ALBERT

Description: Abstract 4476 Chronic graft-versus-host disease (cGVHD) is a serious and frequent complication of allogeneic hematopoietic stem cell transplantation (HSCT). It is not known why some patients develop cGVHD and others do not, but identifying predictive biomarkers would facilitate the development of pre-emptive therapeutic strategies. Investigation into the pathophysiology of cGVHD has revealed different patterns of immune reconstitution in patients who develop cGVHD following HSCT. This suggests that the development of cGVHD occurs as a result of altered immune homeostasis and the inability to establish B and T cell tolerance. We prospectively examined 88 patients with hematologic malignancies following allogeneic HSCT at the Brigham and Woman's Hospital/Dana-Farber Cancer Institute between the years 2004 and 2008 and measured plasma cytokines known to modulate effector T cell, regulatory T cell, and B cell homeostasis and function. Seventy-six patients (86%) received reduced intensity conditioning; 84 (95%) received filgrastim-mobilized peripheral blood stem cells. Median follow-up for all patients was 5 years (range 2.9 to 7.3 years). Fifty-nine percent developed cGVHD. Plasma samples were collected at 1, 3, 6, and 12 months after HSCT and multiplex Luminex bead assays were used to measure levels of the following cytokines: Interferon-γ, IL-2, IL-7, IL-1β, IL-12, TNFα, IL-4, IL-5, IL-6, IL-10, and GM-CSF. Cytokine levels in patients who developed cGVHD were compared to patients who did not develop cGVHD. Results are shown in figure 1. Significantly higher levels of IL-4, IL-6, IL-12, and IL-1β were observed at 1 and/or 3 months after transplantation in patients who subsequently developed cGVHD. IL-4 and IL-6 are characteristic of T-helper-2 (TH2) cellular and humoral immune responses previously associated with cGVHD and fibrogenesis. IL-12 and IL-1β have heterogeneous functions including stimulating interferon-γ and TNFα production, enhancing cytotoxic and helper T cells, and promotion of autoimmunity. IL-1α also stimulates IL-6 production and activates fibroblasts. Finally, GM-CSF was significantly elevated in patients who do not develop cGVHD at 1 year following HSCT (2.5 v 1.46 pg/mL, p=0.017). As shown in figure 1, we observed a general pattern for many cytokines, including IFNγ, IL-2, IL-4, IL-5, IL-6, and IL-1β, in which these cytokines are initially similar in both groups, or lower in patients without cGVHD, but gradually increase over time in patients who do not develop cGVHD. In contrast, cytokine levels remain stable in patients with cGVHD and are generally lower 1 year after transplantation in these patients. These late differences may reflect immune suppressive therapies as well as persistent abnormalities of immune homeostasis. Taken together, these results support the hypothesis that alterations in immune homeostasis early after allogeneic HSCT are significantly associated with the subsequent development of cGVHD. Manipulation of the cytokine environment early after HSCT can modulate immune homeostasis and may be of potential prophylactic value. Administration of homeostatic cytokines late after HSCT may also have therapeutic benefit. Disclosures: No relevant conflicts of interest to declare.

Description: Key Points 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.

Description: 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. Disclosures 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.

Description: 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. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 3036 There is evidence that B cells play a role in the development of chronic graft-versus-host disease (cGVHD), a major cause of non-relapse morbidity and mortality following hematopoeitic stem cell transplantation (HSCT). Elevated levels of B cell activating factor (BAFF) relative to the number of circulating B cells have been associated with the development of cGVHD as well as its response to treatment. To better define the relationship between BAFF levels, the recovery of CD19 B cells, and BAFF/CD19 B cell ratios and cGVHD following HSCT, we prospectively analyzed 440 hematologic malignancy patients who underwent HSCT. Two hundred and fifty four patients (58%) underwent a reduced intensity HSCT; 375 (85%) received filgrastim-mobilized peripheral blood stem cells. Sixty-one percent developed cGVHD, with a median time to development of cGVHD of 221.5 days. Whole blood and plasma samples were collected at 1, 3, 6, and 12 months after HSCT and analyzed by flow cytometry and enzyme linked immunosorbent assay for CD19 B cells and BAFF levels respectively. Results in patients who developed cGVHD were compared to patients who did not develop cGVHD. At 1 month after HSCT, BAFF levels were significantly higher in the no cGVHD cohort compared to the cGVHD cohort (14.78 versus 11.31ng/ml, p=0.003)(see figure below). BAFF levels gradually fell in the no cGVHD cohort while remaining stable in the cGVHD cohort such that they were significantly lower in the no cGVHD cohort by 12 months after HSCT (5.63 versus 9.11ng/ml, p=0.018). In contrast, CD19 B cell count was similar between the two cohorts through 6 months after which point it was higher in the no cGVHD cohort (279.0 versus 119.0 cells/μL at 12 months, p=0.062). This correlated to a significantly decreased BAFF/CD19 B cell ratio in this cohort at 12 months after HSCT (0.03 versus 0.15, p=0.01). Prednisone is known to lower BAFF levels so each cohort was further analyzed based on the concurrent use of prednisone at the time of sample collection. BAFF levels were lower in patients who received prednisone at the time of analysis. BAFF levels fell sharply in both the cGVHD and no cGVHD cohorts on prednisone between 1 and 3 months after HSCT such that there was no significant difference between the two groups. However, within the group of patients who received prednisone at 3 months, patients who did not develop cGVHD had a significantly higher CD19 B cell count at this time than those who subsequently developed cGVHD (88.0 versus 20.5 cells/μL, p=0.037). These results suggest that patients who do not develop cGVHD achieve higher BAFF levels early after HSCT followed by more rapid B cell recovery, which in turn results in the normalization of BAFF levels and BAFF/CD19 B cell ratios. In contrast, B cell recovery is delayed and high BAFF/CD19 B cell ratios persist in patients who develop cGVHD. Disclosures: No relevant conflicts of interest to declare.

Description: 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. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Description: 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 (p3-fold (p4-fold (p

Description: Abstract 48 We recently reported on the use of the proteasome-inhibitor bortezomib as a novel strategy to control acute graft-versus-host-disease (GVHD). We evaluated bortezomib, tacrolimus and mini-methotrexate for GVHD prophylaxis after reduced-intensity conditioning (RIC) hematopoietic stem cell transplantation (HSCT) with unrelated donors mismatched at 1-2 HLA loci (-A, -B, -C, -DRB1). The regimen was safe, with zero non-relapse mortality. There was a 13% incidence of grade II-IV acute GVHD. We analyzed immune reconstitution in the bortezomib cohort (n=24) [Bort] vs. i) 8/8 matched-related donor (-A, -B, -C, -DRB1) RIC HSCT cohort (n=96) [MRD]; ii) 8/8 matched-unrelated donor RIC HSCT cohort (n=139) [MUD]; and iii) 1-2 HLA mismatched-unrelated donor RIC HSCT cohort (n=24) [MMUD]. Comparator groups received sirolimus, tacrolimus and mini-methotrexate GVHD prophylaxis. All received fludarabine/busulfan conditioning and G-CSF mobilized adult-donor peripheral blood stem cells. All groups had rapid hematopoietic engraftment and 〉90% median donor-chimerism by day +45 post-HSCT. They were not different with regards to age (p=0.53), sex (p=0.39) and acute grade II-IV GVHD (p=0.63). There was a difference in the proportion of good-risk disease: [Bort] (46%), [MRD] (21%), [MUD] (14%) and [MMUD] (21%) (p=0.005). Chronic GVHD was also different: [Bort] (41%), [MRD] (39%), [MUD] (53%) and [MMUD] (67%) (p=0.03). Peripheral blood cells incubated with directly conjugated monoclonal antibodies defining distinct lymphocyte populations were analyzed by flow cytometry. Lymphocyte subsets included T (CD3+, CD4+, CD8+) B (CD19+, CD20+) NK (CD56+CD16+) NK-T (CD56+CD3+) and regulatory-T (Treg; CD4+CD25+) cells. Unadjusted analyses revealed no significant differences in B, NK, NK-T or Treg recovery. In contrast, CD3+, CD4+ and CD8+ T-cell reconstitution was significantly higher in the bortezomib group, especially at 3 months post-HSCT. T-cell immune reconstitution parameters are improved after HLA-mismatched RIC HSCT and bortezomib-based GVHD prophylaxis. Importantly this can be accomplished with rapid hematopoietic engraftment and without increased GVHD incidence in patients without HLA-matched donors. Disclosures: Off Label Use: bortezomib for GVHD control.

Description: Key Points 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.