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Published in final edited form as: Am J Transplant. 2023 Feb 4;23(4):549–558. doi: 10.1016/j.ajt.2023.01.016

Low-dose IL-2 promotes immune regulation in face transplantation: A pilot study

Naoka Murakami 1,*, Thiago J Borges 1,2,*, Thet Su Win 3,4, Phammela Abarzua 5, Sotirios Tasigiorgos 3, Branislav Kollar 3,6, Victor Barrera 7, Shannan Ho Sui 7, Jessica E Teague 4, Ericka Bueno 3, Rachael A Clark 4, Christine G Lian 5, George F Murphy 5, Bohdan Pomahac 3,8,**, Leonardo V Riella 1,2,**
PMCID: PMC10318113  NIHMSID: NIHMS1902585  PMID: 36740193

Abstract

Face transplantation is a life-changing procedure for patients with severe composite facial defects. However, it is hampered by high acute rejection rates due to the immunogenicity of skin allograft, and toxicity linked to high doses of immunosuppression. To reduce immunosuppression-associated complications, we, for the first time in face transplant recipients, utilized low-dose interleukin 2 (IL-2) therapy to expand regulatory T cells (Tregs) in vivo and to enhance immune modulation, under close immunological monitoring of peripheral blood and skin allograft. Low-dose IL-2 achieved a sustained expansion (~4–5-fold) of circulating Tregs and a reduction (~3.5-fold) of B cells. Post-IL-2 Tregs exhibited greater suppressive function, characterized by higher expression of TIM-3 and LAG3co-inhibitory molecules. In the skin allograft, Tregs increased after low-dose IL-2 therapy. IL-2 induced a distinct molecular signature in the allograft with reduced cytotoxicity-associated genes (granzyme B and perforin). Two complications were observed during the trial: one rejection event and an episode of autoimmune therapy was able to promote immune regulation in face transplant recipients, but also highlighted challenges hemolytic anemia. In summary, this initial experience demonstrated that low-dose IL-2 related to its narrow therapeutic window. More specific targeted Treg expansion strategies are needed to translate this approach to the clinic.

Introduction

Solid organ transplantation is the treatment of choice for end-stage organ failure. However, transplant recipients suffer from a higher risk of infection and malignancy due to long-term immunosuppression. This challenge is magnified in vascularized composite allograft (VCA) recipients, as these patients require a higher level of immunosuppression due to a nearly 100% rejection rate post-transplantation.1 This elevated rejection rate is in part related to the high immunogenicity of skin allografts.

Recent advances in the understanding of alloimmunity highlight the critical role of regulatory T cells (Treg) in immunoregulation2 with a skewed balance between effector (Teff) and Treg contributing to the allograft rejection in preclinical transplant models. In the clinic, several different approaches have been attempted to offset the Teff and Treg balance and to minimize the long-term immunosuppression burden. Cellular therapies using ex-vivo expanded polyclonal Tregs have been tested in kidney transplantation3 and type 1 diabetes4 with promising outcomes.5 Nevertheless, there are many aspects that we need to address before Treg-based cellular therapy could be widely applied to clinical practice, including the plasticity of Treg that may differentiate into effector cells, the timing of the Treg administration, and the longevity of infused Tregs in vivo.6

Low-dose interleukin-2 (IL-2) administration has been shown to sustain Treg expansion in vivo, and several clinical trials reported clinical benefits of IL-2 in chronic graft-versus-host disease (GVHD)7 and hepatitis C-associated vasculitis.8 Preclinical studies have also shown the benefits of low-dose IL-2 expansion of Tregs in promoting transplant survival in several models including islet, heart and skin transplantation9–11 However, its efficacy and safety in solid organ transplant patients— vascularized composite allograft (VCA) in particular— has not been fully examined, especially in the setting of concomitant immunosuppression use. Here, we report the first two cases of facial transplant recipients who received low-dose IL-2, aiming to assess the safety and feasibility of immunosuppression minimization. While on low-dose IL-2, Tregs were persistently expanded in the periphery, with enhanced suppression function at a per-cell basis. Transcriptional characterization of serial skin biopsies revealed that IL-2 altered transcriptional signatures distinctively in the allografts.

Results

Case description of the first patient

A 57-year-old female received a full-face allograft after a severe animal attack. The patient received induction with thymoglobulin and was maintained on two-agent immunosuppression with tacrolimus and mycophenolic acid after prednisone was discontinued at 4.5 months post-transplant. The patient experienced four acute cell-mediated rejection episodes before IL-2 protocol was initiated (Banff II (2 months post-transplant), Banff II (17 months post-transplant), Banff II/III (30 months post-transplant), and Banff II (47 months post-transplant)). There was no evidence of donor-specific anti-HLA antibodies. Four and a half years post-transplantation, the patient was on a stable regimen of tacrolimus (trough level 6–7 ng/ml) and mycophenolic acid 360 mg twice daily and was started on low-dose IL-2 protocol (Figure 1A). As tacrolimus has been reported to inhibit proliferation and survival of Tregs,12 the patient was converted from tacrolimus to sirolimus (goal trough 6–8 ng/ml) at 54 months post-transplant, and then initiated on daily subcutaneous IL-2 injections. IL-2 was started at 1.5×106 IU/m2 daily.7,8 As a “dose de-escalation” trial design, an incremental 25% dose reduction was allowed for the development of severe hematologic toxicities, such as >4-fold increase of natural killer (NK) cells or eosinophils. In our case, the dose of IL-2 was titrated down to 0.5 ×106 IU/m2/day (0.87 × 106 IU/day) as depicted in Figure 1. The effect of IL-2 was monitored by i) flow cytometric analysis of peripheral blood for Treg, Tfh, NK, and B cell subsets, ii) histological and immunohistochemical analyses of protocol face allograft biopsies, iii) gene expression profiles of allograft (n=8) and native skin (n=2) biopsies by Nanostring platform for 730 genes, and iv) analysis of serum cytokines and chemokines by Luminex. After 17 weeks of IL-2 therapy, the patient was withdrawn from the study due to development of Banff grade 2/3 rejection on the face allograft. Treatment with pulse intravenous solumedrol led to complete resolution of the rejection event.

Figure 1. Characterization of circulating immune cells and clinical timeline of patient 1.

Figure 1.

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(A) Picture of the patient before and after the face transplant. The picture from the post-face transplant has no signs of rejection. (B) The proportion of Treg (% of total CD4+ T cells), (C) NK cells and eosinophils (% of the total white blood cells [WBCs]) in peripheral blood. The gray box represents the period during IL-2 therapy. (D) Doses of IL-2 and other immunosuppressants (tacrolimus, sirolimus, mycophenolate mofetil and prednisone), the timing of skin biopsies, and histology grades of the biopsy are shown. (MIU: million international units, SubQ: subcutaneous, MMF: mycophenolate mofetil).

Peripheral blood immunophenotypic changes with IL-2 administration

Throughout the protocol, the patient’s peripheral blood count and peripheral blood immune phenotype were monitored serially. Specifically, we monitored CD4+ and CD8+ T cells, Treg (CD4+CD25+CD127−) and Tfh subsets of CD4+ T cells, CD19+ B cells, as well as NK cells (CD3−CD19−CD56+) (gating strategy displayed in Figure S1). IL-2 administration significantly expanded Tregs on average 5-fold (Figure 1B). At the same time, however, CD56bright NK cells and eosinophils significantly increased (Figure 1C and Figure S2A) as high as 26% (at 6 weeks) and 21% (at 4 weeks) of peripheral leukocytes, respectively. The ratio of Treg:Teff was 0.052 before IL-2 protocol initiation and 0.22 at 2 weeks post-IL-2-initiation. Despite this increase in effector cells, the patient remained asymptomatic without any clinical signs of rejection. As NK cells have been reported as a potential contributor to acute rejection,13,14 we decreased the IL-2 dose, following the pre-defined protocol (Methods). After the dose reduction, NK cells and eosinophils decreased promptly, whereas the increased proportion of Tregs was maintained (Figure 1B and C). In addition, IL-2 administration resulted in a decrease in B cells (Figure S2B) and Tfh cells (CD4+CXCR5+, Figure S2C) in peripheral blood, consistent with other reports.8

Luminex analysis of serum cytokines/chemokines over time, demonstrated no major changes in the levels of IL-6, TNF-α, IL-5, Granzyme B, CXCL9 and MMP3 (Figure S3) during IL-2 treatment. Although we observed no changes in systemic IL-5 levels, the increase of NK cells and eosinophils was an expected effect of IL-2 due to the expression of high-affinity IL-2Ra (CD25) by NK cells and innate lymphoid cells (ILC)2s, which leads to the local IL-5 production and consequent eosinophil expansion.15

IL-2 therapy increases Tregs’ suppressive function

We next examined how Tregs are functionally different after IL-2 administration. Tregs were flow-sorted from PBMC, and their suppressive function was tested using co-culture with aCD3/aCD28-activated effector T cells (Teff) at different ratios of Treg:Teff. To control the proliferative capacity of Teff, we used Teff which was collected from one specific timepoint throughout the suppression assays. IL-2-expanded Tregs showed a more potent suppressive function than pre-IL-2 Tregs (Figure 2A). To further investigate the molecular mechanisms underlying the superior suppressive capacity of the Tregs induced by the low-dose IL-2 therapy, we evaluated the expression of co-inhibitory molecules and phosphorylated STAT5 (pSTAT5) on the Tregs before, during and after the IL-2 therapy. Co-inhibitory molecules regulate functions of Tregs,16 and STAT5 is a key downstream signaling molecule of IL-2, required for Treg homeostasis.14 17 In our patient, low-dose IL-2 therapy induced the expression of TIM-3 (Figure 2B), ICOS (Figure 2C) and LAG-3 (Figure 2E) in circulating Tregs. We observed no effect of IL-2 therapy on the expression of TIGIT (Figure 2D) or on the ratio of pSTAT5 in Tregs versus Teff (Figure 2F).

Figure 2. Phenotype and function of Tregs during IL-2 therapy (patient 1).

Figure 2.

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(A) Flow-sorted Tregs were co-cultured with Tconv cells in the presence of anti-CD3/CD28 beads. Tregs had enhanced suppression function while on IL-2, compared to pre-IL-2. Treg surface expression of (B) TIM-3, (C) ICOS, (D) TIGIT and (E) LAG3 were higher on IL-2. (F) STAT5 phosphorylation in Tregs did not differ significantly before and after IL-2 therapy. (G) Treg suppression function was similar between rejection vs. non-rejection time points. (H) The proliferation of Teff cells at rejection timepoint than at non-rejection time points (on IL-2). (Siro: sirolimus, Pre-IL-2: 3 weeks before IL-2 initiation, Non-Rej: non-rejection timepoint (8, 16 and 18 weeks on IL-2), Rej: rejection timepoint (17 weeks on IL-2), Teff: T effector).

We next tested whether the epigenetic changes could explain the superior suppression function of IL-2-expanded Treg cells, as epigenetic modifications in the CpG-rich Treg-specific demethylated region (TSDR) are associated with stable FOXP3 gene expression, Treg functional stability and commitment to Treg lineage.18,19 We observed a trend that FOXP3 TSDR methylation on Tregs was lower while on IL-2 therapy, but was not statistically significant (Fig S4A and B). Thus, enhanced Treg function while on IL-2 therapy was likely due to increased expression of co-inhibitory markers.

Despite the expansion of Tregs and their enhanced suppressive capacity, a rejection event occurred while the patient was receiving IL-2. In all ratios tested, IL-2-expanded Tregs at the rejection time point were equally suppressive as at non-rejection time points (Figure 2G). However, we noted that Teff cells were more proliferative when the patient was on-IL-2 than pre-IL-2, and at the rejection time point compared to the non-rejection timepoint (Figure 2H). Therefore, the activation of Teff cells may have contributed to the rejection process.

Transcriptional signature of allograft biopsies on IL-2

To address the effect of IL-2 on the gene expression signature in the allograft, we performed transcriptional analyses of serial skin biopsies using the Nanostring platform (timeline shown in Figure 1). We observed that IL-2 induced a distinct molecular signature in the allograft, including reduced inflammation-associated genes, such as chemokines and chemokine receptors, IL-21, and TNF family members (Figure 3A). It is notable that, even more than 30 weeks after discontinuation of IL-2 administration, allograft revealed a distinct pattern of transcriptional signature from pre-IL-2 or on-IL-2. Genes related to IFN-γ responses were demonstrated to be associated with cellular rejection across different organs, including face transplantation.20,21 We then evaluated whether IL-2 treatment would interfere in the expression of IFN-γ-related genes. We found no differences in the expression of skin CXCL11, CXCL10, NLRC5, PSMB9 and GZMB upon IL-2 treatment (Figure 3B).

Figure 3. Gene expression analysis of skin allograft by Nanostring (patient 1).

Figure 3.

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The expression of 770 immune-related genes was compared in 2 “pre-IL-2” (−12 and −4 weeks), 6 “on IL-2” (4, 8, 11, 13, 15, 17 weeks on IL-2) and 1 “post-IL-2” (11 weeks after discontinuation of IL-2) allograft skin biopsies. (A) Hierarchical clustering of the differentially expressed genes (DEGs). Normalized expression of (B) IFN-g-related genes and (C) FOXP3 gene are shown. Gene Ontology (GO) analysis of the (D) upregulated genes and (E) downregulated genes. (F) Unbiased primary component analysis showed segregated gene expression patterns among pre-IL-2, on IL-2 and post- IL-2.

We next asked whether Tregs remain in the blood during the rejection episode but are no longer in the graft. Skin FOXP3 expression was slightly lower in the rejection time point compared to the non-rejection “on-IL-2” samples (Figure 3C), suggesting that Tregs were decreased in the skin tissue during rejection.

Pathway analyses of the upregulated differentially expressed genes (DEGs) on IL-2 were enriched for NF-kB activation and leukocyte extravasation signaling (Figure 3D). The downregulated DEGs were enriched for cytokine, chemokines, innate receptors and IL-2 signaling (Figure 3E). Further, unbiased principal component analysis (PCA) revealed distinctively segregated patterns in pre-IL-2, on IL-2 and post-IL-2 samples (Figure 3F).

Examination of immune cell compartment in serial skin biopsies

Skin is the most immunogenic component in face transplantation due to its high content of immune cells. The allograft-infiltrating immune cells were characterized by immunohistochemical analyses of serial allograft biopsies using CD3 and Foxp3. Similar to the Treg expansion observed in the peripheral blood, there were more Foxp3+ T cells in both allograft and native skins following IL-2 administration (Figure 4A–C). Moreover, the ratio of Foxp3+/CD3+ cells was significantly higher in biopsies from on-IL-2 time points (Figure 4D). Nanostring analyses revealed that biopsies from on-IL-2 timepoints had less NK cells, B cells and neutrophils transcripts (Figure 4E).

Figure 4. Histopathological analysis of skin allograft biopsies (patient 1).

Figure 4.

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(A) Immunohistochemistry of infiltrating immune cells (CD3+ and Foxp3+ cells) with quantitative analyses shown (B-C). (D) The ratio of Foxp3+ cells/ CD3+ cells was higher while on IL-2. “Pre-IL-2” is from week −3 and “on IL-2” is from week 11 (allograft) 12 (native skin) post-treatment. (E) Immune cell scores calculated by Nanostring nCounter software from samples of Figure 3 showed lower scores of NK cells, B cells and neutrophils while on IL-2.

Case description of the second patient

A 60-year-old male recipient of a partial-face allograft was enrolled as the second patient. At 6 months post-transplant, the patient was on a stable regimen of tacrolimus (trough level 6–8 ng/ml), MMF (1,500 mg/day) and prednisone (5 mg/day) without rejection episodes and was started on the IL-2 protocol (Figure 5A). In the second patient, we modified the protocol based on the experience with the first patient. The second patient was initiated on a much lower dose of IL-2, approximately 30% of the initial dose of IL-2 that was used for the first patient (0.5 × 106 IU/m2). With de-escalation of dosing according to the protocol, the dose 0.28 × 106 IU/m2 every 3 days achieved steady expansion of Tregs without unwanted expansion of NK cells and eosinophils. Additionally, we modified our protocol to avoid mTOR inhibitor conversion. While mTOR inhibitors are beneficial for Treg homeostasis,22–24 mTOR inhibitor conversion was associated with rejection in face transplantation, 25 and we observed more inflammatory cells in the allograft (Banff grade 1) in the first patient after complete conversion to mTOR inhibitor and even before IL-2 initiation. Thus, in our second patient, we avoided mTOR inhibitor, and instead, proceeded with dose minimization of calcineurin inhibitor.26 Once Treg expansion was stable, MMF and prednisone were tapered off at 13 weeks after initiation of IL-2 (Figure 5). The effect of IL-2 was monitored weekly for 10 weeks, and every other week afterward. The patient remained on single immunosuppression with tacrolimus and low-dose IL-2 without any evidence of rejection. However, 1 year after IL-2 initiation (18 months post-transplant), the patient developed severe anemia (Hgb 3.9 g/dL) and thrombocytopenia (65 × 103/uL). Hematological workup was consistent with autoimmune hemolytic anemia (AIHA, high LDH, low haptoglobin, a new warm antibody) and Evans syndrome. Flow cytometry of peripheral blood was without evidence of lymphoma or leukemia. IL-2 was stopped, and the patient was started on a high dose steroid (prednisone 60 mg daily) with taper over 2 months. Hemoglobin and platelet count improved afterward.

Figure 5. Characterization of circulating immune cells and clinical timeline of patient 2.

Figure 5.

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(A) Picture of the patient before and after the face transplant. The picture from the post-face transplant has no signs of rejection. (B) The proportion of Treg (% of total CD4+ T cells), (C) NK cells and eosinophils (% of the total white blood cells [WBCs]) in peripheral blood. The gray box represents the during of the low-dose IL-2 therapy. (D) Doses of IL-2 and other immunosuppressants (tacrolimus, sirolimus, mycophenolate mofetil and prednisone), the timing of skin biopsies, and histology grades of the biopsy are shown. (MIU: million international units, SubQ: subcutaneous, MMF: mycophenolate mofetil).

Peripheral blood immunophenotyping of the second patient

In our second patient, while significantly lower doses of IL-2 were used, Tregs expanded significantly, accounting for ~20% of total CD4+ T cells. After dose de-escalation per protocol, Tregs stabilized ~12% of total CD4+ T cells compared to 3.1% prior to IL-2 initiation (Figure 5B). CD56bright NK cells (35.1%) and eosinophils (14.7%) also significantly expanded and peaked after 2 weeks of IL-2 initial doses (Figure 5C, Figure S5A). Remarkably, those cell populations decreased and stabilized to almost basal levels after IL-2 dose reduction (Figure 5C). The ratio of Treg:Teff was 0.031 at the beginning of the IL-2 therapy and 0.21 at week 2 post-IL-2-initiation. As observed in our first patient, IL-2 treatment decreased circulating B cells (Figure S5B). However, our second patient had an increase in circulating Tfh cells (Figure S5C). Alike patient 1, we observed no changes in the serum levels of IL-6, TNF-α, IL-5, Granzyme B, CXCL9 and MMP3 during IL-2 treatment (Figure S6).

Functionally, as we observed in the first patient, IL-2-expanded Tregs showed more potent suppressive function than pre-IL-2 Tregs (Figure 6A). The expression of TIM-3 (Figure 6B), ICOS (Figure 6C) and TIGIT (Figure 6D) were upregulated on Tregs after IL-2 treatment, though we did not observe an increase in LAG3 expression at this time (Figure 6E). Once again, IL-2 therapy did not affect the ratio of pSTAT5 levels in Tregs versus Tconv (Figure 6F).

Figure 6: Phenotype and function of Tregs during IL-2 therapy (patient 2).

Figure 6:

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(A) Flow-sorted Tregs were co-cultured with Tconv cells in the presence of anti-CD3/CD28 beads. Tregs had enhanced suppression function while on IL-2, compared to pre-IL-2. Treg surface expression of (B) TIM-3, (C) ICOS, (D) TIGIT and (E) LAG3 were higher on IL-2. (F) STAT5 phosphorylation in Tregs did not differ significantly before and after IL-2 therapy. On IL-2 samples were from weeks 9, 20 and 23 post-treatment initiation.

Transcriptional and histological analysis of skin biopsies on IL-2 from the second patient

We next compared gene expression profiles between pre-IL-2 (n = 2) vs on IL-2 biopsy samples (n = 4) from our second patient. A total of 26 genes were significantly up- (24 genes) or downregulated (2 genes, SERPINB2 and PLA2G6). IL-2-treated allografts had a distinct molecular signature compared to pre-IL-2 biopsies (Figure 7A). We found no major changes in the expression of IFN-γ-related genes upon IL-2 treatment (Figure 7B). Pathways analyses showed that upregulated DEGs on IL-2 time points were related to T cell proliferation and TNF-mediated signaling pathway (Figure 7C). Moreover, PCA analysis revealed that pre-IL-2 and on-IL-2 samples had distinct segregated patterns (Figure 7D). Similar to patient 1, immunohistochemical analyses of serial allograft biopsies showed an increased ratio of Foxp3+ cells over total CD3+ T cells in allograft and following IL-2 administration (Figure 7E–H). In contrast to the first patient, biopsies from on-IL-2 timepoints increased NK cells and neutrophils transcripts, with no changes in B cell transcripts (Figure 7I). To better understand how the DEGs correlated with the changes in the histology and cell populations scores, we analyzed the expression of the upregulated genes across different immune cell populations using the My Geneset tool from the Immunological Genome Project (ImmGen).27 We found that the CD3E and IL2RB genes were related to the upregulation of Tregs. Moreover, the increase in NK cell scores correlated with the upregulation of the CD247, IL2RB, PRF1 and TNFSF14 genes, while CSF1R, TNFSF14 and CTSL expression correlated with neutrophils.

Figure 7: Gene expression analysis and histopathology of skin allograft (patient 2).

Figure 7:

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The expression of 770 immune-related genes was compared in 2 “pre-IL-2” (−65 and 0 weeks) and 4 “on IL-2” (4, 12, 16 and 20 weeks on IL-2) allograft skin biopsies. (A) Hierarchical clustering of the differentially expressed genes (DEGs). (B) Normalized expression of IFN-g-related genes. (C) pathway analysis of upregulated genes and (D) unbiased primary component analysis showed segregated gene expression patterns among pre-IL-2 and on IL-2. (E-H) Immunohistochemistry of infiltrating CD3+ and Foxp3+ immune cells. More Foxp3+ T cells in the allograft while on low-dose IL-2 therapy compared to pre-IL-2. (E) Histology images and (F-H) quantitative analyses are shown. “Pre-IL-2” is from week 0 and “on IL-2” is from week 20 post-treatment. (I) Immune cell scores were calculated by Nanostring nCounter software.

Discussion

In this study, we report the first two cases of face transplant recipients who received low-dose IL-2 to promote immune regulation. We demonstrated that subcutaneous administration of IL-2 significantly expanded Tregs in circulation, and those Tregs were more functional than pre-IL-2 on a per-cell basis. Further, we observed that the dosage adjustment of IL-2 and increased interval administration was critical to maintaining a sustained Treg expansion with a minimal increase in NK cells or eosinophils. Gene expression analysis of skin allograft revealed distinct patterns after low-dose IL-2 administration, which persisted even several weeks after discontinuation of IL-2.

One of the main challenges in IL-2 treatment is to determine the Goldilocks dose for each individual, sufficient enough to selectively expand Tregs but not leading to unwanted adverse events, as pointed out by the preceding clinical trials in GVHD.28 Koreth et al. suggested that the ratio of Teff/Treg might be a good predictor of the efficacy of low-dose IL-2.29 It is suggested that the ratio of Treg:Teff >0.07 at baseline and >0.2 at 1 week were predictable for clinical response. In our first patient, the ratio was 0.052 before IL-2 protocol initiation and 0.22 at 2-week post-initiation. The second patient had a ratio of 0.031 at the beginning of the IL-2 therapy and 0.21 at week 2, consistent with his clinical good response to the treatment. In our first patient, while low-dose (1.0 MU/m2/day) IL-2 successfully expanded circulating Tregs, the patient experienced grade 3 rejection 17 weeks after initiation of IL-2, leading to her withdrawal from the study. IL-2 might have stimulated not only Treg but also Teff (Figure 2I) and this Teff activation may have led to the rejection, despite the patient being still on immunosuppression. Notably, two groups reported safety concerns on IL-2 administration in kidney (NCT0241787030) and liver (NCT294949231) transplant recipients. In both cohorts, low-dose IL-2 expanded Treg proportions, but failed to achieve immunosuppression minimization. Additionally. the conversion from CNI to mTOR inhibitor prior to IL-2 initiation might have contributed to acute rejection; it is less potent in suppressing effector immune response and has been associated with rejection in face transplantation.25 Because the per-cell suppression function of circulating Treg in the first patient was comparable at the rejection time point to that of the non-rejection time point, it is also possible that the allograft tissue microenvironment could have contributed to the rejection. In fact, tissue transcriptional analyses revealed a distinctive change in skin biopsy at rejection, including leukocyte extravasation signals, which might have lowered the threshold for Teff to infiltrate the allograft and cause rejection. For the second patient, we started with a lower daily dose of IL-2 (0.5 MU/m2) and the dose was further decreased to 0.281 MU/m2 every 3 days which stabilized the Tregs and decreased NK cells and eosinophils, with no sign of rejection. However, the patient experienced severe autoimmune hemolytic anemia, which improved after discontinuation of IL-2. No autoimmune hemolytic anemia complication has been reported in the many patients treated with low-dose IL-2 for chronic graft-versus-host disease29 (communication with Dr. Koreth). Nonetheless, this autoimmune complication may indicate a potential off-target effect of low-dose IL-2 in expanding self-reactive immune cells.

It is noteworthy that we observed a decrease in B cells in both patients. Though neither of the patients had donor-specific antibodies throughout the course, the formation of antibodies against allograft is a challenge in transplant graft longevity.32 Decrease in B cell population could be seen as another potential benefit of IL-2 treatment as an adjunct therapy to prevent antibody-mediated allograft rejection or antibody-mediated autoimmune disease.33

Transcriptional signatures in allograft skin tissue revealed a significant change in pre-, on- and post-IL-2 treatments. Given that IL-2 is a potent T cell proliferation cytokine, and there was a considerable increase in allograft-infiltrating immune cells seen in immunohistochemistry (Figures 4 and 7), it was not surprising to see the upregulation of T helper cell differentiation while on IL-2. However, as skin tissue contains various resident immune cells at baseline, it is challenging to distinguish the tissue-infiltrating mononuclear cells from the resident cells, or pathogenic from non-pathogenic infiltrating immune cells by histopathology examination without knowing their specific phenotype.21 Since the current Banff grading system defines rejection by quantification of T cell infiltration, without consideration of functional differences of infiltrating lymphocytes such as regulatory cells and effector cells, it may be conceptually reasonable to use additional characterization to define rejection in patients receiving IL-2 therapy without evidence of clinical rejection. On the other hand, it is unexpected and surprising that even >30 weeks after IL-2 treatment, allograft retained a distinct transcriptional signature, so to be called the legacy effect. This provided important insight as no analyses have been done in tissue-based characteristics in cGVHD or HepC-induced vasculitis.

In summary, this report provides critical insights into IL-2-mediated immune biology both in circulation as well as in skin tissue. Low-dose IL-2 administration expands Tregs, though it was associated with a rejection event and an unexpected autoimmune complication. Due to its narrow therapeutic window, it is unlikely that low-dose IL-2 could be translated to larger trials of drug minimization. To overcome the lack of specificity of IL-2 in Treg, novel mutein IL-2 molecules could be used as an alternative to recombinant IL-2. By applying selective mutations on the β-chain of the IL-2 molecule, the mutein IL-2 reduces its ability to activate the IL-2R on effector T cells and NK cells, enhancing its selectivity to Tregs.34,35 Furthermore, coupling of this molecule with an Fc portion of human IgG significantly improves its half-life, showing greater promise in providing more selective targeting of Tregs and potentially minimizing complications in transplanted patients. Our findings provide important insights into the safety, allograft-specific and systemic immunoregulatory effects of low-dose IL-2.

Methods

(Provided in the Supporting Information section)

Supplementary Material

1

Acknowledgments/Funding

This manuscript is dedicated to the memory of Dr. Terry B. Strom. We thank the patients, whose commitment to advancing vascularized composite allotransplant research made this study possible. This study was supported by funding from Department of Defense awards to BP (W81XWH-13-2-0053 and W911QY-09-C-0216) and to LVR (W81XWH-16-1-0647 and W81XWH-21-1-0904). Opinions, interpretations, conclusions and recommendations are those of the author and are not necessarily endorsed by the Department of Defense. NM is supported by the National Institutes of Health (T32DK7527 and K08DK120868). T.J.B. was the recipient of an American Heart Association postdoctoral fellowship (20POST35210659). TSW was supported by the American Society of Transplantation’s Transplantation and Immunology Research Network Fellowship Research Grant. B.K. was the recipient of the Plastic Surgery Foundation Research Fellowship Grant. Biostatistics and bioinformatics support was funded by Harvard Catalyst | The Harvard Clinical and Translational Science Center (NIH award #UL1 RR 025758 and financial contributions from participating institutions). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Abbreviations:

DEGs

differentially expressed genes

GVHD

graft-versus-host disease

ICOS

inducible co-stimulatory molecule

IFN

interferon

IL

interleukin

LAG-3

lymphocyte activation gene-3

MMF

Mycophenolate mofetil

NK

natural killer cells

PCA

principal component analysis (PCA)

Teff

effector T cells

Tfh

T follicular helper cell

TIM-3

T-cell immunoglobulin and mucin domain-3

TNF

tumor necrosis factor

Tregs

regulatory T cells

TSDR

Treg-specific demethylated region

VCA

vascularized composite allograft

Footnotes

Disclosure

The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.

Supporting information statement

Additional supporting information may be found online in the Supporting Information section

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Data availability statement

Data to support the findings in the study are available from the corresponding author upon request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS1902585-supplement-1.docx (2.8MB, docx)

Data Availability Statement

Data to support the findings in the study are available from the corresponding author upon request.

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