Selective lysis of acute myeloid leukemia cells by CD34/CD3 bispecific antibody through the activation of γδ T-cells.

Despite the considerable progress in acute myeloid leukemia (AML) treatment, relapse after allogeneic hematopoietic stem cell transplantation (HSCT) is still frequent and associated with a poor prognosis. Relapse has been shown to be correlated with an incomplete eradication of CD34+ leukemic stem cells prior to HSCT. Previously, we have shown that a novel CD34-directed, bispecific T-cell engager (BTE) can efficiently redirect the T-cell effector function toward cancer cells, thus eliminating leukemic cells in vitro and in vivo. However, its impact on γδ T-cells is still unclear. In this study, we tested the efficacy of the CD34-specific BTE using in vitro expanded γδ T-cells as effectors. We showed that the BTEs bind to γδ T-cells and CD34+ leukemic cell lines and induce target cell killing in a dose-dependent manner. Additionally, γδ T-cell mediated killing was found to be superior to αß T-cell mediated cytotoxicity. Furthermore, we observed that only in the presence of BTE the γδ T-cells induced primary AML blast killing in vitro. Importantly, our results show that γδ T-cells did not target the healthy CD34intermediate endothelial blood-brain barrier cell line (hCMEC/D3) nor lysed CD34+ HSCs from healthy bone marrow samples.

Inducing expression of ICOS-L by oncolytic adenovirus to enhance tumor-specific bi-specific antibody efficacy.

BACKGROUND: Intratumoral injection of oncolytic viruses (OVs) shows promise in immunotherapy: ONCOS-102, a genetically engineered OV that encodes Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) demonstrated efficacy in early clinical trials, enhancing T cell infiltration in tumors. This suggests OVs may boost various forms of immunotherapy, including tumor-specific bi-specific antibodies (BsAbs). METHODS: Our study investigated in vitro, how ONCOS-204, a variant of ONCOS-virus expressing the ligand of inducible T-cell co-stimulator (ICOSL), modulates the process of T cell activation induced by a BsAb. ONCOS-102 was used for comparison. Phenotypic and functional changes induced by combination of different OVs, and BsAb in T cell subsets were assessed by flow cytometry, viability, and proliferation assays. RESULTS: Degranulation and IFNγ and TNF production of T cells, especially CD4 + T cells was the most increased upon target cell exposure to ONCOS-204. Unexpectedly, ONCOS-204 profoundly affected CD8 + T cell proliferation and function through ICOS-L/ICOS interaction. The effect solely depended on cell surface expression of ICOS-L as soluble ICOSL did not induce notable T cell activity. CONCLUSIONS: Together, our data suggests that oncolytic adenoviruses encoding ICOSL may enhance functional activity of tumor-specific BsAbs thereby opening a novel avenue for clinical development in immunotherapeutics.

KIR2DS1 and KIR2DL1-C245 Dominantly Repress NK Cell Degranulation Triggered by Monoclonal or Bispecific Antibodies, whereas Education by Uptuning Inhibitory Killer Ig-related Receptors Exerts No Advantage in Ab-dependent Cellular Cytotoxicity.

NK cell responsiveness to target cells is tuned by interactions between inhibitory NK cell receptors and their cognate HLA class I ligands in a process termed "NK cell education." Previous studies addressing the role for NK cell education in Ab-dependent cellular cytotoxicity (ADCC) show ambiguous results and do not encompass full educational resolution. In this study, we systematically characterized human NK cell CD16-triggered degranulation toward defined human tumor cell lines in the presence of either the mAb rituximab or a recently developed CD34xCD16 bispecific killer engager. Despite positive correlation between killer Ig-related receptor (KIR)-mediated education and CD16 expression, NK cells educated by one or even two inhibitory KIRs did not perform better in terms of ADCC than uneducated NK cells in either missing-self or KIR-ligand matched settings at saturating Ab concentrations. Instead, NKG2A+ NK cells consistently showed more potent ADCC in the missing-self context despite lower levels of CD16 expression. KIR2DS1+ NK cells demonstrated dampened ADCC in both the missing-self and KIR-ligand matched settings, even in the presence of its ligand HLA C2. The lower response by KIR2DS1+ NK cells was also observed when stimulated with a bispecific killer engager. Surprisingly, repression of ADCC was also observed by NKG2A+ NK cells coexpressing the inhibitory KIR2DL1-C245 receptor that confers weak education. In conclusion, our study suggests that NK cell education by inhibitory KIRs does not augment ADCC per se, whereas expression of KIR2DS1 and KIR2DL1-C245 dominantly represses ADCC. These insights add to the fundamental understanding of NK cells and may have implications for their therapeutic use.

Human Vγ9Vδ2 T cell expansion and their cytotoxic responses against cholangiocarcinoma.

Human Vγ9Vδ2 T lymphocytes are regarded as promising effector cells for cancer immunotherapy since they have the ability to eliminate several tumor cells through non-peptide antigen recognition. However, the cytotoxic function and the mechanism of Vγ9Vδ2 T cells leading to specific killing of cholangiocarcinoma cells are yet to be confirmed. In this study, we established a protocol for ex vivo expansion of Vγ9Vδ2 T cells from healthy donors' peripheral blood mononuclear cells by culture with zoledronate and addition of IL-2, and IL-15 or IL-18 or neither. Testing the cytotoxic capacity of cultured Vγ9Vδ2 T cells against cholangiocarcinoma cell lines showed higher reactivity than against control cells. Surface expression of CD107 was detected on the Vγ9Vδ2 T cells, suggesting that these cells limit in vitro growth of cholangiocarcinoma cells via degranulation of the perforin and granzyme pathway. Analysis of molecular signaling was used to demonstrate expression of pro- and anti-survival genes and a panel of cytokine genes in Vγ9Vδ2 T cells. We found that in the presence of either IL-15 or IL-18, levels of caspase 3 were significantly reduced. Also, IL-15 and IL-18 stimulated cells contained cytotoxicity against cholangiocarcinoma cells, suggesting that stimulated Vγ9Vδ2 T cells may provide a feasible therapy for cholangiocarcinoma.

Modulation of lytic molecules restrain serial killing in γδ T lymphocytes.

γδ T cells play a pivotal role in protection against various types of infections and tumours, from early childhood on and throughout life. They consist of several subsets characterised by adaptive and innate-like functions, with Vγ9Vδ2 being the largest subset in human peripheral blood. Although these cells show signs of cytotoxicity, their modus operandi remains poorly understood. Here we explore, using live single-cell imaging, the cytotoxic functions of γδ T cells upon interactions with tumour target cells with high temporal and spatial resolution. While γδ T cell killing is dominated by degranulation, the availability of lytic molecules appears tightly regulated in time and space. In particular, the limited co-occurrence of granzyme B and perforin restrains serial killing of tumour cells by γδ T cells. Thus, our data provide new insights into the cytotoxic arsenal and functions of γδ T cells, which may guide the development of more efficient γδ T cell based adoptive immunotherapies.

T cell receptor excision circles are potential predictors of survival in adult allogeneic hematopoietic stem cell transplantation recipients with acute myeloid leukemia.

Background: Lymphocyte neogenesis from primary lymphoid organs is essential for a successful reconstitution of immunity after allogeneic hematopoietic stem cell transplantation (HSCT). This single-center retrospective study aimed to evaluate T cell receptor excision circles (TREC) and kappa-deleting recombination excision circles (KREC) as surrogate markers for T and B cell recovery, as predictors for transplantation-related outcomes in adult acute myeloid leukemia (AML) patients. Methods: Ninety adult patients diagnosed with AML and treated with HSCT between 2010 and 2015 were included in the study. TREC and KREC levels were measured by quantitative PCR at 1, 3, 6, and 12 months after transplantation. Results: Overall, excision circle levels increased between 3 and 6 months post-HSCT for TREC (p = 0.005) and 1 and 3 months for KREC (p = 0.0007). In a landmark survival analysis at 12 months post-HSCT, TREC levels were associated with superior overall survival (HR: 0.52, 95% CI: 0.34 - 0.81, p = 0.004). The incidence of viral infections within the first 100 days after transplantation was associated with lower TREC levels at 6 months (p = 0.0002). CMV reactivation was likewise associated with lower TREC levels at 6 months (p = 0.02) post-HSCT. KREC levels were not associated with clinical outcomes in statistical analyzes. Conclusions: Results from the present study indicate that TREC measurement could be considered as part of the post-HSCT monitoring to identify AML patients with inferior survival after transplantation. Further prospective studies are warranted to validate these findings.

Gamma delta T-cell reconstitution after allogeneic HCT: A platform for cell therapy.

Allogeneic Hematopoietic stem cell transplantation (allo-HCT) is a curative platform for several hematological diseases. Despite its therapeutic benefits, the profound immunodeficiency associated with the transplant procedure remains a major challenge that renders patients vulnerable to several complications. Today, It is well established that a rapid and efficient immune reconstitution, particularly of the T cell compartment is pivotal to both a short-term and a long-term favorable outcome. T cells expressing a TCR heterodimer comprised of gamma (γ) and delta (δ) chains have received particular attention in allo-HCT setting, as a large body of evidence has indicated that γδ T cells can exert favorable potent anti-tumor effects without inducing severe graft versus host disease (GVHD). However, despite their potential role in allo-HCT, studies investigating their detailed reconstitution in patients after allo-HCT are scarce. In this review we aim to shed lights on the current literature and understanding of γδ T cell reconstitution kinetics as well as the different transplant-related factors that may influence γδ reconstitution in allo-HCT. Furthermore, we will present data from available reports supporting a role of γδ cells and their subsets in patient outcome. Finally, we discuss the current and future strategies to develop γδ cell-based therapies to exploit the full immunotherapeutic potential of γδ cells in HCT setting.

A novel CD34-specific T-cell engager efficiently depletes acute myeloid leukemia and leukemic stem cells in vitro and in vivo.

Less than a third of patients with acute myeloid leukemia (AML) are cured by chemotherapy and/or hematopoietic stem cell transplantation, highlighting the need to develop more efficient drugs. The low efficacy of standard treatments is associated with inadequate depletion of CD34+ blasts and leukemic stem cells, the latter a drug-resistant subpopulation of leukemia cells characterized by the CD34+CD38- phenotype. To target these drug-resistant primitive leukemic cells better, we have designed a CD34/CD3 bi-specific T-cell engager (BTE) and characterized its anti-leukemia potential in vitro, ex vivo and in vivo. Our results show that this CD34-specific BTE induces CD34-dependent T-cell activation and subsequent leukemia cell killing in a dose-dependent manner, further corroborated by enhanced T-cell-mediated killing at the singlecell level. Additionally, the BTE triggered efficient T-cell-mediated depletion of CD34+ hematopoietic stem cells from peripheral blood stem cell grafts and CD34+ blasts from AML patients. Using a humanized AML xenograft model, we confirmed that the CD34-specific BTE had in vivo efficacy by depleting CD34+ blasts and leukemic stem cells without side effects. Taken together, these data demonstrate that the CD34-specific BTE has robust antitumor effects, supporting development of a novel treatment modality with the aim of improving outcomes of patients with AML and myelodysplastic syndromes.

Revisiting the Role of γδ T Cells in Anti-CMV Immune Response after Transplantation.

Gamma delta (γδ) T cells form an unconventional subset of T lymphocytes that express a T cell receptor (TCR) consisting of γ and δ chains. Unlike conventional αß T cells, γδ T cells share the immune signature of both the innate and the adaptive immunity. These features allow γδ T cells to act in front-line defense against infections and tumors, rendering them an attractive target for immunotherapy. The role of γδ T cells in the immune response to cytomegalovirus (CMV) has been the focus of intense research for several years, particularly in the context of transplantation, as CMV reactivation remains a major cause of transplant-related morbidity and mortality. Therefore, a better understanding of the mechanisms that underlie CMV immune responses could enable the design of novel γδ T cell-based therapeutic approaches. In this regard, the advent of next-generation sequencing (NGS) and single-cell TCR sequencing have allowed in-depth characterization of CMV-induced TCR repertoire changes. In this review, we try to shed light on recent findings addressing the adaptive role of γδ T cells in CMV immunosurveillance and revisit CMV-induced TCR reshaping in the era of NGS. Finally, we will demonstrate the favorable and unfavorable effects of CMV reactive γδ T cells post-transplantation.

Characterization of ascites- and tumor-infiltrating γδ T cells reveals distinct repertoires and a beneficial role in ovarian cancer.

The role of γδ T cells in antitumor immunity has been under investigation for the past two decades, but little is known about their contribution to clinical outcomes in patients. Here, we set out to define the clonotypic, phenotypic, and functional features of γδ T cells in peripheral blood, ascites, and metastatic tumor tissue from patients with advanced epithelial ovarian cancer. T cell receptor (TCR) sequencing of the γ chain revealed that tumor-infiltrating γδ T cells have a unique and skewed repertoire with high TCR diversity and low clonality. In contrast, ascites-derived γδ T cells presented a lower TCR diversity and higher clonality, suggesting a TCR-dependent clonal focusing at this site. Further investigation showed that tumor samples had abundant γδ T cells with a tissue-resident, activation-associated phenotype, less usage of Vγ9 and an impaired response to adaptive-associated stimuli, implying an innate-like activation pathway, rather than an adaptive TCR-engaging pathway, at these tumor sites. Furthermore, high γδ T cell cytokine responsiveness upon stimulation was associated with a favorable outcome for patients in terms of both overall survival and reduced residual tumor burden after primary surgery. Last, the functionality of γδ T cells and patient survival were negatively affected by the proportions of CD39-expressing T cells, highlighting the potential of CD39 as a target to improve γδ T cell responses and unleash their antitumor capabilities.

Cryopreservation of buffy coat derived platelets: Paired in vitro characterization using uncontrolled versus controlled freezing rate protocols.

BACKGROUND: Cryopreserved platelets show a reduced recovery and viability after freezing and thawing including several ultrastructural and phenotypic deteriorations compared with liquid-stored platelets. It is suggested that using Controlled-Rate Freezing (CRF) can reduce variability and optimize the functionality profile for cells. The objective of the study is to compare cellular, metabolic, phenotypic and functional effects on platelets after cryopreservation using different freezing rate protocols. STUDY DESIGN AND METHODS: To evaluate the possible effects of different freezing rate protocols a two-experimental study comparing diverse combinations was tested with a pool and split design. Uncontrolled freezing of platelets in materials with different thermal conductivity (metal vs cardboard) was evaluated in experiment 1. Experiment 2 evaluated uncontrolled vs a controlled-rate freezing protocol in metal boxes. All variables were assessed pre and post cryopreservation. RESULTS: Directly after thawing, no major differences in platelet recovery, LDH, ATP, Δψ, CD62P, CD42b, platelet endothelial cell adhesion molecule and sCD40L were seen between units frozen with different thermal conductivity for temperature. In contrast, we observed signs of increased activation after freezing using the CRF protocol, reflected by increased cell surface expression of CD62P, PAC-1 binding and increased concentration of LDH. Agonist induced expression of a conformational epitope on the GPIIb/IIIa complex and contribution to blood coagulation in an experimental rotational thromboelastometry setup were not statistically different between the groups. CONCLUSION: The use of a uncontrolled freezing rate protocol is feasible, creating a platelet product comparable to using a controlled rate freezing equipment during cryopreservation of platelets.

Evaluating Thymic Function After Human Hematopoietic Stem Cell Transplantation in the Personalized Medicine Era.

Hematopoietic stem cell transplantation (HSCT) is an effective treatment option for several malignant and non-malignant hematological diseases. The clinical outcome of this procedure relies to a large extent on optimal recovery of adaptive immunity. In this regard, the thymus plays a central role as the primary site for de novo generation of functional, diverse, and immunocompetent T-lymphocytes. The thymus is exquisitely sensitive to several insults during HSCT, including conditioning drugs, corticosteroids, infections, and graft-vs.-host disease. Impaired thymic recovery has been clearly associated with increased risk of opportunistic infections and poor clinical outcomes in HSCT recipients. Therefore, better understanding of thymic function can provide valuable information for improving HSCT outcomes. Recent data have shown that, besides gender and age, a specific single-nucleotide polymorphism affects thymopoiesis and may also influence thymic output post-HSCT, suggesting that the time of precision medicine of thymic function has arrived. Here, we review the current knowledge about thymic role in HSCT and the recent work of genetic control of human thymopoiesis. We also discuss different transplant-related factors that have been associated with impaired thymic recovery and the use of T-cell receptor excision circles (TREC) to assess thymic output, including its clinical significance. Finally, we present therapeutic strategies that could boost thymic recovery post-HSCT.

Profound Functional Suppression of Tumor-Infiltrating T-Cells in Ovarian Cancer Patients Can Be Reversed Using PD-1-Blocking Antibodies or DARPin® Proteins.

PD-1/PD-L1 blockade has revolutionized the field of immunooncology. Despite the relative success, the response rate to anti-PD-1 therapy requires further improvements. Our aim was to explore the enhancement of T-cell function by using novel PD-1-blocking proteins and compare with clinically approved monoclonal antibodies (mAbs). We isolated T-cells from the ascites and tumor of 17 patients with advanced epithelial ovarian cancer (EOC) and analyzed the effects using the mAbs nivolumab and pembrolizumab and two novel engineered ankyrin repeat proteins (DARPin® proteins). PD-1 blockade with either mAb or DARPin® molecule significantly increased the release of IFN-γ, granzyme B, IL-2, and TNF-α, demonstrating successful reinvigoration. The monovalent DARPin® protein was less effective compared to its bivalent equivalent, demonstrating that bivalency brings an additional benefit to PD-1 blockade. Overall, we found a higher fold increase of lymphokine secretion in response to the PD-1 blockade by tumor-derived T-cells; however, the absolute amounts were significantly lower compared to the release from ascites-derived T-cells. Our results demonstrate that PD-1 blockade can only partially reinvigorate functionally suppressed T-cells from EOC patients. This warrants further investigation preferably in combination with other therapeutics. The study provides an early pilot proof-of-concept for the potential use of DARPin® proteins as eligible alternative scaffold proteins to block PD-1.

TOX is expressed by exhausted and polyfunctional human effector memory CD8+ T cells.

CD8+ T cell exhaustion is a hallmark of many cancers and chronic infections. In mice, T cell factor 1 (TCF-1) maintains exhausted CD8+ T cell responses, whereas thymocyte selection-associated HMG box (TOX) is required for the epigenetic remodeling and survival of exhausted CD8+ T cells. However, it has remained unclear to what extent these transcription factors play analogous roles in humans. In this study, we mapped the expression of TOX and TCF-1 as a function of differentiation and specificity in the human CD8+ T cell landscape. Here, we demonstrate that circulating TOX+ CD8+ T cells exist in most humans, but that TOX is not exclusively associated with exhaustion. Effector memory CD8+ T cells generally expressed TOX, whereas naive and early-differentiated memory CD8+ T cells generally expressed TCF-1. Cytolytic gene and protein expression signatures were also defined by the expression of TOX. In the context of a relentless immune challenge, exhausted HIV-specific CD8+ T cells commonly expressed TOX, often in clusters with various activation markers and inhibitory receptors, and expressed less TCF-1. However, polyfunctional memory CD8+ T cells specific for cytomegalovirus (CMV) or Epstein-Barr virus (EBV) also expressed TOX, either with or without TCF-1. A similar phenotype was observed among HIV-specific CD8+ T cells from individuals who maintained exceptional immune control of viral replication. Collectively, these data demonstrate that TOX is expressed by most circulating effector memory CD8+ T cell subsets and not exclusively linked to exhaustion.

CD8+ γδ T Cells Are More Frequent in CMV Seropositive Bone Marrow Grafts and Display Phenotype of an Adaptive Immune Response.

The role of gamma delta (γδ) T cells in human cytomegalovirus (HCMV) immune surveillance has been the focus of research interest for years. Recent reports have shown a substantial clonal proliferation of γδ T cells in response to HCMV, shedding light on the adaptive immune response of γδ T cells. Nevertheless, most efforts have focused on Vδ2neg γδ T cell subset while less attention has been given to investigate other less common γδ T cell subsets. In this regard, a distinct subpopulation of γδ T cells that expresses the CD8 coreceptor (CD8+ γδ T cells) has not been thoroughly explored. Whether it is implicated in HCMV response and its ability to generate adaptive response has not been thoroughly investigated. In this study, we combined flow cytometry and immune sequencing of the TCR γ-chain (TRG) to analyze in-depth bone marrow (BM) graft γδ T cells from CMV seropositive (CMV+) and CMV seronegative (CMV-) donors. We showed that the frequency of CD8+ γδ T cells was significantly higher in CMV+ grafts compared to CMV- grafts (P < 0.001). Further characterization revealed that CD8+ γδ T cells from CMV+ grafts express Vγ9- and preferentially differentiated from a naive to terminal effector memory phenotype (CD27low/-CD45RO-). In line with these findings, TRG immune sequencing revealed clonal focusing and reduced usage of the Vγ9/JP gene segment in a CMV+ graft. Furthermore, CD8+ γδ T cells showed an enhanced response to TCR/CD3 and cytokine stimulation in contrast to CD8- γδ T cells. We conclude that γδ T cells in BM grafts are reshaped by donor CMV serostatus and highlight the potential adaptive role of CD8+ γδ T cells in HCMV immune response.

Impact of γδ T cells on clinical outcome of hematopoietic stem cell transplantation: systematic review and meta-analysis.

Allogeneic hematopoietic stem cell transplantation (HSCT) using αß T-/B-cell-depleted grafts recently emerged as a transplant strategy and highlighted the potential role of γδ T cells on HSCT outcomes. Our aim was to scrutinize available evidence of γδ T-cell impact on relapse, infections, survival, and acute graft-versus-host disease (aGVHD). We performed a systematic review and meta-analysis of studies assessing γδ T cells in HSCT. We searched PubMed, Web of Science, Scopus, and conference abstracts from inception to March 2019 for relevant studies. We included all studies that assessed γδ T cells associated with HSCT. Data were extracted independently by 2 investigators based on strict selection criteria. A random-effects model was used to pool outcomes across studies. Primary outcome was disease relapse. We also assessed infections, survival, and aGVHD incidence. The review was registered with PROSPERO (CRD42019133344). Our search returned 2412 studies, of which 11 (919 patients) were eligible for meta-analysis. Median follow-up was 30 months (interquartile range, 22-32). High γδ T-cell values after HSCT were associated with less disease relapse (risk ratio [RR], 0.58; 95% confidence interval [95% CI], 0.40-0.84; P = .004; I2 = 0%), fewer viral infections (RR, 0.59; 95% CI, 0.43-0.82; P = .002; I2 = 0%) and higher overall (HR, 0.28; 95% CI, 0.18-0.44; P < .00001; I2 = 0%) and disease-free survivals (HR 0.29; 95% CI, 0.18-0.48; P < .00001; I2 = 0%). We found no association between high γδ T-cell values and aGVHD incidence (RR, 0.72; 95% CI, 0.41-1.27; P = .26; I2 = 0%). In conclusion, high γδ T cells after HSCT is associated with a favorable clinical outcome but not with aGVHD development, suggesting that γδ T cells have a significant effect on the success of HSCT. This study was registered with PROSPERO as #CRD42019133344.

Granulocyte transfusions could benefit patients with severe oral mucositis after allogeneic hematopoietic stem cell transplantation.

BACKGROUND AND OBJECTIVES: Mucositis is a common complication after allogeneic hematopoietic stem cell transplantation (HSCT), and is caused by a combination of conditioning-induced mucosal damage and severe neutropenia. The symptoms include oral and abdominal pain, inability to swallow food and fluids, and severe diarrhoea. Severe mucositis is associated with increased risk of Graft-versus-Host disease and infection. Granulocyte transfusions (GCX) could be a treatment option, and our objective was to study its feasibility and potential benefits. MATERIAL AND METHODS: This retrospective, single-centre study included 30 patients receiving GCX because of severe oral mucositis after HSCT during 2005-2017. Clinical outcome, response to GCX, change in opiate administration and adverse events were studied. RESULTS: Twenty-seven patients received GCX from donors pre-treated with steroids and G-CSF, and three from donors pre-treated with steroids only. Overall response was 83% (24/29 evaluable patients). Fifteen patients reached a complete response. In 14 of 24 responders, a reduction of the administration of opiate pain relief was seen. In eight patients this reduction was ≥50% of the dose. Adverse events (AEs) were reported in 14 cases, and were mild to moderate, and well manageable with symptomatic treatment. No life-threatening or fatal AEs were recorded. CONCLUSIONS: These results indicate that GCX could be a safe and effective treatment for oral mucositis after HSCT with the potential to reduce the necessity of opiate analgesic treatment in this disorder. No severe AEs were seen in this study, but the risk for severe pulmonary AEs after GCX needs to be considered.

Higher response rates in patients with severe chronic skin graft-versus-host disease treated with extracorporeal photopheresis.

INTRODUCTION: Different forms of graft-versus-host disease (GVHD) remain a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT). The prognosis for steroid-refractory chronic GVHD (cGVHD) remains poor. Our aim was to evaluate extracorporeal photopheresis (ECP) treatment in cGVHD patients with different organ involvement to detect subgroups of patients with the best response. MATERIAL AND METHODS: Thirty-four patients who underwent HSCT and developed moderate (n = 7) or severe (n = 27) steroid-refractory or steroid-dependent cGVHD treated with ECP were included in the analysis. A matched cGVHD control patient group untreated with ECP was collected for comparison. RESULTS: Compared to the control group and the stable/progressive disease (SD/PD) patients, individuals with complete/partial remission have higher overall survival and lower transplant-related mortality. Furthermore, patients with complete and partial remission (CR/PR) had significantly higher levels of albumin and platelets after ECP treatment compared to patients with stable or progressive cGVHD (SD/PD). Corticosteroid treatment and other immunosuppressive agents could successfully be tapered in the CR/PR group compared to the SD/PD patients. In this study patients with skin cGVHD are those with the highest rate of CR/PR after ECP treatment. CONCLUSIONS: Our results suggest that ECP treatment is safe and effective for patients with predominantly skin, oral and liver cGVHD.

The importance of graft cell composition in outcome after allogeneic stem cell transplantation in patients with malignant disease.

BACKGROUND: Graft-versus-host disease (GVHD) and relapse remain majobstacles ftreatment success in allogeneic hematopoietic stem cell transplantation (HSCT). In the present study, we evaluated the immune cell profile of the graft to outcome after HSCT. STUDY DESIGN AND METHOD: Flow cytometry data of graft cell subsets [CD34+ , CD3+ , CD19+ , CD4+ , CD8+ , CD3-CD56+ CD16+ , CD4+ CD127low CD25high ] from G-CSF primed peripheral blood stem cell (PBSC) donors was collected retrospectively from 299 patients with hematological malignancies undergoing HSCT between 2006 and 2013. The association to overall survival, transplant-related mortality (TRM), GVHD and probability of relapse was analyzed. Patients outcome from HLA-identical sibling (Sib) (n = 97) and unrelated donors (URD) (n = 202) were analyzed separately as all URD patients received anti-thymocyte globulin (ATG). RESULTS: Five-year overall survival was similar in the two cohorts (68% (Sib) vs 65% (URD)). The relapse incidence was significantly lower in the Sib cohort (24% vs 35%, P = 0.04). Multivariate analysis in the URD group revealed an association between a higher CD8+ dose and less relapse (HR, 0.94; 95%CI, 0.90-0.98; P = 0.006) as well as an association between higher CD34+ dose and both higher TRM (HR, 1.09; 95%CI, 1.02-1.20; P = 0.02) and relapse (HR, 1.09; 95%CI, 1.01-1.17; P = 0.025). The Sib analysis showed an association between a higher graft CD19+ dose and more severe acute GVHD (HR, 1,09; 95%CI, 1.03-1.15; P = 0.003) and TRM (HR, 1.09; 95%CI, 1.01-1.17; P = 0.036). In addition, a higher CD4+ graft content was associated to an increased risk for chronic GVHD (HR, 1.02; 95%CI 1.00-1.04; P = 0.06). CONCLUSION: These data indicate an importance of PBSC dongraft composition in patients with a hematological malignancy.

Immune profiling and identification of prognostic immune-related risk factors in human ovarian cancer.

Suppression of immune reactivity by increased expression of co-inhibitory receptors has been discussed as a major reason as to why the immune system fails to control tumor development. Elucidating the co-inhibitory expression pattern of tumor-infiltrating lymphocytes in different cancer types will help to develop future treatment strategies. We characterized markers reflecting and affecting T-cell functionality by flow cytometry on lymphocytes isolated from blood, ascites and tumor from advanced ovarian cancer patients (n = 35). Significantly higher proportions of CD4+ and CD8+ T-cells expressed co-inhibitory receptors LAG-3, PD-1 and TIM-3 in tumor and ascites compared to blood. Co-expression was predominantly observed among intratumoral CD8+ T-cells and the most common combination was PD-1 and TIM-3. Analysis of 26 soluble factors revealed highest concentrations of IP-10 and MCP-1 in both ascites and tumor. Correlating these results with clinical outcome revealed the proportion of CD8+ T-cells without expression of LAG-3, PD-1 and TIM-3 to be beneficial for overall survival. In total we identified eight immune-related risk factors associated with reduced survival. Ex vivo activation showed tumor-derived CD4+ and CD8+ T-cells to be functionally active, assessed by the production of IFN-γ, IL-2, TNF-α, IL-17 and CD107a. Blocking the PD-1 receptor resulted in significantly increased release of IFN-γ suggesting potential reinvigoration. The ovarian tumor environment exhibits an inflammatory milieu with abundant presence of infiltrating immune cells expressing inhibitory checkpoints. Importantly, we found subsets of CD8+ T-cells with double and triple expression of co-inhibitory receptors, supporting the need for multiple checkpoint-targeting agents to overcome T-cell dysfunction in ovarian cancer.