Treg-targeted IL-2/anti-IL-2 complex controls graft-versus-host disease and supports anti-tumor effect in allogeneic hematopoietic stem cell transplantation

Abstract

Modulating an immune response in opposite directions represents the holy grail in allogeneic hematopoietic stem cell transplantation (allo-HSCT) to avoid insufficient reactivity of donor T cells and hematologic malignancy relapse while controlling the potential development of graft-versus-host disease (GVHD), in which donor T cells attack the recipient’s tissues. IL-2/anti-IL-2 complexes (IL-2Cx) represent a therapeutic option to selectively accentuate or dampen the immune response. In dedicated experimental models of allo-HSCT, including also human cells injected in immunodeficient NSG mice, we evaluated side-by-side the therapeutic effect of two IL-2Cx designed either to boost regulatory T cells (Treg) or alternatively to activate effector T cells (Teff), on GVHD occurrence and tumor relapse. We also evaluated the effect of the complexes on the phenotype and function of immune cells in vivo. Unexpectedly, both pro-Treg and pro-Teff IL-2Cx prevented GVHD development. They both induced Treg expansion and reduced CD8+ T-cell numbers, compared to untreated mice. However, only mice treated with the pro-Treg IL-2Cx, showed a dramatic reduction of exhausted CD8+ T cells, consistent with a potent anti-tumor effect. When evaluated on human cells, pro-Treg IL-2Cx also preferentially induced Treg expansion in vitro and in vivo, while allowing the development of a potent anti-tumor effect in NSG mice. Our results demonstrate the clinical relevance of using a pro-Treg, but not a pro-Teff IL2Cx to modulate alloreactivity after HSCT, while promoting a graft-versus-leukemia effect.

Introduction

Initially identified for its capacity to stimulate T cells in vitro, interleukin-2 (IL-2) has been used in the clinic for boosting effector immune responses for the treatment of metastatic melanoma and renal carcinoma,¹ or for inducing remission maintenance in patients with acute myeloid leukemia,² although with limited (5-15% of responder patients) or non-significant benefit, respectively. Additionally, high-dose IL-2 administration is limited by its high associated toxicity.

Low efficacy of IL-2 in cancer can be explained by (i) an insufficient activation of CD8⁺ T and natural killer (NK) cells, which respond to IL-2 through the intermediate affinity IL-2 receptor, composed of IL-2R|3 and IL-2Ryc,³ and/or (ii) by the undesired activation of regulatory T cells (Treg). Once activated by IL-2, Treg block the anti-tumor immune response.⁴ Treg constitutively express the high affinity IL-2 receptor composed of three subunits: IL-2-Rα, IL-2R|3 and IL-2Ryc,⁴ giving them an advantage over other T cells in the consumption of IL-2, especially in environments with poor IL-2 content. Thus, although IL-2 has a pleiotropic activity, its major role is to favor Treg survival and suppressive functions.⁵ Consequently, low efficacy of IL-2 treatment in cancer is mainly due to low response of CD8⁺ T and NK cells expressing the intermediate affinity IL-2 receptor, as well as the unwanted effect of IL-2 on Treg due to their constitutive high affinity IL-2 receptor expression.

Understanding of the effect of IL-2 on Treg has led to the design of IL-2-based immunosuppressive strategies targeting Treg. Thus, contrary to what was initially proposed, IL-2 administered at low dose represents a novel immunosuppressive drug for the treatment of autoimmune and inflammatory diseases, acting by Treg activation.

Allogeneic hematopoietic stem cell transplantation (alloHSCT) is a particularly interesting situation in which to test the therapeutic efficacy of IL-2. Indeed, the risk associated with insufficient reactivity between donor and recipient is the hematologic malignancy relapse for which allo-HSCT has been performed initially. In this case, high-dose IL-2 therapy could accentuate the graft-versus-leukemia/tumor (GVL/GVT) response through expansion and activation of donor effector T cells (Teff). On the other hand, the major complication of allo-HSCT is the development of life-threatening graft-versus-host disease (GVHD), in which donor T cells attack the recipient’s tissues.⁶ In this situation, low-dose IL-2 therapy could lead to Treg expansion, and thus to GVHD inhibition. Before the paradoxical effect of IL-2 on the immune response was understood, the group of David Sachs^7-9 initially observed that 3 days of low-dose IL-2 administration controlled experimental GVHD, while preserving the GVL/GVT effects. At odds, Shin et al.¹⁰ reported a lack of therapeutic effect of low-dose IL-2 in experimental GVHD. We also studied different preventive or curative approaches based on low-dose IL-2 administration +/- rapamycin, but did not observe any effect on GVHD, including in a model of xenogeneic GVHD using human cells administered to immunodeficient mice.¹¹ In humans, low-dose IL-2 therapy has been administered to dampen inflammation in GVHD,^12-14 but with either no, limited, or not yet evidenced clinical recuperation. In our models, as well as in healthy volunteers or in allo-HSCT patients, low-dose IL-2 administration induced an approximately 1.5-2-fold expansion of Treg without activating conventional T cells (Tconv). This increase in Treg numbers was far away from the desired 20-fold needed to reach a 1:1 Treg:Tconv ratio and consequently, a protective effect of Treg in experimental GVHD, as we and other previously observed.^15-19 Thus, it is actually difficult to conclude on the potential of sole IL-2 administration to prevent GVHD or to induce GVL/GVT effect. Overall, improvements on IL-2-based therapy are needed to increase therapeutic efficacy in both situations. IL-2/anti-IL-2 complexes (IL-2Cx) represent an alternative to enhance IL-2 therapeutic usefulness. Depending on the IL-2 monoclonal antibody (mAb) used to generate the IL2Cx, they can direct IL-2 action to either Teff or to Treg.²⁰ Consequently, some complexes induce dramatic anti-tumor responses by vigorously activating effector immune cells.²¹ Other complexes show a selective effect on Treg expansion, resulting in immune response attenuation and autoimmune control.²² In allo-HSCT, the possibility of modulating donor T-cell alloreactivity in both directions depending on the used complexes remains unsolved.^23-26 Moreover, no study has systematically compared the effect of different complexes side-by-side. Here, we tested two IL-2Cx. One is made with a pro-Treg mAb (i.e., preferentially activating Treg bearing the IL-2Rαβγ, now referred to as Cx25 as CD25 is the IL-2Rα chain). The mAb used in Cx25 binds to the part of IL-2 that normally binds to the IL-2R|3y. As a result, IL-2 effect will be mainly mobilized for Treg that constitutively express CD25. The second one is made with a pro-Teff mAb (i.e., preferentially activating effector IL-2R|3y- bearing cells, such as CD4⁺ conventional T cell, CD8⁺ T cells and NK cells, now referred to as Cx122 as CD122 is the IL-2R|3 chain). The mAb used in Cx122 binds to the part of IL-2 that normally binds to the high affinity IL-2Ra. As a consequence, IL-2 loses its preferential affinity for constitutively expressing CD25 Treg, leading to an improved availability of IL-2 for Tconv. We show that even if both complexes are effective in preventing GVHD, only the pro-Treg complex allows the emergence of the GVL/GVT effect.

Methods

Mice and graft-rareus-host disease models

Female C57BL/6J (B6, H-2^b), BALB/cJ (H-2^d), (B6xDBA2) F1 (B6D2F1, H-2^bxd), (B6xC3H) F1 (B6C3F1, H-2^bxk) from Charles River Laboratories (France) were used at 8-10 weeks of age. NSG (NOD.Cg-Prkdc^scidIL2rg^tm1Wjl/SzJ) mice were obtained from our own breeding and used at 10-12 weeks of age. All experiment protocols were approved by the local ethics committee (authorization no. APAFIS#11511-2017092610086943) and are in compliance with European Union Guidelines. GVHD was induced in B6C3F1 female injection of CD3⁺ T cells from B6 mice as previously described and evaluated three times a week.^27,28

Treatment

Recombinant human IL-2 (rhIL-2, Proleukine, Novartis), anti-CTLA4 (clone 9H10; Bio X Cell), anti-human IL-2 (clone MAB602; Bio-Techne), and anti-human IL-2 (clone 5344.111; BD Biosciences) were purchased. IL-2Cx were prepared by mixing 15,000 UI of rhIL-2 with 4.5 μg of mAb (molar ratio 2:1) and incubated for 30 minutes at 37°C. IL-2Cx is called Cx122 when made using MAB602 clone; and Cx25 when made using 5344.111 clone. IL-2Cx were administered in five or ten intraperitoneal injections from day 0 (d0) to d4 and d7 to d11 or only from d0 to d4 after bone marrow transplant (BMT) according to experiments. Anti-CTLA-4 was administered in three intraperitoneal mAb injections of 200 ug each on d0, d3, and d6 after BMT

Phenotypic analysis of immune cell populations

Spleens were harvested on d12 and splenocytes stained with the antibodies listed in Online Supplementary Table S1. Non-specific binding was blocked using anti-CD16/ CD32 (Miltenyi Biotec). For intracellular cytokine staining, cells were stimulated for 5 hours with phorbol 12-myristate 13-acetate (PMA) 50 ng/mL and ionomycin 1 μg/mL (Sigma– Aldrich, Saint Louis, CA, USA,). Brefeldin A (BD Pharmingen, San Diego, CA, USA) was added for the last 4 hours. Then, cells were fixed and permeabilized with fixation/permeabilization solution (ThermoFischer), following the manufacturer’s instructions. Data acquired with a Canto 2 flow cytometer were compensated and exported into FlowJo (version 10.0.8, TreeStar Inc).

Tumor relapse models

Irradiated recipient B6D2F1 female mice received retro-orbital infusion of P815-GFP mastocytoma cells (2x10⁴ per mouse) +/- bone marrow (BM) and T cells. GVHD symptoms were evaluated three times per week. Blood samples were systematically collected at d14 to identify the presence of tumor cells before tumor apparition at the eye, the site of injection. GFP⁺ cells were detected by flow cytometry analysis. In addition to blood sampling, presence of an eye tumor was assessed daily. Mice found to have tumors were sacrificed as previously described.^27-29

RS4;11-GFP⁺ acute lymphoblastic cells were used to test the possibility for human cells to support a GVL effect in NSG mice. For this, 3x10⁶ CD3⁺ cells contained in peripheral blood mononulcear cells (PBMC) from two healthy donor blood samples (Etablissement Francais du Sang, Créteil, France) and 0.5x10⁶ RS4;11-GFP⁺ were intravenously injected at d0. Mice were treated with Cx25 for 5 days from d2 to d6. At d20 and d30, mice were randomly sacrificed, and BM, spleen, blood and liver were harvested for the exploration of the presence of tumor cells by GFP detection by flow cytometry.

Short-term in vivo xenogeneic human lymphocyte activation

NSG mice were used to compare the effect of IL-2 and Cx25 on human healthy donor immune cells in vivo; 12x10⁶ CD3⁺ cells among PBMC from two healthy donor blood samples were injected at d0 in NSG mice. IL-2 and Cx25 were injected at d0, d1 and d2. After 5 days, their spleens were collected, and analyzed by flow cytometry.

Results

In allogeneic hematopoietic stem cell transplantation, clinical effects are determined by the timing and duration of Cx25 and Cx122 administration, rather than by the monoclonal antibody used to generate the complexes

We performed a series of experiments to evaluate the effects of Cx25 and Cx122 on GVHD development. When administered at the time of BMT, both IL-2Cx reduced clinical manifestations of GVHD, and prolonged survival of grafted mice, compared to untreated mice or mice receiving low doses of IL-2 (Figure 1A). Thus, unexpectedly, Cx25 and Cx122 induced a similar preventive effect on GVHD development when administered at the time of BMT and for a short period. Using the same anti-IL-2 mAb than in our study to generate IL-2Cx, the distinct effector T-cell activation of the Cx122 complex³⁰ or, the pro-Treg effect of Cx25³¹ were previously documented in other clinical settings. We thus questioned whether the indissociable effects observed in our hands with the two IL-2Cx were due to the particular setting of allo-HSCT. We grafted B6D2F1 skins onto B6 recipient mice and treated them for 4 days, initiating the treatments at time of skin grafting. In untreated mice, skin grafts were rapidly rejected at d10 as well as in IL-2 or in Cx122 treated mice. As expected, skin rejection was delayed only in mice treated with Cx25 (Online Supplementary Figure S1). These results are in accordance with the dissociated clinical effects previously described with Cx25 or Cx122 and suggest that the similar protective effects observed in our experiments (Figure 1A) are exclusively due to the particular setting of allo-HSCT.

We then reproduced experiments of BMT but extending the duration of IL-2Cx administration from d0 to d11. It led not only to a loss of their protective effect observed with a short-time treatment, but even to an accentuation of GVHD, and accelerated death (Figure 1B). Prolonged low-dose IL-2 administration did not induce any significant effect on GVHD control. Thus, during allo-HSCT, the timing and/or the duration of IL-2Cx administration seem more important in determining the clinical effect than the different nature of the complexes. These results indicate that short-time administration of IL-2Cx at the time of BMT is more effective than low-dose IL-2 and similarly alleviates GVHD independently of the mAb used to generate the complexes. Cx25 and CX122 are supposed to act on different target cells. However, these two complexes induced comparable effects on GVHD when administered preventively. A possibility to assess whether or not their respective effects depend on Treg is to eliminate them in vivo at the same time as the complexes are administered. For this, we took advantage of our recently published data²⁹ showing that the in vivo administration of an anti-CTLA4 mAb at time of BMT efficiently eliminated Treg. We, therefore, tested the effect of the IL-2Cx when Treg are eliminated by an anti-CTLA4 treatment. In this experiment, both IL-2Cx still fully prevented GVHD, whereas anti-CTLA4 worsened it. Interestingly, the protective effect of both IL-2Cx was reduced when anti-CTLA4 was co-administered, suggesting at least a partial link between IL-2Cx effects on Treg and GVHD attenuation (Online Supplementary Figure S2).

In allogeneic stem cell transplantation, both Cx25 and Cx122 treatments increase the proportion of regulatory T cells

Next, we evaluated the biological effects of both IL-2Cx on immune reconstitution. First, we assessed IL-2Cx effects on immune cell engraftment at d12 (Figure 2A), as by this time four of 21 (19%) mice had died in the untreated control group, 2 of 16 (12.5%) in the IL2-treated group, 3 of 16 (18%) in the Cx122 group, and none in the Cx25 group. By the time the mice were sacrificed, weight curves and clinical scores already differed according to treatment (Figure 2B). Thus, d12 appears to be a tipping point for GVHD. The frequencies of Treg and Teff of recipient mouse origin were only residual, with only few CD4⁺Foxp3⁺, CD4⁺Foxp3^-and CD8⁺Foxp3^- cells expressing the H-2K^k recipient-type molecules at their surface being detected in untreated mice (Figure 2C). Administration of Cx122, and to a lesser extent of Cx25, increased the percentage of recipient CD4⁺ T cells (Foxp3⁺ or Foxp3^-) which, however, remained in very small proportions in the spleen of treated mice. Thus, both complexes induced a slight decrease in the engraftment as evidenced by the persistence of a larger, although minor, proportion of cells of recipient’s origin. Following this observation, we considered that cells collected at d12 were mainly of donor origin (between 95% and 100%) and consequently attested for the biologic and clinical effects observed with both IL-2Cx.

Figure 1.Short treatments with Cx25 or CX122 prevent graft-versus-host disease after allogeneic hematopoietic stem cell transplantation. Lethally irradiated B6C3F1 received semi-allogeneic hematopoietic stem cell transplantation (HSCT) (10x10⁶ bone marrow cells plus 2x10⁶ T cells). Weight and graft-versus-host disease (GVHD) signs were evaluated 3 times a week. (A) Mice were untreated (U.T) (N=21) or treated from day (d)0 to d4 after HSCT with 15,000 IU of human interleukin 2 (hIL-2) alone (N=16) or with Cx25 (N=16) or with Cx122 (N=16). Survival curves and clinical grade histograms are the cumulative data of 3 independent experiments. (B) Mice were untreated (N=15) or treated daily from d0 to d4 then from d7 to d11 after HSCT with 15,000 IU of hIL-2 alone (N=15) or with Cx25 (N=16) or with Cx122 (N=16). Survival curves and clinical grade histograms are the cumulative data of 3 independent experiments. Histograms show area under the curve (AUC) of GVHD manifestations evaluated for each mouse for all the duration of the experiment. The AUC were calculated for the GVHD clinical-grade curve for each mouse and are presented as the mean ± standard error of the mean. Kruskal-Wallis tests were performed. *P<0.05, **P<0.01, ***P<0.001. Kaplan-Meier survival curves were compared using the log-rank test. *P<0.05, **P<0.01, ***P<0.001.

Whereas the absolute numbers of total spleen cells or of T cells were not modified in Cx25 treated mice or slightly decreased for CD8 T cells in Cx122-treated mice compared to untreated mice, we observed that the clinical immunosuppressive effects of both IL-2Cx were compatible with the important decrease in CD8⁺ T cells percentage in Cx25- and Cx122-treated animals, compared to untreated mice; as well as with the increase of the proportion of CD4⁺Foxp3⁺ Treg (Figure 3A, B). Comparable observations were made in the liver of grafted animals, a target organ of GVHD (Online Supplementary Figure S3).

It has been previously observed that a small immunosuppressive CD8⁺Foxp3⁺ T-cell population can emerge early during GVHD both in experimental models^32,33 and in humans.^32,34 Here, we observed that the frequency of spleen CD8⁺Foxp3⁺ T cells increased under IL-2Cx administration. Finally, we observed that the IL-2Cx induced a dramatic reduction of the CD8/Treg ratios both in spleen and liver of grafted animals, likely underlying GVHD attenuation (Figure 3; Online Supplementary Figure 3B).

Figure 2.Impact of IL-2/anti-IL-2 complexes on early graft-versus-host disease and chimerism at day 12 after hematopoietic stem cell transplantation. (A) Lethally irradiated B6C3F1 received 10x10⁶ semi-allogeneic B6 bone marrow (BM) cells and 2x10⁶ B6 T cells. (B) Weight loss and clinical grade were evaluated at day (d)12 in untreated mice (UT) (N=9) or mice treated from d0 to d4 after hematopoietic stem cell transplantation (HSCT) with Cx25 (N=21) or with Cx122 (N=22). Data cumulative from 3 independent experiments are presented as the mean ± standard error of the mean. Kruskal-Wallis tests were performed. *P<0.05, **P<0.01, ***P<0.001. (C) Mice were sacrificed and splenocytes were collected for chimerism analysis in untreated mice (N=5) or mice treated from d0 to d4 after HSCT with Cx25 (N=8) or with Cx122 (N=8). Kruskal-Wallis tests were performed. *P<0.05, **P<0.01, ***P<0.001.

Then, we analyzed the expression of CTLA-4, CD25 and TNFR2 markers of Treg function and fitness (Online Supplementary Figure S4) among the remaining CD4⁺ FOXP3⁺ Treg. Although similar percentages of Treg expressing CTLA-4 were observed, the mean fluorescence intensity (MFI) of CTLA-4 expression was statistically significantly increased after treatment with both IL-2Cx. For CD25, only mice receiving Cx122 showed statistically significantly increased percentages of CD25⁺ Treg, with no major changes in its MFI values. Thus, the remaining Treg in IL-2Cx-treated mice displayed a more activated phenotype, compatible with the observed reduced GVHD. However, this observation must be counterbalanced with the observed decrease in the percentage of Treg expressing TNFR2 in mice treated with the Cx25 (Online Supplementary Figure S4).

The combined use of CD44 and CD62L membrane markers allows distinguishing naïve (CD44^-CD62L⁺), memory effector (CD44⁺CD62L^-) and central memory (CD44⁺CD62L⁺) T cells^35,36 (Online Supplementary Figure S5A). Compared to untreated mice, Cx25 and Cx122 treatments dramatically increased the proportion of CD4⁺ Foxp3^- naïve T cells, whereas CD4⁺ effector memory T cells were decreased. The proportion of CD8⁺ effector memory T cells also decreased with Cx122, but not Cx25 administration. No major modification was observed in the central memory T- cell compartment (Figure 4A). In the liver of grafted animals, CD8⁺ effector memory T cells decreased and CD8⁺ central memory T cells increased under Cx122 treatment (Online Supplementary Figure S6A).

Figure 3.IL-2/anti-IL-2 complexes increase donor regulatory T cells after hematopoietic stem cell transplantation. (A) Lethally irradiated B6C3F1 received 10x10⁶ semi-allogeneic B6 bone marrow (BM) cells and 2x10⁶ B6 T cells. On day (d)12, mice were sacrificed and splenocytes were collected for T-cell analysis. Mice were untreated (U.T) (N=14) or treated from d0 to d4 after hematopoietic stem cell transplantation (HSCT) with Cx25 (N=16) or with Cx122 (N=17). Numbers of spleen cells and CD4⁺ and CD8⁺ T cells (A) and percentages (B) of CD4⁺, CD4⁺Foxp3⁺, CD8⁺, CD8⁺Foxp3⁺ cells among live splenocytes and CD8⁺Foxp3^-/ CD4⁺Foxp3⁺ ratio are shown for each group. Data cumulative from 3 independent experiments are presented as the mean ± standard error of the mean. Kruskal-Wallis tests were performed. *P<0.05, **P<0.01, ***P<0.001.

Additionally, we evaluated the effects of complexes on the expression levels of the anergy/exhaustion T-cell markers PD-1, T-bet and Eomes (Online Supplementary Figure S5B, C).^37,38 Both IL-2Cx induced a decrease in the percentage of splenic CD4⁺Foxp3^- and CD8⁺Foxp3^- PD-1-expressing conventional T cells and of liver CD8⁺Foxp3^- PD-1-expressing conventional T cells, suggesting a global attenuation of T-cell activation, compared to untreated animals (Figure 4B; Online Supplementary Figure S6B). Among the 80% of CD8⁺Foxp3^- T cells that expressed PD-1, more than 70% co-expressed Eomes but not T-bet in untreated mice, attesting a very high fraction of potentially exhausted CD8⁺ T cells. Interestingly, this fraction of cells dramatically dropped in mice treated with Cx25 (Figure 4C). The percentage of cells with the Eomes⁺T-bet ^- phenotype represented 20% of the CD4⁺PD-1⁺ cells and Cx122 treatment negatively impacted this cell population (Figure 4C). Thus, the effect of the complexes seems associated with a reduction in the percentage of activated Teff and a more marked and statistically significant decrease in the fraction of CD8+ T cells with an exhausted phenotype when administering Cx25. Similar observations were made in the liver of grafted animals (Online Supplementary Figure S5C).

IFNγ, TNFα and IL17 are considered as essential players during the cytokine storm associated with GVHD (Online Supplementary Figure S7).⁶ We did not observe any major effects of both IL-2Cx treatments on cytokine production by T e f f, e x c e p t f o r I F N γ production by CD8⁺ cells, with a statistically significant decrease only when Cx122 was used. The production of the three tested cytokines by Treg statistically significantly decreased in Cx25 and Cx122 treated animals, compared to control group (Figure 5). Thus, as soon as d12, the inflammatory status of Treg was dramatically modified when complexes were administered in grafted mice.

Figure 4.IL-2/anti-IL-2 complexes modify the distribution of naïve/memory populations and the activation state of T cells after hematopoietic stem cell transplantation. B6C3F1 mice were lethally irradiated and grafted as for Figures 2 and 3 and were either untreated (U.T) (N=8) or treated from day (d)0 to d4 after hematopoietic stem cell transplantation (HSCT) with Cx25 (N=7) or with Cx122 (N=8). On d12, mice were sacrificed and memory, effector and naïve T cells were analyzed from splenocytes of grafted animals by flow cytometry. (A) Percentage of effector memory (defined as CD44⁺ CD62L^- cells), central memory (defined as CD44⁺ CD62L⁺ cells), and naïve (defined as CD44^- CD62L⁺ cells) cells, among CD4⁺Foxp3^- cells, CD4⁺Foxp3⁺ cells and CD8⁺Foxp3^-cells are shown for each group. (B) PD-1 among CD4⁺ and CD8⁺ T cells according to Foxp3 expression and (C) Eomes and T-bet among CD4⁺Foxp3-PD-1⁺ and CD8⁺Foxp3-PD1⁺ T cells were also analyzed by flow cytometry. Shown are the cumulative data of 2 independent experiments. Untreated mice (N=5), mice treated from d0 to d4 after HSCT with Cx25 (N=8) or with Cx122 (N=8). Data are presented as the mean ± standard error of the mean. Kruskal-Wallis tests were performed. *P<0.05, **P<0.01, ***P<0.001.

Cx25 but not Cx122 prevents graft-versus-host disease while inducing a graft-versus-leukemia/tumor effect

After observing that both complexes inhibit GVHD through different mechanisms of action, we evaluated their ability to induce a GVL effect. We used our recently published model in which BM cells (5x10⁶ per mouse) are engrafted together with a defined number of tumor cells (2x10⁴ P815-GFP cells per mouse) and T cells (1x10⁶ CD3⁺ T cells per mouse) into recipient mice, allowing tumor development without inducing GVHD. In this model, P815 tumors develop in 100% of mice, inexorably leading to 100% of tumor-related mortality by d22, without the development of measurable clinical signs of GVHD (Figure 6A, B). We did not observe any differences in survival, tumor incidence or GVHD clinical grade in mice treated with Cx122, when compared to untreated mice. In marked contrast, Cx25 administration led to the protection of half of the grafted mice from leukemia-induced death. No tumor cells were detected before d20 and, over the duration of the experiment (d60), P815-GFP cells were detected only in 55% of the Cx25-treated mice. Interestingly, the anti-tumor effect in Cx25-treated mice was not associated to increased GVHD clinical score (Figure 6B). Thus, only Cx25, but not Cx122, induces a protective GVL effect in mice without inducing GVHD.

Figure 5.IL-2/anti-IL-2 complexes inhibit pro-inflammatory cytokines production by regulatory T cells after hematopoietic stem cell transplantation. B6C3F1 mice were lethal-ly irradiated and grafted as described above and were either untreated (U.T) (N=14) or treated from day (d)0 to d4 after hematopoietic stem cell transplantation (HSCT) with Cx25 (N=16) or with Cx122 (N=17). On d12, mice were sacrificed and spleno-cytes were collected and then stimulated with PMA/ionomycin and golgi plug for 5 hours before analysis by flow cytometry. TNF-α, IFN-y, and IL-17 expression in CD4⁺Foxp3-, CD4⁺-Foxp3⁺ and CD8⁺Foxp3- T cells are shown for each group of mice. Data are presented as the mean ± standard error of the mean. Kruskal-Wallis tests were performed. *P<0.05, **P<0.01, ***P<0.001.

We recently showed in this same experimental model that anti-CTLA4, but not anti-PD-1 administration, induces a strong GVL effect, but is associated to lethal GVHD, rendering this therapeutic approach not compatible with a clinical use.²⁹ Thus, given that both IL-2Cx efficiently reduce GVHD, we hypothesized that their combination with anti-CTLA4 would represent an ideal situation associating the IL-2Cx GVHD-protective effect with the potent anti-tumor effect of anti-CTLA4 mAb. Combination of anti-CTLA4 with Cx122, did not improve mice survival, tumor incidence or GVHD clinical signs, compared to either IL-2Cx alone. However, the GVL/GVT effect as well as the improved survival obtained with Cx25 were lost when combined with anti-CTLA4 despite an increased alloreactivity, as attested by the statistically significant augmentation of the clinical grade compared to Cx25 alone (Figure 6B). This suggested that the GVL/GVT effect observed with Cx25 was not due to the sole alloreactivity, but rather to a not yet identified mechanism that could partially depend on CTLA4.

Figure 6.Only Cx25 but not Cx122 treatment triggers a graft-versus-leukemia/tumor effect after allogeneic hematopoietic stem cell transplantation but that is reverted when combined with an anti-CTLA4 treatment. (A) Lethally irradiated B6D2F1 recipient mice received 5x10⁶ bone marrow cells, 1x10⁶ T cells and 2x10⁴ P815-GFP tumor cells. Recipient mice were divided into 5 groups: untreated mice (U.T) (N=10), mice treated with 5 injections from day (d)0 to d4 either with Cx25 (N=9) or Cx122 (N=10) alone or combined with 3 injections at d0, d3, and d6 with an anti-CTLA-4 monoclonal antibody (mAb) (N=8 with Cx25) or (N=12 with Cx122). (B) Survival curves, tumor incidence curves and areas under the curve (AUC) are presented for all experimental groups of mice. Shown are the cumulative data of 2 independent experiments. Kaplan-Meier survival curves were compared using the logrank test. *P<0.05, **P<0.01, ***P<0.001. The AUC were calculated for the graft-versus-host disease (GVHD) clinical-grade curve for each mouse and are presented as the mean ± standard error of the mean. To compare AUC, Mann-Whitney tests were performed. *P<0.05, **P<0.01, **P<0.001.

Preclinical considerations before Cx25 therapeutic utilization

Given that Cx25 was the sole IL-2Cx enabling GVHD control concomitant with GVL triggering, we assessed its effects on human immune cells in vitro and in a newly designed humanized mice model of leukemia. Of note, and for translational rational, this Cx25 is made of human IL-2 and anti-human IL-2 mAb. First, we co-cultured in vitro CD2⁺-sorted human cells containing T and NK cells with allogeneic APC and evaluated the effects of IL-2 and Cx25 on cell proliferation (Online Supplementary Figure S8). Addition of IL-2 induced a significant proliferation of CD8⁺ and NK cells, but not of Treg. At odds, Cx25 selectively increased the percentage of Treg.

Next, we evaluated the effect of Cx25 on the initial steps of human T-cell activation in vivo, 5 days after the adoptive transfer of human PBMC containing 12x10⁶ T cells into immunodeficient NSG mice (Figure 7A). At this time point, IL-2 or Cx25 administration did not modify the percentage of human colonizing immune cells (evaluated as percentage of human CD45⁺ cells), in accordance with the similar levels of Ki67⁺ proliferating CD4⁺ and CD8⁺ T cells (Figure 7B). Of note, the percentage of Treg strongly increased in mice treated with Cx25 compared to untreated or IL-2-treated mice. These results suggest that the protective effect of Cx25 evidenced by a preferential expansion of Treg is triggered very early after the adoptive transfer of human cells. Even if the xenograft GVHD model, due to the high xenograft reactivity and the paucity of human cytokines and immune cell growth factors, only partially reflects the physiopathology of human GVHD, it is the only model allowing the in vivo evaluation of therapeutic molecules on human cells before clinical evaluation. This model is based on the administration in immunodeficient NSG mice of a number of human tumor cells mimicking a leukemia relapse and a suboptimal number of human T cells not allowing the development of an anti-leukemic effect. In order to evaluate the GVL effect of the Cx25, we infused NSG mice with the 0.5x10⁶ RS4-GFP lymphoblast T cells, together with human PBMC containing 3x10⁶ T cells, and treated the mice with PBS or Cx25 for 5 consecutive days, starting on d2 after cells injection (Figure 7C). When untreated mice were screened between d20 and d30 (a time line compatible with a leukemia development), RS4-GFP cells were detected in six of eight untreated mice. In marked contrast, no GFP-cells were detected in Cx25-treated mice, attesting of a potent GVL effect.

Discussion

In this work, we comparatively analyzed the effects of IL-2Cx made with two different anti-IL-2 mAb with IL-2, with the aim to modulate the alloreactivity after allo-HSCT. Unexpectedly, both complexes, when administered early and for a short period, attenuated GVHD, resulting in prolonged mice survival in the absence of clinical signs of GVHD (Figure 1A). At odds with our data, the group of Bruce Blazar observed an acceleration of acute GVHD in Cx25-treated mice, and even an inhibition of the protective effect of transferred therapeutic Treg.²⁴ These discordant results could be explained by the nature of the complexes (murine in their article, human in our work), but more probably by the level of histo-incompatibility between donor and recipient mice (fully allogeneic in Blazar et al., semi-allogeneic in our work). In order to confirm this hypothesis, we evaluated the effect of preventive treatment of Cx25 and Cx122 in a second B6 in BALB/c fully allogeneic model. No protective effect was observed (Online Supplementary Figure S9A). Along these lines, they did observe the efficacy of the Cx25 complex, when used in the B6 into B10. BR chronic GVHD model.

In our model, administration of IL-2Cx over a prolonged period of time, not only leads to a loss of their protective effects, but even accentuates GVHD development (Figure 1B). Because GVHD is a highly inflammatory pathology, it is likely that both complexes indiscriminately, activate Teff more than Treg when administered for a prolonged period, thus reflecting their loss of specificity of action. Indeed, we observed a strong increase in CD25 expression on a very high proportion of conventional T cells at d4 after transplantation, compared to those of donor T cells before their injection (Online Supplementary Figure S9B). In accordance, IL-2Cx treatments initiated at d5, when an elevated ratio of conventional T cells already strongly express CD25, has no protective effect on the occurrence of GVHD (Figure 9C). We, therefore, focused the rest of this study on short treatments in order to understand the mechanisms of action of the two IL-2Cx in HSCT and their potential clinical relevance.

The protective properties of both complexes were mainly associated with comparable biological effects on donor T cells (Figure 2B), inducing an efficient control of GVHD as the result of IL-2Cx effect at early time of BMT. In order to evaluate early events that complexes could have on Treg, we performed KI67 staining of donor T cells collected in grafted mice at d4. First, we did not observe major difference on KI67 staining on Treg depending on whether IL-2Cx were administered or not. This suggested that at d4, treatments did not significantly influence the percentage of cycling Treg. However, whereas each mouse was grafted with 2x10⁵ Treg at d0, the number of Treg collected at d4 in IL-2Cx-treated mice tended to increase compared to untreated mice. As the Treg viability were comparable (data not shown), this suggested that IL-2Cx likely induced Treg proliferation (Online Supplementary Figure S9D).

Figure 7.Cx25 induces human regulatory T-cell expansion in vivo and triggers a potent graft-versus-leukemia/tumor effect in a model of xenograft-transplantation. (A) NSG mice were receiving human peripheral blood mononuclear cells (PBMC) containing 12x10⁶ CD3⁺ T cells from 2 healthy volunteer donors, circle or triangle distinguish the 2 donors. IL-2 and Cx25 were injected at day (d)0, d1 and d2 and spleen were harvested at d5 for flow cytometry analysis. (B) The percentage of human CD45⁺ (huCD45⁺) cells, of CD4⁺ and CD8⁺ among huCD45⁺, in the spleen of grafted animals, the percentage of CD4 and CD8 proliferating T cells attested by Ki67 staining as well as the proportion of regulatory T cells (Treg) (CD25⁺Foxp3⁺) among CD4⁺ T cells are shown. Dot blot sum the data with each dot representing 1 mouse. Kruskal-Wallis tests were performed. *P<0.05, ***P<0.001. (C) NSG mice were receiving human PBMC containing 3x10⁶ CD3⁺ T cells from 2 healthy volunteer donors, circle or triangle distinguish the 2 donors; 0,5x10⁶ RS4-GFP⁺ tumor cells were injected in mice at time of human PBMC infusion and were treated or not (U.T) with 5 injection of Cx25 from d2 to d6. Mice were randomly sacrificed at d20 or d30 and spleen, liver, blood and bone marrow were harvested to determine the presence or not of tumor cells by GFP detection. Pie chart represent the presence (grey) or not (white) of RS4-GFP tumor cells.

It should be noted that CD8⁺Foxp3⁺ T cells appear during GVHD development, and increase in mice treated with both IL-2Cx (Figure 3B), suggesting that CD8⁺ Treg arise with increased alloreactivity, as we and others have recently demonstrated.^29,34,39 Furthermore, CD8⁺ Treg represent an attractive therapeutic target since their frequencies are increased when GVHD is controlled. Along these lines, administration of a murine pro-Treg complex in combination with rapamycin was reported to expand CD8⁺Foxp3⁺ Treg cells with protective properties against GVHD.³³ Additionally, the controlled alloreactivity achieved by both IL-2Cx could be attributed to a decrease of CD4⁺ EM T-cell frequencies, an increase of naive T cells, and a global decrease of T-cell activation attested by reduced PD-1 expression by CD4⁺ and CD8⁺ T-cell activation. Concerning Treg functions, both IL-2Cx led to higher CTLA4 expression levels, without increasing the already high percentage of CTLA4-expressing Treg (Online Supplementary Figure S2). In mice developing GVHD, Treg atypically produced TNF-α, IFN-γ and IL-17. Along these lines, it has been described that during autoimmune diseases Treg can convert to pathological, dysregulated Treg with reduced or no immunosuppressive properties.^40,41 Our results prone for IL-2Cx likely halting this process of Treg dysregulation, thus ensuring the maintenance of their suppressive capacity (Figure 5).

IL-2Cx also induced distinct effects on donor T cells. The statistically significant increase in the percentage of CD25⁺ Treg was only observed with the Cx122. Alternatively, the statistically significant decrease in TNFR2 expression was only observed with the Cx25 (Online Supplementary Figure S4). We have recently described that TNFR2 is essential for the suppressive functions of Treg during allo-HSCT.²⁷ Thus, all these observations taken together, it could be concluded that GVHD is more efficiently controlled by Cx25, compared to Cx122. In Cx25-treated mice at d12, Treg seem less mobilized to exert their suppressive effect which is reflected by their less activated/suppressive phenotype. This is consistent with discrete but noticeable differences observed between the two complexes, with the better probability of survival and the weaker clinical score (Figure 1A) obtained with Cx25 when compared to untreated mice (P<0.001), compared to those obtained with Cx122 (P<0.005). We also studied the CD8⁺PD-1⁺EOMES⁺T-bet^- T-cell compartment that identifies an exhausted population of T cells. In GVHD-experiencing mice, it represents nearly 80% of CD8⁺ T cells. Although Cx122 administration tended to reduce this cell population, only Cx25 induced a statistically significant decrease in the proportion of exhausted CD8⁺ T cells. This observation may have important implications regarding the ability of donor cells to mediate a GVL/GVT effect. Indeed, when tumor cells were injected with T cells at the time of BMT, a GVT effect was only observed in mice that were concomitantly protected from GVHD by Cx25 administration. Importantly, this GVT effect was not associated with an increased clinical score of GVHD, suggesting that it could develop partly independently of an increased alloreactivity. At odds, tumor cells were consistently detected in all untreated or Cx122 treated mice. Overall, our study is the first, to our knowledge, to assess side by side the therapeutic effects of IL-2Cx on the occurrence of acute GVHD and the compatibility of such treatment with the development of a GVL/GVT effect. We demonstrate that only Cx25 effectively prevents GVHD while ensuring a GVL/GVT effect through a CTLA4-dependent mechanism. Indeed, co-administration of Cx25 and anti-CTLA4 was incompatible with the maintenance of a GVT effect, despite increased in alloreactivity. Our results are in accordance with the recent description of a method using an orthogonal IL-2/IL-2Rβ system targeting Treg that allows to prevent GVHD while maintaining a GVL effect.⁴² In this work, we made the choice to use human-compatible complexes in order to generate information that could be directly transferred to humans. We first demonstrated that these complexes can be evaluated in murine experimental GVHD. More importantly, this allowed us to directly test the Cx25 candidate complex on human cells, in vitro and in vivo. As observed in the whole murine mice model, Cx25 induced human Treg expansion in vitro at d2 and in vivo at d5 in NSG immunodeficient mice grafted with human PBMC. In the same xenograft-GVHD model, the co-injection of RS4 cells induced leukemia in six of eight mice between d20 and d30, while no leukemic cells were detected when mice were treated with Cx25.

Taken together, our results demonstrate the clinical relevance of using complexes to modulate alloreactivity after HSCT. Due to its protective effects against GVHD and its capacity in inducing the GVL effect, our data sustains the use of Cx25 in a first clinical trial in patient with high-risk to develop GVHD. A secondary objective in these patients would be to assess the rate of leukemic relapse. It would be also interesting in this context to compare the effect of such complex with the direct in vivo injection of an anti-IL-2 mAb, especially since two anti-IL-2 pro-Treg human mAb have been described.^43,44 In this context, IL-2Cx have been described to be directly formed in vivo, and the efficacy of administration of a murine anti-IL-2 mAb in the prevention of GVHD has recently been demonstrated.²⁵

Footnotes

• Received January 6, 2023
• Accepted September 7, 2023

Correspondence

^*AT and CP contributed equally as first authors.

^#EP and JLC contributed equally as senior authors

J.L. Cohen jose.cohen@inserm.fr

Disclosures

EP is a co-founder and consultant for Egle-TX. All other authors have no conflicts of interest to disclose.

Contributions

AT, CP, EP and JLC designed the study. AT, CP, PC and CS performed experiments. AT, CP, PC, AM, ML, SM EP and JLC analyzed the data. AT, CP, EP and JLC wrote the first draft of the manuscript and all authors contributed in the final version.

Data-sharing statement

Data and protocols are available on request to corresponding author.

Funding

AM received a PhD grant from the Université Paris-Est-Créteil (UPEC). This work was fully supported and funded by a common grant from the French Ministry of Health and The French National Cancer Institute (PRTK-2015) (to JLC).

Acknowledgments

We are grateful to the IMRB for providing access to their animal facility team and the flow cytometry platform team for their help.