁶ cells; black circles, n = 5); and CD4⁺CD25⁺ T-cells (10×10⁶ cells, white circles, n = 5) from DSTx4+LTx at 100days post LTx were transferred into lightly irradiated LEW rats one day prior to LTx. *P<0.05 compared with no cell transfer (black triangles).</p

Alterations in different CD4⁺ populations in spleens obtained from LEW rats subjected to DST in the absence or presence of LTx.

<p>(A) Representative flow cytometric analysis of splenocytes harvested from either naïve, DSTx4 alone or DSTx4+LTx rats 100days after LTx. Cells are gated on CD3⁺ CD4⁺ T-cells. Isotype-matched control antibodies were used to set all gates. (B) Percentages of Foxp3⁺, CD25⁺ or CD45RC⁻ T-cells within the CD4⁺ T-cell population in spleens from naive, DST treated or DSTx4+LTx rats. (C) Percentages of Foxp3⁺CD25⁻, Foxp3⁺CD25⁺ or Foxp3⁻CD25⁺ T-cells within the CD4⁺ T-cell population in spleens from naïve, DST treated or DSTx4+LTx rats. The results are expressed as mean±SE from at least three individual experiments for each group. * ^{# ¶} P<0.05 compared with corresponding population in naïve group.</p

Donor specific blood transfusion (DST) induces tolerance to liver allografts.

<p><b>LEW rats were pretreated with 1 ml of heparinized DA blood (DST) once per week for 4 weeks prior to DA liver transplantation (LTx).</b> LTx was performed 7 days following last DST treatment (DSTx4+LTx 7days; black squares, n = 10). DSTx4+LTx 37days (white squares, n = 5) and DSTx4+LTx 107days (white diamonds, n = 4) represent LEW rats pretreated once per week for 4 weeks with DST with LTx performed at 37 or 107 days following last DST treatment. Some animals did not receive DST but did receive LTx (black triangles, n = 7). *P<0.05 compared with LTx alone.</p

Suppressive activity of CD4⁺ or CD8⁺ T-cells from LEW rats subjected to DST alone or DSTx4+LTx <i>in vitro</i>.

<p>(A) CD4⁺ T-cells (1×10⁵) from rats subjected to one or more weekly DST treatments with or without LTx were incubated with donor (DA) irradiated splenocytes (2×10⁵) in the presence of naïve LEW T-cells (1×10⁵) as described above. The dotted line shows the proliferation of T-cells from naïve LEW rats (designated as 100%). DSTx4 alone at 14 and 107 days represent suppressive activity of CD4⁺ T-cells obtained from LEW rats pretreated with 4 weekly DST treatments and then harvested at 14 and 107 days following the last DST treatment. (B) CD8⁺ T-cells obtained from rats subjected to one or more weekly DST treatments with or without LTx were incubated with donor (DA) irradiated splenocytes (2×10⁵) in the presence of naïve LEW T-cells (1×10⁵) as described above. The dotted line shows the proliferation of T-cells from naïve LEW rats (designated as 100%). DSTx4 alone at 14 and 107 days represent suppressive activity of CD8⁺ T-cells obtained from LEW rats pretreated with 4 weekly DST treatments and then harvested at 14 and 107 days following the last DST treatment. Combined results are shown as mean±SE. * and ^# P<0.05 compared with either CD4⁺ T-cells or CD8⁺ T-cells from naïve LEW rat.</p