Chronic 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 IL-2 is efficacious in steroid-refractory cGVHD, with objective responses in >50% of patients, and durable disease control.•IL-2 initiation earlier after cGVHD onset, prior to severe impairment of Treg:Tcon ratios, improves likelihood of clinical response.
The development and maintenance of immune tolerance after allogeneic hematopoietic stem cell transplantation (HSCT) requires the balanced reconstitution of donor-derived CD4 regulatory T cells ...(CD4Tregs) as well as effector CD4 (conventional CD4 T cells CD4Tcons) and CD8 T cells. To characterize the complex mechanisms that lead to unbalanced recovery of these distinct T-cell populations, we studied 107 adult patients who received T-replete stem cell grafts after reduced-intensity conditioning. Immune reconstitution of CD4Treg, CD4Tcon, and CD8 T cells was monitored for a 2-year period. CD3 T-cell counts gradually recovered to normal levels during this period but CD8 T cells recovered more rapidly than either CD4Tregs or CD4Tcons. Reconstituting CD4Tregs and CD4Tcons were predominantly central memory (CM) and effector memory (EM) cells and CD8 T cells were predominantly terminal EM cells. Thymic generation of naive CD4Tcon and CD8 T cells was maintained but thymic production of CD4Tregs was markedly decreased with little recovery during the 2-year study. T-cell proliferation was skewed in favor of CM and EM CD4Tcon and CD8 T cells, especially 6 to 12 months after HSCT. Intracellular expression of BCL2 was increased in CD4Tcon and CD8 T cells in the first 3 to 6 months after HSCT. Early recovery of naive and CM fractions within each T-cell population 3 months after transplant was also strongly correlated with the subsequent development of chronic graft-versus-host disease (GVHD). These dynamic imbalances favor the production, expansion, and persistence of effector T cells over CD4Tregs and were associated with the development of chronic GVHD.
•Homeostatic recovery after allogeneic HSCT favors the production, expansion, and survival of effector T cells over CD4Tregs.•Unbalanced reconstitution of regulatory and effector T-cell subsets contributes to the development of chronic graft-versus-host disease.
Introduction: Cytokines play important roles in the activation, proliferation, differentiation and survival of T cells. Previous studies have revealed that individual cytokines selectively activate ...different T cell populations and also function at specific stages of T cell differentiation. For example, IL-2 supports the development of CD4 regulatory T cells. IL-7 is required for naive conventional CD4 T cell (Tcon) homeostasis, whereas naive CD8 T cell homeostasis requires both IL-7 and IL-15. In contrast, IL-6 promotes Th17 T cell differentiation. The functions of each cytokine are partly defined by the differential expression of specific multi-unit receptors but the selective homeostatic effects of individual cytokines are still incompletely understood.
Methods: We stimulated peripheral blood mononuclear cells from healthy donors with varying concentrations of IL-2, IL-7, IL-15 and IL-6 for 15 min in vitro. Single cell mass cytometry (CyTOF) with a panel of 33 markers was used to simultaneously examine signaling pathways activated by each cytokine in distinct T cell subsets. viSNE, a cytometry analysis tool, was used to visualize high-dimensional cytometry data on a two-dimensional map. Expression of pSTAT5 was used to monitor activation induced by IL-2, IL-7 and IL-15; pSTAT3 was used to monitor activation by IL-6.
Results: In CD4 Tcon, relatively high concentrations of IL-2 (100-1000 IU/ml) are required to induce pSTAT5 (Figure 1). However even at high concentrations, IL-2 activation was selective for memory Tcon subsets. In contrast, IL-7 induced pSTAT5 at very low concentrations (1-10 IU/ml). Although all Tcon were affected, activation was more robust in memory than naive Tcon subsets at all IL-7 concentrations. IL-15 activation of pSTAT5 required at least 10 IU/ml and only memory Tcon subsets were activated even at high IL-15 concentrations. Whereas IL-2, IL-7 and IL-15 preferentially activated memory Tcon subsets, IL-6 selectively activated pSTAT3 in naive and central memory (CM) Tcon subsets at low concentrations (10 IU/ml). At high IL-6 concentrations (100-1000 IU/ml) effector memory (EM) Tcon were also activated.
CD8 T cells (Figure 2) are relatively insensitive to IL-2, and only CM CD8 T cells are activated at high IL-2 concentrations (100 IU/ml). Although all CD8 T cell subsets were activated at very high IL-2 concentrations (1000 IU/ml), pSTAT5 activation remained most evident in CM CD8 T cells. Similar to Tcon, IL-7 induced pSTAT5 in CD8 T cells at very low IL-7 concentrations (1-10 IU/ml). However unlike Tcon, pSTAT5 activation was most prominent in naive and CM CD8 T cells. EM CD8 T cells were activated at higher IL-7 concentrations but TEMRA CD8 T cells were resistant to IL-7 stimulation. IL-15 induced pSTAT5 equally in all CD8 T cell subsets but relatively high concentrations (100-1000 IU/ml) were required. Similar to CD4 Tcon, IL-6 induced selective pSTAT3 activation in naive CD8 T cells. Activation of naive CD8 T cells was observed at low concentrations of IL-6 and both EM and TEMRA were resistant to very high IL-6 concentrations (100-1000 IU/ml).
Conclusion: This detailed analysis of cytokine signaling has identified differential effects of IL-2, IL-7, IL-15 and IL-6 on different subsets of CD4 Tcon and CD8 T cells. Whereas CD4 Treg are activated at low IL-2 concentrations, CD4 Tcon and CD8 T cells are relatively resistant to IL-2. At high IL-2 concentrations, activation was most prominent for memory CD4 Tcon and CM CD8 T cells. In contrast, low concentrations of IL-7 are sufficient to activate both CD4 Tcon and CD8 T cells. Within these populations, memory Tcon and naive CD8 cells were preferentially activated at low IL-7 concentrations. Within the CD8 T cell population, IL-15 activated all subsets equally. Within CD4 Tcon, IL-15 preferentially activates memory subsets. IL-6 acts at low concentrations and primarily on naive cells in both CD4 Tcon and CD8 T cells. In all experiments, these effects do not require TCR antigen activation and therefore reflect the potency and differential activity of homeostatic signals supported by these cytokines. Importantly, high concentrations used for in vitro experiments are not likely achieved in vivo but may reflect toxicities of high dose exogenous cytokine therapies or cytokine release syndromes. In contrast, differential effects observed at low concentrations more likely reflect physiologic homeostatic effects of these cytokines in vivo.
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No relevant conflicts of interest to declare.
Reconstitution of T cell function after allogeneic HSCT is dependent on the balanced recovery of CD4+Foxp3+ regulatory T cells (Treg) and CD4+Foxp3- conventional T cells (Tcon). While Tcon are ...required for effector T cell function, Treg play an essential role in the maintenance of immune tolerance after allogeneic HSCT and prevention of graft versus host disease (GVHD). To examine the reconstitution of Treg and Tcon after HSCT and identify mechanisms that contribute to homeostatic imbalance of these T cell subsets, we undertook a prospective analysis of 188 adult patients (median age 54y) with hematologic malignances who underwent T-cell replete allogeneic HSCT. Patients received either myeloablative (MAC; n=80) or reduced-intensity conditioning (RIC; n=108). GVHD prophylaxis differed in these two cohorts as RIC patients received tacrolimus/methotrexate/sirolimus-based regimens and MAC patients received tacrolimus/methotrexate-based regimens. Serial blood samples (total n=739) obtained at 1, 2, 3, 6, 9 and 12 months after transplant were characterized by flow cytometry with a panel of intracellular and surface markers designed to quantify phenotypically and functionally distinct subsets of Treg, Tcon and CD8 T cells in each sample.
Likely reflecting the prophylactic administration of sirolimus for the first 6 months post-HSCT after RIC conditioning, recovery of absolute CD3+, CD4+ and CD8+ T cell counts was significantly greater after MAC-HSCT throughout the first year after HSCT. Total CD4+ Tcon recovery was significantly decreased in RIC patients at all time points but Treg recovery was significantly lower only in the first 2 months after HSCT. Central memory cells (CM; 45RA-62L+) comprise the majority of Treg and Tcon throughout the first year after HSCT. The percentage of Treg-CM is greater than Tcon-CM and the fraction of CM cells within Treg and Tcon is increased in the RIC cohort (Figure 1A). Effector memory cells (EM; 45RA-62L-) also represent a large fraction of Treg and Tcon. Reconstitution of Treg-EM and Tcon-EM is similar but recovery of this subset appears to be strongly affected by sirolimus (Figure 1B). In RIC patients who receive sirolimus, the percentage of EM cells within Treg and Tcon subsets is significantly lower in the first 6 months after HSCT. Naïve Treg and Tcon (CD45RA+CD62L+) represent a relatively small fraction of recovering CD4 T cells during this period. Reconstitution of naïve CD4 T cells identified as recent thymic emigrants (RTE; CD45RA+CD31+) is markedly different in Treg and Tcon (Figure 1C). Within Tcon, the RTE fraction rapidly recovers to levels observed in healthy donors. In contrast, the fraction of RTE-Treg remains very low, with no evidence of improvement for at least 1 year. For both Treg-RTE and Tcon-RTE, recovery is significantly greater after RIC suggesting that either the reduced-intensity of conditioning or sirolimus is thymus protective.
In vivo proliferation of each T cell subset was monitored by expression of Ki67. As previously observed in healthy donors, proliferation of Treg was significantly greater than Tcon at all time points. This primarily reflects homeostatic proliferation of Treg memory cells since very few naïve Treg are present. Both Treg and Tcon proliferation was higher in the MAC cohort in the first 1-3 months after HSCT. In vivo susceptibility to apoptosis was monitored by expression of pro-survival Bcl-2 and pro-apoptotic CD95 (FAS) expression. All Treg subsets expressed lower levels of Bcl-2 and higher levels of CD95 compared to Tcon. RIC/sirolimus was associated with higher levels of Bcl-2 and lower levels of CD95 predominately in the first 3 months after transplant. This effect was evident in all Treg and Tcon subsets.
These results demonstrate distinctly different patterns of reconstitution of Treg and Tcon after allogeneic HSCT. Reconstitution of Tcon is characterized by rapid recovery of thymic generation, moderate homeostatic proliferation of memory subsets and relative resistance to apoptosis. Reconstitution of Treg is characterized by prolonged impaired thymic generation, high levels of homeostatic proliferation of memory subsets and increased susceptibility to both intrinsic and extrinsic pathways of apoptosis. RIC followed by administration of sirolimus for GVHD prophylaxis appears to selectively delay recovery of Treg and Tcon EM cells while sparing naïve, RTE and CM subsets.
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No relevant conflicts of interest to declare.
Abstract Hematopoietic stem cell transplantation (HSCT) recipients lacking HLA-matched related donors have increased graft-versus-host disease (GVHD) and nonrelapse mortality (NRM). Bortezomib added ...to reduced-intensity conditioning can offer benefit in T cell–replete HLA-mismatched HSCT and may also benefit myeloablative conditioning (MAC) transplants. We conducted a phase II trial of short-course bortezomib plus standard tacrolimus/methotrexate after busulfan/fludarabine MAC in 34 patients with predominantly myeloid malignancies. Fourteen (41%) received 8/8 HLA-matched unrelated donor (MUD) and 20 (59%) received 7/8 HLA-mismatched related/unrelated donor peripheral blood stem cell grafts. Median age was 49 years (range, 21 to 60), and median follow-up was 25 months (range, 11 to 36). The regimen was well tolerated. No dose modifications were required. Neutrophil and platelet engraftment occurred at a median of 14 (range, 10 to 33) and 17 (range, 10 to 54) days, respectively. Median 30-day donor chimerism was 99% (range, 90 to 100), and 100-day grades II to IV and III to IV acute GVHD incidence was 32% and 12% respectively. One-year chronic GVHD incidence was 50%. Two-year cumulative incidence of both NRM and relapse was 16%. Two-year progression-free and overall survival rates were 70% and 71%, respectively. Outcomes were comparable to an 8/8 MUD MAC cohort (n = 45). Immune reconstitution was robust. Bortezomib-based MAC HSCT is well tolerated, with HLA-mismatched outcomes comparable with 8/8 MUD MAC HSCT, and is suitable for randomized evaluation. ( clinicaltrials.gov : NCT01323920 .)
Patients with active chronic graft-versus-host disease (cGVHD) have poor reconstitution of CD4+ CD25+ FOXP3+ regulatory T cells (Tregs), which have broad suppressive activity and are required for the ...maintenance of peripheral tolerance after allogeneic hematopoietic stem cell transplant. Interleukin-2 (IL-2) is a key growth factor for the development, expansion and function of Tregs in vivo. Phase 1 (DFCI 07-083) and phase 2 (DFCI 11-149) studies of daily subcutaneous low-dose IL-2 in patients with refractory cGVHD demonstrated preferential Treg expansion in all patients and objective clinical responses in approximately 50% of participants. In the phase 2 study, 23 participants with clinical benefit (PR or SD with minor response) continued on extended IL-2 therapy, with 6 and 8 patients receiving over 1 and 2 years of low-dose IL-2, respectively. Analysis of phase 1 study patients indicated that IL-2 restored Treg homeostasis through rapid induction of Treg proliferation, increase in thymic Treg neogenesis and increased Treg expression of the anti-apoptotic Bcl-2 protein. Here, we provide novel insights into the immune homeostatic impact of low-dose IL-2 in phase 2 study patients during the initial 12 week treatment period and during extended therapy.
Proliferation within the Treg compartment, measured by Ki67 expression, rapidly increased and peaked within 1 week of IL-2 initiation. Memory Tregs contained a higher fraction of proliferating cells and correspondingly, a rise in central memory (CM) and effector memory (EM) Tregs preceded an increase in naïve Tregs during the initial 12 week treatment period (Figure 1A and 1B). This is consistent with the observation that memory Tregs are more responsive than naive Tregs to activation by IL-2 in vitro. Although CM and EM Tregs continue to represent the predominant subsets of Tregs, there is a brisk expansion of the naïve Treg subset that coincides with increased thymic output of Tregs during extended IL-2 therapy.
Consistent with their greater proliferative potential, CM and EM Tregs expressed lower levels of Bcl-2 compared with naïve Tregs. However, IL-2 promoted higher Bcl-2 expression in all Treg subsets to similar magnitudes during both the initial 12 week treatment period and extended therapy (Figure 2A). Bcl-2 was preferentially increased in Tregs and not in conventional CD4 (Tcon) or CD8 T cells (Figure 2B). As expected, naïve Tregs expressed very low levels of the pro-apoptotic CD95/Fas receptor protein. CD95 expression in CM and EM Tregs peaked during the period of rapid proliferation within the first week of IL-2 therapy and declined as Bcl-2 expression increased. Changes in CD95 expression levels were similar across all T cell populations. Thus, IL-2 leads to preferential expansion and survival of Tregs in cGVHD patients throughout the duration of therapy.
Once IL-2 was discontinued following the initial 12 week treatment period, Treg numbers decreased to pre-treatment baseline levels within 4 weeks, indicating that continuous IL-2 exposure is required for maintenance of enhanced Treg homeostasis. Although patients in the extended duration cohort sustain stably elevated Treg numbers, it is not known whether a long-lasting Treg response is preserved in the absence of exogenous IL-2. Post-IL-2 Treg monitoring results were available for 3 patients in the extended duration cohort. One patient received continuous IL-2 for approximately 3 years and 2 patients stopped after over 1 year of therapy. At 4 weeks after IL-2 discontinuation, 2 of the 3 patients maintained elevated Treg numbers. One patient who stopped IL-2 after 74 weeks due to renal insufficiency had stably elevated Treg numbers and no worsening of cGVHD at 9 months post-IL-2, indicating that some patients may have durable restoration of Treg homeostasis following extended IL-2 therapy (Figure 3).
There were no significant differences in absolute numbers of Treg, Tcon or CD8 subsets between clinical responders and non-responders during the initial 12 week treatment period. Plasma IL-2 and soluble IL-2 receptor levels were also similar between the two groups. Thus, differences in clinical response to IL-2 are likely determined by qualitative differences in Treg or effector T cell function. Functional Treg suppression assays and gene expression profiling studies are in progress to explore this possibility.
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Armand:Bristol-Myers Squibb: Research Funding; Merck: Consultancy, Research Funding; Infinity Pharmaceuticals: Consultancy. Soiffer:Gentium SpA/Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Antin:Gentium S.p.A.: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.
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.
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Koreth: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.
Anti–programmed cell death protein 1 (PD-1) monoclonal antibodies are being increasingly tested in patients with advanced lymphoma. Following treatment, many of those patients are likely to be ...candidates for allogeneic hematopoietic stem cell transplant (HSCT). However, the safety and efficacy of HSCT may be affected by prior PD-1 blockade. We conducted an international retrospective analysis of 39 patients with lymphoma who received prior treatment with a PD-1 inhibitor, at a median time of 62 days (7-260) before HSCT. After a median follow-up of 12 months, the 1-year cumulative incidences of grade 2-4 and grade 3-4 acute graft-versus-host disease (GVHD) were 44% and 23%, respectively, whereas the 1-year incidence of chronic GVHD was 41%. There were 4 treatment-related deaths (1 from hepatic sinusoidal obstruction syndrome, 3 from early acute GVHD). In addition, 7 patients developed a noninfectious febrile syndrome shortly after transplant requiring prolonged courses of steroids. One-year overall and progression-free survival rates were 89% (95% confidence interval CI, 74-96) and 76% (95% CI, 56-87), respectively. One-year cumulative incidences of relapse and nonrelapse mortality were 14% (95% CI, 4-29) and 11% (95% CI, 3-23), respectively. Circulating lymphocyte subsets were analyzed in 17 patients. Compared with controls, patients previously treated with PD-1 blockade had significantly decreased PD-1+ T cells and decreased ratios of T-regulatory cells to conventional CD4 and CD8 T cells. In conclusion, HSCT after PD-1 blockade appears feasible with a low rate of relapse. However, there may be an increased risk of early immune toxicity, which could reflect long-lasting immune alterations triggered by prior PD-1 blockade.
•HSCT after PD-1 blockade is feasible, although may be associated with increased early immune toxicity.•PD-1 blockade may cause persistent depletion of PD1+ T cells and alterations in T-cell differentiation impacting subsequent treatment.
Low-dose daily Interleukin-2 (IL-2) therapy expands regulatory T cells (Treg) in vivo in patients with chronic graft-versus-host disease (cGvHD). Clinical improvement, primarily partial response, is ...seen in 50-60% of those treated. We hypothesized that infusion of fresh Tregs prior to low dose IL-2 may augment clinical and immune responses. In a Phase 1 study, we administered fresh Treg-enriched lymphocyte infusions (Treg-DLI) obtained from the original stem cell donor, without in vitro stimulation or expansion, followed by daily low-dose IL-2 as therapy for cGvHD. Methods: 24 recipients of 7-8/8 HLA-matched allogeneic hematopoietic stem cell transplants with steroid-refractory cGvHD on stable immune suppression received escalating doses Treg-DLI: 5 each at 0.1, 0.3, or 1x106 cells/kg plus an expansion cohort of 9 at 1x106/kg. Non-mobilized leukapheresis products were CD8/CD19 co-depleted, then positively selected for CD25+ Tregs via the CliniMACS system (Miltenyi). IL-2 was administered at 1x106 U/m2/day for 8 weeks after Treg-DLI infusion. Subjects: The first 22 subjects had a median age of 48 (range, 23-68), median of 3 cGvHD sites (range, 1-6), and median of 2 prior therapies in addition to steroids (range, 0 -5). Four had prior IL-2 exposure. Median time from cGvHD diagnosis to enrollment was 26 months (range, 7- 84). Manufacturing: All Treg-DLI products met release criteria. Median CD8 and CD19 log-depletions were 4.5 and 4.6, respectively. Released products contained a median of 92.6% CD4+CD25++ cells and 88.2% CD4+CD25+CD27low Tregs, a 24.8-fold enrichment (range, 12.7-43.4). At the highest targeted dose level, a median of 0.98x106 cells/kg were infused (range, 0.64-1.0). Safety: In the 21 subjects evaluable for safety, there were no infusional AEs, no DLTs, and no cases of relapse. 2 subjects underwent a 50% dose reduction of IL-2 for Grade 2 flu-like symptoms and injection site irritation, at provider discretion. Grade 3 AEs possibly related to therapy were dyspnea and edema in 1 subject. At median follow-up of 26 months, 17 subjects are alive. 2-year survival is 82% (95% CI, 54-95%). Response: At 8 weeks, 2 of 5 subjects in Cohort 1 (1 partial response (PR), 1 stable disease with minor response (SDMR)), 4 of 5 subjects in Cohort 2 (2 PR, 2 SDMR), and 8 of 11 subjects at the highest cell dose (1 complete response(CR), 3 PR, 4 SDMR) demonstrated clinical benefit. Response rate (CR+PR) was 33%; clinical benefit rate (CR+PR+SDMR) was 67%. 11 subjects opted to continue IL-2, for a median of 14.5 months (range, 3-39). Of 4 subjects previously on IL-2 alone, 2 were responders (CR, PR) and 1 each had mixed response and SDMR, respectively. Interestingly, 3 subjects with SDMR at 8 weeks manifested PR on extended IL-2. No impact of Treg-DLI dose was observed. At 6 months, median prednisone dose was 15mg (range, 5-40), down from 30mg daily at baseline (range, 5-80). Correlative Immunology: Treg counts and the ratio of Treg to conventional T cells (Tcon) rose over time. Higher Treg:Tcon ratios at baseline (p=0.08) and at 2 weeks into therapy (p=0.03) were associated with clinical response (Figure 1). Breadth of diversity within Treg T-cell receptor β (TCRβ) sequences (Shannon's entropy) and selective expansions of individual Treg clones (clonality) were low in cGvHD subjects at baseline, as compared to normal donors (p<0.01) and to Treg-DLI products (p<0.001). Treg TCRβ entropy increased in cGvHD subjects after 8 weeks of therapy (p<0.05), and resulting entropy was comparable to normal donors (n=16, Figure 2). This normalization was independent of clinical response and persisted through 6 months (n=8). Treg TCRβ clonality trended upwards at 8 weeks but did not reach levels seen in normal donors. Entropy and clonality of CD8 and Tcon TCRβ populations in cGvHD subjects were comparable to normal donors and did not change with therapy. Conclusions: GMP-grade manufacture ofCD25+ enriched Treg-DLI is feasible. In combination with low-dose IL-2, Treg-DLI is safe and has clinical efficacy in steroid-refractory cGvHD, including in those with inadequate responses to IL-2 alone. TCRβ population diversity specifically in Tregs is normalized with therapy. The incremental benefit of adding Treg-DLI to therapy with IL-2 alone on clinical responses, normalization of immunologic diversity, and functional suppressive activity, including at timepoints beyond 8 weeks, is the subject of ongoing analyses.
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Nikiforow:Kite Therapeutics: Membership on an entity's Board of Directors or advisory committees. Armand:Affimed: Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Infinity: Consultancy; Otsuka: Research Funding; Merck & Co., Inc.: Consultancy, Research Funding; Sequenta/Adaptive: Research Funding; Genmab: Consultancy; Roche: Research Funding; Tensha: Research Funding; Pfizer: Consultancy, Research Funding; Sigma Tau: Research Funding. Antin:Gentium SpA/Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Koreth:Millennium Pharmaceuticals: Research Funding; Prometheus Labs: Research Funding; Kadmon Corp: Membership on an entity's Board of Directors or advisory committees; Takeda Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Amgen Inc.: Consultancy.
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.
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Nikiforow: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.
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