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BackgroundB cells play a pivotal role in regulating the immune response. The induction of B cell-mediated immunosuppressive function requires B cell activating signals. However, the mechanisms by which activated B cells mediate T-cell suppression are not fully understood.MethodsWe investigated the potential contribution of metabolic activity of activated B cells to T-cell suppression by performing in vitro experiments and by analyzing clinical samples using mass cytometry and single-cell RNA sequencing.ResultsHere we show that following activation, B cells acquire an immunoregulatory phenotype and promote T-cell suppression by metabolic competition. Activated B cells induced hypoxia in T cells in a cell–cell contact dependent manner by consuming more oxygen via an increase in their oxidative phosphorylation (OXPHOS). Moreover, activated B cells deprived T cells of glucose and produced lactic acid through their high glycolytic activity. Activated B cells thus inhibited the mammalian target of rapamycin pathway in T cells, resulting in suppression of T-cell cytokine production and proliferation. Finally, we confirmed the presence of tumor-associated B cells with high glycolytic and OXPHOS activities in patients with melanoma, associated with poor response to immune checkpoint blockade therapy.ConclusionsWe have revealed for the first time the immunomodulatory effects of the metabolic activity of activated B cells and their possible role in suppressing antitumor T-cell responses. These findings add novel insights into immunometabolism and have important implications for cancer immunotherapy.

Immune checkpoint therapy has resulted in remarkable improvements in the outcome for certain cancers. To broaden the clinical impact of checkpoint targeting, we devised a strategy that couples targeting of the cytokine-inducible Src homology 2–containing (CIS) protein, a key negative regulator of interleukin 15 (IL-15) signaling, with fourth-generation “armored” chimeric antigen receptor (CAR) engineering of cord blood–derived natural killer (NK) cells. This combined strategy boosted NK cell effector function through enhancing the Akt/mTORC1 axis and c-MYC signaling, resulting in increased aerobic glycolysis. When tested in a lymphoma mouse model, this combined approach improved NK cell antitumor activity more than either alteration alone, eradicating lymphoma xenografts without signs of any measurable toxicity. We conclude that targeting a cytokine checkpoint further enhances the antitumor activity of IL-15–secreting armored CAR-NK cells by promoting their metabolic fitness and antitumor activity. This combined approach represents a promising milestone in the development of the next generation of NK cells for cancer immunotherapy. Key Points: • CRISPR-Cas9 CISH deletion enhances the metabolic fitness and antitumor activity of armored IL-15–secreting CB-derived CAR-NK cells.

This study aimed to analyze the factors associated with outcomes of bone marrow transplantation (UR-BMT) or cord blood stem cell transplantation from unrelated donors (UR-CBT). We assessed the time from diagnosis to transplantation among acute myeloid leukemia (AML) patients with intermediate- or poor-risk cytogenetics to identify the potential clinical efficacy of transplantation. We retrospectively analyzed 5331 patients who received UR-BMT or UR-CBT between 2008 and 2017. Patients were divided into four groups according to time from diagnosis to transplantation: (1) UR-BMT and 5 months (n = 2353), (2) UR-BMT and ≤ 5 months (n = 379), (3) UR-CBT and 5 months (n = 1494), and (4) UR-CBT and ≤ 5 months (n = 1106). There was no difference in overall survival (OS) for transplantation at ≤5 months and 5 months in patients with first complete remission for both UR-BMT and UR-CBT, but OS in patients with primary induction failure (PIF) and transplantation at ≤ 5 months was significantly higher in the UR-CBT group compared with that at5 months (P 0.001). Multivariate Cox regression analysis also showed that transplantation at5 months in patients with PIF was an independent predictor of poorer OS. Therefore, UR-CBT at ≤ 5 months after diagnosis is an alternative option for AML patients with PIF.

To investigate which reduced-intensity conditioning (RIC)/reduced-toxicity conditioning (RTC) is superior for umbilical cord blood transplantation (UCBT) for lymphoid malignancies, we retrospectively compared three widely used RIC/RTC regimens: fludarabine/melphalan/total body irradiation (FM-TBI, n = 524), fludarabine/cyclophosphamide/total body irradiation (FC-TBI, n = 96), and fludarabine/busulfan/total body irradiation or melphalan (FB-based, n = 159). Among patients with acute lymphoblastic leukemia (ALL) (n = 314), there were no differences in overall survival (OS) by conditioning regimen. Among patients with malignant lymphoma (ML) (n = 465), FM-TBI and FC-TBI regimens had similar OS, whereas FB-based regimen had lower OS (hazard ratio [HR], 1.73; P

We retrospectively compared the outcomes of reduced-intensity conditioning (RIC) transplantation from matched related donors (MRD; n = 266), matched unrelated donors (MUD; n = 277), and umbilical cord blood (UCB; n = 513) for mature lymphoid malignancies. The 3-year overall survival rates for the MRD, MUD, and UCB groups were 54%, 59%, and 40%, respectively (P

Glioblastoma multiforme (GBM), the most aggressive brain cancer, recurs because glioblastoma stem cells (GSCs) are resistant to all standard therapies. We showed that GSCs, but not normal astrocytes, are sensitive to lysis by healthy allogeneic natural killer (NK) cells in vitro. Mass cytometry and single-cell RNA sequencing of primary tumor samples revealed that GBM tumor–infiltrating NK cells acquired an altered phenotype associated with impaired lytic function relative to matched peripheral blood NK cells from patients with GBM or healthy donors. We attributed this immune evasion tactic to direct cell-to-cell contact between GSCs and NK cells via αv integrin–mediated TGF-β activation. Treatment of GSC-engrafted mice with allogeneic NK cells in combination with inhibitors of integrin or TGF-β signaling or with TGFBR2 gene–edited allogeneic NK cells prevented GSC-induced NK cell dysfunction and tumor growth. These findings reveal an important mechanism of NK cell immune evasion by GSCs and suggest the αv integrin/TGF-β axis as a potentially useful therapeutic target in GBM.

ORF8 is an accessory protein encoded by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Consensus regarding the biological functions of ORF8 is lacking, largely because the fundamental characteristics of this protein in cells have not been determined. To clarify these features, we herein established an ORF8 expression system in 293T cells. Using this system, approximately 41% of the ORF8 expressed in 293T cells were secreted extracellularly as a glycoprotein homodimer with inter/intramolecular disulfide bonds. Intracellular ORF8 was sensitive to the glycosidase Endo H, whereas the secreted portion was Endo-H-resistant, suggesting that secretion occurs via a conventional pathway. Additionally, immunoblotting analysis showed that the total amounts of the major histocompatibility complex class Ι (MHC-I), angiotensin-converting enzyme 2 (ACE2), and SARS-CoV-2 spike (CoV-2 S) proteins coexpressed in cells were not changed by the increased ORF8 expression, although FACS analysis revealed that the expression of the cell surface MHC-I protein, but not that of ACE2 and CoV-2 S proteins, was reduced by ORF8 expression. Finally, we demonstrate by RNA-seq analysis that ORF8 had no significant stimulatory effects in human primary monocyte-derived macrophages (MDMs). Taken together, our results provide fundamental evidence that the ORF8 glycoprotein acts as a secreted homodimer, and its functions are likely associated with the intracellular transport and/or extracellular signaling in SARS-CoV-2 infection.

Glioblastoma, the most aggressive brain cancer, often recurs because glioblastoma stem cells (GSCs) are resistant to all standard therapies. Here, we show that patient-derived GSCs, but not normal astrocytes, are highly sensitive to lysis by healthy allogeneic natural killer (NK) cellsin vitro. In contrast, single cell analysis of autologous, tissue infiltrating NK cells isolated from surgical samples of high-grade glioblastoma patient tumors using mass cytometry and single cell RNA sequencing revealed an abnormal phenotype associated with impaired lytic function compared with peripheral blood NK cells from GBM patients or healthy donors. This immunosuppression was attributed to an integrin-TGF-β mechanism, activated by direct cell-cell contact between GSCs and NK cells. Treatment of GSC-engrafted mice with allogeneic NK cells in combination with inhibitors of integrin or TGF-β signaling, or withTGF-β receptor 2gene-edited NK cells prevented GSC-induced NK cell dysfunction and tumor growth. Collectively, our findings reveal a novel mechanism of NK cell immune evasion by GSCs and implicate the integrin-TGF-β axis as a useful therapeutic target to eliminate GSCs in this devastating tumor.

Immune checkpoint therapy has produced remarkable improvements in the outcome for certain cancers. To broaden the clinical impact of checkpoint targeting, we devised a strategy that couples targeting of the cytokine-inducible SH2-containing (CIS) protein, a key negative regulator of interleukin (IL)-15 signaling, with chimeric antigen receptor (CAR) engineering of natural killer (NK) cells. This combined strategy boosted NK cell effector function through enhancing the Akt/mTORC1 axis and c-MYC signaling, resulting in increased aerobic glycolysis. When tested in a lymphoma mouse model, this combined approach improved NK cell anti-tumor activity more than either alteration alone, eradicating lymphoma xenografts without signs of any measurable toxicity. We conclude that combining CIS checkpoint deletion with CAR engineering promotes the metabolic fitness of NK cells in an otherwise suppressive tumor microenvironment. This approach, together with the prolonged survival afforded by CAR modification, represents a promising milestone in the development of the next generation of NK cells for cancer immunotherapy.

The establishment of long-lived pathogen-specific T cells is a fundamental property of the adaptive immune response. However, the mechanisms underlying long-term persistence of antigen-specific CD4+ T cells are not well-defined. Here we identify a subset of memory CD4+ T cells capable of effluxing cellular toxins, including rhodamine (Rho), through the multidrug efflux protein MDR1 (also known as P-glycoprotein and ABCB1). Drug-effluxing CD4+ T cells were characterized as CD161+CD95+CD45RA-CD127hiCD28+CD25int cells with a distinct chemokine profile and a Th1-polarized pro-inflammatory phenotype. CD4+CD161+Rho-effluxing T cells proliferated vigorously in response to stimulation with anti-CD3/CD28 beads and gave rise to CD161- progeny in vitro. These cells were also capable of self-renewal and maintained their phenotypic and functional characteristics when cultured with homeostatic cytokines. Multidrug-effluxing CD4+CD161+ T cells were enriched within the viral-specific Th1 repertoire of healthy donors and patients with acute myeloid leukemia (AML) and survived exposure to daunorubicin chemotherapy in vitro. Multidrug-effluxing CD4+CD161+ T cells also resisted chemotherapy-induced cytotoxicity in vivo and underwent significant expansion in AML patients rendered lymphopenic after chemotherapy, contributing to the repopulation of anti-CMV immunity. Finally, after influenza vaccination, the proportion of influenza-specific CD4+ T cells coexpressing CD161 was significantly higher after 2 years compared with 4 weeks after immunization, suggesting CD161 is a marker for long-lived antigen-specific memory T cells. These findings suggest that CD4+CD161+ T cells with rapid efflux capacity contribute to the maintenance of viral-specific memory T cells. These data provide novel insights into mechanisms that preserve antiviral immunity in patients undergoing chemotherapy and have implications for the development of novel immunotherapeutic approaches.

Natural killer (NK) cells are highly heterogeneous, with vast phenotypic and functional diversity at the single-cell level. They are involved in the innate immune response against malignant and virus-infected cells. To understand the effect of NK diversity during immune recovery on the antitumor response after cord blood transplantation (CBT), we used high-dimensional mass cytometry and the metrics of NK cell diversity to study the NK cell repertoire in serial samples from 43 CBT recipients. A higher-diversity index based on single-cell combinatorial phenotypes was significantly associated with a lower risk for relapse after CBT (P = .005). Cytomegalovirus reactivation was a major factor in the development of a more diverse NK repertoire after CBT. Notably, we identified a group of patients whose CB-derived NK cells after transplantation possessed an immature phenotype (CB-NK(im)), characterized by poor effector function and a low diversity index. Frequencies of CB-NK(im) of 11.8% or higher during the early post-CBT recovery phase were highly predictive for relapse (area under the curve [AUC], 0.979), a finding that was validated in a second independent cohort of patients (n = 25; AUC, 0.977). Moreover, we showed that the maturation, diversity, and acquisition of effector function by CB-NK(im) early after CBT were driven by interleukin 15. Our data indicate that the diversity of the NK cell repertoire after CBT contributes importantly to the risk for subsequent relapse. We suggest that the use of diversity metrics and high-dimensional mass cytometry may be useful tools in predicting clinical outcomes and informing the design of therapeutic strategies to prevent relapse after CBT.

Programmed death-ligand 1 (PD-L1) binds to programmed death-1 (PD-1) on activated T cells and contributes to T-cell exhaustion. PD-L1 expressed on antigen-presenting cells (APCs) could be thought to inhibit the induction of Ag-specific cytotoxic T lymphocytes (CTLs) by transducing negative signal into T cells; however, the roles of PD-L1 on APCs have not yet been well examined. Therefore, we evaluated the roles of PD-L1 on APCs in the induction of Ag-specific CTLs. CD3 T cells isolated from cytomegalovirus (CMV)-seropositive healthy donors were stimulated with mature dendritic cells pulsed with CMV pp65-derived HLA-restricted peptides in the presence of anti-PD-L1 blocking antibody. Unexpectedly, PD-L1 blockade resulted in a less efficient induction of CMV-specific CTLs, suggesting that PD-L1 play a positive role in the induction of Ag-specific CTLs. For further evaluations and application to adoptive immunotherapy, we generated K562-based artificial APCs, which were retrovirally transduced with HLA class I molecules and various combinations of CD80/86 and PD-L1. K562/HLA+CD80/86+PD-L1 cells produced significantly higher induction of CMV-specific CTLs than K562/HLA or K562/HLA+CD80/86 cells without causing excessive differentiation or functional exhaustion of the induced CTLs, whereas PD-L1 itself did not have a stimulatory effect. Furthermore, only K562/HLA+CD80/86+PD-L1 cells pulsed with HLA-A*24:02-restricted Wilms tumor 1 (WT1) peptide clearly expanded WT1-specific CTLs from healthy donors. Our findings presumed that PD-L1 expressed on APCs along with CD80/86 enhanced the induction of Ag-specific CTLs probably depending on fine-tuning excessive stimulation of CD80/86, and that K562/HLA+CD80/86+PD-L1 cells has therapeutic potential as a novel type of artificial APCs for adoptive immunotherapy.

Regulatory T cells (Tregs) play a fundamental role in the maintenance of self-tolerance and immune homeostasis. Defects in Treg function and/or frequencies have been reported in multiple disease models. Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder affecting upper and lower motor neurons. Compelling evidence supports a neuroprotective role for Tregs in this disease. Indeed, rapid progression in ALS patients is associated with decreased FoxP3 expression and Treg frequencies. Thus, we propose that strategies to restore Treg number and function may slow disease progression in ALS. In this study, we developed a robust, Good Manufacturing Practice (GMP)-compliant procedure to enrich and expand Tregs from ALS patients. Tregs isolated from these patients were phenotypically similar to those from healthy individuals but were impaired in their ability to suppress T-cell effector function. In vitro expansion of Tregs for 4 weeks in the presence of GMP-grade anti-CD3/CD28 beads, interleukin (IL)-2 and rapamcyin resulted in a 25- to 200-fold increase in their number and restored their immunoregulatory activity. Collectively, our data facilitate and support the implementation of clinical trials of adoptive therapy with ex vivo expanded and highly suppressive Tregs in patients with ALS.

The ability of cord blood transplantation (CBT) to prevent relapse depends partly on donor natural killer (NK) cell alloreactivity. NK effector function depends on specific killer-cell immunoglobulin-like receptors (KIR) and HLA interactions. Thus, it is important to identify optimal combinations of KIR-HLA genotypes in donors and recipients that could improve CBT outcome. We studied clinical data, KIR and HLA genotypes, and NK-cell reconstitution in CBT patients (n = 110). Results were validated in an independent cohort (n = 94). HLA-KIR genotyping of recipient germline and transplanted cord blood (CB) grafts predicted for large differences in outcome. Patients homozygous for HLA-C2 group alleles had higher 1-year relapse rate and worse survival after CBT than did HLA-C1/C1 or HLA-C1/C2 (HLA-C1/x) patients: 67.8% vs 26.0% and 15.0% vs 52.9%, respectively. This inferior outcome was associated with delayed posttransplant recovery of NK cells expressing the HLA-C2-specific KIR2DL1/S1 receptors. HLA-C1/x patients receiving a CB graft with the combined HLA-C1-KIR2DL2/L3/S2 genotype had lower 1-year relapse rate (6.7% vs 40.1%) and superior survival (74.2% vs 41.3%) compared with recipients of grafts lacking KIR2DS2 or HLA-C1. HLA-C2/C2 patients had lower relapse rate (44.7% vs 93.4%) and better survival (30.1% vs 0%) if they received a graft with the combined HLA-C2-KIR2DL1/S1 genotype. Relapsed/refractory disease at CBT, recipient HLA-C2/C2 genotype, and donor HLA-KIR genotype were independent predictors of outcome. Thus, we propose the inclusion of KIR genotyping in graft selection criteria for CBT. HLA-C1/x patients should receive an HLA-C1-KIR2DL2/L3/S2 CB graft, while HLA-C2/C2 patients may benefit from an HLA-C2-KIR2DL1/S1 graft.

Little is known regarding the chimerism status after reduced-intensity conditioning transplantation when bone marrow is used as a stem cell source. We prospectively analyzed lineage-specific chimerism and retrospectively evaluated clinical outcomes in 80 adult patients who underwent unrelated donor bone marrow transplantation (URBMT) with fludarabine plus melphalan (FM) as the conditioning regimen. Mixed donor chimerism (MDC) was seen in 43 and 10 % of patients at days 14 and 28, respectively. Melphalan at ≤130 mg/m(2) was associated with an increased incidence of MDC at day 28 (P = 0.03). Patients with MDC at day 14 showed a marginally increased risk of primary graft failure and a marginally decreased risk of graft-versus-host disease. In multivariate analysis, MDC at day 14 was associated with higher overall mortality (hazard ratio (HR) = 2.1; 95 % confidence interval (CI), 1.1-4.2; P = 0.04) and relapse rate (HR = 3.0; 95 % CI, 1.2-7.5; P = 0.02), but not with non-relapse mortality (HR = 1.8; 95 % CI, 0.70-4.6; P = 0.23). Thus, the FM regimen yields prompt complete donor chimerism after URBMT, but the melphalan dose significantly impacts the kinetics of chimerism. Chimerism status evaluation at day 14 may be instrumental in predicting relapse after URBMT with the FM regimen.

Although recent studies of virus-specific T-cell (VST) therapy for viral infections after allogeneic hematopoietic stem cell transplantation have shown promising results, simple and less time-intensive and labor-intensive methods are required to generate VSTs for the wider application of VST therapy. We investigated the efficacy of anti-CD28 and anti-4-1BB antibodies, which can provide T cells with costimulatory signals similar in strength to those of antigen-presenting cells, in generating VSTs. When peripheral blood mononuclear cells were stimulated with viral peptides together with isotype control, anti-CD28, or anti-4-1BB antibodies, anti-4-1BB antibodies yielded the highest numbers of VSTs, which were on an average 7.9 times higher than those generated with isotype control antibody. The combination of anti-CD28 and anti-4-1BB antibodies did not result in increased numbers of VSTs compared with anti-4-1BB antibody alone. Importantly, the positive effect of anti-4-1BB antibody was observed regardless of the epitopes of the VSTs. In contrast, the capacity of dendritic cells (DCs) to generate VSTs differed considerably depending on the epitopes of the VSTs. Furthermore, the numbers of VSTs generated with DCs were at most similar to those generated with the anti-4-1BB antibody. Generation of VSTs with anti-4-1BB antibody did not result in excessive differentiation or deteriorated function of the generated VSTs compared with those generated with control antibody or DCs. In conclusion, VSTs can be generated rapidly and efficiently by simply stimulating peripheral blood mononuclear cells with viral peptide and anti-4-1BB antibody without using antigen-presenting cells. We propose using anti-4-1BB antibody as a novel strategy to generate VSTs for adoptive therapy.

Ibrutinib, a covalent inhibitor of Bruton Tyrosine Kinase (BTK), is approved for treatment of patients with relapsed/refractory or treatment-naive chronic lymphocytic leukemia (CLL). Besides directly inhibiting BTK, ibrutinib possesses immunomodulatory properties through targeting multiple signaling pathways. Understanding how this ancillary property of ibrutinib modifies the CLL microenvironment is crucial for further exploration of immune responses in this disease and devising future combination therapies. Here, we investigated the mechanisms underlying the immunomodulatory properties of ibrutinib. In peripheral blood samples collected prospectively from CLL patients treated with ibrutinib monotherapy, we observed selective and durable downregulation of PD-L1 on CLL cells by 3 months post-treatment. Further analysis showed that this effect was mediated through inhibition of the constitutively active signal transducer and activator of transcription 3 (STAT3) in CLL cells. Similar downregulation of PD-1 was observed in CD4+ and CD8+ T cells. We also demonstrated reduced interleukin (IL)-10 production by CLL cells in patients receiving ibrutinib, which was also linked to suppression of STAT3 phosphorylation. Taken together, these findings provide a mechanistic basis for immunomodulation by ibrutinib through inhibition of the STAT3 pathway, critical in inducing and sustaining tumor immune tolerance. The data also merit testing of combination treatments combining ibrutinib with agents capable of augmenting its immunomodulatory effects.

Impact of T-cell chimerism on relapse after cord blood transplantation for hematological malignancies: Nagoya Blood and Marrow Transplantation Group study

Subgroup B adenovirus serotype 11 (Ad11) occasionally causes fatal infections in immunocompromised patients. The present study describes a novel Ad11 epitope presented by HLA-A*24:02 that could be used for adoptive immunotherapy. Ten synthetic Ad11 hexon protein-derived nonamer peptides that bound to HLA-A*24:02 were selected by a computer algorithm and MHC stabilization assay. Stimulation of peripheral blood mononuclear cells from HLA-A*24:02+ donors with each of these synthetic peptides induced peptide-specific CD8(+) T-cells for three peptides. Testing the reactivity of these peptide-specific CD8(+) T-cells against various target cells confirmed that peptide TYFNLGNKF is naturally processed in Ad11-infected cells and is presented by HLA-A*24:02. Emergence of TYFNLGNKF-specific CD8(+) T-cells coincided with the clearance of adenoviruses in a patient with Ad11 disease. Importantly, TYFNLGNKF-specific CD8(+) T-cells were suggested to be not serotype cross-reactive. The novel HLA-A*24:02-restricted Ad11 epitope could be used for anti-Ad11 adoptive immunotherapy and to monitor immunity to Ad11 using MHC tetramers.

Iron overload is a common complication in allogeneic hematopoietic cell transplantation (HCT). We studied the prevalence of iron overload using serum ferritin from 122 allogeneic HCT survivors who had survived a median of 1259 (range 134–4261) days. We also quantified iron overload by determining non-transferrin-bound iron (NTBI), which reflects iron overload more directly than ferritin, and compared the results with those of the ferritin assay. Fifty-two patients (43 %) showed hyperferritinemia (HF) (serum ferritin >1000 ng/mL), and there was a moderate correlation between serum ferritin and the number of transfused red blood cell units (ρ = 0.71). In multivariate analyses, HF was a significant risk factor for liver dysfunction (P = 0.0001) and diabetes (P = 0.02), and was related to a lesser extent with performance status (P = 0.08). There was a significant correlation between serum ferritin and NTBI (ρ = 0.59); however, the association of NTBI with these outcomes was weaker than that of serum ferritin. In conclusion, serum ferritin is a good surrogate marker of iron overload after allogeneic HCT, and reflects organ damage more accurately than NTBI.

The establishment of long-lived pathogen-specific T cells is a fundamental property of the adaptive immune response. However, the mechanisms underlying long-term persistence of antigen-specific CD4

Background Because of the less graft-facilitating effect by bone marrow (BM), we need to assess a dosage of conditioning more accurately particularly in combination with reduced-intensity conditioning. Thus we examined that modified continual reassessment method (mCRM) is applicable for deciding appropriate conditioning of allogeneic BM transplantation. Patients and methods The conditioning regimen consisted of i.v. fludarabine (125 mg/m2) plus an examination dose of i.v. melphalan. The primary endpoint was a donor-type T-cell chimerism at day 28 with successful engraftment defined as >90% donor cells. Five patients per dose level were planned to be accrued and chimerism data were used to determine the next dose. Results Seventeen patients were enrolled at doses between 130 and 160 mg/m2. The dose was changed from 160 to 130 mg/m2 (second level) after five full-donor chimerisms. With one patient of 0% chimera in the second level, the dose was increased to 135 mg/m2 (third level). Following five full-donor chimerisms in the third level, the study was complete as projected. Conclusions mCRM was shown to be a relevant method for dose-finding of conditioning regimen. The melphalan dose of 135 mg/m2 was determined as the recommended phase II dose to induce initial full-donor chimerism.

Partial human leukocyte antigen (HLA)-mismatched hematopoietic stem cell transplantation (HSCT) is often performed when an HLA-matched donor is not available. In these cases, CD8(+) or CD4(+) T cell responses are induced depending on the mismatched HLA class I or II allele(s). Herein, we report on an HLA-DRB1*08:03-restricted CD8(+) CTL clone, named CTL-1H8, isolated from a patient following an HLA-DR-mismatched HSCT from his brother. Lysis of a patient Epstein-Barr virus-transformed B cell line (B-LCL) by CTL-1H8 was inhibited after the addition of blocking antibodies against HLA-DR and CD8, whereas antibodies against pan-HLA class I or CD4 had no effect. The 1H8-CTL clone did not lyse the recipient dermal fibroblasts whose HLA-DRB1*08:03 expression was upregulated after 1 week cytokine treatment. Engraftment of HLA-DRB1*08:03-positive primary leukemic stem cells in non-obese diabetic/severe combined immunodeficient/γc-null (NOG) mice was completely inhibited by the in vitro preincubation of cells with CTL-1H8, suggesting that HLA-DRB1*08:03 is expressed on leukemic stem cells. Finally, analysis of the precursor frequency of CD8(+) CTL specific for recipient antigens in post-HSCT peripheral blood T cells revealed a significant fraction of the total donor CTL responses towards the individual mismatched HLA-DR antigen in two patients. These findings underscore unexpectedly significant CD8 T cell responses in the context of HLA class II.

The effects of macrophage activation on the outcome of allogeneic hematopoietic SCT (allo-HSCT) have yet to be fully examined. A total of 70 adult patients who received a first allo-HSCT for hematological diseases were studied. We counted the number of hemophagocytic cells in BM clot sections on day +14±7, and analyzed its impact on subsequent outcome. In all, 23 patients were diagnosed as having increased numbers of hemophagocytic cells (HP group), whereas 47 were not (non-HP group). The HP group was not associated with an increased incidence of acute or chronic GVHD, but was associated with worse hematopoietic recovery than the non-HP group. The 2-year OS for the HP group and the non-HP group was 30 and 65% (P

CTLA-4 is a negative regulator of activated T cells and the association of CTLA-4 polymorphisms with autoimmune diseases and transplant outcome has been reported. We evaluated the effect of donor CTLA-4 polymorphisms on outcome after allogeneic hematopoietic SCT (HSCT). We analyzed 147 Japanese HLA-matched sibling recipients and their donors who had undergone allogeneic HSCT. Genotyping of three single-nucleotide polymorphisms in CTLA-4 (-318, +49, CT60) was performed using TaqMan-PCR. According to the international HapMap database, only these three CTLA-4 haplotypes, classified as C-G-G, C-A-A and T-A-G, are present in the Japanese population. In this study, percentage expression of the C-G-G, C-A-A and T-A-G haplotypes was 59.5, 30.6 and 9.9%, respectively. Recipients of the C-A-A haplotype donor showed a significantly lower risk of relapse (HR: 0.54, 95% CI: 0.30-0.97, P=0.040) and a trend toward higher OS (HR: 0.61, 95% CI: 0.36-1.0, P=0.054) than did recipients of a donor without the C-A-A haplotype. The presence or absence of the C-A-A haplotype did not affect GVHD or non-relapse mortality. As the presence of the C-A-A haplotype reduced relapse risk and improved survival after allogeneic HSCT, this CTLA-4 haplotype may provide useful information for donor selection.

Corticosteroids are often used following allogeneic hematopoietic stem cell transplantation (HSCT) to control complications such as graft-versus-host disease (GVHD). However, there is some concern that corticosteroids may suppress the graft-versus-leukemia effect and increase leukemia relapse. To evaluate the effect of corticosteroids on relapse, we analyzed 112 adult patients who received their first allogeneic HSCT for acute myeloid leukemia at our institution between 1997 and 2007. Fifty-seven patients (50.9%) received corticosteroid therapy. Patients who had corticosteroid therapy included higher proportion of patients who developed GVHD. In multivariate analysis, with corticosteroid administration entered as a time-dependent covariate, corticosteroid administration was not a risk factor for relapse (p = 1.00, hazard ratio [HR] 1.00, 95% confidence interval [CI] 0.53-1.88), but it was associated with higher non-relapse mortality (NRM) (p < 0.001, HR 55.5, 95% CI 7.42-416) and lower overall survival (p < 0.001, HR 2.68, 95% CI 1.56-4.61). The higher NRM associated with corticosteroid administration was mainly due to the increased deaths caused by the complications themselves, which required corticosteroid therapy. The findings of this study indicate the importance of controlling complications after allogeneic HSCT. The strategy of refraining from indispensable corticosteroid therapy because of the excessive concerns about relapse should be avoided.

Early deaths after allogeneic stem cell transplantation (allo-SCT) are of major concern. On the assumption that both decreased and increased basal metabolism might relate to early deaths, we analyzed the risk factors for overall survival to days 30 (OS30) and 60 (OS60). The Harris-Benedict equation was used to calculate basal metabolism. Comparing a patient's basal metabolism (PBM) calculated from pretransplant body weight with the standard basal metabolism (SBM) calculated from standard body weight (body mass index (BMI) = 22), we defined the basal metabolic ratio (BMR) as a parameter (BMR = PBM/SBM). We retrospectively analyzed 360 adult patients transplanted between 1997 and 2006 at a single center in Japan. A multivariate analysis of OS30 showed risk factors to be: BMRor = 0.95 (low BMR; LBR) (P = 0.01), BMR1.05 (high BMR; HBR) (P = 0.005) and non-complete remission (non-CR) (P 5 0.001), whereas a multivariate analysis of OS60 showed those risk factors to be: LBR (P = 0.02), HBR (P = 0.04), non-CR (P = 0.002), and performance statusor = 1 (P = 0.01). OS30 and OS60 were found to be favorable in 0.95BMRor = 1.05 (average BMR; ABR) (96.8 and 90.3% for ABR, 87.1 and 76.2% for LBR, and 87.8 and 81.1% for HBR). In conclusion, BMR could prove to be a predictor of early death after allo-SCT.

High-dose chemotherapy followed by autologous stem cell transplantation (ASCT) is a standard therapy for newly diagnosed multiple myeloma. Combinations of recently proposed prognostic factors such as cytogenetics and international scoring system (ISS) may be useful to predict prognosis after ASCT. This study evaluated 60 consecutive patients who underwent ASCT in four institutes. The median age of patients was 57 years old. Cytogenetic analyses of bone marrow at diagnosis detected metaphase abnormalities in 9 of 51 patients and interphase abnormalities in six of 35 patients (17p13 deletion, t(4;14) and t(14;16)). Seventeen patients had ISS stage 3 at diagnosis. Twenty-five patients who had any of these risk factors were defined as high risk. All patients were conditioned with high-dose melphalan. With a median follow-up of 3.4 years, overall survival and event-free survival at 3 years were significantly worse in high-risk patients (48% vs. 97%; P = 0.0005 and 16% vs. 37%; P = 0.038, respectively) despite the higher CR plus VGPR rate among high-risk patients. In addition, survival at 1 year after progression was significantly worse in high-risk patients despite salvage chemotherapy containing thalidomide (32% vs. 100%, P = 0.0001). Combinations of cytogenetics and ISS could readily predict prognosis. Quality of response is a poor surrogate marker for ultimate outcome. High-risk patients may need more effective treatment.

Background: NK cells play a crucial role in the antitumor immune response and are involved in controlling tumor formation, progression and metastases. Glioblastoma (GBM) is the most devastating brain tumor, associated with very poor prognosis. While immunotherapy emerged as a promising approach for anti cancer therapy, GBM appears immune resistant. Here, we studied the role of NK cells in targeting glioblastoma and possible mechanisms of NK immune evasion. Methods: NK cells were isolated from freshly resected GBM tissue, matching peripheral blood and the blood of healthy controls. Flow cytometry was used to characterize the cells and their ability to produce cytokines, and chromium release assay was perfumed to assess their cytotoxicity. In vitro assays were performed by co-culturing GBM stem cells (GSCs) with healthy donor NK cells. Results: We found that whereas NK cells were abundant in primary GBM tissue and could efficiently target GBM stem cells (GSCs), GBM infiltrating NK cells (TiNKs) displayed an abnormal phenotype with downregulation of many activating receptors including CD16, NKG2D, DNAM, NKp30, NKp46, 2B4 and NKG2C and upregulation of inhibitory proteins such as PD-1. This inhibitory phenotype was associated with impaired NK cell function when compared with NK cells isolated from the peripheral blood of patients and healthy donors. GSC-NK cell-cell contact resulted in release of TGF-β by GCSs, which in turn led to NK dysfunction through constitutive activating of the p-Smad pathway. TGF-β activation, in turn, is partially mediated by the matrix metalloproteases MMP-2 and MMP-9, secreted by GSCs upon contact with NK cells and enhanced upon TGF-β exposure. We demonstrated that inhibition of the TGF-β axis, in particular by the small molecule inhibitor, galunisertib, can prevent GSC-induced NK cell dysfunction but is unable to inactivate the p-Smad pathway, thus, cannot reverse existing dysfunction. Conclusions: Our results indicate that NK-GBM cross-talk plays an important role in tumor escape and highlight the importance of developing future adoptive transfer therapies with the intent of limiting tumor escape from antitumor immunity. Note: This abstract was not presented at the meeting. Citation Format: Hila Shaim, Abdullah Alsuliman, Konrad Gabrusiewicz, Jun Wei, John Yu, Rafet Basar, May Daher, Lucila Kerbauy, Mayela Mendt, Muharrem Muftuoglu, Li Li, Enli Liu, Nobuhiko Imahashi, Sonny Ang, Young Gi, Pinaki Banerjee, David Marin, Richard Champlin, Elizabeth Shpall, Amy Heimberger, Katayoun Rezvani. TGF-β is a key mediator of NK cell dysfunction in gliolastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2949. doi:10.1158/1538-7445.AM2017-2949

In allo-stem cell transplantation (SCT), it is unclear whether donor-specific anti-HLA Abs (DSAs) can actually mediate graft rejection or if they are simply surrogate markers for the cellular immunity that causes graft rejection. Here, we first analyzed a case of cord blood allograft rejection in which DSA and cytotoxic T lymphocyte (CTL) specific for donor HLA-B*54:01 were detected at the time of graft rejection. Both the DSA and CTL inhibited colony formation by unrelated bone marrow mononuclear cells sharing HLA-B*54:01, suggesting that the humoral and cellular immune responses were involved in the graft rejection. Interestingly, the DSA and CTL were also detected in cryopreserved pre-transplant patient blood, raising a hypothesis that the presence of anti-HLA Abs could be an indicator for corresponding HLA-specific T cells. We then evaluated the existence of HLA-specific CD8(+) T cells in other patient blood specimens having anti-HLA class I Abs. Interferon-γ enzyme-linked immunospot assays clearly confirmed the existence of corresponding HLA-specific T-cell precursors in three of seven patients with anti-HLA Abs. In conclusion, our data demonstrate that integrated humoral and cellular immunity recognizing the same alloantigen of the donor can mediate graft rejection in DSA-positive patients undergoing HLA-mismatched allo-SCT. Further studies generalizing our observation are warranted.

Macrophage infiltration of skin GVHD lesions correlates directly with disease severity, but the mechanisms underlying this relationship remain unclear and GVHD with many macrophages is a therapeutic challenge. Here, we characterize the macrophages involved in GVHD and report that dexamethasone palmitate (DP), a liposteroid, can ameliorate such GVHD by inhibiting macrophage functions. We found that host-derived macrophages could exacerbate GVHD in a mouse model through expression of higher levels of pro-inflammatory TNF-α and IFN-γ, and lower levels of anti-inflammatory IL-10 than resident macrophages in mice without GVHD. DP significantly decreased the viability and migration capacity of primary mouse macrophages compared to conventional dexamethasone in vitro. DP treatment on day 7 and day 14 decreased macrophage number, and attenuated GVHD score and subsequent mortality in a murine model. This is the first study to provide evidence that therapy for GVHD should be changed on the basis of infiltrating cell type.

Myelodysplastic syndromes (MDS) are a group of hematopoietic disorders affecting the myeloid lineage, characterized by cytopenias and clonal evolution to AML. Immune responses against MDS, partly mediated by NK cells, have the potential to affect disease progression. However, immune evasion remains an important barrier. NK cells express both activating and inhibitory killer immunoglobulin-like receptors (KIRs) that interact to regulate NK effector function. Based on the number and distribution of inherited KIR genes, individuals can be classified in two broad haplotypes. Haplotype A comprises a single activating KIR (aKIR) gene (KIR2DS4), while haplotype B incorporates various combinations of aKIR genes (up to 6). We hypothesized that the aKIR gene repertoire may be useful in refining predictions of clinical outcomes in MDS. Thus, we studied the variations in aKIR gene content and haplotype in MDS and their relationship to the risk of AML transformation and patient outcomes. We first compared the number of aKIR genes in 108 MDS patients treated at MDACC with that in 139 HSC donors. The median number of aKIR genes was significantly lower in MDS than controls: 2 (range 0-6) vs 3 (range 0-6), p=0.001. Functional studies revealed that compared to healthy controls, NK cells from MDS patients demonstrate less Interferon gamma production (p We next examined the influence of KIR haplotype on the risk of AML transformation and outcomes in the cohort of 108 MDS patients (28 KIR haplotype A and 80 KIR haplotype B patients). On multivariate analysis, cytogenetic risk group and KIR haplotype were identified as independent predictors of MDS progression to AML. The relative risk of MDS-AML transformation in patients with haplotype A vs. haplotype B was 2.67 (1.13-6.31) (p=0.02). Similarly, cytogenetic risk group, IPSS and KIR haplotype independently predicted survival. MDS patients with KIR haplotype A had worse adjusted PFS (RR with 95%CI 2.96 [1.59-5.52], p=0.001) and OS (2.25 [1.17-4.31], p=0.02), compared to patients with haplotype B. These novel findings may help identify a subgroup of MDS patients with a high risk of disease progression and poor outcomes, who would likely benefit from adoptive NK cell therapy. Disclosures Kantarjian: ARIAD: Research Funding; Bristol-Myers Squibb: Research Funding; Amgen: Research Funding; Pfizer Inc: Research Funding; Delta-Fly Pharma: Research Funding; Novartis: Research Funding.

T cell exhaustion is characterized by coordinated expression of a series of negative checkpoint receptors such as programmed death-1 (PD-1), 2B4, CD160 and TIGIT, resulting in T cell dysfunction and immune evasion. Under physiological states, these inhibitory molecules maintain self-tolerance and prevent autoimmunity by applying a break on cytotoxic T cells. In cancer, T-cells exhibit features of T-cell exhaustion including increased expression of PD-1, 2B4 and CD160, coupled with reduced T cell proliferation, altered synapse formation and impaired cytotoxicity. Although the role of the PD-1/PD-L1 axis in mediating T cell defects in chronic lymphocytic leukemia (CLL) is well-studied, the contribution of other checkpoint molecules such as 2B4, CD160 and TIGIT in mediating tumor-induced immune dysfunction remains to be determined. Checkpoint inhibitors have provided a paradigm-shifting approach to cancer treatment. We hypothesized that the expression levels of checkpoint receptors on T-cells, as well as the "fitness" of the T cell compartment may provide a prognostic stratification system to predict response to checkpoint inhibitors in CLL. To determine if the number of inhibitory receptors per cell and their expression level may identify patient-to-patient differences that may not be easily deciphered using conventional research tools, we performed a detailed single-cell analysis of the T-cell repertoire, using 40-parameter mass cytometry (CyTOF) in 12 untreated CLL and 12 healthy controls. Consistent with previous reports, we found that expression of 2B4 (43.7% vs 30.8%), PD1 (28.8% vs 21%) and CD160 (17% vs. 9.7%) was significantly higher on CLL CD8+ T cells compared to healthy controls. In addition, CD8+T cells in CLL expressed higher levels of TIGIT (48.2% vs 25.2%), CD57 (43.9% vs 17.9%) and KLRG1 (49.5% vs. 29.7%). We clearly distinguished 2 patterns of exhaustion marker distribution in CLL. In one group of patients, the expression of checkpoint receptors was similar to that seen in healthy controls, whereas in the second group, CD8+ T-cells expressed higher levels of PD1, 2B4, TIGIT, CD160 as well as markers of terminal differentiation such as CD57 and KLRG1. Compared to healthy donors, CLL was characterized by an inversion in the CD4:CD8 ratio. Interestingly, CD8+ T cells in patients with a low CD4:CD8 ratio (defined as Next, we compared the number of checkpoint molecules expressed per CD8+ T-cells in CLL patients versus healthy donors. Whereas a similar proportion of CD8+ T-cells in CLL (mean 19.56%, range 18.34-31.73%) and healthy donors (mean 22.13%, 14.17-41.19%) expressed one inhibitory receptor, a significantly higher proportion of CLL patients expressed 2 and more inhibitory receptors (mean 28.4, range 10.52-48.78%) compared to healthy controls (mean 15.38%, range 9.67-21.94%). PD-1 was mostly co-expressed with TIGIT, although TIGIT+PD-1+CD4+ and CD8+ T-cells were higher in CLL compared to healthy controls (12.9% vs 7.1%). Interestingly the predominant population of PD-1+CD8+ T cells in CLL was also positive for 2B4 and TIGIT, whereas expression of TIGIT was more diverse and was seen in association with PD-1, 2B4, KLRG1 or CD57. Taken together, our findings indicate a remarkable heterogeneity in the expression patterns of inhibitory molecules on CD8+ and CD4+ T-cells in CLL. While CLL patients with a normal CD4:CD8 ratio expressed comparable levels of inhibitory molecules to that seen in healthy controls, a low CD4:CD8 ratio was indicative of higher expression of checkpoint molecules. On a per cell basis, CLL CD8+ T cells expressed more inhibitory receptors compared to healthy controls, suggesting that certain patients may benefit from combinational use of checkpoint molecules. A more detailed data and analysis, including transcription and functional profile of exhausted CLL T cells, will be presented in the meeting. Disclosures Wierda: Abbvie: Research Funding; Novartis: Research Funding; Acerta: Research Funding; Gilead: Research Funding; Genentech: Research Funding. Jain:Incyte: Research Funding; Pharmacyclics: Consultancy, Honoraria, Research Funding; Abbvie: Research Funding; Infinity: Research Funding; BMS: Research Funding; Genentech: Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; ADC Therapeutics: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria; Seattle Genetics: Research Funding; Celgene: Research Funding; Servier: Consultancy, Honoraria; Novimmune: Consultancy, Honoraria.

Allogeneic hematopoietic SCT (allo-HCT) from matched sibling donor (MSD) is recommended for younger patients with intermediate cytogenetic risk AML in first CR (CR1), whereas the role of alternative donor transplants in these patients is unknown. We retrospectively analyzed 605 patients with intermediate-risk AML, who received myeloablative allo-HCT in CR1. The 4-year OS for MSD (n=290) and matched unrelated donor (MUD; n=141) was 65% and 68% (P=0.50), respectively. In multivariate analysis, MUD had a similar risk of overall mortality as MSD (hazard ratio=0.90; 95% confidence interval, 0.62-1.30; P=0.58), whereas older age, female donor/male recipient (FDMR) combination, and requiring more than one course of induction chemotherapy to achieve CR1 were poor prognostic factors for OS. Thus, OS after MUD HCT with sex combinations other than FDMR was significantly higher than that after MSD HCT from female donors to male recipients (4-year OS 72% versus 55%, P=0.04). These results suggest that HCT, not only from MSD, but also from MUD, should be considered in younger patients with intermediate-risk AML in CR1, and that the donor-recipient sex combination is more important than the donor type in donor selection.

Patients with CLL experience generalized immune suppression, susceptibility to infections and secondary malignancies that likely involve complex bi-directional interactions between leukemic cells, components of the tumor microenvironment and immune effectors. CLL cells are capable of secreting IL-10 and exhibit regulatory functions comparable to that of normal B10 cells, a regulatory B cell subset that suppresses effector T-cell function through STAT3-mediated production of IL-10. However, the underlying mechanisms governing IL-10 production by CLL cells are not fully understood. The chemokine CXCL12 is constitutively secreted by bone marrow stroma in CLL, and binds CXCR4 to direct chemotaxis, support tumor survival and activate various signaling pathways, including STAT3. Thus, we investigated if CXCL12 can enhance IL-10 production by activating the STAT3 pathway in CLL. Using peripheral blood mononuclear cells (PBMC) from 24 CLL patients who had not received therapy for ≥2 years, we showed that CXCL12 can enhance IL-10 production by CLL cells by activating S727-STAT3. This effect was CXCR4-mediated since blocking the CXCR4-CXCL12 interaction with a blocking antibody abolished CXCL12-induced IL-10 production. Addition of the STAT3 inhibitor curcubitacin to the culture also abrogated CXCL12-induced IL-10 production, confirming an important role for S727-STAT3 as a mediator of CXCL12-CXCR4-induced IL-10 production by CLL cells. We next determined if activation of the CXCR4-CXCL12-STAT3-IL10 pathway in CLL is important in mediating their immunoregulatory function. Culture of primary CLL withCXCL12 induced significantly more suppression of CD3+ T cell function, including TNF-α, IFN-γ and IL-2 production, and CD107a degranulation, compared to CD3+ T cells cultured with untreated CLL cells or with CXCL12 alone. The addition of IL-10 blocking antibody to the co-culture completely reversed T cell dysfunction, supporting an important for IL-10 in CLL-mediated T-cell suppression. IL-10 has been reported to induce T cell suppression through phosphorylation of Y705-STAT3.. Blocking IL-10 also prevented CLL-induced phosphorylation of Y705-STAT3 in T cells, confirming an important role for CLL-derived IL-10 in activation of Y705-STAT3 and induction of T cell dysfunction. Lenalidomide is an immune-modulatory drug with clinical efficacy in CLL and was recently reported to inhibit STAT3 phosphorylation. To investigate if lenalidomide can inhibit CXCL12-induced STAT3 phosphorylation, we treated CLL cells with lenalidomide and measured p-S727-STAT3 levels. Exposure of CLL cells to 10µM/ml lenalidomide prevented CXCL12-induced increase in p-S727-STAT3 and resulted in significant reduction in the IL-10 response by CLL cells. Lenalidomide also suppressed IL-10-induced Y705-STAT3 phosphorylation in healthy T cells, thus reversing CLL-induced T cell dysfunction. We next confirmed the in vivo relevance of our findings using PBMC cryopreserved from patients treated with lenalidomide monotherapy (NCT00535873). When compared to pretreatment samples, CLL cells from on-treatment samples produced less IL-10 and showed significantly improved T cell function. We thus conclude that the capacity of CLL to produce IL-10 is mediated by the CXCL12-CXCR4-STAT3 pathway and may contribute to immunodeficiency in patients. Lenalidomide can reverse CLL-induced immunosuppression through multiple mechanisms that involve abrogation of the CXCL12-CXCR4-S727STAT3-mediated IL-10 response by CLL B cells and prevention of IL-10-induced phosphorylation of Y705-STAT3 in T cells. Disclosures Estrov: incyte: Consultancy, Research Funding. Wierda:Glaxo-Smith-Kline Inc.: Research Funding; Celgene Corp.: Consultancy. Rezvani:Pharmacyclics: Research Funding.

INTRODUCTION: The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib is a covalent inhibitor of BTK, a member of the B-cell receptor (BCR) signaling pathway and induces objective clinical responses in the majority of CLL patients (Byrd et al., NEJM 2013). Interestingly this drug also inhibits L2-inducible T cell kinase (ITK), an essential enzyme for the development and effector function of Th2 and Th17 cells, and has been shown to shift the balance towards a Th1 response. The purpose of the study was to determine how ibrutinib influences the Th1/Th17/T regulatory cell response, expression of immune checkpoint blockade molecules on T cells and the functional pathogen-specific T cell recovery. METHODS: Here we present data from a clinical trial of ibrutinib versus ibrutinib + rituximab in previously treated patients (NCT02007044). Peripheral blood and serum were collected at baseline, 3 months and 6 months during therapy. Multicolor flow cytometry was used to characterize B cell subsets, T-cell subsets, expression of PD-1, PD-L1 and CTLA-4 and T-cell effector function. For statistical analysis of pre-treatment to on-treatment measurements the paired Student t-test was used. RESULTS: Here we report on the phenotypic and functional recovery of immune subsets in 41 CLL patients treated with ibrutinib (n=17) or ibrutinib + rituximab (n=25). Both PD-1 and PD-L1 were expressed at high levels on CLL cells. Interestingly, by 3 and 6 months, there was a significant decrease in PD-1 expression from a pre-treatment median of 15% to 4% (at 3 months) and 3% (at 6 months; P CONCLUSION: Based on these data we propose that ibrutinib therapy can modulate the T cell response through multiple mechanisms which include (i) direct inhibition of ITK and skewing of the T cell response toward a Th1 profile; (ii) reduction in PD1/PDL1 expression on B and T cells and (iii). suppression of Tregs. It is unclear how ibrutinib influences the PD1/PDL1 interaction and whether this is a direct effect of the drug on essential components of B and T cell-receptor signaling or whether it is an indirect effect related to a reduction in the leukemia burden. Mechanistic studies to understand how ibrutinib modulates the PD1/PL1 axis are currently underway. Figure 1. Ibrutinib therapy is associated with a reduction in PDL1 expression on the surface of CD19+ B cells. Figure 1. Ibrutinib therapy is associated with a reduction in PDL1 expression on the surface of CD19+ B cells. Figure 2. Ibrutinib therapy is associated with a reduction in PD1 expression on the surface of CD3+ T cells. Figure 2. Ibrutinib therapy is associated with a reduction in PD1 expression on the surface of CD3+ T cells. Disclosures Burger: Pharmacyclics LLC, an AbbVie Company: Research Funding. Wierda:Glaxo-Smith-Kline Inc.: Research Funding; Celgene Corp.: Consultancy. Rezvani:Pharmacyclics: Research Funding.

Programmed death-ligand 1 (PD-L1) binds to programmed death-1 (PD-1) on T cells and contributes to T cell exhaustion during chronic infections. Moreover, it has been shown that engagement of PD-1 on T cells by PD-L1 on tumor cells is associated with the immune escape of tumors, and recent clinical trials have highlighted the anti-tumor efficacy of blockade of the PD-1/PD-L1 pathway. Along with costimulatory molecules such as CD80 and CD86, PD-L1 is also highly expressed on dendritic cells (DCs), which are professional antigen-presenting cells (APCs) that are widely used to generate antigen-specific cytotoxic T lymphocytes (CTLs) for adoptive immunotherapy. However, the roles of PD-L1 on APCs have not been well examined. Thus, we evaluated the roles of PD-L1 on APCs in the induction of cytomegalovirus (CMV)-specific CTLs (CMV-CTLs). First, peripheral blood mononuclear cells (PBMCs) were obtained from five CMV-seropositive healthy donors (HLA-A*02:01, n = 3; A*24:02, n = 1; B*07:02, n = 1). Mature DCs were generated from adherent PBMCs by stimulation with GM-CSF/IL-4 and maturational cytokines (TNF-a, IL-1b, IL-6, and PGE2), and freshly isolated CD3+ T cells were stimulated with mature DCs pulsed with CMV pp65-derived HLA-restricted peptides (NLVPMVATV for HLA-A*02:01, QYDPVAALF for A*24:02, and TPRVTGGGAM for B*07:02) in the presence or absence of anti-PD-L1 blocking antibody. After 14 days of culture, the presence of anti-PD-L1 antibody resulted in a less efficient induction of CMV-CTLs (Figure 1), suggesting that PD-L1 might play a positive role in the induction of antigen-specific CTLs. For further evaluation, we generated K562-based artificial APCs (aAPCs), which were retrovirally transduced with HLA class I molecules and various combinations of CD80/86 and PD-L1 (named K562, K562+CD80/86, K562+PD-L1, and K562+CD80/86+PD-L1). CD3+ T cells isolated from nine CMV-seropositive healthy donors (HLA-A*02:01, n = 4; A*24:02, n = 4; B*07:02, n = 1) were stimulated weekly for 28 days using HLA-restricted CMV peptide-pulsed K562-based aAPCs. K562+CD80/86+PD-L1 led to significantly higher induction of CMV-CTLs than K562 or K562+CD80/86 (Figure 2). Since the original K562 cells slightly express CD80, we completely knocked out the CD80 expression in the cells using the CRISPR/Cas9 system, and then we transduced the cells with or without PD-L1 (named CD80KO K562+PD-L1 and CD80KO K562, respectively) to determine whether PD-L1 itself has a stimulatory effect on the induction of CMV-CTLs. CD80KO K562 and CD80KO K562+PD-L1 induced the same amount of CMV-CTLs, suggesting that PD-L1 itself does not have a stimulatory effect for the induction of CMV-CTLs. Phenotypic analysis showed that CMV-CTLs induced by K562+CD80/86+PD-L1 contained more CD45RA- CD62L+ central memory T cells, which persist longer and are more effective in vivo after adoptive transfer in CTL therapy, than CMV-CTLs induced by other K562-based aAPCs. In addition, when compared with CMV-CTLs induced by other K562-based aAPCs, CMV-CTLs induced by K562+CD80/86+PD-L1 did not express higher levels of exhaustion markers such as PD-1 and TIM-3, and exhibited similar levels of IFN-gamma and IL-2 production and CD107a expression in response to the specific peptide stimulation. Furthermore, to evaluate the effect of K562-based aAPCs on the induction of tumor antigen-specific CTLs, CD3+ T cells were isolated from three HLA-A*24:02-positive healthy donors and stimulated with HLA-A*24:02-restricted WT1 peptide-pulsed K562-based aAPCs weekly. After four stimulations, only K562+CD80/86+PD-L1 could clearly expand the WT1-specific CTLs from all of the three donors (Figure 3). In conclusion, our findings demonstrate that PD-L1 expressed on APCs along with CD80/86 enhanced the induction of antigen-specific CTLs without causing excessive differentiation or functional exhaustion of the induced CTLs. These results suggest that stimulation with K562+CD80/86 might result in the activation-induced cell death of antigen-specific CTLs and that PD-L1 might be involved in fine-tuning excessive stimulation of CD80/86. Further analyses to determine the mechanisms of our findings are warranted. Our results also highlight the possibility that K562+CD80/86+PD-L1 has therapeutic potential as novel aAPCs for the generation of CTLs for adoptive immunotherapy. Disclosures Kiyoi: Takeda Pharmaceutical Co., Ltd.: Research Funding; Pfizer Inc.: Research Funding; Teijin Ltd.: Research Funding; Taisho Toyama Pharmaceutical Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Yakult Honsha Co.,Ltd.: Research Funding; MSD K.K.: Research Funding; Alexion Pharmaceuticals: Research Funding; Novartis Pharma K.K.: Research Funding; Astellas Pharma Inc.: Consultancy, Research Funding; Japan Blood Products Organization: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; FUJIFILM RI Pharma Co.,Ltd.: Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; FUJIFILM Corporation: Patents & Royalties, Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Consultancy, Research Funding; Bristol-Myers Squibb: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Mochida Pharmaceutical Co., Ltd.: Research Funding.

Hemophagocytic syndrome (HPS) induced by uncontrolled macrophage activation and subsequent graft failure is a frequent and prominent complication after allogeneic stem cell transplantation (allo-SCT), a cause of severe morbidity and death, and a therapeutic challenge. Liposome-incorporated dexamethasone, dexamethasone palmitate (DP), shows greater efficacy against macrophages as compared to dexamethasone sodium phosphate (DSP). Based on our findings that DP achieves significantly larger decrease than DSP on the viability of primary human macrophages compared in vitro, we tested the effects of DP in patients with HPS. A decrease in number of macrophages in the bone marrow and prevention of engraftment failure were observed in all patients without any severe complications. In conclusion, these data provide a rationale for testing DP as a first-line treatment for patients with HPS after allo-SCT.

Objective: Herpes zoster is the most common infection in patients treated with bortezomib-containing regimens for multiple myeloma. Some clinical trials have reported on the use of acyclovir prophylaxis to decrease the incidence of herpes zoster. However, the appropriate acyclovir dose and duration of prophylaxis remain unclear. The primary objective of this study was to evaluate the efficacy of continuous oral 200 mg/day acyclovir prophylaxis and the secondary objective was to determine the risk factors for developing herpes zoster. Methods: We collected medical information from consecutive patients who received bortezomib with or without acyclovir prophylaxis for relapsed or refractory multiple myeloma at our hospital and retrospectively analyzed the efficacy of acyclovir prophylaxis and the parameters for predicting the risk factors for developing herpes zoster. The definition of acyclovir prophylaxis was oral continuous administration of 200 mg of once daily, without cessation, during the entire period of bortezomib treatment. Results: Six of the 33 patients in the study developed herpes zoster during bortezomib treatment. No varicella-zoster virus reactivation was observed in the 19 patients in the acyclovir prophylaxis group. The incidence of herpes zoster was significantly higher in the group that did not receive acyclovir prophylaxis (43%, 6 of 14 patients) than in the group that did (0%, 0 of 19; P ¼ 0.003). The predictive factors for varicella-zoster virus reactivation were male sex (P ¼ 0.035) and the use of acyclovir (P ¼ 0.003). Conclusions: Continuous prophylaxis by oral 200 mg/day acyclovir in multiple myeloma patients receiving bortezomib treatment is effective and sufficient in preventing herpes zoster.

Early relapse is a parameter that affects clinical outcomes in relapsed diffuse large B cell lymphoma (DLBCL). The prognostic value of lymphopenia following the completion of first-line therapy and the relationship between lymphopenia and early relapse are unknown. Therefore, we studied the role of absolute lymphocyte count (ALC) on early relapse. We retrospectively analyzed de novo DLBCL patients who were treated with rituximab-containing treatment between 2003 and 2010. The median age at the time of diagnosis of 59 DLBCL patients was 71 years. We identified no association between ALC at diagnosis and ALC following the completion of first-line therapy. Among all patients analyzed, 13 (22%) patients were confirmed to exhibit early relapse. Low ALC following the completion of first-line therapy was significantly associated with early relapse by univariate analysis [hazard ratio (HR) = 4.05; 95% confidence interval (CI), 1.11–14.73; P = 0.02] and multivariate analysis (HR = 4.66; 95% CI, 1.24–17.48; P = 0.023). The low ALC group tended to have worse outcomes than the high ALC group with lower rates of progression-free survival (66% and 74%, respectively; P = 0.13) and overall survival (74% and 86%, respectively; P = 0.09), but these differences did not reach statistically. Lymphopenia following the completion of first-line therapy can be used as a marker to predict early relapse.

The prognosis of patients with Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph(+)ALL) relapsing after allogeneic hematopoietic stem cell transplantation is dismal. Here we describe a patient with post-transplant relapse of Ph(+)ALL, who has remained in complete remission (CR) for 30 months after relapse. A 55-year-old woman with Ph(+)ALL received allo-HSCT from an unrelated donor during first CR. The conditioning regimen consisted of fludarabine+melphalan, and graft-versus-host disease (GVHD) prophylaxis consisted of tacrolimus and short-term methotrexate. She achieved and maintained molecular remission without developing GVHD after transplantation, but suffered a hematologic relapse on day 871. She received imatinib-combined chemotherapy, and again achieved molecular remission. Since the completion of imatinib-combined chemotherapy, she has been receiving imatinib monotherapy. Although it has been reported that chemotherapy and imatinib are effective only transiently in patients with relapsed Ph(+)ALL, our patient has remained in molecular remission for 30 months after post-transplant relapse at the time of this report. Our case suggests that by continuing imatinib after the induction of molecular remission by imatinib-combined chemotherapy, the antileukemic activity of imatinib could achieve durable remission in combination with the graft-versus-leukemia effect. However, this needs to be investigated in studies involving a large number of patients.

Corticosteroids are often used following allogeneic hematopoietic stem cell transplantation (HSCT) to control complications such as graft-versus-host disease (GVHD). However, there is some concern that corticosteroids may suppress the graft-versus-leukemia effect and increase leukemia relapse. To evaluate the effect of corticosteroids on relapse, we analyzed 112 adult patients who received their first allogeneic HSCT for acute myeloid leukemia at our institution between 1997 and 2007. Fifty-seven patients (50.9%) received corticosteroid therapy. Patients who had corticosteroid therapy included higher proportion of patients who developed GVHD. In multivariate analysis, with corticosteroid administration entered as a time-dependent covariate, corticosteroid administration was not a risk factor for relapse (p = 1.00, hazard ratio [HR] 1.00, 95% confidence interval [CI] 0.53-1.88), but it was associated with higher non-relapse mortality (NRM) (p0.001, HR 55.5, 95% CI 7.42-416) and lower overall survival (p0.001, HR 2.68, 95% CI 1.56-4.61). The higher NRM associated with corticosteroid administration was mainly due to the increased deaths caused by the complications themselves, which required corticosteroid therapy. The findings of this study indicate the importance of controlling complications after allogeneic HSCT. The strategy of refraining from indispensable corticosteroid therapy because of the excessive concerns about relapse should be avoided.