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Research Highlights

Husain, Irma MD1; Luo, Xunrong PhD, MD1

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doi: 10.1097/TP.0000000000003325
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Phase I Trial of Donor-derived Modified Immune Cell Infusion in Kidney Transplantation

Morath C, Schmitt A, Kleist C, et al. J Clin Invest. 2020;130:2364–2376.

Cellular therapies are increasingly employed for the purpose of donor-specific immunomodulations for transplantation. Interestingly, in spontaneously tolerant kidney transplant recipients, immune signature studies have shown a characteristic B-cell phenotype of increased immune regulatory capabilities mediated by interleukin-10 and tumor necrosis factor-ß (termed “regulatory B cells” or Bregs).1 However, in several bone marrow chimera (BMC)–based tolerance induction protocols currently in clinical trials,2 the role of Bregs is less clear. In this phase I clinical trial,3 the authors took a non-BMC approach and used donor-derived modified immune cell (MIC) infusions in living donor kidney transplant recipients to test their safety, and possible efficacy and mechanisms in promoting donor-specific hyporesponsiveness.

In this single-center phase I trial, 10 living kidney transplant donor and their ABO compatible preemptive recipient pairs were recruited, all of whom had a PRA of <20%, negative crossmatches and Chronic Kidney Disease stage 4 and 5. Donors underwent leukapheresis to collect peripheral blood mononuclear cells, which were then treated with mitomycin C, washed and infused to the recipients. Recipients were divided into 3 groups: (A) 1.5 × 106 MICs/kg, day 2, n = 3; (B) 1.5 × 108 MICs/kg, day 2, n = 3; and (C) 1.5 × 108 MICs/kg, day 7, n = 4. No induction therapy was used. Posttransplantation standard immunosuppression was routine and consisted of cyclosporine A, enteric-coated mycophenolate sodium and methylprednisolone, with the exception of complete steroid withdrawal in group C around day 135. MIC infusions were extremely well tolerated; rejection episodes, positive de novo donor-specific anti-HLA antibodies or donor chimerism or were absent during the 30-day study phase and up to 1 year thereafter. Detailed immunological testing was, however, only performed in group C patients. At 1-year posttransplantation, peripheral T cells from group C recipients showed an almost complete absence of response to donor-specific stimulation, while third-party responses were preserved. Intriguingly, the authors stated that such donor-specific hyporesponsiveness was transiently reverted by common infections such as upper respiratory tract infections and urinary tract infections, but spontaneously reestablished following resolution of the infection. Furthermore, in quantifying peripheral cell populations in group C recipients, the authors found that the percentage of CD19+CD24hiCD38hi Bregs increased significantly after a month and peaked on day 180 to a remarkable 40% of all circulating CD19+ B cells, a finding that was absent in either group A or group B recipients. The majority of Bregs were also interleukin-10 producing, and their presence correlated with high-serum levels of interleukin-10 and tumor necrosis factor-ß.

Several important aspects regarding the use of MIC can now be addressed in order to broaden its scope of clinical applications: (1) on the timing of infusion of MIC. If the efficacy of MIC could be optimized for a timing that is closer to transplantation and possibly even after transplantation, its utility could be expanded to the transplantation of deceased donor organs; (2) on the possibility of banking of donor cells and utility of repetitive MIC dosing. As this therapy progresses to phase II and III trials with a goal of complete withdrawal of immunosuppression, maximizing the efficacy of MIC posttransplant could be highly relevant; (3) the applicability of MIC in allosensitized transplant recipients. Identified mechanisms of action of MIC may support their potential efficacy in recipients with preexisting cellular and humoral memory responses.4

In summary, the current study shows that a non-BMC approach is safe and can potentially be effective in inducing donor-specific hyporesponsiveness for transplantation.

CRISPR Screen in Regulatory T Cells Reveals Modulators of Foxp3

Cortez JT, Montauti E, Shifrut E, et al. Nature. 2020. doi:10.1038/s41586-020-2246-4

Foxp3 is a key transcription factor that determines the functionality of regulatory T cells (Treg).1 Consequently, the ability to manipulate Foxp3 levels in Treg would permit a tunable Treg function versatile for a wide range of therapeutic purposes. To do so, understanding critical regulators of Foxp3 in Treg appears of paramount importance. In the present study,2 the authors developed a pooled CRISPR-screening platform to achieve this goal. Specifically, they selected 489 known nuclear factors that have optimized single-guide RNAs from the Mouse CRISPR Knockout Pooled Library (the “Brie library”) and used a retroviral vector to introduce this selective library into Foxp3GFP Treg. Using green fluorescence marking for the the level of Foxp3 expression, the authors were able to identify many Foxp3 positive and negative regulators.

Among the top hit, positive regulators were Usp22, a deubiquitinase within the chromatin-modifying complex SAGA. To definitely demonstrate the role of Usp22 in Treg function, the authors first showed that Treg-specific ablation of Usp22 resulted in a marked decrease in Foxp3 protein levels as well as the frequency of natural Treg (nTreg), and that Treg were functionally less suppressive. Interestingly, induced Treg (iTreg) also exhibited a similar impairment, although effects were less pronounced with increasing levels of ambient TGF-β. The authors further demonstrated that Usp22 controlled Foxp3 production at both transcriptional (via chromatin modifications) and posttranslational (via protein ubiquitination and degradation) levels. Interestingly, in addition to Foxp3 itself, they found that many Foxp3-bound chromatin regions in Treg were also modified by Usp22-mediated ubiquitination, underscoring a broader role of Usp22 in controlling the Treg chromatin landscape. Several in vivo disease models were then used to confirm the role of Usp22 in supporting Treg functions. Particularly, Treg-specific deficiency of Usp22 resulted in spontaneous autoimmune lymphocytic infiltrations in multiple organs, including the kidney, lung, colon, and liver. Moreover, more severe disease scores were observed in models of experimental autoimmune encephalomyelitis and colitis. In tumor models, on the other hand, Treg-specific deficiency of Usp22 enhanced antitumor cytotoxic lymphocyte responses, reducing tumor volume. Among the top hit, negative regulators were Rnf20, an E3 ubiquitin ligase. The dichotomous roles of deubiquitination versus ubiquitination of Usp22 and Rnf20 manifested in their epistatic control of key chromatin regions in Treg including that of Foxp3.

This tour de force study has identified an intriguing landscape of positive and negative regulators of Foxp3, and therefore presented a powerful tool set with which Treg functions can be fine-tuned to benefit specific therapeutic purposes. Within the identified regulators, Usp22 and Rnf20 were selected for close examinations for their positive and negative regulatory effects on Foxp3 and the broader Treg chromatic landscape. It would be highly informative to determine which identified factors function independently and if simultaneous manipulations will be additive or synergistic in enhancing or inhibiting Treg function. This study is also timely and most relevant for the field of transplantation, particularly with the growing interest in Treg-based immunotherapies and the potential utilization of chimeric antigen receptor Treg.3 A combinatorial approach of engineering donor-specificities into Treg with fine-tuning their suppressive function has the potential to generate “super-Treg” with exquisite specificities that would greatly advance our ability to minimize transplant rejection and promote transplant tolerance.

In summary, the current study marks an important step in our pursuit of Treg-based therapies for various clinical indications.

REFERENCES

1. Newell KA, Asare A, Kirk AD, et al.; Immune Tolerance Network ST507 Study GroupIdentification of a B cell signature associated with renal transplant tolerance in humans. J Clin Invest. 2010; 120:1836–1847
2. Kawai T, Leventhal J, Wood K, et al. Summary of the Third International Workshop on Clinical Tolerance. Am J Transplant. 2019; 19:324–330
3. Morath C, Schmitt A, Kleist C, et al. Phase I trial of donor-derived modified immune cell infusion in kidney transplantation. J Clin Invest. 2020; 130:2364–2376
4. Schmitz R, Fitch ZW, Schroder PM, et al. B cells in transplant tolerance and rejection: friends or foes? Transpl Int. 2020; 33:30–40

REFERENCES

1. Rudensky AY. Regulatory T cells and Foxp3. Immunol Rev. 2011; 241:260–268
2. Cortez JT, Montauti E, Shifrut E, et al. CRISPR screen in regulatory T cells reveals modulators of Foxp3. Nature. 2020
3. MacDonald KG, Hoeppli RE, Huang Q, et al. Alloantigen-specific regulatory T cells generated with a chimeric antigen receptor. J Clin Invest. 2016; 126:1413–1424
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