Ultra Low-Dose IL-2 for GVHD prophylaxis after Allogeneic Hematopoietic Stem Cell Transplantation mediates Expansion of Regulatory T Cells Without Diminishing Anti-viral and Anti-leukemic Activity

Abstract

Purpose

Graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (alloSCT) has been associated with low numbers of circulating CD4⁺CD25⁺FoxP3⁺ regulatory T-cells(T_regs). Because T_regs express high levels of the IL-2 receptor, they may selectively expand in vivo in response to doses of IL-2 insufficient to stimulate T-effector T-cell populations, thereby preventing GvHD.

Experimental Design

We prospectively evaluated the effects of ultra low-dose (ULD) IL-2 injections on T_reg recovery in pediatric patients after alloSCT and compared this recovery with T_reg reconstitution post alloSCT in patients without IL-2. Sixteen recipients of related(n=12) or unrelated(n=4) donor grafts received ULD-IL-2 post HSCT (100,000–200,000 IU/m² 3×weekly), starting <day30 and continuing for 6–12weeks.

Results

No grade 3/4 toxicities were associated with ULD-IL-2. CD4⁺CD25⁺FoxP3⁺ T_regs increased from a mean of 4.8%(range, 0–11.0%) pre IL-2 to 11.1%(range,1.2–31.1%) following therapy, with the greatest change occurring in the recipients of MRD transplants. No IL-2 patients developed grade II-IV aGvHD, compared to 4/33(12%) of the comparator group who did not receive IL-2. IL-2 recipients retained T-cells reactive to viral and leukemia antigens, and in the MRD recipients, only 2/13(15%) of the IL-2 patients developed viral infections versus 63% of the comparator group (p=0.022).

Conclusions

Hence, ULD-IL-2 is well-tolerated, expands a T_reg population in vivo, and may be associated with a lower incidence of viral infections and GvHD.

INTRODUCTION

In patients with leukemia, those who develop GvHD after allogeneic HSCT may have a lower incidence of leukemia relapse than patients not developing GvHD, a finding that suggests residual leukemic cells present after the preparatory regimens can be eliminated by donor cellular mechanisms. Unfortunately, this graft-versus-leukemia (GvL) effect is often associated with graft versus host disease (GvHD) a leading cause of morbidity and mortality occurring in 30 to 60% of patients, with mortality reaching 50%. A hallmark of acute GvHD is the activation and expansion of alloreactive T cells.(1) Although alloreactive T cells present in a SCT graft are responsible for GvHD, removal of T cells entirely may compromise engraftment by increasing the risk of rejection by the residual recipient immune system, increasing infection risks, and reducing GvL.(2) Therefore, a balance must be maintained between the benefits of donor T cells (GvL, infection control) and their risks (GvHD).

Accumulating evidence suggests that lower levels of circulating regulatory T cells (T_regs) following alloSCT in humans are associated with a higher incidence of GvHD. A causal link for this association is suggested by murine models that show infusion of donor T_regs at the time of transplant, or shortly thereafter, prevents GvHD while maintaining the GvL effect. Several groups are therefore evaluating the adoptive transfer of ex vivo generated and expanded donor-derived T_regs to prevent or treat GvHD, but expansion of functional T_regs requires expensive and extensive cell sorting and cell culture.(3–5) Moreover, the most specific T_reg marker, FoxP3, is intracellular, rendering impossible CD4⁺ CD25⁺ FoxP3⁺ based cell-sorting. It is also unclear whether natural T_regs expanded in vitro have the same immunological properties in vivo compared to "naturally occurring" T_regs. Finally, the effect of infusing expanded T_regs on the recovering humoral and cellular immune response to leukemia antigens and to viruses and bacteria is unknown.

Since the identification of the IL-2 receptor alpha chain (CD25) as a marker for T_regs,(6) the role of IL-2 in maintaining T_reg homeostasis and suppressive function has become increasingly clear.(7) Here we explored the use of ultra low dose IL-2 as GVHD prophylaxis in pediatric and adult patients after alloSCT. We found that ultra-low doses of IL-2 (100,000 units subcutaneously x3 weekly) significantly increased circulating CD4⁺ CD25⁺ FoxP3⁺ Tregs without precipitating GvHD or incapacitating the cell-mediated response to viral or leukemic antigens.

METHODS

Patients

Subjects under 70 years of age who met standard criteria to receive an alloSCT from a matched related donor or unrelated donor were eligible for this clinical trial. Individuals were eligible for treatment if their Karnofsky/Lanksy score was ≥ 50. Patients with severe intercurrent infection, severe organ dysfunction or GvHD > grade II were ineligible. All protocols were approved by the Baylor College of Medicine IRB. The study was also registered with Clinical trials.gov NCT00539695.

Transplant conditioning regimens

The IL-2 treated patients (n=16) received standardized TBI-based conditioning regimens for patients undergoing transplant for malignant disease as previously published.(8) (9) Patients receiving alternative donor grafts also received alemtuzumab. As additional GvHD prophylaxis, all patients received targeted doses of calcineurin inhibitor FK506 (Tacrolimus) with mini-MTX (5mg/m² on days +1, 3, 6 and 11) following our transplant standard operating procedures.

Administration of ultra low-dose (ULD) IL-2

This was a Phase II study to evaluate safety and efficacy of low-dose IL-2 in the prevention of severe (grade III or IV) acute GVHD in alloSCT recipients. We used a fixed ULD of IL-2. Between day 7 – 30 (median 28 days) post alloSCT, recombinant human IL-2 (Proleukin®; Novartis) was started and continued for 12 weeks. Patients received 1–2×10⁵ units/m²/dose subcutaneously three times weekly (generally Monday/Wednesday/Friday) for the first 6 weeks. If this dose was tolerated, patients could continue to receive IL-2 at the same dose for an additional 6 weeks. T_reg numbers were measured and GvHD assessed weekly while on IL-2 (generally prior to the Wednesday dose), and monthly thereafter for 1 year. Patients were evaluated monthly for 1 year for acute or chronic GvHD. If a patient developed greater than grade II GvHD while on IL-2, therapy was halted and patients were treated using standard institutional guidelines. Patients were routinely monitored for viral infections according to our institutional SOPs. All patients were regularly monitored for disease status according to our institutional SOP including: (i) morphologic analysis of bone marrow samples to assess “conventional” remission status and (ii) minimal residual disease analyses of marrow and peripheral blood samples using chromosomal markers. As an additional measure of disease recurrence, recipients were regularly monitored for (iii) the level of donor chimerism in myeloid and lymphoid cells in the blood and marrow, as per standard institutional protocols.

Monitoring patients who did not receive IL-2

To determine the pattern of recovery of T_regs and the rates of GvHD in recipients of alloSCTs who did not receive IL-2, we studied a contemporaneous group of recipients on our IRB-approved immune monitoring protocols, receiving the same conditioning regimens, (and the same GVHD prophylaxis and supportive care elements) as the IL-2 study patients but who were either not approached or declined participation in the IL-2 study. Kinetics of T_reg recovery and other immune reconstitution studies were determined at time points post alloSCT comparable with IL-2 recipients. An intention to treat analysis was performed, and only patients who would have otherwise been eligible for the IL-2 study by day 28 were included in the data collection, which was the day when the majority of the study patients started IL-2. Blood was drawn monthly after SCT for 6 months, then every 3 months until 12 months. PBMCs were frozen and the samples batched. These patients were also evaluated for GvHD monthly for 3 months and then every 3 months until 12 months similar to patients who received IL-2.

Immunization of both the related donor and recipient before transplantation to produce high and sustained levels of antibody protection

We immunized a subset of donor and recipient pairs with tetanus toxoid (TT) one week pre-transplant since such dual immunization consistently produces high level and sustained TT antibody responses when such antigen primed donor B cells are transferred into an antigen-containing environment. Related donors received tetanus toxoid 7–10 days prior to stem cell harvest, while selected transplant recipients were given the toxoid 7–10 days prior to stem cell transplant. Recipients received a TT booster 3 months post alloSCT. The impact of IL-2 on humoral immune responses was evaluated by measuring tetanus antibody IgG on serum samples obtained pre- and post-IL-2 infusion using a quantitative ELISA. A patient with a post-vaccination to pre-vaccination ratio of less than 1.5 was considered a non-responder, a ratio of 1.5 to less than 3.0, a limited responder, and a ratio of 3.0 or greater, a normal responder.

Evaluating immune reconstitution of T cells after alloSCT

Flow cytometric analyses were used to quantify T cells (CD3, CD4, CD8) and T-cell subsets, including naïve and memory T cells, and NK cells. Specifically, we evaluated the percentage of CD4 and CD8 T cells, NK cells (CD56+CD3−), naïve (N; CD45RA+CCR7+) T cells, central-memory (CM; CD45RO+CCR7+) T cells, effector-memory (EM; CD45RO+CCR7−) T cells and terminally differentiated effector (TEMRA; CD45RA+CCR7−) T cells.

Evaluation of T_regs post SCT

PBMC were isolated from patients using Ficoll-Hypaque density-gradient centrifugation and frozen in liquid nitrogen enabling batching of tests to minimize inter-assay variation. To determine T_reg phenotype, we used the intracellular FoxP3 Treg kit (eBioscience) and CD4 and CD25 fluorescent antibodies. (BD Biosciences) All analyses used a FACScalibur flow cytometer (BD Biosciences) and CellQuest Pro^™ software. Absolute lymphocyte counts were measured to determine the absolute frequency of T_regs.

Evaluation of T_reg function

T_reg induced suppression was demonstrated by effects on thymidine uptake in mixed lymphocyte reactions. PBMC were separated into CD4⁺ CD25⁺ and CD4⁺ CD25⁻ fractions using fluorescent cell-sorting or magnetic bead separation. The CD4⁺ CD25⁻ fraction (responder cells) were plated into a 96-well plate coated with anti-CD3 (OKT3). The CD4⁺ CD25⁺ T_reg fractions were added at a 1:1 ratio and incubated at 37°C. On day 3, each well was pulsed with 1 µCi of ³H-thymidine. After 18 hours of additional incubation, plates were harvested and processed for counting.

Evaluating antiviral and antileukemia activity

Twenty to 40mL of blood was taken at monthly intervals post alloSCT to measure the frequencies of virus-specific T cells. T-cell frequencies were measured in ELISPOT assays as previously described, (10) using a cocktail of peptides (pepmixes) specific for viral antigens: CMV-pp65 and CMV-IE1, Adhexon and Adpenton and the EBV antigens BZLF1, EBNA1, EBNA3A, EBNA3B, EBNA3C, LMP1, and LMP2. In patients with myeloid and lymphoid malignancies we measured the precursor frequency of cytotoxic T lymphocytes (CTL) specific for the leukemia/lymphoma associated antigens MAGE A4, Survivin, PRAME and WT-1 using elispot assays using pepmix pulsed PBMC. (11, 12)

Statistical considerations

We utilized the Bryant and Day two-stage design incorporating toxicity and efficacy considerations to calculate sample size. (13) We considered the treatment not effective if T_reg expansion occurred in less than 50% of subjects. A treatment-related serious adverse event (SAE) was defined as any grade 3 or 4 toxicity considered related to IL-2 as indicated by the criteria listed in the National Cancer Institute (NCI) Common Toxicity Criteria version 3.0 (http://ctep.info.nih.gov/ctc3/ctc.htm). Percentages of T_reg expansion, IL-2 related SAE events and acute GvHD were summarized descriptively. Summary statistics, including mean and medians, were calculated for the subsets of T cells. Immune reconstitution of T_regs in alloSCT patients was compared with patients who received the same conditioning but did not receive IL-2. The change in T_reg and general immune reconstitution pre and one-month post transplant was compared between the patients who received IL-2 and those who did not. We analyzed the difference in changes by a two sample t-test. Acute GVHD and viral infection rates were compared between groups using Fisher’s exact test. T-cell specific reactivity to at least one leukemia associated antigen was compared between groups using Wilcoxon rank-sum test. Survival data were analyzed by Kaplan-Meier method and the comparisons between groups were performed using the log-rank test. Overall survival (OS) was calculated from the time of transplant to death from any cause or censored at last follow-up. Relapse-free survival (RFS) was calculated from the time of transplant to the date of relapse, death, or last follow-up, whichever occurred first.

RESULTS

Patient Characteristics and GVHD

We treated 16 patients with ULD IL-2 for 6–12 weeks after HSCT (12 matched related donor (MRD) and 4 matched unrelated donor (MUD) SCT; Table 1). Follow-up ranged from 5–40 months. Median age was 13 years and all patients had underlying hematologic malignancies (ALL (n=14), AML (n=1) and NHL (n=1)). Adverse effects after IL-2 administration were all grade I only, and included muscle aches, arthralgias, fatigue, nausea and decreased appetite. However, although there were no significant adverse events, seven patients elected not to complete the full 12 weeks of therapy because of aversion to the injections given three times a week, a potential limitation of a pediatric only study. To evaluate the tempo of immune reconstitution of T_regs in alloSCT patients who did not receive IL-2, we collected immune reconstitution and clinical data at monthly intervals after transplant from 16 recipients of related donor grants (Supplemental Table 1) and 18 recipients of unrelated donor grafts (Supplemental Table 2). The median age of the MRD cohort was 8.5 years with all but two patients (myelofibrosis and lymphoma) receiving their transplant for ALL. In the MUD cohort, the median age was 9 years with all but two patients (CML and SCAEBV) receiving their transplant for ALL. These patients received the same transplant conditioning regimens to the IL-2 study patients and only patients who would have been eligible to receive IL-2 by day 28 after HSCT were included in the analysis.

Table 1.

Recipients of ULD- IL-2

Age (yrs)	Sex	Race	Disease/Stage	Donor Type	Duration of IL-2	Current Disease Status	Acute GvHD while on IL-2
9	F	HISP	ALL/CR2	MRD	12 weeks	Remission	None
15	F	HISP	AML/CR1	MRD	12 weeks	Remission	None
17	M	WHITE	NHL/CR1	MRD	12 weeks	Remission	Grade 1 skin
11	M	HISP	ALL/CR2	MRD	10 weeks	Remission	None
7	M	WHITE	ALL/CR2	MRD	10 weeks	Died Relapse	None
11	F	WHITE	ALL/CR2	MRD	8 weeks	Remission	None
14	M	HISP	ALL/CR1	MRD	12 weeks	Remission	none
12	M	HISP	ALL/CR1	MRD	12 weeks	Remission	Grade 1 skin
8	F	HISP	ALL/CR1	MRD	12 weeks	Remission	None
15	F	HISP	ALL/CR2	MRD	12 weeks	Remission	None
15	F	HISP	ALL/CR1	MRD	6 weeks	Remission	None
8	F	HISP	ALL/CR1	MRD	6 weeks	Remission	None
14	M	WHITE	ALL/CR2	MUD	12 weeks	Died Relapse	Grade 1 skin
6	M	BLACK	ALL/CR1	MUD	6 weeks	Remission	None
17	M	WHITE	ALL/CR1	MUD	10 weeks	Remission	None
16	M	HISP	ALL/CR1	MUD	12 weeks	Remission	None

Race: HISP = Hispanic

Disease/Stage: ALL= acute lymphoblastic leukemia, CML= chronic myeloid leukemia, CAEBV= chronic active EBV, CR= complete remission, PR= partial remission

Donor type: MUD= matched unrelated donor. MRD= matched related donor

TRM: Transplant relapsed mortality

To determine whether patients with IL-2 developed severe acute GVHD, all patients were followed for signs and symptoms of GVHD in both the IL-2 and control cohorts. No episodes of grade II to IV acute GvHD occurred in any recipient who received ULD IL-2, with only 3/16 (19%) patients developing grade I acute GVHD; 2 recipients of MRD grafts (2/12,17%) and 1 of a MUD graft (1/4,25%). In contrast, of the 16 recipients of MRD grafts who did not receive IL-2, six (38%) developed grade I GVHD and two (13%) developed grade II-IV GVHD. Further, of the 18 recipients of MUD grafts who did not receive IL-2, five patients (28%) developed GVHD with three grade I, one grade II and one grade IV (Figures 1A–B and Table 1 and Supplemental Tables 1–2). Although the study was not powered to evaluate the effects of ULD IL-2 on chronic GVHD, only one patient (MRD recipient) who received ULD IL-2 developed with chronic GVHD (skin). In contrast, of the patients who did not received IL-2, four MRD recipients developed chronic GVHD (skin; hepatic; skin and oral; skin and liver) and two MUD recipients developed chronic GVHD (both skin).

Figure 1. Incidence of acute GvHD.

The pie charts represent the percentage of patients who were recipients of MRD and MUD grafts who did not receive ULD-IL-2 (A) versus patients who did receive ULD-IL-2 (B). No incidences of Grade III-IV were seen in IL-2 recipients

Administration of ULD IL-2 rapidly increased T_regs after alloSCT

Increases in the frequency of CD4⁺ CD25⁺ FoxP3⁺ T_regs from baseline were seen in all recipients who received ULD-IL-2 after starting IL-2 (Figure 2A). Flow cytometric analysis showed their percentage of CD4⁺CD25⁺FoxP3⁺ T_regs increased from a mean of 4.8% (range, 0–11.0%) pre IL-2 to 11.1% (range, 1.2–31.1%) following 6 weeks of IL-2 therapy (Figure 2A). T_regs in the peripheral blood of the IL-2 recipients increased 1 week after starting IL-2 (to 10.5%) compared to recipients of MRD grafts who did not receive IL-2 (mean 5.8%; p=0.031) (Figures 2B–C). By contrast, in vivo expansion of T_regs was absent in the unrelated graft recipients given ULD IL2, whose preparative regimen had included serotherapy with the T CD52 antibody alemtuzumab. All these patients had minimal detectable Treg numbers pre IL-2 (absolute Treg numbers ranged from 0–1 × 10³ cells/µL). However, even in these patients, there was a trend to higher T_reg in the IL-2 patients compared to the control group at 3 months post SCT (2 months post IL-2) (Figures 2D–E). Combining the data for both groups, the Treg:Tcon ratio increased to a median of 1.3 times the baseline value at 3 months (range 0–29).

Figure 2. Immune reconstitution of CD4⁺ CD25⁺ FoxP3⁺ regulatory T cells in patients receiving ULD-IL-2.

Percent Tregs as demonstrated by flow cytometry in patients receiving MRD (A–C) vs MUD (D–E) grafts with or without ULD IL-2. Data shown from 1–6 months post HSCT. (F–G) Representative suppression assays from two patients receiving IL-2 post HSCT. PBMCs sorted for CD4⁺ CD25⁻ (responder cells) and CD4⁺ CD25^bright (T_regs). Responder cells were stimulated with OKT3 for 3 days and proliferation detected using thymidine uptake assay.

Administration of ULD IL-2 rapidly restored functional T_regs after alloSCT

The T_regs obtained from the recipients of MRD grafts receiving IL-2 suppressed alloreactive responses in vitro since 3H thy incorporation by activated T cells (CD4⁺ CD25⁻) stimulated with allogeneic OKT3 blasts was markedly suppressed by the addition of patient T_regs (CD4⁺ CD25^hi Tregs) (Figures 2F–G). To determine whether the T_regs produced after IL-2 administration were of donor origin, PBMC were analyzed for donor chimerism by fluorescence in situ hybridization for sex chromosomes in sex-mismatched transplants and short tandem repeats measurement in same sex pairs. All patients were 100% donor during ULD IL-2 administration suggesting that these expanded cells were of donor origin (data not shown).

No effect of ULD IL-2 on NK or CD8+ T-cells immune reconstitution post SCT

Since recovery of CD8+ T-cells, CD56+/CD3neg NK cells and CD56+/CD3+ NK-like T-cells may play a critical role in engraftment and defense against relapse, we compared immune reconstitution in the treatment and control groups. In the recipients of MUD grafts, no significant differences in recovery of any cell subset was seen between treatment and control groups likely related to recent serotherapy use (data not shown). There was a significant rise of NK cells in the IL-2 treatment cohort at 1 week after starting therapy (Supplemental Figure 1A) and a concomitant fall in CD8+ T cells (Supplemental Figure 1B). These differences had disappeared by day 35. No significant differences were observed in absolute numbers or percentages of NK-like T cells (CD3+/56+) CD4+ CD3+ T-cells, or CD19+ B-cells (Supplemental Figures 1C–E).

Administration of ULD-IL-2 can blunt humoral but not cellular immune responses

To determine whether the rise in Tregs in patients who received ULD-IL-2 was associated with blunted antibody-mediated anti-bacterial responses after alloSCT we immunized related matched related donor and recipient pairs with tetanus toxoid one week pretransplant. As shown in Figures 3A–B, we enrolled 3 patients in the treatment group and 4 controls. Patients who received IL-2 also received a 3 month tetanus booster. Figure 3B shows blunting of the tetanus antibody response in all 3 IL2 treated patients; in contrast, three control patients responded well to the vaccine and the single non-responder relapsed shortly after immunization.

Figure 3. In vivo T_reg expansion may negatively affect humoral immunity.

Donor-recipient pairs were immunized with tetanus toxoid 1 week pre-SCT and received a booster at 3 months post-SCT. Compared to patients who did not receive ULD-IL-2 (A), the patients who received ULD IL-2 (B) showed blunting of tetanus antibody response post-booster.

ULD IL-2 therapy does not impair virus-specific cell mediated immunity and viral infection rates were low

We next evaluated the cell mediated immune recovery to viral antigens: adenovirus hexon and penton (Figure 4A), BK virus VP1 and large T antigen (Figure 4B), CMV IE1 and pp65 (Figure 4C) and EBV antigens BZLF1, EBNA1, EBNA3A, EBNA3B and EBNA3C (Figure 4D). We detected specific reactivity against all viral antigens in all recipients of MRD grafts early after SCT indicating equal recovery of virus–specific cellular immunity in the treatment and control groups. (Figures 4A–D). We next evaluated virus infection rates, defined as culture positive infection or PCR positivity in plasma or urine requiring treatment. In the MRD recipients, only 2/13 (15%) of the IL-2 patients developed viral infections (RSV and parainfluenza) as compared to 10/16 (63%) of the untreated patients (HHV6, VZV, BK virus, EBV, CMV). Moreover, in the recipients of MUD grafts, 17/18 (95%) of patients without IL-2 developed viral infections versus 2/6 (33%) of the IL-2 treated group (p=0.007) suggesting that IL-2 therapy does not increase (and may reduce) the risk of developing virus infections (Figure 4E–F).

Figure 4. Virus specific immunity is not negatively affected by the administration of ULD-IL-2.

Virus specific immune reconstitution was evaluated in individual patients who received MRD grafts and either received ULD-IL-2 (orange triangles) or did not receive ULD-IL-2 (blue triangles) post HSCT. Peripheral blood T-cells were incubated with Adenovirus hexon/penton pepmix (A) and Large T BK virus pepmix (B) and CMVpp65 and IE1 pepmix (C) and BZLF1, EBNA1–3 and LMP1–2 EBV pepmixes (D). The number of IFN-γ spot-forming cells (SFC) per 2×10⁵ mononuclear cells was measured in ELISPOT assays. The orange triangles represent patients who received ULD-IL-2 and blue trianges represent patients who did not receive ULD- IL-2. Note there was no difference in the frequencies of virus specific T cells in either group. This preservation of virus specific immunity corresponded to similar infection rates observed in the untreated “control” group (E) versus the ULD IL-2 treatment group (F).

Comparable GvL effects in treatment and control groups

To detect tumor-specific T-cells the PBMC were stained with HLA A2-restricted tetramers (Figure 5A–B). T-cell specific reactivity to at least one leukemia associated antigen (MAGE, PRAME Survivn or WT1) was detectable in in IFN-γ Elispot assay in recipients of MRD grafts in both treatment and control groups (Figures 5C–D). No significant differences in IFNg spot forming cells were observed between the treatment (n=9) and control (n=6) patients following stimulation with MAGE (p=0.862), PRAME (p=0.563), Survivin (p=0.907) or WT1 (p=0.862) and there was no significant difference in overall survival (Figure 5E) or relapse-free survival (Figure 5F) between IL-2 treatment group and non-IL2 group who received either MRD or MUD SCT, respectively.

Figure 5. Tumor specific T cells are detectable in recipients of IL-2 with no increase in relapse rates.

(A) Leukemia specific T cells targeting WT1 are detectable by tetramer assay in an HLA A2+ patient with ALL who received IL-2 versus no IL-2 (B) after a MRD SCT suggesting that tumor specific immunity is preserved even when IL-2 is administered to increase T_regs in vivo. Further, peripheral blood T cells were incubated with leukemia/lymphoma antigen pepmixes (MAGE A4, survivin, WT-1 and PRAME) and the number of IFN-γ SFC per 2×10⁵ mononuclear cells was measured in ELISPOT assays in MRD patients who received IL-2 (C) versus no IL-2 (D). No significant differences in IFNg spot forming cells were observed between the treatment and control patients following stimulation with each leukemia associated antigen (p>0.05). Two-year overall (E) and relapse free (F) survival for patients who received a MRD graft treated with or without IL-2 showed no significant differences as with recipients of MUD grafts with verus without IL-2 post HSCT.

DISCUSSION

The purpose of this study was to administer ultra low-dose IL-2 to patients post allo SCT to evaluate the effect on T_reg immune reconstitution with the ultimate goal of preventing moderate to severe (grades 2 to 4) acute GvHD without increasing the risk for viral infection or relapse. Patients receiving ULD-IL2 showed a marked expansion of functionally suppressive T_regs in vivo compared to controls, without significant GvHD. We also noted a lower infection rate in the treatment group and comparable relapse rates. These results suggest that increasing T_regs using ULD IL-2 may be beneficial for outcome after HSCT.

Recombinant high dose human IL-2 has been used therapeutically for many years, most often in adult cancers and HIV. To reduce toxicity while retaining beneficial immunomodulatory effects, more recent clinical trials conducted in patients with HIV or solid tumors (e.g. renal cell carcinoma or melanoma) have used IL2 in doses 1–2 log lower than the original studies, (14–16) Low-dose IL-2 (usually between 6×10⁵ units/m² to 9×10⁶ units/dose) has also been given following SCT to augment antitumor immunity. Low-dose regimens are safe and well tolerated,(17–21) and although not measurably enhancing GvL, there was no evidence that the risk of GvHD was increased, and some evidence to suggest the incidence and severity of this complication was in fact reducede.(18)

The investigators also observed that patients receiving low-dose IL-2, also had expanded CD4⁺ CD25⁺ FoxP3⁺ populations. This possible link between IL2-augmented CD4⁺ CD25⁺ FoxP3⁺ T cells and inhibition of GvHD is supported by a recent study showing that low-dose IL-2 infusions in adult patients with steroid refractory chronic GvHD increased circulating T_regs and reduced the severity of their cGvHD. (22, 23) We therefore developed a study that exploited the high expression of the IL2-CD25 receptor on the Treg population to prospectively administer ultra low dose IL-2 (1×10⁵ units/m²) to pediatric patients after allogeneic stem cell transplantation and measure the effects on T reg and other immune reconstitution, and assess the effects of this regimen on GvHD, infection and relapse.

In pediatric recipients of MRD transplants, the administration of ULD IL-2 was associated with a rapid (1–2 weeks) doubling in the number of circulating Tregs, indicating early amplification of this population. By contrast, IL-2 mediated effects on Treg numbers were both delayed and reduced in the recipients of MUD grafts a patient group who had received the CD52 antibody Alemtuzumab as part of their conditioning regimen. Alemtuzemab may persist at lymphodepleting levels beyond 4 weeks in stem cell transplant recipients (24) after HSCT, which likely reduces the pool of pre-existing IL-2 responsive Treg and depletes any newly emergent cells with this functional phenotype. Only by 3 months post transplant did we observe a rise in Treg in the MUD ULD-IL-2 group, and even then the differences from controls was a trend without reaching conventional statistical significance. Nonetheless, in the MUD groups as in the MRD patients, the incidence of GvHD and of viral infections was lower in individuals receiving IL-2 than in untreated controls so that even in the presence of alemtuzumab sufficient T_regs may be present to suppress GvHD while preserving virus specific cell mediated immunity. These findings suggest that shorter acting immunosuppression, for example with horse ATG, might allow superior Treg recovery in this subset of ULD-IL2- treated patients.

Although this study was not designed to show a causal connection between the enhancement of Treg and the reduction of GvHD, growing evidence suggests a pivotal role for Tregs in the modulation of GvHD. Donors with higher natural frequencies of Tregs produce less GvHD in their recipients,(25) while increasing the proportion of T regs in the stem cell transplant reduces GVHD in mismatched SCT recipients.(26) Moreover, Tregs infused post SCT can prevent GvHD in both murine models and in preliminary studies in man. (27, 28) Hence, our findings add further weight to a central role of Tregs in the control of GvHD.

Current immune suppressive agents target both regulatory and effector T-cells, thereby potentially affecting both alloreactive and anti-viral/bacterial T-cell responses. The consequence is an increased risk of life threatening infection that may offset the benefit from reduced GvHD. Murine models had suggested that increasing T_regs in recipients after allo SCT may substantially decrease the risk of GVHD without affecting responses to infectious agents (or tumors).(29) In this study we saw that patients treated with ULD-IL2 preserved their virus-specific immune responses measured in vitro, and had low rates of viral, fungal and bacterial infections in vivo. Other studies, however, had shown that administration of IL-2 reduced the humoral immune response to vaccine antigens. (30) Our studies confirmed this paradoxical activity, with preservation of anti-viral activity in vitro and in vivo (judged by a low rate of viral infections) even in patients in whom the humoral immune response to tetanus toxoid antigen was diminished. This disparity suggests that Th2 –associated antibody responses may be more susceptible to control by IL2-induced human Treg than anti-viral responses that consist predominantly of Th1 cytotoxic T cell responses, but the mechanism for this differential effect remains poorly explained.(31, 32) While Th1 cellular immune reconstitution is critical for protection against viruses, Th2 associated humoral immunity may be critical for protection against bacteria including pneumococcus, and it is possible that a larger series of patients would indeed show an increased risk for such infections, which may then require either antibiotic prophylaxis or administration of routine IV immunoglobulin.

Detection of WT-1 and PRAME-specific T cells in the peripheral blood of patients with lymphoid and myeloid malignancies after alloSCT strongly correlates with a low relapse rate after transplant, likely because these antigens are overexpressed by malignant target cells.(33–35) Because IL-2 has a biphasic dose response curve, in which low doses favor a Treg response and a high dose Th1 activation, there is a concern that the Treg induced by ULD IL-2 will increase Treg that also suppress the anti-tumor response, leading to increased relapse rates. Our study shows that ULD-IL-2 can increase regulatory T cells without reducing tumor-specific immunity or increasing the relapse rate. This difference in effects on alloreactivity compared to the effects on antitumor (and antiviral) immunity may reflect the differences in the cell of origin of each response. The virus specific populations are derived from memory T cell populations, as are WT1 and PRAME specific T cells which circulate in small numbers even in healthy individuals.(36–38) By contrast, most alloreactive T cells able to stimulate GvHD may reside in the naïve population, which may have greater sensitivity to inhibition by Treg. (39) (32)

Our findings highlight the potential of ULD IL-2 as an effective form of GvHD prophylaxis that may preserve immune responses to infectious agents and malignancy and the approach seems well suited to further study.

TRANSLATIONAL RELEVANCE.

Allogeneic stem cell transplant (alloSCT) is recognized as the treatment of choice for several hematologic malignancies. However, graft-versus-host disease (GvHD) is one of the major adverse consequences of the procedure. GvHD occurs in approximately 30 to 70% of patients undergoing alloSCT increasing morbidity and mortality, as well as the cost of care. Standard prophylactic therapies are often ineffective and lead to significant complications including organ damage and impaired immune recovery with resultant life-threatening infections and an increased risk of relapse. Thus, more effective and less toxic therapies are urgently needed to prevent GvHD, while still preserving the virus specific and the graft versus tumor effect post SCT. We have directly translated a novel strategy to prevent GvHD by expanding donor-derived Tregs in vivo using ULD IL-2 in patients after alloSCT. Hence, this innovative approach has the potential to prevent GVHD without increasing morbidity and mortality for virus infections and relapse.