Low-Dose IL-2 for In Vivo Expansion of CD4⁺ and CD8⁺ Regulatory T Cells in Nonhuman Primates

Introduction

Regulatory T cells (Tregs) play a critical role in a variety of human physiologic and pathologic processes including autoimmunity (1), cancer (2) and solid organ transplantation (3). Treg immunotherapy has been of great interest in the treatment of autoimmune disease or organ/cellular transplant recipients. Significant progress has been made in methods to expand Treg ex vivo (4), which has been applied to reduce the incidence or severity of graft versus host disease (GVHD) in postbone-marrow transplant recipients (5). In vivo expansion of Tregs is an alternative Treg immunotherapy, and methods to expand Tregs in vivo have also been studied. Among various cytokines, IL-2 has been identified as an essential cytokine for development and expansion of Tregs (6), which was also found to expand functional Tregs with moderate to lower dose (7–10). Saadoun et al. recently reported successful treatment of hepatitis C virus-induced vasculitis by expanding Tregs by low-dose IL-2 treatment (11). Most recent studies by Koreth et al. demonstrated that low-dose IL-2 treatment induced preferential and sustained Treg expansion in bone-marrow transplant recipients with clinical improvements of GVHD (12). However, to date, the in vivo effects of low-dose IL-2 on Tregs have only been shown in rodent models or severely immunocompromised patients. In this study, aiming at possible application for organ/cellular transplantation, we evaluated low-dose IL-2 administration for in vivo expansion of Tregs in naïve cynomolgus monkeys. Our study demonstrated that low-dose IL-2 therapy significantly expanded both CD4⁺ and CD8⁺ functional Tregs in vivo with limited expansion of non-Treg cells in nonhuman primates (NPH).

Material and Methods

Suppression assay

20×10³ of PBMCs were incubated in medium containing RPMI 1640 (Mediatech, Inc., Manassas, VA) and 10% monkey plasma, with stimulation of plate-bound CD3-Ab (SP34, 10 μg/mL) and soluble CD28-Ab (28.2, 1 μg/mL) in the presence of different ratios (1:1, 1:1/4 and 1:1/16) of CD4⁺CD25^high cells sorted by a FACS Aria (BD Biosciences, Bedford, MA). CD4⁺CD25^dim cells were used as a control. Cells were incubated for 110 hours, and during the last 24 hours, plates were pulsed and proliferation was evaluated by incorporation of (³H) thymidine as previously reported (13). In some experiments, CD3⁺CD8⁺CD25^high cells and CD3⁺CD8⁻CD25^dim cells were sorted as CD8Treg. (Figure 3B).

Results

Low-dose IL-2 injection expanded CD4⁺and CD8⁺T regulatory cells in vivo

In the peripheral blood, normal adult (age 3–6) cynomolgus monkeys (n = 7) were found to have 4.4 ± 2.0% of CD4⁺CD25⁺ Foxp3⁺ cells among CD4⁺ cells (Figure 1A). These cells were further divided by the expression of CD45RA, according to the recent studies in human Tregs (14). Similar to human Tregs, resting Treg (rTreg, CD45RA⁺Foxp3^low), activated Treg (aTreg, CD45RA⁻Foxp3^high) and nonsuppressive T cells comprise 40 ± 3%, 19 ± 8% and 42 ± 16% among CD4⁺Foxp3⁺ cells, respectively (Figure 1B).

During the course of daily subcutaneous injections of IL-2 (1 million IU/m² BSA) into five naïve monkeys for four weeks, there was significant expansion of absolute numbers of Tregs in the peripheral blood (Figure 1C and D). During IL-2 treatment, no adverse effects, including capillary leak syndrome, were observed. High Treg counts were maintained as long as IL-2 was administered but quickly returned to the pretreatment level after discontinuation of IL-2. In contrast, such expansion of Tregs was not observed with lower dose of IL-2 (0.1 and 0.6 million IU/m² BSA; Figure 1D). The majority of these proliferating cells were aTreg, CD45RA⁻Foxp3^highCTLA4⁺ CD62L^low and KI-67 positive (Figure 1E), which have been reported to be most immunosuppressive (14). IL-2 treatment also significantly expanded CD8⁺ Tregs in vivo (Figure 1F). In normal cynomolgus monkeys, CD8⁺Foxp3⁺ cells are of a minor population, typically comprising less than 0.5% of CD8⁺ cells (absolute count 2–4 cells/μl), but they were increased to as high as 30/mm³ during low-dose IL-2 treatment (Figure 1G).

Effects of low-dose IL-2 on non-Treg cells

Since normal values of lymphocyte subsets vary for each monkey, we analyzed relative increase of each cell population to the pretreatment level to compare the effect of low-dose IL-2 on Tregs and non-Treg cell populations in five monkeys (Figure 2). There was no significant expansion observed in CD8⁺ memory T cells and NK cells. The most significant expansion was observed in CD4⁺CD25⁺Foxp3⁺ Tregs (p < 0.0001), which were maintained 14–17 fold higher than pretreatment levels during IL-2 treatment. Although less significant, CD4⁺CD25⁺Foxp3⁻ (p < 0.0001) and CD8⁺ Tregs (p < 0.0001) also showed 5.8 ± 1.8 and 9.3 ± 3.0 fold expansion, respectively. Expansion of CD4⁺CD25⁺Foxp3⁺ was rapid, within 1 week after IL-2 treatment but CD4⁺CD25⁺Foxp3⁻ and CD8⁺ Tregs tended to increase after 2 weeks of IL-2 treatment. Significant, but less than threefold expansion of CD4⁺ memory T cells (p = 0.0002), CD4 (p = 0.017) and CD8 (p = 0.009) naïve cells were also observed (Figure 2).

Discussion

IL-2 was originally identified as a potent growth factor for antigen-activated T cells and NK cells, thereby enhancing innate and adaptive immunity. These properties have led to the use of high-dose IL-2 for treatment of metastatic renal cell carcinoma and malignant melanoma. However, IL-2 often induced immunosuppressive effects rather than enhancing immune responses especially with low-dose treatment (15,16). In clinical settings, low-dose IL-2 treatment had been applied to patients with HIV infection in an attempt to improve CD4⁺ cell counts and to expand NK cell expansion and augment innate and adaptive immune responses. Unfortunately, IL-2 treated patients did not experience a better clinical outcome (7) and further studies revealed significant expansion of functional Tregs in these patients (8, 9). In pediatric patients with sarcoma, low-dose IL-2 was administered with autologous lymphocyte infusions after cyclophosphamide-based chemotherapy, which also resulted in significant expansion of functional Tregs (10). Most recently, Koreth et al. applied low-dose IL-2 treatment in the treatment of chronic GVHD in bone-marrow transplant recipients and observed preferential and sustained Treg expansion with clinical improvements of GVHD (12). Since Tregs constitutively express all three subunits of the high affinity IL-2R (α, β and γ), they can presumably respond to physiologically low doses of IL-2. Low-dose IL-2 may also induce JAK/STAT signaling rather than PI3K signaling, which preferentially expand Treg rather than conventional effector T cells (17,18). Therefore, depending on the dose, IL-2 treatment may potentially be useful for immunotherapy by expanding Tregs.

In the current study, using naïve cynomolgus monkeys, we studied low-dose IL-2 treatment for in vivo expansion of Tregs. The dose of IL-2 was determined based on a clinical study with low-dose IL-2 treatment (12,18), which was approximately 1/100–1/200 of the dose that has been used for treatment of malignancies (600 000–720 000 IU/kg every 8 hours) (19). Our preliminary studies also showed no effects in lymphocytes by 0.1 million IU/m² and limited effects with 0.6 million IU/m² of IL-2 (Fig. 1D). Therefore, we estimated that the minimal dose of IL-2 that can expand Tregs would be 0.6–1.0 million IU/m².

We found that daily low-dose IL-2 injections markedly expanded absolute counts of both CD4⁺ and CD8⁺Tregs with limited expansion of other T cells and NK cells. The majority of these expanded Tregs were CD45RA⁻FOXP3^highCTLA4⁺ Ki67⁺, which is considered the most immunosuppressive subpopulation among Tregs (14). From the observations that these Tregs were highly proliferative and their expansion was less significant in the lymph nodes or bone marrow (data not shown), we speculate that these expanded Tregs originated from either preexisting resting Tregs or nonregulatory CD4⁺ T cells in the peripheral blood. However, we cannot conclude how much Tregs were originated from naturally occurring Tregs in this study. Since these expanded Tregs have significant suppressive function, in vivo expansion of Tregs by low-dose IL-2 may be useful for induction therapy or even for tolerance induction in organ/cellular transplantation. However, it may be difficult to apply for treatment of rejection, since low-dose IL-2 did expand non-Tregs (CD4⁺CD25⁺Foxp3-). Further studies of concomitant immunosuppressive medications are necessary to ensure exclusive expansion of Tregs without the activation of conventional effector T cells.

Disclosure

The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation. This manuscript was also not prepared or funded by any commercial organization.