Proliferation tracing with single-cell mass cytometry optimizes generation of stem cell memory-like T cells.

Selective differentiation of naive T cells into multipotent T cells is of great interest clinically for the generation of cell-based cancer immunotherapies. Cellular differentiation depends crucially on division state and time. Here we adapt a dye dilution assay for tracking cell proliferative history through mass cytometry and uncouple division, time and regulatory protein expression in single naive human T cells during their activation and expansion in a complex ex vivo milieu. Using 23 markers, we defined groups of proteins controlled predominantly by division state or time and found that undivided cells account for the majority of phenotypic diversity. We next built a map of cell state changes during naive T-cell expansion. By examining cell signaling on this map, we rationally selected ibrutinib, a BTK and ITK inhibitor, and administered it before T cell activation to direct differentiation toward a T stem cell memory (TSCM)-like phenotype. This method for tracing cell fate across division states and time can be broadly applied for directing cellular differentiation

Comprehensive Immune Monitoring of Clinical Trials to Advance Human Immunotherapy.

The success of immunotherapy has led to a myriad of clinical trials accompanied by efforts to gain mechanistic insight and identify predictive signatures for personalization. However, many immune monitoring technologies face investigator bias, missing unanticipated cellular responses in limited clinical material. We present here a mass cytometry (CyTOF) workflow for standardized, systems-level biomarker discovery in immunotherapy trials. To broadly enumerate immune cell identity and activity, we established and extensively assessed a reference panel of 33 antibodies to cover major cell subsets, simultaneously quantifying activation and immune checkpoint molecules in a single assay. This assay enumerates ≥98% of peripheral immune cells with ≥4 positively identifying antigens. Robustness and reproducibility are demonstrated on multiple samples types, across two research centers and by orthogonal measurements. Using automated analysis, we identify stratifying immune signatures in bone marrow transplantation-associated graft-versus-host disease. Together, this validated workflow ensures comprehensive immunophenotypic analysis and data comparability and will accelerate biomarker discovery

Comprehensive Immune Monitoring of Clinical Trials to Advance Human Immunotherapy

The success of immunotherapy has led to a myriad of clinical trials accompanied by efforts to gain mechanistic insight and identify predictive signatures for personalization. However, many immune monitoring technologies face investigator bias, missing unanticipated cellular responses in limited clinical material. We present here a mass cytometry (CyTOF) workflow for standardized, systems-level biomarker discovery in immunotherapy trials. To broadly enumerate immune cell identity and activity, we established and extensively assessed a reference panel of 33 antibodies to cover major cell subsets, simultaneously quantifying activation and immune checkpoint molecules in a single assay. This assay enumerates >= 98% of peripheral immune cells with >= 4 positively identifying antigens. Robustness and reproducibility are demonstrated on multiple samples types, across two research centers and by orthogonal measurements. Using automated analysis, we identify stratifying immune signatures in bone marrow transplantation-associated graft-versus-host disease. Together, this validated workflow ensures comprehensive immunophenotypic analysis and data comparability and will accelerate biomarker discovery

Proliferation tracing with single-cell mass cytometry optimizes generation of stem cell memory-like T cells.

Selective differentiation of naive T cells into multipotent T cells is of great interest clinically for the generation of cell-based cancer immunotherapies. Cellular differentiation depends crucially on division state and time. Here we adapt a dye dilution assay for tracking cell proliferative history through mass cytometry and uncouple division, time and regulatory protein expression in single naive human T cells during their activation and expansion in a complex ex vivo milieu. Using 23 markers, we defined groups of proteins controlled predominantly by division state or time and found that undivided cells account for the majority of phenotypic diversity. We next built a map of cell state changes during naive T-cell expansion. By examining cell signaling on this map, we rationally selected ibrutinib, a BTK and ITK inhibitor, and administered it before T cell activation to direct differentiation toward a T stem cell memory (TSCM)-like phenotype. This method for tracing cell fate across division states and time can be broadly applied for directing cellular differentiation

2009

Sex-mismatched hematopoietic cell transplantation is linked to increased graft-versus-host disease and mortality in myeloablative conditioning. Here we evaluated outcomes of 1,041 adult transplant recipients at two centers between 2006 and 2013 and investigated how the effect of sex-mismatching differed in myeloablative, reducedintensity, and non-myeloablative total lymphoid irradiation with anti-thymocyte globulin conditioning. Among patients who underwent myeloablative conditioning, male recipients with female donors had increased chronic graft-versus-host disease (hazard ratio 1.83, P<0.01), increased non-relapse mortality (hazard ratio 1.84, P=0.022) and inferior overall survival (hazard ratio 1.59, P=0.018). In contrast, among patients who received reduced-intensity conditioning, male recipients with female donors had increased acute graft-versus-host disease (hazard ratio 1.96, P<0.01) but no difference in non-relapse mortality or overall survival. Among the patients who underwent total lymphoid irradiation with anti-thymocyte globulin, male recipients with female donors showed no increase in graft-versus-host disease or non-relapse mortality. Notably, only in the cohort receiving total lymphoid irradiation with antithymocyte globulin were male recipients with female donors significantly associated with reduced relapse (hazard ratio 0.64, P<0.01), and allo-antibody responses against H-Y antigens were predictive of reduced relapse. In the cohort given total lymphoid irradiation with anti-thymocyte globulin, the graft-versus-leukemia effect resulted in superior overall survival in recipients of sex-mismatched grafts (HR 0.69, P=0.037). In addition, only in the cohort treated with total lymphoid irradiation with anti-thymocyte globulin were female recipients with male donors associated with reduced relapse (hazard ratio 0.59, P<0.01) and superior survival (hazard ratio 0.61, P=0.014) compared with sex-matched pairs. We conclude that the risks and benefits of sex-mismatched transplants appear to differ according to conditioning strategy and this could affect donor selection. Risks and benefits of sex-mismatched hematopoietic cell transplantation differ according to conditioning strategy ABSTRACT © F e r r a t a S t o r t i F o u n d a t i o n H. Nakasone et al. 1478 haematologica | 2015; 100 Methods Patients We reviewed clinical data of 1,041 adults who received peripheral blood stem cell transplants between 2006 and 2013 at Stanford University Hospital (n=749) and Karolinska University Hospital (n=292). Recipients were treated for a number of primary diseases including acute myeloid leukemia, myelodysplastic syndrome, chronic lymphocytic leukemia, non-Hodgkin lymphoma, and others. Our study excluded indications for which TLI-ATG is not used such as acute lymphoblastic leukemia. Haploidentical/HLA-mismatched related-HCT patients and recipients who received GVHD prophylaxis other than cyclosporine and tacrolimus were also excluded. Plasma samples were collected from recipients who survived without disease relapse for at least 3 months after Female→Male HCT at Stanford University Hospital and were cryopreserved until use. Written informed consent was given for the cryopreservation and analyses of blood samples, in accordance with the Declaration of Helsinki. This study was approved by the institutional review board of Stanford University. Definitions of categories The details of category definitions are described in the Online Supplementary Methods. Briefly, conditioning regimens were classified into three groups: TLI-ATG, reduced intensity conditioning (RIC), and myeloablative conditioning (MAC). The TLI-ATG conditioning protocol (n=430) has already been reported. 21 Detection of H-Y antibodies in Female→Male hematopoietic cell transplantation The details are described in the Online Supplementary Methods. Briefly, using plasma samples collected 3 months after HCT, anti- Statistical analysis The statistical methods are described in detail in the Online Supplementary Methods. Briefly, the cumulative probabilities of each event were estimated by the Gray method, considering competing risks. Overall survival from HCT was estimated with a 95% confidence interval (CI) by the Kaplan-Meier method and compared by log-rank test. Since our primary aim was to address the difference of impact of sex-mismatching among different conditioning regimens, we assessed effects of sex-mismatched HCT within the TLI-ATG, RIC, and MAC groups, separately. Multivariate analyses were performed using a Cox proportional hazard model. Since overall survival of sex-matched HCT was not significantly different between Female→Female and Male→Male transplants in the whole cohort, we considered both of these types of transplants as one group of sex-matched HCT. Two-tailed Pvalues <0.05 are considered statistically significant. All analyses and data management were performed using Stata version 12.0 (StataCorp, College Station, TX, USA), and EZR. 23 Results Patients' characteristics In general, conditioning intensity is selected based on patients' characteristics, such as age and co-morbidity, and donor selection follows the choice of conditioning intensity. There are, therefore, many different characteristics, including age, disease, and CMV serostatus that vary among conditioning types (Online Overall survival according to sex-mismatch In agreement with previously published reports, in the MAC group, Female→Male HCT was significantly associated with inferior overall survival compared with the other combinations (HR 1.59; P=0.018) (Figures 1A and 2A). In the RIC group, there was no difference in overall survival according to sex-mismatch ( In the TLI-ATG group, the overall survival of sex-mismatched HCT recipients was better than that of sexmatched HCT recipients (P=0.0071) ( Incidences of acute and chronic graft-versus-host disease according to sex-mismatch In the MAC group, there was no difference in incidences of grade II-IV acute GVHD according to sex-mismatch ( Among patients who survived more than 100 days after HCT without relapse, in the MAC group, Female→Male HCT recipients experienced chronic GVHD more frequently than donor-recipient combinations (P=0.041) ( In summary, in the TLI-ATG group, sex-mismatched HCT did not have an impact on either grade II-IV acute GVHD or on chronic GVHD, while Female→Male HCT was associated with acute GVHD in the RIC group and chronic GVHD in the MAC group. Non-relapse mortality according to sex-mismatch In the MAC group, Female→Male HCT recipients had increased non-relapse mortality compared with donorrecipient combinations (P=0.049) ( © F e r r a t a S t o r t i F o u n d a t i o n In summary, Female→Male HCT was associated with an increased risk of non-relapse mortality in the MAC group, but not in the TLI-ATG or RIC group. Disease relapse according to sex-mismatch While no impact of sex-mismatch on relapse was observed in the MAC or RIC group (Figures 2A,B and 4D,E), in the TLI-ATG group, the incidence of relapse was higher following sex-matched HCT than after sex-mismatched HCT (P=0.0012) ( In summary, sex-mismatched HCT was associated with a reduced risk of relapse in the TLI-ATG group, while no association was observed in the MAC or RIC group. H. Nakasone et al. 1480 haematologica | 2015; 100(11) Detection of H-Y antibodies 3 months after hematopoietic cell transplantation and disease relapse of Female→Male transplant recipients within the group conditioned with total lymphoid irradiation and antithymocyte globulin In the TLI-ATG group, the benefit of sex-mismatched HCT on overall survival seems due to the reduced relapse rate 14 We, therefore, focused on the H-Y antibody response in Female→Male HCT and explored whether the detection of H-Y antibodies 3 months after HCT could predict reduced relapse in Female→Male HCT with TLI-ATG. Excluding patients with myelodysplastic syndrome and chronic lymphocytic leukemia because of the absence of a GVL benefit by sex-mismatch in the TLI-ATG group (Online H-Y antibodies were also tested for in 28 patients in the MAC group. The distribution of H-Y antibodies was not different between the TLI-ATG and MAC groups ( Other assessments Impact of HLA-mismatch on survival of recipients of unrelated transplants conditioned with total lymphoid irradiation and antithymocyte globulin Given the results of sex-mismatched transplants, we next assessed whether TLI-ATG patients could benefit from HLA-mismatched HCT or not, focusing on unrelated transplants. In the TLI-ATG group, recipients of HLA-mismatched HCT had comparable overall survival to that of HLA-matched HCT recipients (Online Supplementary A B C D E F © F e r r a t a S t o r t i F o u n d a t i o n saw in sex-mismatched HCT. Further investigation is required before drawing any conclusions. Impact of conditioning strategies within the group of sex-mismatched hematopoietic cell transplant recipients Many clinical parameters, such as age, disease, and comorbidity drive clinicians to select conditioning intensity before considering the choice of donor. We, therefore, decided to base the bulk of our evaluation on sex-mismatched HCT within the three conditioning intensity regimens. Nonetheless, we next evaluated the effects of conditioning intensity. In the overall cohort, recipients in the TLI-ATG group had comparable overall survival to those in the MAC group (Online Discussion In general, conditioning intensity is selected based on age, co-morbidity, and disease status. Since donor selection usually follows the choice of conditioning intensity, the effect of having a sex-mismatched donor should be considered within each conditioning strategy. We found that sex-mismatched HCT, especially Female→Male HCT, was an important factor affecting survival, GVHD, and relapse, and that the impact differed according to conditioning strategy and warrants consideration in donor selection algorithms. Specifically, we found that patients undergoing TLI-ATG conditioning benefit from sex-mismatched GVL allo-immunity without the detrimental non-relapse mortality associated with GVHD. It has been widely reported that Female→Male HCT is associated with an increased incidence of chronic GVHD and decreased overall survival. 1-4 However, as summarized in 1482 haematologica | 2015; 100(11) A D E F © F e r r a t a S t o r t i F o u n d a t i o n Impact of sex-mismatched transplants differs by conditioning haematologica | 2015; 100(11) 1483 low non-relapse mortality. Since TLI-ATG is devoid of chemotherapy cytotoxicity, conditioning provides little direct anti-tumor benefit. Rather, the successful outcome of TLI-ATG transplants depends mostly on an allo-immune GVL benefit. We hypothesized that the increased allo-reactive immunity in sex-mismatched HCT could result in an enhanced GVL effect without increased GVHD or non-relapse mortality in the TLI-ATG cohort. The apparent GVL benefit from sexmismatched donors in the TLI-ATG group supports our hypothesis and will be considered during donor selection. Pre-clinical studies in animal models have suggested that TLI-ATG can increase the proportion of host natural killer T cells, leading to expansion of donor CD4 + regulatory T cells through interleukin-4 pathways. 24,25 These regulatory T cells are thought to have a potential not only to suppress fatal acute GVHD but also to preserve the antitumor effect of cytotoxic T cells. H-Y humoral immunity is an allogeneic response of female donors against the proteins encoded on the Y chromosome of male recipients and is considered an important model of allo-immunity. While Female→Male HCT is an established risk factor for chronic GVHD in the MAC group, the impact of sex-mismatching on acute GVHD has been controversial. Our results show that the improved GVL benefit in the TLI-ATG group associated not only with Female→Male but also Male→Female HCT. The concept of female-specific gene expression has been suggested before and might play a role here. For example, there may be several genes expressed only in female cells, such as XIST, a gene that A B C © F e r r a t a S t o r t i F o u n d a t i o n inactivates one of the X chromosomes, which is not observed in male cells. 32 These phenomena, observed exclusively in females, may become candidate anti-tumor immune targets under a unique T-cell environment induced by TLI-ATG, although the real reason for the GVL benefit from Male→Female HCT remains unclear. One potential confounder to consider is that in the TLI-ATG group, the rate of CMV seropositivity tended to be higher in Male→Female HCT patients Our study may have some limitations because of its retrospective nature. The study cohort had heterogeneous disease backgrounds and relapse incidence might depend on diseases and their status. We, therefore, explored the impact of sex-mismatch in each disease category as a subgroup analysis and found that the GVL benefit by sex-mismatch of TLI-ATG was limited in specific diseases such as acute myeloid leukemia and lymphoma other than chronic lymphocytic leukemia. One possible explanation is that expression of H-Y antigens might vary according to type of disease or individual leukemic cells. Loss of the Y chromosome is frequently observed in myelodysplastic syndrome (10-15%). 36,37 The defect of H-Y antigens in tumor cells with loss of Y chromosome may result in a reduced GVL effect by sex-mismatch, although the real reason has yet to be elucidated. Furthermore, MAC and RIC groups included various conditioning regimens and ATG usage in these conditioning regimens may also affect the impact of sex-mismatch. MAC Female→Male recipients had equivalent survival as sex-matched recipients when ATG or alemtuzumab was included (Online Supplementary In summary, the benefits and risks of sex-mismatched transplants differ according to conditioning strategy. Recipients of TLI-ATG conditioning preferentially benefit from sex-mismatched HCT with significantly reduced relapse rates and improved overall survival. The presence of H-Y antibodies 3 months after HCT, as representative alloantibodies demonstrated the association with reduced relapse in the TLI-ATG group. This could affect donor selection, such that sex-mismatching might be preferred in TLI-ATG patients and avoided in myeloablative transplant recipients. Our results suggest that there is the possibility of developing and employing new strategies to enhance GVL effects in TLI-ATG and non-myeloablative conditioning without increasing toxicity. PR2013-0022 and KF2013-0011, Marianne and Marcus Wallenbergs Foundation 2013.0117, Stockholm City Council (ALF) 20140451, and Swedish Cancer Foundation Mattsson/CF 2014-2016 © F e r r a t a S t o r t i F o u n d a t i o n Acknowledgments © F e r r a t a S t o r t i F o u n d a t i o

Culturing of human peripheral blood cells reveals unsuspected lymphocyte responses relevant to HIV disease

Recombinant HIV-Tat (Tat) induces extensive apoptosis in peripheral blood mononuclear cells (PBMCs) cultured in typical CO2 incubators, which are equilibrated with air (21% O2). However, as we show here, Tat apoptosis induction fails in PBMCs cultured at physiological oxygen levels (5% O2). Under these conditions, Tat induces PBMCs to divide, efficiently primes them for HIV infection, and supports virus production by the infected cells. Furthermore, Tat takes only 2 h to prime PBMCs under these conditions. In contrast, PHA/IL-2, which is widely used to prime cells for HIV infection, takes 2–3 days. These findings strongly recommend culturing primary cells at physiological oxygen levels. In addition, they suggest HIV-Tat as a key regulator of HIV disease progression