Ultra low-dose IL-2 for GVHD prophylaxis after allogeneic hematopoietic stem cell transplantation mediates expansion of regulatory T cells without diminishing antiviral and antileukemic activity

Abstract

Purpose: GVHD after allogeneic hematopoietic stem cell transplantation (alloSCT) has been associated with low numbers of circulating CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs). Because Tregs express high levels of the interleukin (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 Treg recovery in pediatric patients after alloSCT and compared this recovery with Treg 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 hematopoietic stem cell transplantation (HSCT; 100,000-200,000 IU/m(2) ×3 per week), starting <day 30 and continuing for 6 to 12 weeks.

Results: No grade 3/4 toxicities were associated with ULD IL-2. CD4(+)CD25(+)FoxP3(+) Tregs 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 matched related donor (MRD) transplants. No IL-2 patients developed grade 2-4 acute GVHD (aGVHD), compared with 4 of 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 of 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 Treg population in vivo, and may be associated with a lower incidence of viral infections and GVHD.

Trial registration: ClinicalTrials.gov NCT00539695.

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

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.

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.

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).

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.