Graft-Specific Regulatory T Cells for Long-Lasting, Local Tolerance Induction

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Seltrecht et al. tested the ability of alloantigen-activated Foxp3-transduced CD4+ T cells to induce tolerance using a mouse model of skin transplantation. The authors studied the survival of C57BL/6 mice skin transplant on Balb/c host. They showed that enforced Foxp3 expression in allograft-activated CD4+ T cells increased skin allograft survival by up to 40% over three months. Furthermore, when Foxp3-enforcement was done in allograft-activated CD4+ T cells derived from F1 generated from the cross of Balb/C and C57BL/6 mice, about 80% of grafts survived.  Overall, this is a well-designed study with appropriate controls. Below are some points that need attention to improve the quality and readability of the manuscript. 

1. What is the transduction efficiency? What proportion of the transduced cells were positive stable Foxp3 expression? Did the authors test the Foxp3 stability by transferring transduced cells into mice with allograft and determine that Foxp3 expression and Treg phenotype are stable, and did not lead to effector transdifferentiation?

2. Fig. 1C. Over 50% of Balb/C responders produced IFN-gamma when stimulated by allogenic cells. Do authors test if they are effector cell origin or any contribution from ex-Tregs (Foxp3-transduced T cells)? This will address the stability issue. 

3 Line. In line with our hypothesis, ------(data not shown), even alloprimed T cells----. I encourage the authors to show this data to improve clarity and readability. similarly, the authors should show data wherever (data not shown) is used.   

4. What changes does Rapamycin treatment may have on cTregs? Any positive change that the authors anticipate?  

5. Figure 3. A group of cTregs without Thy1.2 and but with rapamycin treatment would be worth trying, as this will test whether modulating endogenous effector T cells without needing to deplete them will have any positive effect on graft survival. 

 

Author Response

1. What is the transduction efficiency? What proportion of the transduced cells were positive stable Foxp3 expression? Did the authors test the Foxp3 stability by transferring transduced cells into mice with allograft and determine that Foxp3 expression and Treg phenotype are stable, and did not lead to effector transdifferentiation?

Thank you for your important note that this has not been explained. The transduction rate ranged between 30-70%. Sorting was carried out so that all transferred cells were transduced. We have also added this information to the manuscript.

2. Fig. 1C. Over 50% of Balb/C responders produced IFN-gamma when stimulated by allogenic cells. Do authors test if they are effector cell origin or any contribution from ex-Tregs (Foxp3-transduced T cells)? This will address the stability issue. 

We have not shown this as suggested, but on the one hand, BALB/c responders without cTregs produce more IFN-g, therefore cTregs cannot produce IFN-gamma. And on the other hand, we have shown in Henschel et al. (Journal of Autoimmunity, 2023) in a different context that this is not the case.

3 Line. In line with our hypothesis, ------(data not shown), even alloprimed T cells----. I encourage the authors to show this data to improve clarity and readability. similarly, the authors should show data wherever (data not shown) is used.   

We have added these data as suggested. These are named Figure 1C, 1D, 2C, and 2E.

4. What changes does Rapamycin treatment may have on cTregs? Any positive change that the authors anticipate?  

Rapamycin treatment has the same effect on cTregs as in all clinical protocols for human or murine Tregs or expanded human nTregs:

1. Enhancement of Treg Stability and Function: Rapamycin enhances the stability and suppressive function of Tregs. This occurs partly through the promotion of a transcriptional program that favors the expression of Foxp3, a critical transcription factor for Treg development and function.
2. Preferential Proliferation: Rapamycin selectively promotes the proliferation of Tregs over conventional T cells. This selective expansion is crucial in contexts where maintaining a high ratio of Tregs to effector T cells is beneficial, such as in autoimmune diseases or preventing transplant rejection.
3. Metabolic Shift: Tregs under the influence of rapamycin may undergo a metabolic shift towards increased fatty acid oxidation. This metabolic pathway is less energy-efficient than glycolysis, which is predominantly used by effector T cells, but supports the long-term survival and function of Tregs.
4. Increased expression of Treg-specific markers: Rapamycin treatment has been associated with increased expression of markers such as CTLA-4 and GITR, which are involved in the suppressive function of Tregs.
5. Resistance to Pro-Inflammatory Cytokine Environments: Rapamycin-treated Tregs may show increased resistance to environments rich in pro-inflammatory cytokines, which can destabilize Tregs and diminish their suppressive capabilities.

 

5. Figure 3. A group of cTregs without Thy1.2 and but with rapamycin treatment would be worth trying, as this will test whether modulating endogenous effector T cells without needing to deplete them will have any positive effect on graft survival. 

We attempted to transfer Tregs to different settings. It is essential to generate a niche for Tregs; otherwise, they will not sufficiently engraft. We also tested different niche induction protocols with thy1.2 as described, anti-CD3, and anti-CD25. To keep a long story short, Thy1.2 is essential.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors created alloantigen-specific Treg by converting antigen-specific CD4+ Teffs to adopt a stable Treg phenotype and genotype by forcing ectopic expression of Foxp3 (cTregs). Using allogeneic mice skin transplantation models, the authors demonstrated that the adaptive transfer of the cTregs prior to allogeneic skin transplantation could prolong graft survival. The cTregs from F1 mice showed a better suppressive effect compared to allogeneic cTregs, which indicates that Treg function may be affected by MLC sharing between donors and recipients.

The effects of the adaptive transfer of cTregs are not perfect, therefore, authors combined the treatment with anti-Thy1.1 antibodies and rapamycin as the initial treatment. The combination of three treatments could lead to permanent allograft engraftment in the immunocompetent mice recipients.

The experiments were well conducted, however, it would be good to add some more explanations to understand the figures.

1.      ELISPOT methods were not described in the materials and methods. It is also not explained how to calculate the percent response. The authors should add the explanation to the materials and methods.

2.      Proliferation test and killing test (cytotoxicity test) were mentioned in the materials and methods. However, no data are shown in the results.

3.      Figure1 B, All recipient mice were reconstituted with Balb/c Teffs. Some mice received F1 cTregs but these were also reconstituted with Balb/c Teffs. It could be good to mention clearly, that all recipient mice received the same type of Balb/c Teffs.

4.      Please add the methods for statistical analysis in the materials and methods.

5.      Figure 2, the authors mention the homing effect of cTregs on the grafts. Do the mice show tolerance against the specific donor antigens if given one more transplant from the same strain of donor? If the authors perform the re-transplant experiments, please comment on the results.

6.      In the line 254-255, the authors cited reference 16, which is an abstract in the American Transplantation meeting, should be replaced. A search on Pubmed for this article did not give any hit.  Maybe it could be replaced with: Renal allograft rejection is prevented by adoptive transfer of anergic T cells in nonhuman primates. Bashuda H, Kimikawa M, Seino K, Kato Y, Ono F, Shimizu A, Yagita H, Teraoka S, Okumura K.J Clin Invest. 2005 Jul;115(7):1896-902. doi: 10.1172/JCI23743. Epub 2005 Jun 9.

7.      In the discussion, the authors mentioned that ‘post-transplant cyclophosphamide is not feasible after liver transplantation’. This is not true. Todo et.al. reported that cyclophosphamide was used in their living donor liver transplantation to reduce WBC amount. Seven out of ten patients were successfully withdrawn immunosuppression 18 months after liver transplantation. None of them had severe adverse effects by this treatment. (reference by Todo should be added; Todo S et.al. A pilot study of operational tolerance with a regulatory T-cell-based cell therapy in living donor liver transplantation. Hepatology. 2016 Aug;64(2):632-43)

                                           

Author Response

1. ELISPOT methods were not described in the materials and methods. It is also not explained how to calculate the percent response. The authors should add the explanation to the materials and methods.

We apologize and have added this information to the Methods section. The graph has been changed slightly for a better understanding.

2. Proliferation test and killing test (cytotoxicity test) were mentioned in the materials and methods. However, no data are shown in the results.

We apologize for this error. We have deleted this section accordingly.

3. Figure1 B, All recipient mice were reconstituted with Balb/c Teffs. Some mice received F1 cTregs but these were also reconstituted with Balb/c Teffs. It could be good to mention clearly, that all recipient mice received the same type of Balb/c Teffs.

We have added this information to the results, figure legends, and methods to avoid any misunderstandings.

4. Please add the methods for statistical analysis in the materials and methods.

This section already exists under “data analysis.”

5. Figure 2, the authors mention the homing effect of cTregs on the grafts. Do the mice show tolerance against the specific donor antigens if given one more transplant from the same strain of donor? If the authors perform the re-transplant experiments, please comment on the results.

Homing to the graft means that the tolerance can be transferred along with the graft. The results are shown in Figure 2C. This means that although there was only one reconstitution with Teffs, there was no graft rejection because specific Tregs were already in the graft.

6. In the line 254-255, the authors cited reference 16, which is an abstract in the American Transplantation meeting, should be replaced. A search on Pubmed for this article did not give any hit.  Maybe it could be replaced with: Renal allograft rejection is prevented by adoptive transfer of anergic T cells in nonhuman primates. Bashuda H, Kimikawa M, Seino K, Kato Y, Ono F, Shimizu A, Yagita H, Teraoka S, Okumura K.J Clin Invest. 2005 Jul;115(7):1896-902. doi: 10.1172/JCI23743. Epub 2005 Jun 9.

We are sorry for this. We have no idea of what happened here in our endnote library. We have changed reference 16 and added the suggested literature.

7. In the discussion, the authors mentioned that ‘post-transplant cyclophosphamide is not feasible after liver transplantation’. This is not true. Todo et.al. reported that cyclophosphamide was used in their living donor liver transplantation to reduce WBC amount. Seven out of ten patients were successfully withdrawn immunosuppression 18 months after liver transplantation. None of them had severe adverse effects by this treatment. (reference by Todo should be added; Todo S et.al. A pilot study of operational tolerance with a regulatory T-cell-based cell therapy in living donor liver transplantation. Hepatology. 2016 Aug;64(2):632-43)

Thank you for your comment. We have added the reference to our manuscript and changed the sentence.