Administration Of Low-dose Interleukin-2 Research Articles

Assessment of SLEDAI score changes in systemic lupus erythematosus patients under low-dose interleukin-2 therapy: A meta-analysis

BackgroundInterleukin-2 is being investigated as a potential therapeutic option in systemic lupus erythematosus (SLE), but the current findings regarding its effectiveness remained inconclusive. ObjectivesTo assess the association between the administration of low-dose interleukin-2 and the SLE Disease Activity Index (SLEDAI) score among SLE patients. MethodsA meta-analysis was conducted by gathering articles from Scopus, Pubmed, and Embase databases (PROSPERO registration ID: 451971). Essential data from each study was extracted, and the impact of administering low-dose interleukin-2 on the SLEDAI score in SLE patients was assessed using the inverse variance method. ResultsWe conducted an analysis of 7 articles involving 194 patients. Our study revealed that the administration of low-dose interleukin-2 was associated with a decrease in the SLEDAI score, showing an average reduction of −4.3 points from baseline. Furthermore, when comparing SLE patients treated with standard of care plus low-dose interleukin-2 to those receiving standard of care alone, we observed a significantly greater improvement in the SLEDAI score, with an average difference of 2.91 points. ConclusionOur results indicate the potential beneficial impact of low-dose interleukin-2 in reducing disease activity and enhancing outcomes in SLE management.

Low-Dose Interleukin-2 Restores Human Regulatory T Cell Function, in Vitro, As a Potential in the Handle of Graft Versus Host Disease (GvHD)

BackgroundCurrently, isolation of natural regulatory T cells (nTregs) and in vitro -expanded nTregs was shown to be an effective therapy to GVHD patients. However, shortage of nTregs in peripheral blood and time consumption of expansion in vitro may eventually limit the clinical application. Conversely, induction regulatory T cells (iTregs) can be generated in vitro from naïve T cells and to a large number of iTregs in short time. The activity of iTregs to suppress the responder T cells should be maintained for longer time, then may improve the GVHD therapeutic efficiency. So far, paracrine uptake of soluble mediators as would intensify the Tregs activity on T cell suppression, especially the interleukin-2 (IL-2) is essential for CD4+CD25+ Tregs function. In clinical, low-dose IL-2 administration was suggested to induce the functional Tregs in vivo, and improve the therapy of cGVHD. Therefore, IL-2 may improve iTregs function and expansion during short time, in vitro, then to ensure the successful acute GVHD therapy.AimAs we know, iTreg cells would decay after a period of time, in vivo or in vitro . If we want to maintain a certain number of iTregs for the GVHD treatment, it is best to provide iTregs to the patient more than once. In order to provide more number of effective iTregs for clinical use, we try to develop a method for induction and expansion of more iTregs via IL-2 restores Tregs in vitro .MethodsHuman PBSC were prepared from peripheral blood of health donor by Ficoll-Hypaque density gradient centrifugation. Naïve T cells were isolated by negative selection. The harvested naïve T cells were cultured and stimulated under cytokines-containing RPMI1640 medium. The flow chart was shown in figure. For cell proliferation and activity maintenance, the cells were cultured under IL-2 supplemented medium. The harvested cells were analyzed by flow cytometry with fluorescence-conjugated CD antibodies, including CD4, CD25, CD127 and FoxP3. The iTregs suppression function were analyzed via MLR assay.ResultsWith our purpose, provide more effective iTreg cells should be efficient for clinical application. Amplification of the T cell number can obtain more iTreg cells; therefore, cultivation of the T cells under IL-2-containing medium would stimulate the cell proliferation to about 4-fold on the 10th day. After the first cytokines [IL-2, TGFb-1 and retinoic acid (RA)] stimulation, we harvested the iTregs, and then, keep the cells in low-dose IL-2 only medium for another 5 days to expand and maintain activity of the T cells. On the 10th day, naïve T cell would be performed with anti-CD3/CD28 antibodies and with cytokines supplement for the further induction. The IL-2 expanded-T cells would be further induced to iTregs. FoxP3 gene expression was as a functional iTregs indicator, and showed the highly expression on the 5th- and 15th- day iTergs but lower on the 10th-day. It indicated that the iTregs would be restored for application within a short period under low-dose IL-2 medium. Besides, we could harvest more iTregs after the low-dose IL-2 incubation, because of the cell expansion during IL-2 medium incubation for 5 days. Using MLR assay, we have confirmed all of these functional iTregs to suppress the responder T cells.ConclusionOur study showed that after the activation and induction of naïve T cells, we could maintain the cells under IL-2 containing medium. Then, after the second induction, we could restore the iTreg cells. Therefore, we may provide more of the functional iTregs to GVHD patients, continuously. The result should develop a novel-cell based approach for potentially reducing the risk of acute GVHD within short time. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Low-dose interleukin-2-loaded nanoparticle effect on NK and T-reg cell expression in experimentally induced type 1 diabetes mellitus.

IntroductionType 1 diabetes mellitus is an autoimmune disorder characterized by inflammatory damage to pancreatic β cells resulting in loss of insulin secretion. In autoimmune type 1 diabetes mellitus (T1D) natural killer cells (NK) initiate pancreatic islets cell lyses in autoimmune T1D. Loss of T regulatory cells (Treg) at disease onset facilitates the activation and accumulation of NKs in the pancreatic microenvironment. A proper low-dose interleukin 2 (IL-2) could enhance Tregs and enforce control and regulation of pro-inflammatory NKs.AimThis relation needs to be studied to improve therapeutic strategies aimed at resetting the balance between Tregs and proinflammatory cells.Material and methodsWe used novel formulations of low-dose IL-2 loaded on chitosan nanoparticles. The study included 116 T1D BALB/c mice experimentally induced by streptozotocin, divided into groups. Their splenocytes were maintained in a short-term culture for assessment of expression of CD4+FOXP3+ Treg and NKp46+NK by both flow cytometry and enzyme-linked immunoassay (ELISA). Morphological, immunohistochemical, and morphometrical analyses were done. In vitro suppressor assay was used to assess the suppressor effect of Treg cells after exogenous IL-2 treatment.ResultsNK cell expression, NKp46 level, and NK cell functions were modulated more in mice injected with IL-2-loaded chitosan nanoparticles than in other groups. A statistical inverse correlation was found between Treg and NK cell expression in IL-2-loaded chitosan with 0.3 µIU (p = 0.047), and this correlation was related to FOXP3 expression on Treg cells. The modified expression of NK and NKp46 was noticed in mice injected with 0.3 µIU for longer duration (3 weeks) (p < 0.001), but the NK functions did not show any significant changes with prolonged treatment.ConclusionsProlonged administration of low-dose IL-2 results in the vigorous expression of NKp46, indicating a significant role of Tregs in NK stimulation and motivation. Low-dose IL-2 selectively modulates NKp46 NK and FOXP3+ Tregs and increases their expression.

An Adaptive Control Scheme for Interleukin-2 Therapy.

SummaryRegulatory T cells (Treg) are suppressor cells that control self-reactive and excessive effector conventional T helper cell (Tconv) responses. Breakdown of the balance between Tregs and Tconvs is a hallmark of autoimmune and inflammatory diseases. Interleukin-2 (IL-2) is a growth factor for both populations and subtle leverage to restore the healthy immune balance in IL-2 therapy. By using a mechanistic mathematical model, we introduced an adaptive control strategy to design the minimal therapeutic IL-2 dosage required to increase and stabilize Treg population and restrict inflammatory response. This adaptive protocol allows for dose adjustments based on the feedback of the immune kinetics of the patient. Our simulation results showed that a minimal Treg population was required to restrict the transient side effect of IL-2 injections on the effector Tconv response. In silico results suggested that a combination of IL-2 and adoptive Treg transfer therapies can limit this side effect.

Targeted Activation of T Cells with IL-2-Coupled Nanoparticles.

Interleukin-2 (IL-2) is a T cell growth factor particularly required in regulatory T cell maintenance and memory T cell responses. High-dose IL-2 treatment was the first FDA-approved immunotherapy for cancer, while low-dose IL-2 administration has shown promise in allograft rejection and autoimmune and inflammatory diseases. However, its pleiotropic nature and the existence of IL-2 receptors with different binding affinity limit its therapeutic application. For an improved clinical applicability of the cytokine, a targeted receptor assignment must, therefore, be achieved. Nanoparticles allow controlling the location and dose of immunomodulating compounds and to specifically address specific receptors through targeted drug binding. In this review article we discuss the IL-2 biology and current clinical application with regard to nanoparticle-based IL-2-mediated manipulation of T cell responses in autoimmunity, chronic inflammation, and cancer.

Interleukin-2 drives cortisol secretion in an age-, dose-, and body composition-dependent way.

Background:Interleukin-2 (IL-2), one of the proinflammatory cytokines, is used in the treatment of certain malignancies. In some studies, transient increases in cortisol and ACTH secretion occurred. Thus, this agent may be used as an experimental probe of adrenal cortisol secretion.Objective:This study quantifies the effects of low and moderate doses of IL-2 on cortisol secretion and assesses the modulation by age, dose and body composition.Site:Mayo Clinical Translational Research Unit.Subjects:Study comprised 35 healthy men, 17 young and 18 older.Methods:Randomized prospective double-blind saline-controlled study of IL-2 administration in two doses with concurrent 10-min blood sampling for 24 h.Outcome measures:Deconvolution analysis and approximate entropy of cortisol secretion.Results:Low-dose IL-2 administration increased nocturnal pulsatile cortisol secretion from 1460 ± 160 to 2120 ± 220 nmol/L/8 h in young subjects and from 1680 ± 105 to 1960 ± 125 nmol/L/8 h (treatment P < 0.0001, but more in young than older, P = 0.02). Comparable results were obtained for total cortisol secretion (P treatment <0.0001, age effect P = 0.005). The higher IL-2 dose caused a large increase in young (P < 0.0001), but not in older (P = 0.90) subjects. This dose also increased approximate entropy from 0.877 ± 0.041 to 1.024 ± 0.049 (P = 0.008), pointing to reduced secretory orderliness. Incremental cortisol (nocturnal) secretion correlated negatively with visceral fat mass (R = −0.41, P = 0.019).Conclusion:In healthy men, IL-2 injection drives pulsatile cortisol secretion in a dose-dependent way in young, but not older, individuals and erodes cortisol secretory orderliness at a higher dose in young subjects. Cortisol responses are diminished with increasing abdominal visceral fat mass.

Functional Effects of Low-Dose IL-2 in Patients with Chronic Graft Versus Host Disease

Introduction: CD4+CD25+FoxP3+ regulatory T cells (Treg) have broad suppressive activity and play a central role in the maintenance of immune tolerance and prevention of chronic graft-versus-host disease (cGVHD) after allogeneic hematopoietic stem cell transplantation (HSCT). Interleukin-2 (IL-2) is a key growth factor for Treg and we previously reported that daily administration of low-dose IL-2 in patients with refractory cGVHD induces selective expansion of Treg and NK cells. No expansion of CD4 conventional T cells (Tcon) or CD8 T cells was noted. In a Phase 2 study, 64% of patients had clinical responses. Predictors of clinical response included younger age, early IL-2 initiation and a higher Treg:Tcon ratio at study baseline and at week 1 of IL-2 therapy. However, there were no significant differences in overall Treg expansion or plasma IL-2 levels between responders and non-responders during the initial 12 week treatment period. We therefore asked whether functional or gene expression differences in Treg or effector T cells could distinguish clinical response to IL-2.Methods: In vitro Treg suppression assays were performed using cryopreserved patient samples obtained at study baseline and week 6 of IL-2 therapy. CD4+CD25+CD127- Treg were cultured with CellTrace Violet-labeled CD4+CD25- Tcon in the presence of stimulating antibody-coated beads. Proliferation was measured as CellTrace Violet dilution by flow cytometry after 4 days of incubation. All patient samples were tested against the same healthy donor (HD) Treg and Tcon. Differential gene expression between clinical responders and non-responders was examined by RNA Seq analysis of sorted Treg, Tcon, CD8 and NK cells at study baseline and week 4 of IL-2 therapy.Results: To compare Treg cell function between response groups, we tested the ability of patient Treg to suppress the proliferation of HD Tcon. To determine whether there were qualitative differences in effector T cells between the response groups, we measured suppression of patient Tcon by HD Treg. At study baseline, clinical responders appear to have higher Treg suppressive function although the differences between the response groups were not significant due to small sample size (Figure 1A). Patient Tcon in responders and non-responders were similarly suppressed by HD Treg. At week 6 of IL-2 therapy, Treg from non-responders showed significant improvement in suppressive activity against HD Tcon, whereas there was no significant change in suppressive function of Treg from responders (Figure 1B). Thus, despite the lack of clinical improvement in cGVHD symptoms, non-responder Treg show both numeric increase and functional improvement in response to IL-2 therapy. To identify other cell-intrinsic differences that distinguish clinical response to IL-2, we performed RNA Seq on sorted Treg, Tcon, CD8 and NK cells in 3 responders and 3 non-responders. Differentially expressed genes were identified using the DEseq package. There were very few differentially expressed genes between response groups within the pre-treatment Treg, CD8 and NK cell samples (Figure 2). In contrast, 93 genes (92 upregulated, 1 downregulated) with greater than 4-fold difference in expression levels were identified in pre-treatment Tcon samples between responders and non-responders. The top 10 gene ontogeny terms associated with these differentially expressed genes include cell-cell adhesion and ion transport processes. By week 4 of IL-2 therapy, the gene expression profiles of responder and non-responder cells were very similar. There were no differentially expressed genes within Treg, and only 5 within Tcon.Conclusions: In addition to increasing absolute Treg numbers, daily low-dose IL-2 improves Treg suppressive function in patients who do not exhibit clinical cGVHD improvement. Interestingly, RNA Seq results suggest that the determinants of clinical response do not lie within the Treg. Instead, the greatest differences between response groups were detected in pre-treatment CD4 Tcon, implying that lack of clinical response reflects resistance of CD4 effector cells to suppression mediated by Treg in vivo. Further analysis of differentially expressed Tcon genes may help elucidate the mechanisms of resistance to IL-2 therapy in patients with cGVHD. [Display omitted] [Display omitted] DisclosuresKoreth:LLS: Research Funding; prometheus labs inc: Research Funding; kadmon corp: Membership on an entity’s Board of Directors or advisory committees; amgen inc: Consultancy; millennium pharmaceuticals: Research Funding; takeda pharmaceuticals: Membership on an entity’s Board of Directors or advisory committees. Armand:Otsuka: Research Funding; Roche: Research Funding; BMS: Consultancy, Research Funding; Infinity: Consultancy; Merck & Co., Inc.: Consultancy, Research Funding; Sequenta: Research Funding; Sigma Tau: Research Funding; Tensha: Research Funding. Soiffer:Kiadis: Membership on an entity’s Board of Directors or advisory committees; Juno: Membership on an entity’s Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity’s Board of Directors or advisory committees. Ritz:Kiadis: Membership on an entity’s Board of Directors or advisory committees.

Prophylactic use of low-dose interleukin-2 and the clinical outcomes of hematopoietic stem cell transplantation: A randomized study

ABSTRACTLeukemia relapse and chronic graft-versus-host disease (cGVHD) are still major obstacles of allogeneic hematopoietic stem cell transplantation (allo-HSCT). The numbers and activity of natural killer (NK) and T-regulatory cells can be increased post-transplantation by exposure to interleukin-2 (IL-2). We tested whether administering low-dose IL-2 would decrease leukemia relapse while reducing cGVHD after allotransplantation. This controlled, open-label randomized trial included 90 recipients of allotransplants. Subjects were randomized in a 1:1 ratio to either receive or not receive low-dose IL-2 during the early post-transplantation period. Patients in the IL-2 arm received a subcutaneous injection of low-dose IL-2 (1×106 U/d) on day 60 after allo-HSCT. IL-2 was administered daily for 14 d followed by a 14-d hiatus. The primary endpoint was the cumulative incidence of leukemia relapse (CIR). Three-year CIRs for the IL-2 arm and control arm were 23% (range 16–30%) and 11% (range 6–15%; p = 0.20), respectively. Minimal residual disease-positive (MRD+) tests were more common in the IL-2 arm compared to the control arm (36% [range 29–44%] vs. 15% [range 10–20%], p = 0.03). The cumulative incidence of moderate-to-severe chronic GVHD (cGVHD) was lower in the IL-2 arm compared to the control arm (33% [range 26–39%] vs. 57% [range 49–64%), p = 0.02). Therefore, the 3-y GVHD-free and GVHD progression-free survival (GPFS) rates were significantly higher in the IL-2 arm compared to the control arm (47% [range 39–55%] vs. 31% [range 25–38%], p = 0.048). Blood Tregs, NK cells, and NK-cell cytotoxicity were increased in subjects in the IL-2 arm between 3 mo and 6 mo post-transplantation. Administration of low-dose IL-2 during the immediate post-transplantation period was associated with a higher GPFS but did not decrease the CIR.

Low-Dose IL-2 in the Treatment of Lupus.

Recent extensive research on interleukin-2 (IL-2)/IL-2 receptor (IL-2R) biology has revealed its critical role in the regulation of immune tolerance by influencing regulatory T (Treg) cell functions and survival. Since in vivo low-dose IL-2 administration in humans has been confirmed to be safe and effective in expanding Treg, it is likely that it may be considered for the treatment of several autoimmune diseases including systemic lupus erythematousus (SLE). A recent clinical trial demonstrated the safety and efficacy of low-dose IL-2 treatment on SLE. In SLE, T cells show aberrant function such as deficient IL-2 production and abnormal signaling events. Expansion of Treg by IL-2 represents a specific strategy to control self-tolerance; however, restoration of abnormal immune function and responses should be addressed more carefully in patients with SLE considering the complexity of disease etiology and pathogenesis.

IL-2 Induces Selective Activation of Helios-Positive Regulatory T Cells and CD56bright NK Cells in Vitro and in Patients with Chronic Gvhd Receiving Low-Dose IL-2 Therapy

▪Introduction: CD4+ FoxP3+ CD25+ regulatory T cells (Treg) play a central role in the maintenance of immune tolerance and prevention of chronic graft-versus-host disease (cGVHD) after allogeneic stem cell transplantation (SCT). Treg constitutively express high-affinity interleukin-2 (IL-2) receptors and murine models have established that IL-2 is a critical homeostatic regulator of Treg in vivo. We previously reported that daily administration of low-dose IL-2 in patients with cGVHD induces selective expansion of Treg and NK cells and results in clinical improvement in approximately 50% of patients. However, the mechanisms responsible for these selective effects and the influence of IL-2 therapy on other lymphocytes have not been established due to the limited resolution of traditional cell analytic methods such as flow cytometry.Methods: Single cell mass cytometry (CyTOF) with a panel of 33 markers was used to simultaneously examine the phenotypic and functional effects of low-dose IL-2 on lymphocyte populations in vitro and in vivo. The analytic panel included 22 cell surface markers to identify distinct T, B and NK cell subsets and 11 intracellular markers to measure functional status and activation of specific signaling pathways. viSNE, a cytometry analysis tool, was used to visualize high-dimensional cytometry data on a two-dimensional map.Results: In unstimulated lymphocytes from healthy donors, constitutive expression of CD25 (IL-2Ra) at high levels was restricted to Treg and CD56bright NK cells. Central memory (CM) and effector memory (EM) subsets of conventional CD4 T cells (Tcon) and CM CD8 T cells expressed low levels of CD25. Within the Treg population, the highest expression of CD25 was closely associated with expression of Helios transcription factor. Helios+ Treg also express higher levels of FoxP3, HLA-DR and CD95 and lower levels of BCL2 compared to Helios- Treg. To examine responses to IL-2, we stimulated peripheral blood mononuclear cells (PBMC) from healthy donors with IL-2 for 15 min in vitro (Figure 1). At low IL-2 concentrations (1 to 10 IU/ml), pSTAT5 was preferentially activated in Treg. Notably, pSTAT5 activation was more robust in memory Treg than naïve Treg and in Helios+ Treg than Helios- Treg. In addition, we observed activation of pSTAT5 in CD56bright NK cells at low concentrations of IL-2 (10 IU/ml). Higher IL-2 concentrations (100-1000 IU/ml) were required to activate pSTAT5 in Tcon, CD8 T cells and CD56dim NK cells. At high IL-2 concentrations, pSTAT5 was activated in all Treg, NK, Tcon and CD8 subsets. To examine the response to IL-2 in vivo, we examined PBMC from 14 patients with chronic GVHD receiving daily low-dose IL-2 using the same CyTOF panel of markers. Without additional in vitro stimulation, pSTAT5 expression was increased preferentially in Helios+ Treg. Peak pSTAT5 expression occurred 1 week after starting IL-2 and decreased with continued IL-2 therapy. Similarly, increased expression of FoxP3, CD25, HLA-DR and Ki67 occurred primarily in Helios+ Treg with peak expression at 1 week. At later time points during IL-2 therapy, changes in Treg included increased expression of CD95, CTLA4, PD-1, BIM and BCL2. Although there was no activation of pSTAT5 in CD4 Tcon and CD8 T cells, expression of PD-1 increased in effector memory subsets of Tcon and CD8 T cells 1 week after starting IL-2 therapy. Selective expansion of CD56bright NK cells was also noted, with peak activation at 1 week. No other changes were noted in Tcon, CD8 T cells and B cells. All changes observed during IL-2 therapy returned to baseline levels 4 weeks after treatment was stopped. However, examination of PBMC from 8 patients who received continuous daily low-dose IL-2 therapy for approximately 1 year showed that all of the changes noted above persisted during extended therapy.Conclusion: Comprehensive analysis of T, B and NK cells from healthy donors revealed that low concentrations of IL-2 result in selective activation of Helios+ Treg and CD56bright NK cells. Higher concentrations of IL-2 are required for activation of CD4 Tcon, CD8 T cells and CD56dim NK cells. Identical populations are activated in patients with cGVHD receiving daily low-dose IL-2 and these functional effects persist during extended IL-2 therapy. Although the function of Helios transcription factor is not well defined, Helios expression identifies those Treg most primed to respond to low concentrations of IL-2 in vitro and in vivo. [Display omitted] DisclosuresArmand:Infinity Pharmaceuticals: Consultancy; Merck: Consultancy, Research Funding; Bristol-Myers Squibb: Research Funding. Antin:Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Gentium S.p.A.: Membership on an entity's Board of Directors or advisory committees. Soiffer:Gentium SpA/Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

Host Immune Status Determines the Effects of Therapeutic Interleukin-2 Administration: Enhancement of GVL or Induction of Tolerance?

Interleukin-2 (IL-2) has a central role in immune tolerance thorough maintaining the homeostasis of CD4+CD25+Foxp3+ regulatory T cell (Treg). We recently reported that administration of low-dose IL-2 could preferentially enhance Treg in vivo and suppress clinical manifestations of chronic GVHD after allogeneic hematopoietic stem cell transplantation (NEJM2011). On the other hand, IL-2 is also necessary for the development of cytotoxic T cell function and has been used for the systemic immune therapy to amplify anti-tumor immunity. Thus, IL-2 administration after transplantation can induce the bipolar effects, namely, enhancement of Graft-versus-Leukemia effect (GVL effect) or prevention of Graft-versus-Host Disease (GVHD). For the appropriate therapeutic use of IL-2 for different purpose, we need to elucidate the factors to predict the effect of IL-2, however, the determinants except for the IL-2 dosage has not been characterized. To address this issue, we explored the impact of the immunological reconstitution after transplant on the effect of IL-2 therapy. First, we examined the in vivo reactivity to exogenous IL-2 in different lymphocyte subsets; CD8+ T cells, CD4+ conventional T cells (Tcon) and CD4+ regulatory T cells (Treg). We purified CD62L+ naïve lymphocytes and CD62L- lymphocytes from B6 spleen by cell-sorting and labeled with CFSE, adaptively transferred cells into irradiated B6 host and subcutaneously administrated 5000 IU of IL-2 or control vehicle from day1 to 5. At day6, spleen cells were harvested and the in vivo proliferation of each lymphocyte were evaluated by CFSE dilution assay. Proliferation of Tcons was just limited but both CD8 T cells and Tregs showed vigorous proliferations in response to IL-2. As expected, IL-2 induced the proliferation of CD62L- activated/memory CD8 T cells more than CD62L+ naïve CD8 T cells. In contrast, oppositely to CD8+ subsets, IL-2 induced the proliferation of CD62L+ naïve Tregs more than CD62L- Tregs, suggesting naïve Treg might be “primed” naturally and be ready to respond to IL-2 without antigenic TCR stimulation. Next, we examined the balance between naïve and memory phenotype in each lymphocyte subsets after allogeneic HSCT using murine BMT model. Lethally irradiated B6D2F1 mice were transplanted with 5x106 spleen cells from the CD45.2 B6 mice together with 5x106 TCD-BM from CD45.1 B6 donors. The balance between graft-derived cells (CD45.2) and BM-derived cells (CD45.1) in CD8+ T cell, CD4+ Tcon and Treg were monitored separately at day7, 14, 21,28 and 35. In the early phase of transplant, graft-derived cells showing CD62- were predominant, peaked at day21 and thereafter decreased in each subset. After day28, BM-derived cells generally emerged in each subset. Using this model, we compared the effects of IL-2 administration in the early phase (Day5-12) and in the late phase (Day35-42). Interestingly, IL-2 administration of daily 5000 IU of IL-2 in the early phase resulted in the dominant expansion of circulating CD62-CD44+ effector/memory CD8 T cell without Treg increase (CD8+T; 162 cells vs 75 cells/uL, P<0.05: Treg; 0.42 cells vs 0.48 cells/uL, NS). In contrast, the same dose of IL-2 administration in the late phase resulted in the vigorous increase of CD62+ Treg without CD8 T cell increase (CD8+T; 205 cells vs 185 cells/uL, NS: Treg; 57.1 cells vs 21.0 cells/uL, P<0.05). GVL experiments using 2.5x104 host-type P815 leukemia cells, that were uniformly lethal to the recipients of syngeneic BMT by day 15 after BMT, showed that IL-2 injection from day5 delayed the leukemia relapse (P<0.05), indicating IL-2 enhanced CD8+ CTL might mediated anti-tumor immunity. In conclusion, these data suggests that not only the dose of IL-2 but also host immune status at IL-2 administration is a critical factor to determine the in vivo effect of IL-2 therapy. From the point of view, the adjusted dose and timing of IL-2 infusion based on the detailed immune monitoring might enable to optimize and personalize this cytokine therapy. Our findings provide important information for developing therapeutic strategies to modulate immune cells in vivo and promote well-balanced immune recovery after transplantation. DisclosuresNo relevant conflicts of interest to declare.

Low-Dose IL-2 Reduces Mitochondrial Priming and Increases Bcl2 Expression in CD4 Memory Regulatory T Cells

CD4+CD25+Foxp3+ regulatory T cells (Treg) play a critical role in establishment of immune tolerance and prevention of graft versus host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (HSCT). The recovery and maintenance of Treg after HSCT is dependent on homeostatic factors including the generation of naïve Treg from hematopoietic precursor cells, the proliferation and expansion of mature Treg and the survival of Treg in vivo. We previously reported that Treg in healthy donors are more susceptible to mitochondrial apoptotic priming than conventional T cells (Tcon). Mitochondrial priming is increased after hematopoietic stem cell transplantation in all T cell subsets and particularly in patients with chronic GVHD. Interleukin-2 (IL-2) is a critical homeostatic cytokine that is required for Treg development, expansion, activity and survival. We previously demonstrated that daily administration of low-dose IL-2 resulted in selective expansion of Treg in vivo, clinical improvement of chronic GVHD (Koreth et al. NEJM 2011), increased Treg Bcl2 expression and increased Treg resistance to apoptosis in vitro (Matsuoka et al. SciTM, 2013). These observations led us to undertake a more detailed analysis of apoptotic pathways in functionally and phenotypically distinct Treg subsets including naïve Treg, recent thymic emigrants (RTE) Treg and memory Treg.To examine the mechanisms whereby IL-2 affects susceptibility to apoptosis in each Treg subset, we used a functional flow cytometry-based assay (apoptotic profiling) to measure mitochondrial membrane depolarization in response to a panel of pro-apoptotic BH3 peptides (BIM, BID, BAD, NOXA, PUMA, BMF, HRK). This assay allowed us to compare “priming”, which we define as susceptibility to BH3 peptide-induced mitochondrial membrane depolarization, in naïve, RTE and memory subsets. We also examined cytoplasmic expression of Bcl2 as an additional measure of susceptibility to apoptosis in each subset. In resting blood obtained from healthy donors (n=25), memory Treg were more “primed” than either naïve or RTE subsets when exposed to several BH3 peptides (BID, NOXA, PUMA, BMF and the combination of BAD+NOXA) (Figure 1). Memory Treg were also found to have decreased expression of Bcl2 compared to naïve or RTE subsets. Thus, memory Treg in healthy individuals are more susceptible to apoptosis than other Treg subsets through the mitochondrial pathway (Figure 1).To examine the functional effects of IL-2 on T cell homeostasis, Treg and Tcon were purified by cell sorting and cultured for 5 days with different concentrations of IL-2 (0, 10, 100, 1000 U/ml). Low-concentration IL-2 (10 U/ml) lowered apoptotic priming and increased Bcl2 expression in all Treg subsets. Similar effects were observed in all Tcon subsets, but higher concentrations of IL-2 (≥100 U/ml) were required to decrease apoptotic priming and increase Bcl2 expression in Tcon subsets. The peak effect of IL-2 on Treg priming occurred on day 3 but these effects appeared more rapidly in naïve and RTE Treg subsets than in memory Treg. However, the effect of IL-2 on apoptotic priming persisted for a longer period in memory Treg than in naïve and RTE Treg subsets. The peak effect of IL-2 on Bcl2 expression occurred on day 3 in all Treg subsets. To compare the effect of IL-2 on Treg and Tcon subsets, we examined apoptotic priming and Bcl2 expression after 3 day incubation with different IL-2 concentrations (0.001 - 1000 U/ml). Very low-concentration IL-2 (1 U/ml) lowered priming and increased Bcl2 expression in memory Treg, but higher concentrations of IL-2 (≥10 U/ml) were required to increase Bcl2 expression and decrease priming in naïve and RTE Treg. All Tcon subsets showed a similar pattern of response but higher concentrations of IL-2 (≥100 U/ml) were required.Memory Treg are the predominant regulatory population in patients with cGVHD and the high level of priming in this subset contributes to the inability to maintain adequate numbers of CD4 Treg after HSCT. In healthy donors, low concentrations of IL-2 are sufficient to reduce mitochondrial priming and increase Bcl2 expression in memory Treg. IL-2 has similar effects on Tcon but 10-100 fold higher concentrations of IL-2 are required. These concentration-dependent effects of IL-2 likely contribute to the selective expansion and persistence of Treg in patients with cGVHD receiving daily low-dose IL-2 therapy after HSCT. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Low-Dose IL-2 Induces Bcl2 Expression and Resistance To Apoptosis In CD4 Regulatory T Cells

CD4+CD25+Foxp3+ regulatory T cells (Treg) play an important role in the maintenance of self-tolerance and immune homeostasis. Interleukin-2 (IL-2) is critical for Treg development, expansion, activity and survival. Lack of either IL-2 or IL-2Rα (CD25) results in Treg deficiency and Treg impairment is associated with loss of tolerance, autoimmunity, and chronic GVHD. We previously demonstrated that daily administration of low-dose IL-2 resulted in selective expansion of Treg in vivo and clinical improvement of chronic GVHD (Koreth et al. NEJM 2011). We also reported that low-dose IL-2 enhanced Treg resistance to FAS-mediated apoptosis through the extrinsic pathway (Matsuoka et al. Sci Transl Med 2013). However susceptibility of Treg to apoptosis through the intrinsic pathway has not been examined. To examine the mechanisms whereby IL-2 affects susceptibility of Treg to apoptosis through the intrinsic pathway, we used a functional flow cytometry-based assay (BH3 profiling) to measure mitochondrial membrane depolarization in response to a panel of pro-apoptotic BH3 peptides (BIM, BID, BAD, NOXA, PUMA, BMF, HRK). Mitochondria that are more sensitive to the BH3 peptides are more 'primed' for apoptosis, and more sensitive to pro-apoptotic signaling. This assay allowed us to compare “priming” which we define as susceptibility to BH3 peptide-induced mitochondrial membrane depolarization in Treg and Tcon. We also examined cytoplasmic expression of Bcl-2, BclxL, Mcl1, Bim, XIAP, Ki67 and Staurosporine (STS) induced apoptosis as additional measures of susceptibility to apoptosis and proliferation in each subset. In resting blood obtained from healthy donors (n=25), Treg were more “primed” than Tcon when exposed to several BH3 peptides (Bim, Bad, PUMA and BMF). This BH3 peptide pattern suggests that the anti-apoptotic protein, Bcl2 plays a primary role in the priming of these cells. Analysis of pro- and anti-apoptotic proteins revealed that Treg expressed higher levels Bim and BclxL and lower levels of Bcl2 and XIAP than Tcon, but there was no significant difference in Mcl1. Treg expressed higher levels of Ki67 and were more susceptible to both STS- and FAS-induced apoptosis than Tcon. Thus, Treg in healthy individuals have higher proliferative activity and are more susceptible to apoptosis than Tcon through both mitochondrial and death receptor pathways.To examine the functional effects of IL-2 on T cell homeostasis, Treg and Tcon were purified by cell sorting and cultured with different concentrations of IL-2 (10, 100, 1000 IU/ml) with and without TCR stimulation (1ug/ml anti-CD3 + 1ug/ml anti-CD28). BH3 priming was measured after 3 days and results were compared to cells cultured in media alone. Low-concentration IL-2 (10 U/ml) lowered BH3 priming and increased Bcl2 expression in Treg. Similar effects were observed in Tcon, but higher concentrations of IL-2 (>100 U/ml) were required to increase Bcl2 expression and decrease BH3 priming in Tcon. TCR stimulation alone induced higher BH3 priming in Tcon but had little effect in Treg. The combination of TCR stimulation plus IL-2 increased BH3 priming in both Tcon and Treg. TCR stimulation increased proliferation only in Tcon, but the TCR + IL-2 combination increased proliferation in both Tcon and Treg. Thus, IL-2 alone reduced BH3 priming in unstimulated cells but the TCR + IL-2 combination induced higher proliferation and higher BH3 priming. STS-induced apoptosis assays revealed that low-dose IL-2 reduced susceptibility to apoptosis only in Treg. IL-2 did not change the expression of BclxL, Mcl1, and Bim but XIAP expression was increased in both Treg and Tcon. To confirm the effects of low-dose IL-2 on Treg priming were mediated by Bcl2, we examined the effect of ABT-199, a selective Bcl2 inhibitor on BH3 priming in Treg and Tcon. As shown in Figure 1, ABT-199 enhanced priming and spontaneous apoptosis of both Treg and Tcon. IL-2 had no effect on ABT-199-induced priming or apoptosis in Tcon. In contrast, IL-2 reversed the effects of ABT-199 on Treg priming and apoptosis providing further evidence that the inhibition of intrinsic pathway apoptosis mediated by IL-2 in Treg is dependent on Bcl2. [Display omitted] Disclosures:Letai:AbbVie: Consultancy; Dana-Farber Cancer Institute: Patents & Royalties.

Limitations of IL-2 and Rapamycin in Immunotherapy of Type 1 Diabetes

Administration of low-dose interleukin-2 (IL-2) alone or combined with rapamycin (RAPA) prevents hyperglycemia in NOD mice. Also, low-dose IL-2 cures recent-onset type 1 diabetes (T1D) in NOD mice, partially by boosting pancreatic regulatory T cells (Treg cells). These approaches are currently being evaluated in humans. Our objective was to study the effect of higher IL-2 doses (250,000–500,000 IU daily) as well as low-dose IL-2 (25,000 IU daily) and RAPA (1 mg/kg daily) (RAPA/IL-2) combination. We show that, despite further boosting of Treg cells, high doses of IL-2 rapidly precipitated T1D in prediabetic female and male mice and increased myeloid cells in the pancreas. Also, we observed that RAPA counteracted IL-2 effects on Treg cells, failed to control IL-2–boosted NK cells, and broke IL-2–induced tolerance in a reversible way. Notably, the RAPA/IL-2 combination failure to cure T1D was associated with an unexpected deleterious effect on glucose homeostasis at multiple levels, including β-cell division, glucose tolerance, and liver glucose metabolism. Our data help to understand the therapeutic limitations of IL-2 alone or RAPA/IL-2 combination and could lead to the design of improved therapies for T1D.

Effects of cyclophosphamide and IL-2 on regulatory CD4+ T cell frequency and function in melanoma patients vaccinated with HLA-class I peptides: impact on the antigen-specific T cell response

The frequency and function of regulatory T cells (Tregs) were studied in stage II–III melanoma patients who were enrolled in a phase II randomized trial of vaccination with HLA-A*0201-modified tumor peptides versus observation. The vaccinated patients received low-dose cyclophosphamide (CTX) and low-dose interleukin-2 (IL-2). Tregs were analyzed in the lymph nodes (LNs) of stage III patients who were undergoing complete lymph node dissection and in peripheral blood mononuclear cells (PBMCs) collected before vaccination and at different time points during the vaccination period. The LNs of the vaccinated patients, which were surgically removed after two rounds of vaccination and one dose of CTX, displayed a low frequency of Tregs and a less immunosuppressive environment compared with those of the untreated patients. The accurate time-course analysis of the PBMCs of patients enrolled in the vaccination arm indicated a limited and transient modulation in the frequencies of Tregs in PBMCs collected after low-dose CTX administration and a strong Treg boost in those PBMCs collected after low-dose IL-2 administration. However, a fraction of the IL-2-boosted Tregs was functionally modulated to a Th-1-like phenotype in the vaccinated patients. Moreover, low-dose IL-2 promoted the concomitant expansion of conventional activated CD4+ T cells. Despite the amplification of Tregs, IL-2 administration maintained or further increased the number of antigen-specific CD8+ T cells that were induced by vaccination as demonstrated by the ex vivo human leukocyte antigen-multimer staining and IFN-γ ELISpot assays. Our study suggests that the use of CTX as a Treg modulator should be revised in terms of the administration schedule and of patients who may benefit from this drug treatment. Despite the Treg expansion that was observed in this study, low-dose IL-2 is not detrimental to the functional activities of vaccine-primed CD8+ T cell effectors when used in the inflammatory environment of vaccination.Electronic supplementary materialThe online version of this article (doi:10.1007/s00262-013-1397-7) contains supplementary material, which is available to authorized users.

Apoptotic Pathways of Human Regulatory T Cells in Chronic Graft Versus Host Disease (cGVHD) After Allogeneic Hematopoietic Stem Cell Transplantation (HSCT)

Abstract 225CD4+CD25+Foxp3+ regulatory T cells (Treg) are known to play a central role in the maintenance of self-tolerance and immune homeostasis. After allogeneic stem cell transplantation, impaired recovery of Treg is associated with the development of cGVHD. Interleukin-2 (IL-2) is a critical regulator of Treg development, expansion and survival and lack of IL-2 results in Treg deficiency. In patients with cGVHD, we previously demonstrated that Treg proliferate at high levels but this subset is also highly susceptible to apoptosis leading to inadequate Treg numbers (Matsuoka et al. JCI 2010). We also reported that low-dose IL-2 administration resulted in selective expansion of Treg in vivo and clinical improvement of cGVHD (Koreth et al. NEJM 2011). To identify mechanisms responsible for increased Treg susceptibility to apoptosis in cGVHD we used a new flow cytometry-based assay to measure mitochondrial membrane depolarization in response to a panel of pro-apoptotic BH3 peptides (BIM, BID, BAD, NOXA, PUMA, BMF, HRK). This assessment allowed us to compare BH3 peptide-induced mitochondrial membrane depolarization (“priming”) in different T cell subsets, including CD4 Treg, conventional CD4 T cells (CD4 Tcon), and CD8 T cells. Expression of Bcl-2, CD95 and Ki67 were also studied in each T cell subset. We studied peripheral blood samples from 36 patients with hematologic malignancies (median age 59 yr) who are > 2 years post HSCT (27 patients with cGVHD and 9 patients without cGVHD) and 15 patients who received daily subcutaneous IL-2 for 8 weeks for treatment of steroid-refractory cGvHD. Severity of cGVHD was classified according to NIH criteria.In patients without cGVHD, BH3 priming was similar in all 3 T cell subsets (CD4 Treg, CD4 Tcon and CD8). In patients with cGVHD, CD4 Treg were more primed than CD4 Tcon when challenged with BIM, BAD, PUMA, BMF and the combination of BAD + NOXA peptides (p<0.01 – 0.0001). Treg were more primed than CD8 T cells when challenged with PUMA peptide (p<0.0001), but priming in Treg and CD8 T cells was similar for other BH3 peptides in patients with cGVHD. We also compared BH3 priming of each T cell subset in patients with different grades of cGVHD. When challenged with BH3 peptides, Treg, Tcon and CD8 T cells were less primed in patients with severe cGVHD. In patients with cGVHD, Treg expressed higher levels of Ki-67, higher levels of CD95 and lower levels of Bcl-2 than Tcon. Expression of CD95 did not vary with severity of GVHD in any T cell subset, but expression of Bcl-2 was significantly increased in all subsets in patients with severe cGVHD. Increased BH3 priming and high expression of CD95 indicate that Treg are more susceptible to apoptosis than Tcon in cGVHD. However, both Treg and Tcon become less primed and Bcl-2 levels increase in severe cGVHD suggesting that these cells are less susceptible to mitochondrial pathway apoptosis. Since the total number of Treg and Tcon are significantly reduced in patients with cGVHD, these findings suggest that the remaining circulating cells are relatively resistant to mitochondrial pathway apoptosis. CD95 expression in Treg remains high indicating no change in death receptor pathway apoptosis. Daily treatment with low-dose IL-2 for 8 weeks selectively expands Treg in vivo in patients with severe cGVHD. As the number of Treg increase, BH3 profiling shows that these cells gradually become more primed and therefore more susceptible to mitochondrial pathway apoptosis. Taken together, these studies help define the complex and distinct pathways that regulate survival in different T cell subsets and changes in these pathways that occur in patients with chronic GVHD. These pathways play important roles in the maintenance of T cell homeostasis and targeting these complex pathways can provide new opportunities to promote immune tolerance after allogeneic HSCT. Disclosures:No relevant conflicts of interest to declare.

IL-2 reverses established type 1 diabetes in NOD mice by a local effect on pancreatic regulatory T cells

Regulatory T cells (T reg cells) play a major role in controlling the pathogenic autoimmune process in type 1 diabetes (T1D). Interleukin 2 (IL-2), a cytokine which promotes T reg cell survival and function, may thus have therapeutic efficacy in T1D. We show that 5 d of low-dose IL-2 administration starting at the time of T1D onset can reverse established disease in NOD (nonobese diabetic) mice, with long-lasting effects. Low-dose IL-2 increases the number of T reg cells in the pancreas and induces expression of T reg cell–associated proteins including Foxp3, CD25, CTLA-4, ICOS (inducible T cell costimulator), and GITR (glucocorticoid-induced TNF receptor) in these cells. Treatment also suppresses interferon γ production by pancreas-infiltrating T cells. Transcriptome analyses show that low-dose IL-2 exerts much greater influence on gene expression of T reg cells than effector T cells (T eff cells), suggesting that nonspecific activation of pathogenic T eff cells is less likely. We provide the first preclinical data showing that low-dose IL-2 can reverse established T1D, suggesting that this treatment merits evaluation in patients with T1D.

Central Role of Defective Interleukin-2 Production in the Triggering of Islet Autoimmune Destruction

The dynamics of CD4(+) effector T cells (Teff cells) and CD4(+)Foxp3(+) regulatory T cells (Treg cells) during diabetes progression in nonobese diabetic mice was investigated to determine whether an imbalance of Treg cells and Teff cells contributes to the development of type 1 diabetes. Our results demonstrated a progressive decrease in the Treg cell:Teff cell ratio in inflamed islets but not in pancreatic lymph nodes. Intra-islet Treg cells expressed reduced amounts of CD25 and Bcl-2, suggesting that their decline was due to increased apoptosis. Additionally, administration of low-dose interleukin-2 (IL-2) promoted Treg cell survival and protected mice from developing diabetes. Together, these results suggest intra-islet Treg cell dysfunction secondary to defective IL-2 production is a root cause of the progressive breakdown of self-tolerance and the development of diabetes in nonobese diabetic mice.

Perioperative adjuvant biotherapy in high-risk resected cutaneous melanoma: the results of 5 years of follow-up

A feasibility study was conducted to establish the safety and, to some extent, the effectiveness of a new approach of perioperative adjuvant biotherapy in patients with resected cutaneous melanoma. The candidates for this study included patients with primary cutaneous melanoma greater than 1 mm deep, those with resectable regional lymph node (LN) metastases and patients with resectable distant metastases. Interleukin-2 was administered 1 week before definitive surgery as 22 million IU, and again 1 week after the surgery. This was followed by interferon alpha-2b, 10 million IU three times a week for 4 weeks. Fifty-six patients were studied. The program was well tolerated with low, mainly symptomatic, grade I-II toxicity, occasionally with grade III toxicity. Patients' compliance was good. The 5-year survival data were expressed by Kaplan-Meier analysis, and compared with matched historical controls by the log-rank method. The results suggested an improvement in disease-free survival (P=0.021) and a disease-specific overall survival (P=0.05), but not in overall survival, owing to all causes of death (P=0.089). The consequent administration of low-dose interleukin-2 and interferon, initiated preoperatively on outpatient bases, resulted in several constitutional symptoms that were self-limiting and did not delay surgery. No surgical complications related to this approach were observed. This program was well tolerated in all age groups, and the results suggested some survival benefits when compared with matched historical controls.

Chemo-immunotherapy regimen with gemcitabine + FOLFOX 4 (GOLF) followed by subcutaneous (sc) granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-2 (IL-2). Results from a multicenter phase II trial in colon carcinoma patients

3587 We have recently described a poly-chemotherapy (GOLF) regimen that: A) is active in colon carcinoma patients as second line of therapy; B) induces high levels of necrosis and apoptosis in colon cancer cells; C) up-regulates the expression and release of heat shock proteins (HSP)-70 and -90 and tumour-associated antigens (2,3); and D) down-regulates tumour cell resistance to the death signals of cytotoxic-T-lymphocytes.These effects represented the rationale for projecting GOLFIG regimen. Here we describe the results of a multi-center translational phase II trial designed to evaluate the toxicity, anti-tumour activity of a novel regimen designated as GOLFIG-1, composed by the GOLF poly-chemotherapy followed by the subcutaneous (sc.) administration of GM-CSF and low-dose IL-2 in colorectal carcinoma patients. The study involved 37 patients (21M and 16F, mean age 62.5 years), 24 of whom had received a previous line of treatment, and 24 had liver involvement. All the patients received biweekly chemotherapy with gemcitabine (1g/m2, day 1 and 15), oxaliplatin (85 mg/m2, day 2 and 16), levo-folinic acid (100 mg /m2, day 1, 2, 15, 16) and 5-Fluorouracil (400 mg/m2 as a bolus, and 800 mg/m2 as 24 hour infusion, days 1, 2, 15, 16). These patients also received sc GM-CSF (100 μg, day 3 to 8) followed by sc IL-2 (0.5 X 106 IUs twice a day from day 9 to 14 and from 17 to 29). The treatment was well tolerated and very active in colon carcinoma patients, with high objective response (64.9%) and disease control rates (97.3%), with an average time to progression of 12.94 months (CI 95%: 9.98–15.91). An immunological study confirmed the immunological response to colon carcinoma antigen, a significant reduction in suppressive regulatory T lymphocytes (CD4+CD25+T-reg) and a significant reduction of VEGF levels reported in a previous study. In conclusion, these results suggest that the GOLFIG regimen exerts strong immunological and anti-tumour activity in colorectal cancer patients. A randomized phase III trials aimed to compare the efficacy of GOLFIG-1 with FOLFOX-4 regimen in patients with advanced colorectal carcinoma is presently ongoing. No significant financial relationships to disclose.