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Purpose: Metastatic breast cancer (MBC) response to allogeneic lymphocytes requires donor T-cell engraftment and is limited by graft-versus-host disease (GVHD). In mice, type-II–polarized T cells promote engraftment and modulate GVHD, whereas type-I–polarized T cells mediate more potent graft-versus-tumor (GVT) effects. This phase I translational study evaluated adoptive transfer of ex vivo costimulated type-I/type-II (T1/T2) donor T cells with T-cell–depleted (TCD) allogeneic stem cell transplantation (AlloSCT) for MBC.Experimental Design: Patients had received anthracycline, taxane, and antibody therapies, and been treated for metastatic disease and a human leukocyte antigen (HLA)-identical–sibling donor. Donor lymphocytes were costimulated ex vivo with anti-CD3/anti-CD28 antibody–coated magnetic beads in interleukin (IL)-2/IL-4–supplemented media. Patients received reduced intensity conditioning, donor stem cells and T1/T2 cells, and monitoring for toxicity, engraftment, GVHD, and tumor response; results were compared with historical controls, identically treated except for T1/T2 product infusions.Results: Mixed type-I/type-II CD4+ T cells predominated in T1/T2 products. Nine patients received T1/T2 cells at dose level 1 (5 × 106 cells/kg). T-cell donor chimerism reached 100% by a median of 28 days. Seven (78%) developed acute GVHD. At day +28, five patients had partial responses (56%) and none had MBC progression; thereafter, two patients had continued responses. Donor T-cell engraftment and tumor responses appeared faster than in historical controls, but GVHD rates were similar and responders progressed early, often following treatment of acute GVHD.Conclusion: Allogeneic T1/T2 cells were safely infused with TCD-AlloSCT, appeared to promote donor engraftment, and may have contributed to transient early tumor responses. Clin Cancer Res; 17(21); 6878–87. ©2011 AACR.

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Purpose: Multiple myeloma is a usually incurable malignancy of plasma cells. New therapies are urgently needed for multiple myeloma. Adoptive transfer of chimeric antigen receptor (CAR)–expressing T cells is a promising new therapy for hematologic malignancies, but an ideal target antigen for CAR-expressing T-cell therapies for multiple myeloma has not been identified. B-cell maturation antigen (BCMA) is a protein that has been reported to be selectively expressed by B-lineage cells including multiple myeloma cells. Our goal was to determine if BCMA is a suitable target for CAR-expressing T cells.Experimental Design: We conducted an assessment of BCMA expression in normal human tissues and multiple myeloma cells by flow cytometry, quantitative PCR, and immunohistochemistry. We designed and tested novel anti-BCMA CARs.Results: BCMA had a restricted RNA expression pattern. Except for expression in plasma cells, BCMA protein was not detected in normal human tissues. BCMA was not detected on primary human CD34+ hematopoietic cells. We detected uniform BCMA cell-surface expression on primary multiple myeloma cells from five of five patients. We designed the first anti-BCMA CARs to be reported and we transduced T cells with lentiviral vectors encoding these CARs. The CARs gave T cells the ability to specifically recognize BCMA. The anti-BCMA-CAR–transduced T cells exhibited BCMA-specific functions including cytokine production, proliferation, cytotoxicity, and in vivo tumor eradication. Importantly, anti-BCMA-CAR–transduced T cells recognized and killed primary multiple myeloma cells.Conclusions: BCMA is a suitable target for CAR-expressing T cells, and adoptive transfer of anti-BCMA-CAR–expressing T cells is a promising new strategy for treating multiple myeloma. Clin Cancer Res; 19(8); 2048–60. ©2013 AACR.

PDF file - 90K, Anti-bcma2 transduced T cells from Myeloma Patient 4 produced TNF and IFN in a BCMA-specific manner.BCMA protein in solution does not block recognition of BCMA+ target cells by anti-bcma2-transduced T cells in vitro.Human BCMA protein was detected by ELISA in the serum of NSG mice with RPMI8226 tumors, but human BCMA protein was not detected in the serum of a control mouse that did not have a tumor.

Supplementary Methods, Figure Legends 1-8 from Tumor-Evoked Regulatory B Cells Promote Breast Cancer Metastasis by Converting Resting CD4+ T Cells to T-Regulatory Cells

Supplementary Figure 2 from Tumor-Evoked Regulatory B Cells Promote Breast Cancer Metastasis by Converting Resting CD4+ T Cells to T-Regulatory Cells

PDF file - 126K, Activity of tBregs is efficiently blocked by CpG-ODN

PDF file - 119K, Targeted tBreg inactivation with BLC-arp/CpG-ODN

PDF file - 172K, tBregs express low levels of 4-1BBL

PDF file - 96K, In vivo inactivation of tBregs with BLC-arp/CpG-ODN induces antitumor T cell responses

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Supplementary Figure 8 from Tumor-Evoked Regulatory B Cells Promote Breast Cancer Metastasis by Converting Resting CD4+ T Cells to T-Regulatory Cells

Supplementary Figure 6 from Tumor-Evoked Regulatory B Cells Promote Breast Cancer Metastasis by Converting Resting CD4+ T Cells to T-Regulatory Cells

Supplementary Figure 3 from Tumor-Evoked Regulatory B Cells Promote Breast Cancer Metastasis by Converting Resting CD4+ T Cells to T-Regulatory Cells

Supplementary Figure 7 from Tumor-Evoked Regulatory B Cells Promote Breast Cancer Metastasis by Converting Resting CD4+ T Cells to T-Regulatory Cells

Supplementary Figure 5 from Tumor-Evoked Regulatory B Cells Promote Breast Cancer Metastasis by Converting Resting CD4+ T Cells to T-Regulatory Cells

PDF file - 206K, Regulatory activity is mostly retained in human tBregs expressing low levels of CD20

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Supplementary Figure 4 from Tumor-Evoked Regulatory B Cells Promote Breast Cancer Metastasis by Converting Resting CD4+ T Cells to T-Regulatory Cells

Background: Despite advances in therapy of Hodgkin's lymphoma (HL), a proportion of patients will not respond or relapse. The authors had previously identified CD25, IL-2Rα, as a target for systemic radioimmunotherapy of HL since most normal cells do not express CD25, but it is expressed by a minority of Hodgkin/Reed-Sternberg (HRS) cells and most Tregs rosetting around HRS cells. Study Design and Treatment: This was a single institution, nonrandomized, open-label phase I/II trial of radiolabeled (90)Y-daclizumab, an anti-CD25 monoclonal antibody, BEAM (carmustine, etoposide, cytarabine, and melphalan) conditioning treatment followed by autologous hematopoietic stem cell transplant (ASCT). Four patients with refractory and relapsed HL were treated in this trial with 3 patients receiving a single dose of 564.6–574.6 MBq (90)Y-daclizumab and the fourth patient receiving two doses of 580.9–566.1 MBq (90)Y-daclizumab followed by high-dose chemotherapy and ASCT. Results: All 4 evaluable patients treated with (90)Y-daclizumab obtained complete responses (CRs) that are ongoing 4.5–7 years following their stem cell transplant. The spectrum and severity of adverse events were mild and more importantly none of the patients, including several with multiple therapies before this treatment, developed the myelodysplastic syndrome. Discussion: Targeting by daclizumab was not directed primarily at tumor cells, but rather the nonmalignant CD25-expressing T cells adjacent to the HRS cells and (90)Y-daclizumab provided strong enough β emissions to kill CD25-negative tumor cells at a distance by a crossfire effect. Furthermore, the strong β irradiation killed normal cells in the tumor microenvironment. Conclusions: (90)Y-daclizumab (anti-CD25), high-dose BEAM chemotherapy and ASCT was well tolerated and yielded sustained complete remissions in all 4 patients with recurrent HL patients who completed their treatment. Significance: Despite advances, a proportion of patients with HL will not have a CR to their initial treatment, and some with CRs will relapse. They demonstrated that the addition of (90)Y-daclizumab into the preconditioning regimen for refractory and relapsed HL patients with high-dose BEAM chemotherapy and ASCT provided sustained CRs in the 4 patients studied. Two of these patients were highly refractory to multiple prior treatments with bulky disease at entry into this study, including 1 patient who never entered a remission and had failed 6 different therapeutic regimens. Despite the small number of patients treated in this study, the sustained clinical benefit in these patients indicates a highly effective treatment. The daclizumab was directed primarily not at HRS cells themselves but toward nonmalignant T cells rosetting around malignant cells. (90)Y provided strong β emissions that killed antigen nonexpressing tumor cells at a distance by a crossfire effect. Furthermore, the strong β radiation killed normal cells in the tumor microenvironment that nurtured the malignant cells in the lymphomatous mass. The present study supports expanded analysis of (90)Y-daclizumab as part of the regimen of ASCT in patients with refractory and relapsed HL.

Anti-CD19 chimeric antigen receptor (CAR)-expressing T cells are an effective treatment for B-cell lymphoma, but often cause neurologic toxicity. We treated 20 patients with B-cell lymphoma on a phase I, first-in-human clinical trial of T cells expressing the new anti-CD19 CAR Hu19-CD828Z ( NCT02659943 ). The primary objective was to assess safety and feasibility of Hu19-CD828Z T-cell therapy. Secondary objectives included assessments of blood levels of CAR T cells, anti-lymphoma activity, second infusions and immunogenicity. All objectives were met. Fifty-five percent of patients who received Hu19-CD828Z T cells obtained complete remission. Hu19-CD828Z T cells had clinical anti-lymphoma activity similar to that of T cells expressing FMC63-28Z, an anti-CD19 CAR tested previously by our group, which contains murine binding domains and is used in axicabtagene ciloleucel. However, severe neurologic toxicity occurred in only 5% of patients who received Hu19-CD828Z T cells, whereas 50% of patients who received FMC63-28Z T cells experienced this degree of toxicity (P = 0.0017). T cells expressing Hu19-CD828Z released lower levels of cytokines than T cells expressing FMC63-28Z. Lower levels of cytokines were detected in blood from patients who received Hu19-CD828Z T cells than in blood from those who received FMC63-28Z T cells, which could explain the lower level of neurologic toxicity associated with Hu19-CD828Z. Levels of cytokines released by CAR-expressing T cells particularly depended on the hinge and transmembrane domains included in the CAR design. In a first-in-human, phase I trial in patients with B-cell lymphoma, CD19 CAR T cells with fully human binding domains exhibit lower neurologic toxicity, but similar clinical activity, to previously tested CD19 CAR T cells with murine binding domains.

Allogeneic blood or marrow transplantation (BMT) is a potentially curative therapy for patients with primary immunodeficiency (PID). Safe and effective reduced-intensity conditioning (RIC) approaches that are associated with low toxicity, use alternative donors, and afford good immune reconstitution are needed to advance the field. Twenty PID patients, ranging in age from 4 to 58 years, were treated on a prospective clinical trial of a novel, radiation-free and serotherapy-free RIC, T-cell-replete BMT approach using pentostatin, low-dose cyclophosphamide, and busulfan for conditioning with post-transplantation cyclophosphamide-based graft-versus-host-disease (GVHD) prophylaxis. This was a high-risk cohort with a median hematopoietic cell transplantation comorbidity index of 3. With median follow-up of survivors of 1.9 years, 1-year overall survival was 90% and grade III to IV acute GVHD-free, graft-failure-free survival was 80% at day +180. Graft failure incidence was 10%. Split chimerism was frequently observed at early post-BMT timepoints, with a lower percentage of donor T cells, which gradually increased by day +60. The cumulative incidences of grade II to IV and grade III to IV acute GVHD (aGVHD) were 15% and 5%, respectively. All aGVHD was steroid responsive. No patients developed chronic GVHD. Few significant organ toxicities were observed. Evidence of phenotype reversal was observed for all engrafted patients, even those with significantly mixed chimerism (n = 2) or with unknown underlying genetic defect (n = 3). All 6 patients with pre-BMT malignancies or lymphoproliferative disorders remain in remission. Most patients have discontinued immunoglobulin replacement. All survivors are off immunosuppression for GVHD prophylaxis or treatment. This novel RIC BMT approach for patients with PID has yielded promising results, even for high-risk patients.

NK effector cells expressing a CAR construct may be used to target T-lineage markers. In this work, we compared the activity of a NK-specific CAR-NK and a CAR-T framework when expressed on NK effector cells to target CD3 and CD5 in T-cell malignancies. Our results show that CD3-CAR-T is more active than CD5-CAR-T to eliminate malignant T cells in vitro, however, CD3-CAR-T were less efficient to eliminate tumor cells in vivo, while CD5-CAR-T had antitumor activity in a diffuse xenograft model. Lack of in vivo efficacy correlated with downregulation of CD3 levels in target T cells after coculture with CD3-CAR effector cells. The CAR-NK framework greatly improved the efficacy of CARs leading to increased degranulation, cytokine secretion and elimination of the tumor xenograft by CD5-CAR-NK effector cells. Finally, all CAR constructs were similarly effective to eliminate malignant T cells in vitro. Our results show that the NK-CAR framework improves the activity of CARs in NK cells and that CD5 would be a better target than CD3 for T-cell malignancies, as dynamic downregulation of target expression may affect in vivo efficacy.

The chemokine receptor CCR9 equips T cells with the ability to respond to CCL25, a chemokine that is highly expressed in the thymus and the small intestine (SI). Notably, CCR9 is mostly expressed on CD8 but not on CD4 lineage T cells, thus imposing distinct tissue tropism on CD4 and CD8 T cells. The molecular basis and the consequences for such a dichotomy, however, have not been fully examined and explained. Here, we demonstrate that the forced expression of CCR9 interferes with the tissue trafficking and differentiation of CD4 T cells in SI intraepithelial tissues. While CCR9 overexpression did not alter CD4 T cell generation in the thymus, the forced expression of CCR9 was detrimental for the proper tissue distribution of CD4 T cells in the periphery, and strikingly also for their terminal differentiation in the gut epithelium. Specifically, the differentiation of SI epithelial CD4 T cells into immunoregulatory CD4

Alloreactivity forms the basis of allogeneic hematopoietic cell transplantation (HCT), with donor derived T cell response to recipient antigens mediating clinical responses either in-part or entirely. These encompass the different manifestations of graft vs. host disease (GVHD), infection risk as well as disease response. Whilst the latter is contingent upon disease biology and thus may be less predictable, the former two are more likely to be directly proportional to the magnitude of donor derived T cell recovery. Herein we explore the quantitative aspects of immune cell recovery following allogeneic HCT and clinical outcomes in two cohorts of HLA matched allograft recipients who received rabbit anti-thymocyte globulin (ATG) on different schedules (days -9 to -7 vs. -3 to -1). Monocyte as well as donor derived T cell (ddCD3) recovery was superior in those given ATG early in their course (days -9/-7). This difference was related to a more rapid rate of ddCD3 recovery, largely driven by CD3+/8+ cells in the first month following transplantation. Early monocyte recovery was associated with later T cell recovery and improved survival. In contrast rapid and early ddCD3 expansion out of proportion to monocyte recovery was associated with a high likelihood of acute GVHD and poor survival. This analytic methodology demonstrates that modelling ‘early-term immune reconstitution' following HCT yields insights that may be useful in management of post-transplant immunosuppression and adaptive cellular therapy to optimize clinical outcomes.

Post-transplantation cyclophosphamide (PTCy) recently has had a marked impact on human allogeneic hematopoietic cell transplantation (HCT). Yet, our understanding of how PTCy prevents graft-versus-host disease (GVHD) largely has been extrapolated from major histocompatibility complex (MHC)-matched murine skin allografting models that were highly contextual in their efficacy. Herein, we developed a T-cell-replete, MHC-haploidentical, murine HCT model (B6C3F1→B6D2F1) to test the putative underlying mechanisms: alloreactive T-cell elimination, alloreactive T-cell intrathymic clonal deletion, and suppressor T-cell induction. In this model and confirmed in four others, PTCy did not eliminate alloreactive T cells identified using either specific Vβs or the 2C or 4C T-cell receptors. Furthermore, the thymus was not necessary for PTCy's efficacy. Rather, PTCy induced alloreactive T-cell functional impairment which was supported by highly active suppressive mechanisms established within one day after PTCy that were sufficient to prevent new donor T cells from causing GVHD. These suppressive mechanisms included the rapid, preferential recovery of CD4+CD25+Foxp3+ regulatory T cells, including those that were alloantigen-specific, which served an increasingly critical function over time. Our results prompt a paradigm-shift in our mechanistic understanding of PTCy. These results have direct clinical implications for understanding tolerance induction and for rationally developing novel strategies to improve patient outcomes.

Invariant NKT (iNKT) cells are thymus-generated innate-like T cells, comprised of three distinct subsets with divergent effector functions. The molecular mechanism that drives the lineage trifurcation of immature iNKT cells into the NKT1, NKT2, and NKT17 subsets remains a controversial issue that remains to be resolved. Because cytokine receptor signaling is necessary for iNKT cell generation, cytokines are proposed to contribute to iNKT subset differentiation also. However, the precise roles and requirements of cytokines in these processes are not fully understood. Here, we show that IL-2Rβ, a nonredundant component of the IL-15 receptor complex, plays a critical role in both the development and differentiation of thymic iNKT cells. While the induction of IL-2Rβ expression on postselection thymocytes is necessary to drive the generation of iNKT cells, surprisingly, premature IL-2Rβ expression on immature iNKT cells was detrimental to their development. Moreover, while IL-2Rβ is necessary for NKT1 generation, paradoxically, we found that the increased abundance of IL-2Rβ suppressed NKT1 generation without affecting NKT2 and NKT17 cell differentiation. Thus, the timing and abundance of IL-2Rβ expression control iNKT lineage fate and development, thereby establishing cytokine receptor expression as a critical regulator of thymic iNKT cell differentiation.

Background: Bronchiolitis obliterans syndrome (BOS) is a severe manifestation of chronic graft-versus-host disease (cGVHD) following hematopoietic cell transplantation (HCT). Montelukast interrupts cysteinyl leukotriene activity and may diminish the activation and homing of cells to bronchioles and subsequent fibrosis. Methods: We performed a prospective phase II trial to test whether montelukast altered lung decline for patients with BOS after HCT in a single arm, open-label, multi-institutional study with primary endpoints of: i) FEV1 stability or improvement ( 5% FEV1 improvement, clinically meaningful improvements were seen in the Lee scores of breathing, energy, and mood, and in the Human Activity Profile, and 6MW for

BACKGROUND: Bronchiolitis obliterans syndrome (BOS) is a severe manifestation of chronic graft-versus-host disease (cGVHD) following hematopoietic cell transplantation (HCT). Montelukast interrupts cysteinyl leukotriene activity and may diminish the activation and homing of cells to bronchioles and subsequent fibrosis. OBJECTIVE: We performed a prospective phase II trial to test whether montelukast altered lung decline for patients with BOS after HCT. STUDY DESIGN: We performed a single arm, open-label, multi-institutional study with primary endpoints of: i) FEV1 stability or improvement (

Lymphopenia-induced homeostatic proliferation (LIP) is a critical mechanism for restoring T cell immunity upon lymphodepleting insults or infections. LIP is primarily driven by homeostatic cytokines, such as IL-7 and IL-15, but not all T cells respond with the same efficiency to homeostatic proliferative cues. Although CD8 T cells vigorously proliferate under lymphopenic conditions, naive CD4 T cells are substantially impaired in their response to homeostatic cytokines, and they fail to fully expand. In this study, we show that the availability of IL-2Rβ (CD122), which is a receptor subunit shared by IL-2 and IL-15, affects both the cytokine responsiveness and the LIP of naive CD4 T cells in the mouse. The enumeration of surface IL-2Rβ molecules on murine naive CD4 and naive CD8 T cells revealed a 5-fold difference in IL-2Rβ abundance. Notably, it was the limited availability of IL-2Rβ that impaired CD4 T cell responsiveness to IL-15 and suppressed their LIP. As such, forced IL-2Rβ expression on CD4 T cells by transgenesis bestowed IL-15 responsiveness onto naive CD4 T cells, which thus acquired the ability to undergo robust LIP. Collectively, these results identify IL-2Rβ availability as a new regulatory mechanism to control cytokine responsiveness and the homeostatic proliferation of murine CD4 T cells.

The only current curative treatment for chronic lymphocytic leukemia (CLL) is allogenic hematopoietic stem cell transplantation. Chimeric antigen receptor treatment targeting CD19 for CLL achieved some complete responses, suggesting the need for alternative or combinational therapies to achieve a more robust response. In this work, we evaluated CAR-T cells specific for Siglec-6, an antigen expressed in CLL, as a novel CAR-T cell treatment for CLL. We found that detection of SIGLEC6 mRNA and Siglec-6 protein is highly restricted to placenta and immune cells in other tissues and it is not expressed in hematopoietic stem cells. We generated CAR-T cells specific for Siglec-6 based on the sequence of the fully human anti-Siglec-6 antibody (JML1), which was identified in a CLL patient that was cured after allo-hematopoietic stem cell transplantation (alloHSCT), and observed that it specifically targeted CLL cells in vitro and in a xenograft mouse model. Interestingly, a short hinge region increased the activity of CAR-T cells to target cells expressing higher Siglec-6 levels but similarly targeted CLL cells expressing lower Siglec-6 levels in vitro and in vivo. Our results identify a novel CAR-T cell therapy for CLL and establish Siglec-6 as a possible target for immunotherapy.

Understanding the homeostatic mechanism of invariant natural killer T (ιNKT) cells is a critical issue in ιNKT cell biology. Because IL-15 is required for the thymic generation of ιNKT cells, IL-15 has also been considered necessary for the homeostasis of peripheral ιNKT cells. Here, we delineated the in vivo cytokine requirement for ιNKT cells, and we came to the surprising conclusion that IL-7, not IL-15, is the homeostatic cytokine for ιNKT cells. Employing a series of experimental mouse models in which the availability of IL-7 or IL-15 were manipulated in peripheral tissues, either by genetic tools or by adult thymectomy and cytokine pump installation, we demonstrate that the abundance of IL-7, and not IL-15, limits the size of the peripheral ιNKT cell pool. These results establish a new cytokine requirement for ιNKT cells and reveal a previously unappreciated competition for IL-7 between ιNKT cells and conventional αβ T cells.

Understanding the homeostatic mechanism of invariant natural killer T (iNKT) cells is a critical issue in iNKT cell biology. Because interleukin (IL)-15 is required for the thymic generation of iNKT cells, IL-15 has also been considered necessary for the homeostasis of peripheral iNKT cells. Here, we delineated the in vivo cytokine requirement for iNKT cells, and we came to the surprising conclusion that IL-7, not IL-15, is the homeostatic cytokine for iNKT cells. Employing a series of experimental mouse models where the availability of IL-7 or IL-15 was manipulated in peripheral tissues, either by genetic tools or by adult thymectomy and cytokine pump installation, we demonstrate that the abundance of IL-7, and not IL-15, limits the size of the peripheral iNKT cell pool. These results redefine the cytokine requirement for iNKT cells and indicate competition for IL-7 between iNKT and conventional αβ T cells.

Background: Post-transplantation cyclophosphamide (PTCy) is widely used for graft-versus-host-disease (GVHD) prophylaxis after hematopoietic cell transplantation (HCT). However, the standard PTCy dose (50 mg/kg/day) and timing (given on days +3 and +4) were largely extrapolated from murine skin allografting models, and these have never been rigorously tested to determine if they are optimal. We have shown in murine HCT models that an intermediate PTCy dose of 25 mg/kg/day on days +3/+4 is superior at preventing severe GVHD compared with 50 mg/kg/day on days +3/+4 (Wachsmuth et al., JCI. 2019). Furthermore, PTCy 25 mg/kg/day given on day +4 alone is equivalent to 25 mg/kg/day given on days +3/+4 at preventing severe GVHD (Wachsmuth et al. BBMT. 2019). Thus, the standard clinical dosing may be higher than necessary and potentially come at the cost of increased toxicity, delayed engraftment, and impaired immune reconstitution. Methods: This is a single institutional prospective phase I/II study (NCT03983850) to de-escalate PTCy exposure for adult patients with hematologic malignancies. All patients received myeloablative conditioning with IV daily busulfan/fludarabine, HLA-haploidentical bone marrow, and GVHD prophylaxis with PTCy (dose and timing based on Dose Level (DL)), mycophenolate mofetil (days +5 to +35), and sirolimus (days +5 to +80). The first 5 patients received PTCy 50 mg/kg/day on days +3/+4 (standard dosing, Dose Level 1) for comparative data. This was followed by a 3+3 dose de-escalation design testing 25 mg/kg/day on days +3/+4 (experimental, Dose Level 2) and 25 mg/kg on day +4 only (experimental, Dose Level 3), followed by a phase II expansion cohort at the better experimental dose level. The primary endpoint and dose limiting toxicity for the dose de-escalation was grade III-IV acute GVHD. Results: Phase I enrolled 19 patients, and the phase II expansion has enrolled 13 of 14 patients, 9 of whom have sufficient follow-up (60 days) to be considered evaluable for the primary endpoint; median follow-up of survivors evaluable for the primary endpoint is 288 (range 60-676) days. Patient and disease characteristics and outcomes for these patients are summarized in Table 1. No grade III-IV acute GVHD was seen at either experimental DL, and there have been no cases of grade II-IV acute GVHD at DL2 (Figure 1). Based on more reliable early engraftment (see below) but less intense and shorter duration of engraftment fevers, DL2 was taken to phase II. Engraftment was faster at the experimental DLs but was most consistent with DL2. Median neutrophil engraftment was at day 14 at DL2 compared with day 19 at the standard dosing (p=0.0004) (Figure 2A). Median platelet engraftment was at day 23 at DL2 compared with day 33 at the standard dosing (p=0.026) (Figure 2B). Correspondingly, transfusional requirements were lessened with DL2 (Figure 2C-D). Engraftment syndrome, manifesting as fever, rash, and mild transaminitis, occurred in 1 patient at DL2 and 2 patients at DL3, but resolved rapidly without intervention in all cases. Primary graft failure was seen in 1 patient at DL2, and relapse prior to engraftment was seen in 1 patient at DL3. Mucositis was less severe and shorter in duration for both experimental DLs when compared with standard PTCy dosing (Figure 2E). CMV reactivation requiring pre-emptive therapy was less frequently seen after lower PTCy dosing (Figure 3A). Symptomatic BK virus-associated cystitis in at-risk patients was shorter in duration for patients receiving lower PTCy; median duration was 74 days for the standard DL versus 26 days at DL2 and 7 days at DL3 (Figure 3B). At DL2, there have been 3 cases to date of chronic GVHD requiring systemic immunosuppression among the 13 engrafting patients with at least 100 days of follow-up. Conclusions: De-escalating PTCy exposure is feasible and effective in maintaining protection against severe acute GVHD while promoting more rapid engraftment and less early post-transplant toxicity. Two-day dosing of 25 mg/kg/day PTCy appears to allow for more consistent early engraftment and protection against protracted engraftment fevers compared with day +4 only. Longer follow-up and comparative studies are needed to understand if de-escalated PTCy is superior to the standard dosing schedule. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare. OffLabel Disclosure: The use of cyclophosphamide as graft-versus-host disease prophylaxis

A rapid and selective method for the quantitation of neurotransmitters, l-Glutamic acid (GA) and γ–Aminobutyric acid (GABA), was developed and validated using gas chromatography-tandem mass spectrometry (GC–MS/MS). The novel method utilized a rapid online hot GC inlet gas phase sample derivatization and fast GC low thermal mass technology. The method calibration was linear from 0.5 to 100 μg/mL, with limits of detections of 100 ng/mL and 250 ng/mL for GA and GABA, respectively. The method was used to investigate the effects of deletion of organic anion transporter 1 (Oat1) or Oat3 on murine CNS levels of GA and GABA at 3 and 18 mo of age, as compared to age matched wild-type (WT) animals. Whole brain concentrations of GA were comparable between WT, Oat1(−/−), and Oat3(−/−) 18 mo at both 3 and 18 mo of age. Similarly, whole brain concentrations of GABA were not significantly altered in either knockout mouse strain at 3 or 18 mo of age, as compared to WT. These results indicate that the developed GC–MS/MS method provides sufficient sensitivity and selectivity for the quantitation of these neurotransmitters in mouse brain tissue. Furthermore, these results suggest that loss of Oat1 or Oat3 function in isolation does not result in significant alterations in brain tissue levels of GA or GABA.

Predicting the duration of systemic therapy in patients with chronic graft-versus-host disease (cGVHD) is of critical clinical importance when counseling patients and for treatment planning. cGVHD characteristics associated with this outcome have not been studied in severely affected patients. The National Institutes of Health (NIH) cGVHD scoring provides a standardized set of organ severity measures that could represent clinically useful and reproducible predictive characteristics. We analyzed 227 previously treated patients most with moderate (n = 54) or severe (n = 170) cGVHD defined by NIH criteria who were prospectively enrolled in a natural history protocol (NCT00092235). Patients received a median of 4 prior systemic therapy regimens and were seen at the NIH for a single time-point visit and were then monitored for survival and ability to discontinue cGVHD systemic therapy. With a median follow-up of 71.1 months, the cumulative incidence of systemic therapy discontinuation was 9.5% (95% confidence interval, 6.0% to 13.9%) at 2 years and 27.7% (95% confidence interval, 20.9% to 34.8%) by 5 years after the initial visit. Factors associated with a higher incidence of immunosuppression discontinuation included lower NIH global severity (P = .019) and lung (P = .030) scores and less extensive deep sclerosis (37% body surface area, P = .024). Lower patient- and clinician-reported 0 to 10 severity NIH scores and noncyclosporine prophylaxis regimens were also associated with higher incidence of immunosuppression discontinuation (P .05). In conclusion, we found low success rates for immune suppression discontinuation in previously treated patients who were severely affected with cGVHD. NIH scoring and clinical measures provide new standardized disease-specific tools to predict discontinuation of systemic therapy.