Tcon Ratio Research Articles

Allogeneic cord blood regulatory T cells can resolve lung inflammation

Background aimsCD4+CD25+CD127lo regulatory T cells (Tregs) are responsible for maintaining immune homeostasis. Tregs can be rendered defective and deficient as a result of the immune imbalance seen in lung injury, and such dysfunction can play a major role in continued tissue inflammation. The authors hypothesized that adoptive therapy with healthy allogeneic umbilical cord blood (UCB)-derived Tregs may be able to resolve inflammation. ResultsEx vivo-expanded UCB Tregs exhibited a unique phenotype with co-expression of CD45RA+CD45RO+ >80% and lung homing markers, including CD49d. UCB Tregs did not turn pathogenic when exposed to IL-6. Co-culture with increasing doses of dexamethasone led to a synergistic increase in UCB Treg-induced apoptosis of conventional T cells (Tcons), which translated into significantly higher suppression of proliferating Tcons, especially at a lower Treg:Tcon ratio. Multiple injections of UCB Tregs led to their preferential accumulation in lung tissue in an immune injury xenogenic model. A significant decrease in lung resident cytotoxic CD8+ T cells (P = 0.0218) correlated with a sustained decrease in their systemic distribution compared with controls (P < 0.0001) (n = 7 per arm) as well as a decrease in circulating human soluble CD40 ligand level (P = 0.031). Tissue architecture was preserved in the treatment arm, and a significant decrease in CD3+ and CD8+ burden was evident in immunohistochemistry analysis. ConclusionsUCB Treg adoptive therapy is a promising therapeutic strategy for treatment of lung injury.

Extra-Corporeal Photopheresis Plus Low-Dose Interleukin-2 for Steroid-Refractory Chronic Graft-Vs.-Host Disease: Efficacy, Safety and Immune Correlates

CD4+CD25+FOXP3+ regulatory T cells (Treg) are dominant suppressors of innate and adaptive immune effector cells that enhance tolerance. Treg augmentation can control immune activation underlying chronic graft-versus-host disease (cGVHD). In prior studies, daily subcutaneous (SC) low-dose interleukin-2 (IL-2) therapy for 8-12 weeks induced preferential Treg expansion in-vivo, with objective clinical responses in over half of evaluable participants (NEJM 2011, Blood 2016). Alternative immunomodulatory approaches like extra-corporeal photopheresis (ECP) also have efficacy in cGVHD and may function via Treg augmentation. Combining ECP plus IL-2 could offer therapeutic synergy.We report on a prospective single arm phase 2 trial of twice-weekly ECP (weeks 1-16) plus sequential low-dose IL-2 (1x106 IU/m2/d SC, weeks 9-16) for treatment of steroid-refractory cGVHD. The study enrolled 25 participants (48% male, 88% HLA-matched, 100% PBSC grafts, 52% prior aGVHD). Median participant age was 62 years (range, 34-76). Median time from HSCT and from cGVHD onset to start of treatment was 919 days (range, 177-2842) and 524 days (range, 58-2697) respectively. Participants had a median of 3 cGVHD organ sites (range, 2-5). The median follow-up time in survivors is 16 months (range, 2.1-26).ECP plus IL-2 was well tolerated: 1 participant withdrew due to IL-2 adverse effects (AE) (myalgia, fatigue), and 3 were additionally unevaluable (1 cardiac arrest; 1 parainfluenza infection, 1 still undergoing treatment). None required IL-2 dose reduction. There were four grade 3 AEs during the 16-week study (1 participant had dehydration/hypotension/syncope, possibly IL-2-related;1 had ECP-related anemia). None experienced relapse. At week 16, cGVHD objective partial responses (PR) were documented in 14 of 21 evaluable participants (67%). None had cGVHD progression. Response sites included skin (n=9/18), joint/fascia/muscle (n=7/16), liver (n=1/1), lung (n=2/9), and GI tract (n=3/5). 18 participants with clinical benefit (PR, and stable disease with minor response) proceeded to extended ECP plus IL-2 therapy.Immunologically, 8 weeks of ECP alone did not induce significant changes in buffy coat WBC or absolute lymphocyte count (ALC). Peripheral blood CD3 T cell counts/µL fell, with a median CD8 and CD4 conventional T cell (Tcon) decrement of -109 (Q1, Q3: -220, -13; p<0.001) and -68 (Q1, Q3: -244, -19; p=0.023), respectively, at week 8 (Figure 1A). There was an early fall in Treg cell counts/µL, with a median decrement of -4 (Q1, Q3: -9, 0; p=0.033) at week 4, without change in NK or B cell counts or Treg:Tcon ratio. The addition of low-dose IL-2 thereafter induced a >5-fold increase in median Treg count/µL (p<0.001), from a baseline of 9 (Q1, Q3: 5, 17) at week 8 to a peak of 72 (Q1, Q3: 22, 186) at week 10, plateauing at 50 (Q1, Q3: 33, 71) at week 16 (Figure 1A). No significant change in CD4 Tcon or B cell count was noted. NK cell count increased 1.7-fold (p=0.009). The median Treg:Tcon ratio increased 4-fold (p<0.001): from a baseline of 0.06 (Q1, Q3: 0.03, 0.08) at week 8 to a peak of 0.26 (Q1, Q3: 0.12, 0.42) at week 12, and remained elevated at 0.17 (Q1, Q3: 0.14, 0.34) at week 16 (Figure 1B). Treg count and Treg:Tcon ratio remained elevated in responders during extended ECP plus IL-2 therapy.Immunologically, clinical responders had a higher median Treg count/µL vs. non-responders after 1 and 6 weeks of IL-2 therapy (21 vs. 11, p<0.001; 73 vs. 29, p=0.016 respectively). Treg:Tcon ratio at week 2 and 6 after IL-2 therapy was also higher in responders (0.29 vs. 0.15, p=0.038; 0.28 vs. 0.13, p=0.016 respectively). Applying our previous cutoffs (Blood 2016), Treg:Tcon ratio ≥0.2 after 1 week of IL-2 was also predictive of clinical response (p=0.05).In summary, in steroid-refractory cGVHD, ECP was associated with a fall in peripheral CD8, CD4 Tcon, and Treg counts. The addition of low-dose IL-2 thereafter induced profound Treg and NK cell augmentation. Clinical responses (PR) to combination ECP plus IL-2 therapy were documented in 67% of the evaluable participants. Those with early rise in Treg count and Treg:Tcon ratio after the addition of IL-2 were likelier to benefit. However, the overall clinical efficacy of ECP plus IL-2 did not appear markedly higher than low-dose IL-2 alone. [Display omitted] DisclosuresKoreth:Prometheus Labs: Research Funding; Kadmon Corp: Membership on an entity's Board of Directors or advisory committees; Millennium Pharmaceuticals: Research Funding; Takeda Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Amgen Inc.: Consultancy. Armand:Bristol-Myers Squibb: Consultancy, Research Funding; Merck & Co., Inc.: Consultancy, Research Funding; Sequenta/Adaptive: Research Funding; Sigma Tau: Research Funding; Tensha: Research Funding; Affimed: Research Funding; Roche: Research Funding; Pfizer: Consultancy, Research Funding; Genmab: Consultancy; Infinity: Consultancy; Otsuka: Research Funding. Antin:Gentium SpA/Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

Individual Patient Dose-Escalated Low-Dose Interleukin-2 for Steroid-Refractory Chronic Graft-Vs.-Host Disease in Children and Adults: Safety, Efficacy and Immune Correlates

CD4+CD25+FoxP3+ regulatory T cells (Treg) are broadly suppressive and play a central role in the prevention of chronic graft-versus-host disease (cGVHD) after allogeneic hematopoietic cell transplantation (HCT). Daily subcutaneous (SC) low-dose interleukin-2 (IL-2) at a fixed dose of 1x106 IU/m2/day in adult patients with refractory cGVHD induces selective Treg expansion and leads to clinical responses in 50-60% of evaluable participants (NEJM 2011, Blood 2016). During daily fixed-dose IL-2 therapy, plasma IL-2 levels rose rapidly by week 1, but subsequently decreased over time. The lower IL-2 levels were associated with a decline in Treg proliferation despite daily IL-2 administration (Sci Transl Med 2013), possibly due to IL-2 sequestration via binding to constitutively-expressed high affinity IL-2 receptors on Treg. We reasoned that IL-2 dose escalation at the time of anticipated fall in plasma IL-2 levels would avoid tachyphylaxis and maximize IL-2 induced Treg numbers in vivo.We conducted a phase 1 trial of individual patient IL-2 dose escalation over 8 weeks in adult and pediatric patients with steroid-refractory cGVHD. Daily SC IL-2 was initiated at 0.67x106 and 0.33x106 IU/m2/day in the adult and pediatric cohorts, respectively. Each participant had dose escalations at weeks 2 and 4 to a maximum dose of 2x106 IU/m2/day in adults and 1x106 IU/m2/day in children. We studied 18 HCT recipients (10 adult: 100% HLA-matched, 100% PBSC grafts, 40% prior aGVHD; 8 pediatric: 87.5% HLA-matched, 87.5% BM grafts, 28.6% prior aGVHD). Median age was 57 years (range, 33-71) in the adult cohort and 12 years (range, 5-22) in the pediatric cohort. The median time from HCT and from cGVHD onset to start of IL-2 treatment was 3 years (range, 1-12.1) and 597 days (range, 234-4336) respectively. Participants had a median of 2 cGVHD organ sites (range, 1-5) and 3 prior cGVHD therapies (range, 1-4) at enrollment. The cohorts did not differ in terms of time since cGVHD onset and number of failed therapies prior to enrollment.Individually dose-escalated IL-2 was well tolerated in children with 1 participant requiring dose reduction for electrolyte imbalances. In the adult cohort, 1 participant withdrew due to cGVHD progression, 1 required dose reduction for injection site reactions and 2 were unevaluable (1 IL-2 hold >1 month, 1 non-compliance). Seven grade 3 AEs were reported for six participants (electrolyte imbalance, vomiting, infection, increased ALT and soft tissue neoplasm); none relapsed. At week 8, objective cGVHD responses (PR) were seen in 7 of 8 pediatric participants (88%), but in only 1 of 8 evaluable adult participants (13%). cGVHD response sites included skin (n=4), joint/fascia/muscle (n=1), lung (n=4), and GI tract (n=2). Eight pediatric and 4 adult participants with clinical benefit (PR or SD with minor response) continued extended duration IL-2 therapy up to 120 weeks.Plasma IL-2 levels were higher in adults at weeks 1 and 2, but immune responses were similar between the 2 cohorts. Dose escalated IL-2 induced a >10-fold increase in median Treg count/µL that peaked at week 6. There were no significant changes in CD4 Tcon, CD8 T cell or B cell count. NK cell count increased >5-fold by week 8. The median Treg:Tcon ratio increased 6-fold from a baseline of 0.05 (Q1-Q3, 0.024-0.114) to a week 5 peak of 0.312 (Q1-Q3, 0.203-0.37). Compared with fixed daily dosing of IL-2 (Blood 2016), the individually dose escalated IL-2 schedule had a slower initial rise in Treg number and Treg:Tcon ratio (Figure 1). This likely reflects the lower starting doses in this study. The maximum IL-2 dose in the adults was twice the previously defined MTD but did not improve Treg expansion. Despite lower IL-2 doses, pediatric patients had a superior clinical response and a trend toward a better immunologic response compared with the adults.We demonstrate for the first time that low-dose IL-2 is safe in children with advanced steroid-refractory cGVHD and results in a high clinical response rate. A maximum dose of 1x106 IU/m2/day was well tolerated by pediatric patients in the extended duration phase and durably maintained Treg numbers above baseline. Five pediatric patients that continued IL-2 therapy for >20 weeks have reduced or discontinued steroid therapy. In adults, intrapatient dose escalation above fixed-dose MTD was not clinically tolerated and did not result in greater Treg expansion or clinical improvement relative to fixed daily dosing. [Display omitted] DisclosuresNikiforow:Kite Therapeutics: Membership on an entity's Board of Directors or advisory committees. Armand:Sequenta/Adaptive: Research Funding; Affimed: Research Funding; Roche: Research Funding; Merck & Co., Inc.: Consultancy, Research Funding; Infinity: Consultancy; Genmab: Consultancy; Bristol-Myers Squibb: Consultancy, Research Funding; Otsuka: Research Funding; Pfizer: Consultancy, Research Funding; Tensha: Research Funding; Sigma Tau: Research Funding. Antin:Gentium SpA/Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Koreth:Prometheus Labs: Research Funding; Amgen, Inc: Consultancy.

Regulatory T Cells for Acute Graft Versus Host Disease Prevention

BACKGROUND GVHD remains a major cause of morbidity and mortality after allo HSCT. The past few years have shown a number of successful advances in cellular therapies for the treatment of hematologic malignancies. It is logical to expand the use of these therapies earlier, in strategies aimed at prevention. Several studies showed that regulatory T cells (Treg) can reduce the incidence of aGVHD. METHODS Studies were identified using electronic databases (Medline, Embase, and Cochrane). Preclinical studies were excluded. Data analysis was performed using GraphPad Software 8.4.2. RESULTS 160 patients were identified from 7 clinical studies. 85% of the patients had acute leukemia and were in complete remission at the time of transplant. Tregs were isolated from either UCB or PB. Tregs were expanded ex-vivo using IL-2, CD3/CD28 ab coated beads and K562 cells in three studies (n=40). 1.Ex-vivo expanded Treg: In a phase I study (Brunstein et al) of UCB-derived Tregs in 23 pts undergoing double umbilical cord transplantation, Tregs were cultured with antiCD3/anti CD28 and IL-2. Ex-vivo expanded Tregs were given on day+1 and day+15. Eighteen pts received Treg cells at dose of &amp;gt;3x106 cells/kg. Grade II-IV aGvHD was seen in 39% of pts who received ≥3x106 cells/kg vs 43% in historical controls. In this study, CsA+MMF or sirolimus+MMF were used for GvHD prophylaxis. In a follow up study of 11 patients, Tregs were markedly expanded using K562 cells. Higher Treg doses were achieved up to 100x106cells/kg and the median proportion of CD4+CD127-FOXP3+ within the infused products was 87%. Grade II-IV aGVHD was 9% at 100 days vs 45% in control group. Treg cells were not detected in the peripheral blood after 14 days of infusion. In a phase II study (Kellner et al; Zeng et al), three of six pts undergoing PB MUD received ex-vivo expanded UCB-derived Tregs 1x106/kg at day-1. All developed aGVHD due to the high donor Tcon cells in the graft with donor Tcon:UCB Treg ratio as high as 356:1. Sixth patient was given 1x107 cells/kg Tregs in the presence of anti CD3/28 microbeads and IL-2 with Treg:Tcon ratio of 1:40. No aGVHD was observed in this patient compared to the others who received Treg at the dose of 1x106cells/kg. 2.Unexpanded Treg: In a study (Di Ianni et al) involving 26 high risk leukemia pts undergoing haplo-transplant with TBI based conditioning, 21 pts were infused with donor Tregs at a dose of 2x106 cells/kg and five pts were infused with Treg at a dose of 4x106 cells/kg on day -4, followed by Tcon infusion ranging from 0.5x106 to 2x106 cells/kg on day 0. No post-transplant immune suppression was given. Grade II-IV aGVHD was observed in 2 out of 26 patients who received 4x106cells/kg Tregs and 2x 106cells/kg Tcons. No GVHD was observed in pts who received 2x106cells/kg Tregs followed by 0.5-1x106 cells/kg Tcons. A phase II study by the same group (Martelli et al) reported on an additional 17 pts who received Tregs (2x106cells/kg) at day-4 co-infused with Tcon (1x106cells/kg) at day 0. Six of 41 (15%) evaluable pts developed grade II-IV aGVHD (vs 11% in historical cohort) with two pts requiring a short course of immunosuppression. Incidence of relapse was lower in Treg group than historical controls (5% vs 21%). In a phase I/II study (Meyer et al), 12 pts with hematological malignancies who underwent HLA-matched transplantation received purified Tregs (1-3x106cells/kg) on day0 followed by Tcon (1-3x106cells/kg) on day+2. GVHD was seen in 80% of pts with 50% being grade III in pts who received frozen Tregs. Upon revising protocol, 7 pts were given fresh Treg. The patients also received either tacrolimus or sirolimus for immunosuppression. None of 7 pts in revised cohort developed GVHD. Recent study applied hyper-fractionated TBI to the conditioning regimen in 12 pts out of 40 to further reduce leukemia relapse in Treg/Tcon based haplo-HSCT. Treg was administered at the dose of 2x106cells/kg and Tcon at 1x106cells/kg. No post-transplant GvHD prophylaxis was given. Grade II-IV aGVHD was observed in 12 pts. CONCLUSION These studies suggest that Treg therapy may represent an effective prevention strategy against acute GvHD (figure 1). Stimulated Treg expansion strategies and higher doses of Treg appear to be associated with lower incidence of aGVHD. Treg infusion was well-tolerated without serious adverse events. Large prospective clinical trials are warranted in order to establish the optimal infusion dose, optimal expansion strategies and dosing schedule. Disclosures No relevant conflicts of interest to declare.

NK Cell Impairment after Haploidentical Allogeneic Hematopoietic Cell Transplant in Patients Receiving Post-Transplant Cyclophosphamide (PTCy)

Haploidentical hematopoietic cell transplantation (haplo-HCT) with post-transplant cyclophosphamide (PTCy) as graft versus host disease (GVHD) prophylaxis is frequently used for patients who do not have HLA-identical donors. However, the effect of PTCy on immune reconstitution (IR) has not been studied extensively in large prospective cohorts, despite a high incidence of early viral infections. We quantified IR in 60 patients after haplo-HCT with PTCy, mycophenolate mofetil and tacrolimus (TAC) and compared results to 34 patients with 8/8 HLA matched related or unrelated donors (MD) receiving TAC and methotrexate for GVHD prophylaxis. Both groups received reduced intensity conditioning for hematologic malignancies. Samples were prospectively collected at 1, 2, 3, 6, 9, 12, 18, 24 and 30 months after HCT and analyzed using multi-color flow cytometry to characterize distinct lymphocyte populations. Peripheral blood NK cells 1 month after PTCy were characterized using CyTOF and a panel of 35 metal-labeled monoclonal antibodies and compared to age-matched healthy donor NK cells. Recovery of all T cell subsets (CD4Tcon, CD4Treg, CD8) was significantly reduced in the PTCy cohort compared to MD early after HCT (Figure 1A, B, C). Despite delayed CD4Treg recovery, the Treg:Tcon ratio was significantly higher early after HCT in the PTCY cohort due to relatively deeper depression in CD4Tcon (0.18 vs 0.06, p<0.0001 at 1 month, 0.12 vs 0.06, p=0.0004 at 3 months, Figure 1D). Absolute NK cells were lower at 1 month after PTCy (Figure 1E), entirely due to reduced numbers of CD56^dimCD16⁺ NK cells (Figure 1G). Subsequently, CD56^dim NK cells remained significantly lower in the PTCy group until 3 months after HCT. In contrast, recovery of CD56^brightCD16⁻ NK cells was significantly increased in the PTCy cohort at 2, 3 and 6 months after HCT (p<0.01. Figure 1F). The distribution of NK subsets 1 month post-HCT was also significantly different between two cohorts due to a higher frequency of immature NK cells in the PTCY cohort (Figure 1H). CyTOF analysis performed 1 month after haplo-HCT showed that NK cells expressed higher level of activating receptor NKp46, CD95, granzyme-B and inhibitory receptor NKG2A and lower expression of CD57 compared to HD (Figure 2). Functional assays showed that PTCy NK cells displayed reduced degranulation and release of IFNγ after activation compared to HD. These functions were rescued after priming with low dose IL-15 in vitro. In summary, the effect of PTCy on IR was most pronounced at 1 month with significantly delayed recovery of CD3, CD4Tcon, CD4Treg, CD8 and CD56^dim NK cells. After PTCy, NK cells display an immature phenotype and functional impairment that is at least partly corrected by priming with low dose IL-15. Impairment of NK and T cell recovery early after PTCy-based HCT underscores the need to adopt novel strategies to overcome immune defects induced by this platform.

Activation of Regulatory T Cells with IL-2 and TNF-α to Promote Homing to the Gut and to Improve Acute GvHD Prevention

INTRODUCTION Adoptive immunotherapy with donor conventional T cells (Tcons) and CD4+CD25+FOXP3+regulatory T cells (Tregs) is used to enhance immune reconstitution in clinical trials of haploidentical transplantation. Donor Tregs are infused to prevent adverse alloreactions mediated by Tcons against normal tissues, which cause GvHD, in the absence of any post transplant immune suppression. This strategy proved to be effective in preventing leukemia relapse and chronic GvHD, allowing for ~70% event-free survival in patients with acute leukemia (Pierini et al. submitted). However, acute GvHD occurs in ~30% of the patients. We are investigating if ex-vivo stimulation of Tregs with a combination of low-dose IL-2 and TNF-α before adoptive transfer could improve acute GvHD prevention. Tregs express higher levels of the TNF receptor 2 (TNFR2) compared with Tcons and TNFR2 expression defines a Treg subpopulation with maximal suppressive capacity. Recent studies showed that activation of TNFR2 signaling in mouse Tregs reduced GvHD lethality in mouse models. METHODS Human CD4+or CD4+CD25+T cells were incubated for 60 hours with IL-2 (10 UI/ml) with or without TNF-α (20 ng/ml). Treg proliferation in the presence of allogeneic irradiated peripheral blood mononuclear cells (PBMC), and Treg-mediated suppression of Tcon proliferation were evaluated by CFSE assay. Colon biopsies from patients with suspected GvHD were stained with anti-CD3 and anti-FOXP3 antibodies. RESULTS We developed a translatable in vitro system for improving human Treg function by adding a short term stimulation with low-dose IL-2 and TNF-α (IL-2/TNF) in the selection process. CD4+T cells were primed with IL-2/TNF before CD25 positive selection. When total CD4+T cells were stimulated with IL-2/TNF, CD25 expression was selectively upregulated on CD4+FOXP3+Tregs, but not on CD4+FOXP3- T cells. IL-2/TNF-primed Tregs maintained TNFR2 expression and expressed higher levels of FOXP3 and CD25 compared with Tregs stimulated with IL-2 alone (p&lt;.05). They also had enhanced proliferation in response to allogeneic PBMC (p&lt;.05) and suppressed Tcon proliferation more efficiently (p&lt;.01). Whole transcript expression analyses showed IL-2/TNF priming increased the expression of genes associated with Treg activation, proliferation and function compared to IL-2 alone, such us OX-40, 4-1BB, CD69, CCR8, CD39 genes and components of the NF-κB signaling pathway. We analyzed GvHD clinical signs of the last 20 patients who were diagnosed with grade II-IV acute GvHD after Treg/Tcon-based haploidentical transplantation at our institution. Nineteen presented with early-onset gut GvHD and 40% of those patients did not have any skin involvement. We hypothesized that preferential targeting to the gut was the consequence of defective homing of Tregs to the gut. Compared with Tcons, Tregs express higher levels of skin homing receptors (such as CCR4 and CLA-4), but lower levels of the gut homing receptor α4β7-integrin. We analyzed the infiltration of Tregs and Tcons in 31 colon biopsies from patients with suspected GvHD. The number of Tregs was higher in patients who were not diagnosed with acute GvHD compared with those who were (158.3/mm2 vs 86.3/mm2, p&lt;.05). A similar trend was observed for the Treg:Tcon ratio (0.27 vs 0.15, p&lt;.05). These results suggest that homing of Tregs to the gut plays an important role in the effectiveness of gut GvHD prevention. We also found that IL-2/TNF priming selectively upregulated α4β7-integrin expression on Tregs compared with freshly isolated cells (p&lt;.01). Experiments in humanized mouse models are underway to ascertain if IL-2/TNF priming improves Treg homing to the gut and site-specific GvHD prevention. CONCLUSION The present study shows that activation of TNFR2 signaling in human Tregs enhances their proliferation and suppressive function. It also suggests that Treg suppressive function exerted in periphery could be critical for tissue-specific GvHD control. Moreover, IL-2/TNF-priming could promote homing of Tregs to the gut and their capability to inhibit gut GvHD. Thus, IL-2/TNF-priming may be critical to optimize the ongoing clinical trial of adoptive immunotherapy with Tregs for GvHD prevention. Future studies will ascertain if IL-2/TNF-priming of Tregs can also allow for the infusion of higher doses of Tcons to further enhance immune reconstitution and leukemia killing without increasing the risk of GvHD. Disclosures No relevant conflicts of interest to declare.

Allogeneic Cord Blood Regulatory T Cells Can Prevent Graft Vs. Host Disease and Preserve Graft Vs Leukemia Effect: Update on Phase I/II Clinical Trial

Previously, we presented the results of a phase I trial of using cord blood (CB) derived regulatory T cells (Tregs) at a dose of 1x106 cells/kg in the prevention of graft vs. host disease (GVHD) in five patients undergoing allogeneic stem cell transplant (SCT). We now present an update of those patients and also the outcome of a single patient treated with CB Tregs at higher dose of 1x107 cells/kg. At the last follow up of 3.5 years, 5 of 6 patients are alive, in complete remission, without GVHD and off immune-suppression (table 1). One patient died of head injury at day 45 post-transplant without GVHD. None of the patients relapsed in spite of having high risk disease including Flt3+ acute myeloid leukemia (AML) (pt#1); refractory mycosis fungoides/ sezary syndrome (pt#2); relapsed refractory multiple myeloma including two autologous SCT (pt#3); myeloid sarcoma (pt#4); AML with complex cytogenetics (pt#5). Specifically, the sixth patient (treated at CB Tregs cell dose of 1x107 cells/kg) had a diagnosis of lymphoid blast crisis of chronic myelogenous leukemia (CML) and underwent allogeneic peripheral blood (PB) matched unrelated donor (MUD) SCT in second chronic phase with the conditioning regimen of fludarabine (40 mg/m2/d on day -5 to -2) and melphalan (140mg/m2 on day -2) and received CB Tregs at a dose of 1x107 cells/kg on day -1. The CB Tregs were manufactured at the MD Anderson GMP facility. Tregs were isolated from 4 out of 6 HLA matched CB unit with a post-thaw total nucleated cell (TNC) count of 1295 x 106 cells with 90% recovery. Enrichment of CD25+ Treg cells was accomplished with directly conjugated anti-CD25 magnetic microbeads and the LS column (Miltenyl) based selection. After selection, the total no. of isolated CB Tregs was 17.6 x 106 cells. These cells were ex-vivo expanded in culture for a total of 14 days in the continued presence of CD3/28 microbeads and interleukin-2. On the day of harvest, a total of 1359 x 106 Treg cells were generated with 96% viability. Based on patient's weight of 67 kg, a total of 67x107 CB Treg cells were infused. The expanded CB Tregs were also evaluated for functionality by using an in vitro suppression assay, where the stimulated CD4+ Tcon cells stained with CellTrace Violet and Tregs were added into a 96-well plate at a 1:1 and 1:2 ratio and incubated for three days at 37oC. As shown in figure 1A, the clinically manufactured CB Tregs were functional and exerted 98% suppression of the proliferating Tcon cells. Subsequently, the patient received PB MUD graft on day 0. The graft had a TNC count of 1900 x106 cells where the CD3+ T cells were at a dose of 400 x106 cells/kg resulting in a Treg: Tcon ratio of 1:40, a significantly lower number of Tregs compared to other published clinical trials using Treg prophylaxis for GVHD. There was no infusion reaction related to the CB Treg infusion, the patient engrafted on day+12 with 100% donor chimerism on day +30. The patient did not develop any grade II-IV GVHD and was off immune suppression by +6 months. At the time of last follow up of 2 years and 4 months, the patient remains alive, in complete remission and without GVHD. We compared the inflammatory cytokine profile as well as GVHD biomarkers for pt #6 to the rest of the five patients who received the lower dose of CB Tregs at 1x106 cells/kg (with up to 400 times Tcon cells in the graft), where 4 patients had developed acute GVHD. The inflammatory biomarkers of IL-6 (fig 1B) and IL-8 (fig 1C) for pt#6 were low to undetectable compared to the other patients. Similarly, the GVHD biomarkers of ST2 (fig 1D); MIG/ CXCL9 (fig 1E) and follistatin (fig 1F) were also low to undetectable in pt#6 compared to the rest. At the time of last follow up, all patients had resolved acute GVHD and were off-immune suppression and in complete remission. Based on these data, we conclude that CB Treg dose of 1x107 cells/kg may be able to prevent GVHD without alleviating the graft vs leukemia effect. Disclosures Popat: Jazz: Consultancy; Incyte: Research Funding; Bayer: Research Funding. Nieto:Affimed: Consultancy; Astra-Zeneca: Research Funding; Novartis: Research Funding; Affimed: Research Funding. Ciurea:Kiadis Pharma: Membership on an entity's Board of Directors or advisory committees, Other: stock holder; MolMed: Membership on an entity's Board of Directors or advisory committees; Spectrum: Membership on an entity's Board of Directors or advisory committees; Miltenyi: Research Funding. Qazilbash:Amgen: Consultancy, Other: Advisory Board; Genzyme: Other: Speaker; Bioclinical: Consultancy; Autolus: Consultancy. Champlin:Actinium: Consultancy; Johnson and Johnson: Consultancy; Sanofi-Genzyme: Research Funding. Parmar:Cellenkos Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Individual Patient Dose-Escalated Low-Dose Interleukin-2 for Steroid-Refractory Chronic Graft-Vs.-Host Disease in Children and Adults: Safety, Efficacy and Immune Correlates

Daily low-dose interleukin-2 (IL-2) at a fixed dose of 1 × 106 IU/m2/day leads to preferential expansion of CD4+CD25+CD127-Foxp3+ regulatory T cells (Treg) and clinical responses in 50-60% of adult patients with steroid-refractory chronic GVHD. During fixed-dose IL-2 therapy, plasma IL-2 levels rise rapidly, but decrease over time despite continued daily administration. Lower IL-2 levels correlate with decreased Treg proliferation, possibly due to IL-2 sequestration via binding to high-affinity IL-2 receptors on Treg. We reasoned that IL-2 dose escalation at the time of anticipated fall in plasma IL-2 levels would avoid tachyphylaxis and enhance Treg expansion. We conducted a phase 1 trial of individual patient IL-2 dose escalation over 8 weeks in adult and pediatric patients with steroid-refractory cGVHD. Daily SC IL-2 was initiated at 0.67 × 106 and 0.33 × 106 IU/m2/day in the adult and pediatric cohorts, respectively. Each participant had dose escalations at weeks 2 and 4 to a maximum dose of 2 × 106 IU/m2/day in adults and 1 × 106 IU/m2/day in children. We studied 21 participants (10 adult, 11 children) with median ages of 57 (range, 33-71) and 12 years (range, 3-22) in the adult and pediatric cohorts, respectively. The cohorts did not differ in terms of time since cGVHD onset, number of organ sites involved or failed therapies prior to enrollment. Individually dose-escalated IL-2 was well tolerated in children with 1 patient requiring dose reduction for electrolyte imbalances. In the adult cohort, 1 patient withdrew for cGVHD progression, 4 required dose reduction for injection site reactions and 2 were unevaluable. At week 8, objective cGVHD responses (PR) were seen in 8 of 11 pediatric patients (73%), but in only 1 of 8 evaluable adult patients (13%). Response sites included skin (n=4), joint/fascia/muscle (n=1), lung (n=5), and GI tract (n=2). 11 pediatric and 4 adult participants with clinical benefit (PR or SD with minor response) continued extended duration IL-2 therapy. 9 pediatric patients on IL-2 therapy were able to wean steroid therapy with a median dose reduction of 67% at 6 months (Fig. 1). Children had a superior immunologic response despite lower IL-2 doses. The median Treg:Tcon ratio at week 8 was significantly higher in the pediatric cohort (0.42 vs 0.21, p = 0.02). Both cohorts had increased NK cell numbers, with no significant changes in CD4Tcon, CD8 T cell or B cells (Fig. 2). Pediatric patients had greater thymic output of naive Treg as well as a larger fraction of PD-1-expressing effector memory Treg at baseline and during IL-2 therapy (Fig. 3), which has been correlated with improved Treg expansion and survival. We show for the first time that low-dose IL-2 is safe in children with advanced steroid-refractory cGVHD and results in a high clinical response rate. In adults, dose escalation above fixed-dose MTD did not improve Treg expansion or clinical response relative to fixed daily dosing.

Effect of Sirolimus on Immune Reconstitution Following Myeloablative Allogeneic Stem-Cell Transplantation: A Post-Hoc Analysis of a Randomized Controlled Trial Comparing Sirolimus/Tacrolimus with Tacrolimus/Methotrexate (BMT CTN 0402)

Introduction:Although allogeneic hematopoietic cell transplantation (HCT) is a potentially curative therapy for hematologic neoplasms, one of its limiting toxicities continues to be graft versus host disease, both acute and chronic (aGVHD, cGVHD). Sirolimus is a mammalian target of rapamycin (mTOR) inhibitor which has been found to be effective in GVHD prophylaxis, in combination with calcineurin inhibitors like tacrolimus. The impact of sirolimus on immune reconstitution has not been comprehensively investigated in-vivo thus far. We now present a post-hoc analysis of the randomized study BMT-CTN 0402, examining the effect of sirolimus on immune subsets post-transplant. We further examine the association between different lymphocyte subsets and outcomes post-transplant in each arm.Methods:BMT-CTN 0402 was a randomized trial (n=304) which compared two GVHD prophylaxis regimens, sirolimus/tacrolimus (Sir/Tac) versus tacrolimus/methotrexate (Tac/MTX), in AML/ALL/MDS patients, undergoing myeloablative HLA-matched transplantation. There was no difference in 114-day GVHD free survival (primary end-point) as well as acute or chronic GVHD, relapse or overall survival between arms. 264/304 patients had available samples for the current post-hoc analysis. Blood samples were collected at 1,3, 6, 12 and 24 months post-HCT. Multi-parameter flow cytometry was performed at the project lab (Esoterix Clinical Trials Services) in a blinded fashion, and results were compared between arms. Multivariable Cox regression models, treating each phenotypic parameter as a time dependent variable, were constructed to study impact of reconstitution on clinical outcomes.Results:There were no significant differences in patient and transplant characteristics between the Sir/Tac and Tac/MTX arms in this analysis. Absolute lymphocyte count (ALC), CD3+ and CD4+ counts were significantly decreased in the Sir/Tac arm upto 3 months post-HCT while CD8+ cells recovered even slower (upto 6 months) in this arm (Figure 1, Panels A,B and C). Interestingly there was no difference in the absolute number of regulatory T-cells (Tregs/CD4+ CD25+) between arms at any point post-HCT (Figure 1, Panel D). However the Treg:Tcon ratio was significantly greater in the Sir/Tac arm in the first 3 months post-HCT (Figure 1, Panel E). B-lymphocyte recovery was significantly compromised in the Sir/Tac arm from 1 to 6 months post-HCT (Figure 1, Panel F) while NK cells reconstitution was not affected in the sirolimus arm. In the outcomes analysis, higher numbers of CD3+, CD4+. CD8+ and Tregs were associated with better overall survival. Neither Treg numbers nor Treg:Tcon ratio correlated with GVHD.Conclusion:Sir/Tac has a more profound T-cell suppressive effect than the standard of care combination of Tac/MTX in the early post-transplant period, and particularly compromises recovery of CD8+ T-cells with potential implications in the prevention of aGVHD. Sirolimus when used in-vivo with tacrolimus does not result in increased absolute numbers of Tregs, however it does have a beneficial effect on the Treg:Tcon balance in the first 3 months after transplantation, which may be important in the context of cGVHD prevention. Calcineurin-inhibitor free, sirolimus containing GVHD prophylaxis strategies, incorporating other novel agents, should be investigated further to maximize the favorable effect of sirolimus on Treg:Tcon balance in the post-transplant immune repertoire. Sirolimus significantly compromises B-cell recovery in the first 6 months post-HCT with potential complex effects on cGVHD which merit further study.Acknowledgments:Support for this study was provided by grant #U10HL069294 to the Blood and Marrow Transplant Clinical Trials Network from the National Heart, Lung, and Blood Institute and the National Cancer Institute, along with contributions by Wyeth Pharmaceuticals Inc. BMT CTN 0402 biospecimens were obtained from the NHLBI Biologic Specimen and Data Repository Information Coordinating Center (BioLINCC). The content is solely the responsibility of the authors and does not necessarily represent the official views of the above mentioned partiesAdditional support was provided by the Division of Allergy, Immunology, and Transplantation, National Institute of Allergy and Infectious Diseases for the ancillary study, ‘GVHD Prophylaxis - Immune Reconstitution.‘ [Display omitted] DisclosuresSoiffer:Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Antin:Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

Low-Dose Interleukin-2 Therapy Activates Circulating T Follicular Regulatory Cells and Suppresses Circulating T Follicular Helper Cells in Patients with Chronic Gvhd

Introduction: T follicular helper (TFH) and T follicular regulatory (TFR) cells play important roles in the regulation of B-cell immunity. While TFH promote B cell functions in the germinal center (GC), TFR function as negative regulators of the GC response. Previous studies in murine models established that TFH and GC B cells are required for the development of chronic graft-versus-host disease (cGVHD). We previously reported that circulating TFH (cTFH) were more functionally activated in patients with active cGVHD compared with patients with no cGVHD. Low-dose IL-2 therapy has been shown to selectively expand CD4Treg and improve cGVHD symptoms. In the current study, we examined the effects of IL-2 therapy on cTFH and circulating TFR (cTFR) in patients with steroid resistant cGVHD.Methods: Single cell mass cytomtery (CyTOF) was performed on cryopreserved peripheral blood mononuclear cells (PBMC) from healthy donors and 17 adult patients with active cGVHD receiving daily low-dose IL-2 therapy (Koreth et al. Blood 2016). A panel of 35 metal-tagged monoclonal antibodies 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 lymphocyte subsets and 13 intracellular markers to measure functional status and activation of specific signaling pathways. Before staining for surface and intracellular antigens, serial samples from individual patients were barcoded to ensure uniformity of analysis. viSNE was used to visualize of high-dimensional data on a two-dimensional map and quantify single cell mass cytometry data.Results: In PBMC from healthy donors, expression of CD25 (IL-2Rα), CD95, CTLA-4, BLIMP-1 and GITR was higher in cTFR compared with cTFH. To examine the response to IL-2 in vitro, PBMC from healthy donors were stimulated with IL-2 for 15 minutes (Figure 1A). At low IL-2 concentrations (1 to 10 IU/mL), phospho-STAT5 (p-STAT5) was selectively activated in cTFR compared with cTFH. At high IL-2 concentrations (100 to 1,000 IU/mL), p-STAT5 was activated in both cTFR and cTFH. To examine the response to IL-2 in vivo, we used mass cytometry to examine serial PBMC samples from cGVHD patients receiving daily low dose IL-2 therapy (1x106 IU/M2/day). Selective expansion of cTFR was noted after 1 week of treatment and cTFR expansion remained stable for the 12 week duration of therapy. Expanded cTFR increased expression of p-STAT5, FoxP3, BCL6, HLA-DR (Figure 1B) and CD25, CD95, CTLA-4, ICOS, Ki67 and Helios 1 week after starting IL-2. cTFR:cTFH ratio increased rapidly after starting low dose IL-2 and paralleled the increased Treg:Tcon ratio (Figure 1C). Activated TFH and TFR can be identified by expression of ICOS and PD-1. The expansion of ICOS+PD-1+ cTFR was evident after 1 week of IL-2 and remained elevated at the end of therapy. In contrast, ICOS+PD-1+ cTFH increased 1 week after starting IL-2 therapy but subsequently decreased and fell below baseline 6 and 12 weeks after starting IL-2 (Figure 1D). Activated ICOS+PD-1+ cTFR expressed higher levels of p-STAT5, BCL-6, FoxP3, HLA-DR and CD25 during low dose IL-2 therapy. In contrast, these functional markers were not increased in ICOS+PD-1+ cTFH during IL-2 therapy (Figure 1B).Conclusion: Single cell mass cytometry analysis revealed that daily low dose IL-2 therapy induces selective activation and increased expression of functional proteins in ICOS+PD-1+ cTFR. In contrast, activated ICOS+PD-1+ cTFH were suppressed during IL-2 therapy. The selective activation of cTFR and suppression of cTFH provide a mechanism whereby low dose IL-2 therapy can promote B cell tolerance as well as T cell tolerance in patients with cGVHD. [Display omitted] DisclosuresForcade:Neovii: Other: Travel grant. Koreth:Amgen Inc.: Consultancy; Prometheus Labs: Research Funding; Kadmon Corp: Membership on an entity's Board of Directors or advisory committees; Millennium Pharmaceuticals: Research Funding; Takeda Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Nikiforow:Kite Therapeutics: Membership on an entity's Board of Directors or advisory committees. Armand:Infinity: Consultancy; Bristol-Myers Squibb: Consultancy, Research Funding; Otsuka: Research Funding; Tensha: Research Funding; Sequenta/Adaptive: Research Funding; Genmab: Consultancy; Affimed: Research Funding; Sigma Tau: Research Funding; Merck & Co., Inc.: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Roche: Research Funding. Cutler:Bristol-Myers Squibb: Consultancy; Pfizer: Consultancy; Kite: Consultancy; Pharmacyclics: Consultancy; Incyte: Consultancy; Astellas: Consultancy.

Amg 592 Is an Investigational IL-2 Mutein That Induces Highly Selective Expansion of Regulatory T Cells

Background: Low-dose interleukin-2 (IL-2) therapy expands regulatory T cells (Tregs) and provides clinical benefit for inflammatory diseases; however, concomitant increases in conventional effector T cells (Tcon) and natural killer (NK) cells may result in toxicity. AMG 592 is an investigational IL-2 mutein designed for greater Treg selectivity and longer half-life compared with recombinant IL-2 (aldesleukin). In preclinical studies and a phase 1, double-blind, placebo (PBO)-controlled first-in-human (FIH) study, we investigated the safety and tolerability of AMG 592 and its effects on expansion of Treg, Tcon, and NK-cells.Methods: AMG 592 activity was assessed as in vitro phosphorylated STAT5 (pSTAT5) in primary human peripheral blood mononuclear cells (PBMC). Treg, Tcon, and NK-cell expansion and cytokine production were assessed after culture of pre-stimulated human PBMC with increasing doses of AMG 592 or aldesleukin. The in vivo effects of AMG 592 on body temperature, C-reactive protein (CRP), and peripheral blood Treg, Tcon, and NK numbers were evaluated in cynomolgus monkeys after escalating single SC doses of AMG 592 or 5 consecutive daily SC doses of aldesleukin. In the FIH study, healthy volunteers in 8 dose cohorts received a single SC ascending dose of AMG 592 (n=6 per dose) or placebo (n=2 per dose) and were evaluated for safety and tolerability, pharmacokinetics (PK), pharmacodynamics, and cytokine production for 28 days.Results: In human PBMC cultures, AMG 592 caused more selective Treg response (pSTAT5 and proliferation) and lower production of pro-inflammatory cytokines than aldesleukin. Dose-dependent expansion of FoxP3+ Tregs in aldesleukin-treated cynomolgus monkeys was accompanied by increased body temperature and CRP; however, AMG 592 caused Treg expansion without significant effects on body temperature or CRP. In the ongoing FIH study, AMG 592 was well tolerated, with no serious adverse events (AEs). The most common AE across treatment arms was grade 1 painless erythema at or near the injection site that resolved without treatment. Preliminary PK results indicate dose-related increases in AMG 592 serum exposure. AMG 592 caused a robust, dose-dependent expansion of Tregs relative to Tcon in all treated individuals. Expanded Tregs had increased levels of CD25 and Foxp3, and were enriched for recent thymic emigrants. At the highest dose, the increase in the Treg:Tcon ratio peaked at day 8 (~4-fold vs baseline) and remained elevated up to day 29. Treg expansion by AMG 592 was highly selective, with no directional change in NK-cell numbers and minimal increase in Tcon. Moreover, there were no increases in the serum pro-inflammatory cytokines IL-6, TNFα, or IFN-γ above the limits of detection.Conclusion: AMG 592 caused a dose-dependent, selective expansion of Tregs in healthy volunteers. The lack of pro-inflammatory cytokines and reduced markers of inflammation by AMG 592 compared with aldesleukin suggests a wider therapeutic margin. The sustained Treg elevation compared with aldesleukin suggests less frequent dosing may be feasible. Further investigation of the ability of AMG 592 to restore immune homeostasis by Tregs in inflammatory and autoimmune diseases is warranted. DisclosuresTchao:Amgen Inc.: Employment, Equity Ownership. Gorski:Amgen Inc.: Employment, Equity Ownership. Yuraszeck:Amgen Inc.: Employment, Equity Ownership. Sohn:Amgen Inc.: Employment, Equity Ownership. Ishida:Amgen Inc.: Employment, Equity Ownership. Wong:Amgen Inc.: Employment, Equity Ownership. Park:Amgen Inc.: Employment, Equity Ownership.

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.

TNF-α priming enhances CD4+FoxP3+ regulatory T-cell suppressive function in murine GVHD prevention and treatment

AbstractCD4+CD25+FoxP3+ regulatory T cells (Tregs) have been shown to effectively prevent graft-versus-host disease (GVHD) when adoptively transferred in murine models of hematopoietic cell transplantation and in phase 1/2 clinical trials. Critical limitations to Treg clinical application are the paucity of cells and limited knowledge of the mechanisms of in vivo function. We hypothesized that inflammatory conditions in GVHD modify Treg characteristics and activity. We found that peripheral blood of recipient animals during acute GVHD (aGVHD) induces Treg activation and enhances their function. The serum contains high levels of tumor necrosis factor-α (TNF-α) that selectively activates Tregs without impacting CD4+FoxP3− T cells. TNF-α priming induces Treg in vivo proliferation, whereas it limits the ability of CD4 and CD8 conventional T cells (Tcons) to proliferate and induce GVHD. TNF-α–primed Tregs prolong animal survival as compared with unprimed Tregs when used at an unfavorable Treg:Tcon ratio, demonstrating enhanced in vivo efficacy of TNF-α–primed Tregs. Because TNF-α is produced by several immune cells during inflammation, our work elucidates aspects of the physiologic mechanisms of Treg function. Furthermore, TNF-α priming of Tregs provides a new tool to optimize Treg cellular therapies for GVHD prevention and treatment.

Efficacy, durability, and response predictors of low-dose interleukin-2 therapy for chronic graft-versus-host disease

AbstractChronic graft-versus-host disease (cGVHD) is associated with inadequate reconstitution of tolerogenic CD4+CD25+FOXP3+ regulatory T cells (Tregs). Previous phase 1 studies identified a low daily dose of interleukin-2 (IL-2) that was well tolerated, did not exacerbate alloimmunity, augmented Treg in vivo, and was associated with improvement of active cGVHD. In the current phase 2 study, 35 adults with steroid-refractory cGVHD received daily IL-2 (1 × 106 IU/m2) for 12 weeks. Median time from transplantation and cGVHD onset was 616 days (range, 270-2145 days) and 317 days (range, 28-1880 days), respectively. Two patients withdrew and 5 required IL-2 dose reductions due to side effects. Twenty of 33 evaluable patients (61%) had clinical responses at multiple cGVHD sites (liver, skin, gastrointestinal tract, lung, joint/muscle/fascia). Three patients (9%) had progressive cGVHD. Compared with pretreatment levels, Treg and natural killer cell counts rose >fivefold (P < .001) and >fourfold (P < .001), respectively, without significant change in conventional CD4 T cells (Tcons) or CD8 T cells. The Treg:Tcon ratio rose >fivefold (P < .001). Clinical responders initiated IL-2 earlier (508 vs 917 days after transplantation, P = .005; 249 vs 461 days after cGVHD onset; P = .03). Treg:Tcon ratios ≥0.07 at baseline and ≥0.2 at week 1 also predicted clinical response (P = .003; P = .0003, respectively). After a 4-week treatment hiatus, clinical responders were eligible to continue IL-2 therapy indefinitely. During 2 years of extended IL-2 therapy, clinical and Treg immune responses persisted, while Tcon count and Treg:Tcon ratio gradually normalized. Low-dose IL-2 provides durable clinical improvement in active cGVHD and extended therapy is well-tolerated.

Low-Dose Interleukin-2 for Steroid-Refractory Chronic Graft-Vs.-Host Disease: Phase 2 and Long Term Efficacy, Safety and Immune Correlates

Chronic graft-versus-host disease (cGVHD) after allogeneic hematopoietic cell transplantation (HCT) results from incomplete reconstitution of immune tolerance. CD4+CD25+FOXP3+ regulatory T cells (Treg) are required for tolerance and function as dominant suppressors of innate and adaptive immune effector cells. In our prior phase 1 cGVHD study daily subcutaneous (SC) low-dose interleukin-2 (IL-2) for 8 weeks induced Treg expansion in vivo and objective clinical responses in 12 of 23 evaluable participants (NEJM 2011). We now report on a phase 2 trial of daily low-dose SC IL-2 at 1x106 IU/m2/d for 12 weeks in steroid-refractory cGVHD.The study comprised 35 HCT recipients (51% male, 91% HLA-matched PBSC grafts). Median participant age was 51 years (range, 22-72). Median time from HCT and from cGVHD onset to start of IL-2 treatment was 616 days (range, 270-2145) and 252 days (range, 28-1880) respectively. Participants had a median of 4 cGVHD organ sites (range, 1-7), and 2 concurrent cGVHD therapies (range, 1-3) at enrollment. The median baseline prednisone dose was 20 mg (range, 2.5-50). The median follow-up in survivors was 21 months (range, 4-35).12 week low dose IL-2 was well tolerated: 2 participants withdrew and 5 required IL-2 dose reduction for constitutional AE (n=6) and thrombocytopenia (n=1); 1 had Gr 3 infection (bacteremia); and none experienced relapse. At week 12, objective cGVHD responses (PR) were documented in 21 of 33 evaluable participants (64%). Two (6%) had cGVHD progression. cGVHD response sites included skin (n=9), joint/fascia/muscle (n=4), liver (n=7), lung (n=3), and GI tract (n=4). Overall 2-year OS/PFS was 91% (responders 94%; non-responders 83%). 23 participants with clinical benefit (PR or SD with minor response) proceeded on extended IL-2 therapy.Immunologically, low dose IL-2 induced a >4-fold increase in median Treg count/µL (p<0.001): a rapid rise from a baseline of 17.1 (Q1-Q3, 8.6-40.6) to a week 4 peak of 137.9 (Q1-Q3, 51.8-188) and subsequent stabilization with a week 12 count of 104.1 (Q1-Q3, 53.9-167.1). No significant change in CD4 conventional T cell (Tcon), CD8 T cell, or CD20 B cell count was noted. NK cell count increased >3-fold (p<0.001). The median Treg:Tcon ratio increased >4-fold (p<0.001): a rapid rise from baseline of 0.06 (Q1-Q3, 0.05-0.13) to a week 2 peak of 0.35 (Q1-Q3, 0.26-0.48) that remained elevated through a week 12 ratio of 0.31 (Q1-Q3, 0.27-0.39) (Figure). Treg count and Treg:Tcon ratio declined during 4 weeks off IL-2 and rose thereafter on restarting IL-2.Clinical responders were younger (50 vs. 61.5 years, p=0.01) and initiated IL-2 earlier (499 vs. 903 days post HCT, p=0.015). Responders had a higher median Treg:Tcon ratio at study baseline (0.09 vs. 0.06, p=0.052) and at week 1 of IL-2 (0.3 vs. 0.14, p=0.01). Combining phase 1 and 2 data, Treg:Tcon ratios of ≥0.07 at baseline and ≥0.2 at week 1 of IL-2 were highly predictive of clinical response (p=0.007; p=0.0013 respectively).The combined phase 1 and 2 extended IL-2 cohort comprised 35 participants with a median follow up of 16.2 months (range, 4.1-66.8), with 20 and 12 participants receiving over 1 and 2 years of IL-2 respectively. Extended daily low dose IL-2 was well tolerated, and only 4 participants had Gr 3 AEs deemed IL-2 related: lung infection (n=1), arthralgia (n=1), and injection site induration (n=2). 5 participants required IL-2 dose reduction and 1 had hematologic malignancy relapse. Clinical responses were typically sustained during taper of concomitant immunosuppression. Treg augmentation persisted for the duration of IL-2 therapy. Tcon count slowly recovered to normal levels and Treg:Tcon ratio gradually normalized over a 2 year period. There was no change in CD8 count. The median steroid taper was 50% (range, -20-100).In summary, daily low dose IL-2 therapy induced profound Treg enhancement, and clinical responses in over half of refractory cGVHD patients. Early clinical response predictors suggest IL-2 is more effective earlier in the cGVHD course and when starting numbers of Treg are higher. Sustained clinical and immunologic response during extended IL-2 was documented. Long term tolerance induction with daily low dose IL-2 is a promising and feasible strategy. Optimizing IL-2 clinical response by further augmenting Treg and the Treg;Tcon ratio early in the course of cGVHD is worth exploring. [Display omitted] DisclosuresKoreth:Prometheus Laboratories Inc: Research Funding; Millennium Pharmaceuticals Inc: Research Funding; Takeda Pharmaceuticals Inc: Membership on an entity’s Board of Directors or advisory committees. Off Label Use: Low-dose Interleukin-2 for immune tolerance. Chen:Bayer Pharmaceuticals, Inc.: Other, Research Funding. Avigan:Astex Pharmaceuticals : Research Funding.

Putting Out the Fire

Low-dose interleukin-2 suppresses graft-versus-host disease.

Interleukin-2 and Regulatory T Cells in Graft-versus-Host Disease

Dysfunction of regulatory T (Treg) cells has been detected in diverse inflammatory disorders, including chronic graft-versus-host disease (GVHD). Interleukin-2 is critical for Treg cell growth, survival, and activity. We hypothesized that low-dose interleukin-2 could preferentially enhance Treg cells in vivo and suppress clinical manifestations of chronic GVHD. In this observational cohort study, patients with chronic GVHD that was refractory to glucocorticoid therapy received daily low-dose subcutaneous interleukin-2 (0.3×10(6), 1×10(6), or 3×10(6) IU per square meter of body-surface area) for 8 weeks. The end points were safety and clinical and immunologic response. After a 4-week hiatus, patients with a response could receive interleukin-2 for an extended period. A total of 29 patients were enrolled. None had progression of chronic GVHD or relapse of a hematologic cancer. The maximum tolerated dose of interleukin-2 was 1×10(6) IU per square meter. The highest dose level induced unacceptable constitutional symptoms. Of the 23 patients who could be evaluated for response, 12 had major responses involving multiple sites. The numbers of CD4+ Treg cells were preferentially increased in all patients, with a peak median value, at 4 weeks, that was more than eight times the baseline value (P<0.001), without affecting CD4+ conventional T (Tcon) cells. The Treg:Tcon ratio increased to a median of more than five times the baseline value (P<0.001). The Treg cell count and Treg:Tcon ratio remained elevated at 8 weeks (P<0.001 for both comparisons with baseline values), then declined when the patients were not receiving interleukin-2. The increased numbers of Treg cells expressed the transcription factor forkhead box P3 (FOXP3) and could inhibit autologous Tcon cells. Immunologic and clinical responses were sustained in patients who received interleukin-2 for an extended period, permitting the glucocorticoid dose to be tapered by a mean of 60% (range, 25 to 100). Daily low-dose interleukin-2 was safely administered in patients with active chronic GVHD that was refractory to glucocorticoid therapy. Administration was associated with preferential, sustained Treg cell expansion in vivo and amelioration of the manifestations of chronic GVHD in a substantial proportion of patients. (Funded by a Dana-Farber Dunkin' Donuts Rising Star award and others; ClinicalTrials.gov number, NCT00529035.).

Regulatory T cells control HIV replication in activated T cells through a cAMP-dependent mechanism

AbstractWe hypothesized that regulatory T cells (Tregs) could play a beneficial role during HIV infection by controlling HIV replication in conventional T cells (Tcons). Purified Tregs and Tcons from healthy donors were activated separately. Tcons were infected with the X4 or R5 HIV strains and cultured with or without autologous Tregs. Coculture of Tcons and Tregs resulted in a dose-dependent inhibition of Tcon infection, which was significant when a 1:1 Treg:Tcon ratio was used. Treg suppression of HIV infection was largely mediated by contact-dependent mechanisms. Blockage of cytotoxic T-lymphocyte–associated antigen-4 did not significantly reduce Treg function. In contrast, Tregs acted through cAMP-dependent mechanisms, because the decrease of cAMP levels in Tregs, the blockade of gap junction formation between Tregs and Tcons, the blockage of CD39 activity, and the blockage of protein kinase A in Tcons all abolished Treg-mediated suppression of HIV replication. Our data suggest a complex role for Tregs during HIV infection. Although Tregs inhibit specific immune responses, their inhibition of HIV replication in Tcons may play a beneficial role, particularly during early HIV infection, when the effector immune cells are not yet activated. Such a protective role of Tregs could have a profound impact on infection outcome.

Regulatory and Naïve T Cells in Unmanipulated Donor Grafts Are Not Associated with Acute Graft Vs Host Disease in Matched Sibling Transplants for AML

Acute graft-versus-host disease (aGVHD) occurs in approximately 20–50% of matched-related stem cell transplants (HCT). The presence of increased frequencies of CD4+CD25+ regulatory T cells (Treg) in murine donor grafts has been shown to ameliorate aGVHD. In similar models, several groups have demonstrated that the transfer of equivalent numbers of naïve T cells (vs. various memory subsets) confers a relatively greater risk for aGVHD. To test the hypothesis that natural variations in donor graft naïve and Treg content would be associated with the incidence of aGVHD in graft recipients, we conducted a detailed analysis of donor graft T cells within unmanipulated grafts of 70 SCT recipients transplanted for AML/MDS following the administration of a uniform fludarabine/busulfan conditioning regimen. Median follow up time was 38 months. 19% (n=13) of recipients experienced grades II–IV aGVHD (n=3 grades III–IV). Cryopreserved donor grafts samples were analyzed using 9-color flow cytometry (with a panel including a viability dye and MAbs recognizing CD4, CD8, CD25, CD127, CD45RA, CD27, Foxp3, and Ki-67). Tregs were defined as being CD4+CD25+CD127loFoxp3+. CD45RA and CD27 were used to measure naïve (CD45RA+CD27+) and memory conventional CD4+ and CD8+ T cells. In addition, the proliferating fraction of all cells was defined by Ki-67 nuclear-antigen expression. We conducted analyses based on donor graft T cells assessed both continuously and by quartiles, with similar results. No significant associations were found between aGVHD incidence and total Tregs (HR 1.01, 95%CI 0.9–1.2, p 0.8), nor with proliferating Tregs (HR 1.2, 95%CI 0.2–5.9, p 0.8). Since murine models typically fix the dose of Tregs vs. conventional T cells (Tcon), we also analyzed GVHD risk by donor graft Treg:Tcon ratio, finding no association (HR 1.03, 95%CI 0.8–2.1, p 0.3). Although patients in the highest Treg:Tcon quartile received Treg doses known in vitro to induce suppression (>1:21.5), there was no reduction in aGVHD (HR 1.5 Quartile 4 vs others, 95%CI 0.5–4.9, p 0.5). Additionally, no significant association with aGVHD was found with total naïve T cells (HR 1, 95%CI 0.9–1.0, p 0.9), naïve CD4+ (HR 1, 95%CI 0.9–1.03, p 0.6), or naïve CD8+ cells (HR 0.98, 95%CI 0.9–1.03, p 0.6). Because naïve:memory cell ratios were also fixed in murine models demonstrating the importance of naïve T cells in GVHD, we also assessed naïve:memory T cell ratios within donor grafts with respect to recipient aGVHD. No reduction in aGVHD was observed considering naïve:memory ratios analyzed continuously (HR 1.1, 95%CI 0.3–4.2, p 0.8) or in the quartile receiving the lowest naïve:memory ratio (HR 0.5 Quartile 1 vs others, 95%CI 0.1–2.2, p 0.3). Our results demonstrate that donor graft Treg and naïve T cell content, assessed in the context of unmanipulated PBSC transplantation in matched siblings, does not predict recipient aGVHD incidence. These data suggest that the therapeutic efficacy of Treg enrichment and/ or naïve T cell depletion may require grafts to be manipulated to supraphysiologic levels for maximal therapeutic benefit.

Radioimmunoassay of human serum thyrotrophin.

The double antibody radioimmunoassay of serum thyroid-stimulating hormone (TSH) allows measurement of circulating levels of the hormone in most normal subjects. The serum TSH level in normal subjects is 1.6 +/- 0.8muU/ml. Patients with non-toxic goitre and acromegaly have normal TSH levels. Values are always raised in hypothyroid patients (with primary thyroid disease) and are significantly lowered in those with hyperthyroidism. Of the many stimuli used in an attempt to raise TSH levels in normal adult subjects only three-synthetic thyrotrophin-releasing hormone, ethinyloestradiol, and carbimazole plus iodides-have been effective. The major clinical application of the TSH immunoassay lies in the diagnosis of minor degrees of hypothyroidism. An impaired response of serum TSH to synthetic thyrotrophin-releasing hormone should also help in the diagnosis of hypopituitarism affecting TSH production.