T Cell Activators Exhibit Distinct Downstream Effects on Chimeric Antigen Receptor T Cell Phenotype and Function.

T cell activation is an essential step in chimeric Ag receptor (CAR) T (CAR T) cell manufacturing and is accomplished by the addition of activator reagents that trigger the TCR and provide costimulation. We explore several T cell activation reagents and examine their effects on key attributes of CAR T cell cultures, such as activation/exhaustion markers, cell expansion, gene expression, and transduction efficiency. Four distinct activators were examined, all using anti-CD3 and anti-CD28, but incorporating different mechanisms of delivery: Dynabeads (magnetic microspheres), TransAct (polymeric nanomatrix), Cloudz (alginate hydrogel), and Microbubbles (lipid membrane containing perfluorocarbon gas). Clinical-grade lentiviral vector was used to transduce cells with a bivalent CD19/CD22 CAR, and cell counts and flow cytometry were used to monitor the cells throughout the culture. We observed differences in CD4/CD8 ratio when stimulating with the Cloudz activator, where there was a significant skewing toward CD8 T cells. The naive T cell subset expressing CD62L+CCR7+CD45RA+ was the highest in all donors when stimulating with Dynabeads, whereas effector/effector memory cells were highest when using the Cloudz. Functional assays demonstrated differences in killing of target cells and proinflammatory cytokine secretion, with the highest killing from the Cloudz-stimulated cells among all donors. This study demonstrates that the means by which these stimulatory Abs are presented to T cells contribute to the activation, resulting in differing effects on CAR T cell function. These studies highlight important differences in the final product that should be considered when manufacturing CAR T cells for patients in the clinic.

Effect of Meteorological Factors, Air Pollutants on Daily Hospital Admissions for Ischemic Heart Disease in Lanzhou, China.

BACKGROUND: Meteorological factors and air pollutants are believed to be associated with cardiovascular disease. Ischemic heart disease (IHD) is a major public health issue worldwide. Few studies have investigated the associations among meteorological factors, air pollutants and IHD daily hospital admissions in Lanzhou, China. METHODS: We conducted a distributed lag non-linear model (DLNM) on the basis of five years data, aiming at disentangling the impact of meteorological factors and air pollutants on IHD hospital admissions. All IHD daily hospital admissions recorded from January 1, 2015 and December 31, 2019 were obtained from three hospitals in Lanzhou, China. Daily air pollutant concentrations and meteorological data were synchronously collected from Gansu Meteorological Administration and Lanzhou Environmental Protection Administration. Stratified analyses were performed by sex and two age-groups. RESULTS: A total of 23555 IHD hospital admissions were recorded, of which 10477 admissions were for coronary artery disease (CAD), 13078 admissions were for acute coronary syndrome (ACS). Our results showed that there was a non-linear (J-shaped) relationship between temperature and IHD hospital admissions. The number of IHD hospital admissions were positively correlated with NO2, O3, humidity and pressure, indicating an increased risk of hospital admissions for IHD under NO2, O3, humidity and pressure exposure. Meanwhile, both extremely low (-12ºC) and high (30ºC) temperature reduced IHD hospital admissions, but the harmful effect increased with the lag time in Lanzhou, China, while the cold effect was more pronounced and long-lasting than the heat effect. Subgroup analysis demonstrated that the risk on CAD hospital admissions increased significantly in female and <65 years of age at -12ºC. CONCLUSION: Our findings added to the growing evidence regarding the potential impact of meteorological factors, air pollutants on policymaking from the perspective of hospital management efficiency.

Fractional flow reserve measured via left internal mammary artery after coronary artery bypass grafting: Two case reports.

BACKGROUND: The fractional flow reserve (FFR) has made the treatment of coronary heart disease more precise. However, there are few reports on the measurement of FFR via the left internal mammary artery (LIMA). Herein, we described the determination of further treatments by measuring FFR via the LIMA in 2 cases after coronary artery bypass grafting (CABG). CASE SUMMARY: Case 1 was a 66-year-old male who was admitted due to "chest tightness after CABG." The patient underwent CABG 7 years prior due to coronary heart disease. Coronary artery angiography showed complete occlusion of the left anterior descending artery (LAD), and subtotal occlusion of the third segment of the right coronary artery. On arterial angiography, there was 85% stenosis at the distal end of the anastomosis of the LIMA-LAD graft. FFR via LIMA was determined at 0.75. Thus, balloon dilation was performed in Case 1. FFR after balloon dilation was 0.94. Case 2 was a 60-year-old male who was admitted due to "chest tightness after CABG." The patient underwent CABG 6 years prior due to coronary heart disease. There was 60% segmental stenosis in the middle segment of LAD and 75% anastomotic stenosis. FFR measured via LIMA was 0.83 (negative); thus the intervention was not performed. Case 2 was given drug treatments. At the 3-mo follow-up, there was no recurrence of chest tightness or shortness of breath in both cases. They are currently under continual follow-up. CONCLUSION: We provided evidence that FFR measurement via grafted blood vessels, especially LIMA, after CABG is a good method to determine the intervention course.

Association between ambient temperature and cardiovascular diseases related hospital admissions in Lanzhou, China.

Cardiovascular disease (CVD) remains the leading cause of morbidity and mortality worldwide, ranking first in the global disease burden. Evidence on association between temperature and cardiovascular disease is insufficient and inconsistent in developing countries. In this study, a distributed lag nonlinear model (DLNM) was used to determine the association between daily mean temperature and cardiovascular diseases (CVD) related admission in Lanzhou 2015-2019. We included 41,389 patients with CVD in this study. The relative risk (RR) of CVD admission increased significantly with temperature in lag 5-10 days, and we found harvesting effect of temperature in the study, shown as decreased RR in lag 15-30 days. The maximum RR was 1.15 (95% confidence interval [CI]: 1.03-1.30), corresponding to 24 °C. Both cold and heat effects of temperature could impact the CVD admission. Compared with the 25th percentile of temperature (2 °C), the cumulative relative risk (cumRR) of extreme cold (-5 °C, the 2.5th percentile of the temperature) was 0.69 (95% CI: 0.51-0.94) in lag 0-14, whereas the cumRR of moderate cold (-2 °C, the 10th percentile) was 0.83 (95% CI:0.71-0.97). Compared with the 75th percentile of temperature (20-°C), the cumRR of extreme heat (27 °C, the 97.5th percentile) was 0.93 (95% CI: 0.78-1.10) in lag 0, whereas the cumRR of moderate heat (24 °C, the 90th percentile) was 1.01 (95% CI: 0.94-1.08). In the stratified analysis, cold decreased RR significantly in female and ≥65 years, whereas heat increased it more obviously in male and ≥65 years. Ambient temperature and CVD admissions were positively associated, with the harvesting effect. Our findings demonstrate the adaption of residents in Lanzhou to cold temperature. Public and environmental policies and measures aimed at moderate heat may minimize CVD burden effectively.

Optimizing a fully automated and closed system process for red blood cell reduction of human bone marrow products.

BACKGROUND AIMS: Hematopoietic stem cell transplantation using bone marrow as the graft source is a common treatment for hematopoietic malignancies and disorders. For allogeneic transplants, processing of bone marrow requires the depletion of ABO-mismatched red blood cells (RBCs) to avoid transfusion reactions. Here the authors tested the use of an automated closed system for depleting RBCs from bone marrow and compared the results to a semi-automated platform that is more commonly used in transplant centers today. The authors found that fully automated processing using the Sepax instrument (Cytiva, Marlborough, MA, USA) resulted in depletion of RBCs and total mononuclear cell recovery that were comparable to that achieved with the COBE 2991 (Terumo BCT, Lakewood, CO, USA) semi-automated process. METHODS: The authors optimized the fully automated and closed Sepax SmartRedux (Cytiva) protocol. Three reduction folds (10×, 12× and 15×) were tested on the Sepax. Each run was compared with the standard processing performed in the authors' center on the COBE 2991. Given that bone marrow is difficult to acquire for these purposes, the authors opted to create a surrogate that is more easily obtainable, which consisted of cryopreserved peripheral blood stem cells that were thawed and mixed with RBCs and supplemented with Plasma-Lyte A (Baxter, Deerfield, IL, USA) and 4% human serum albumin (Baxalta, Westlake Village, CA, USA). This "bone marrow-like" product was split into two starting products of approximately 600 mL, and these were loaded onto the COBE and Sepax for direct comparison testing. Samples were taken from the final products for cell counts and flow cytometry. The authors also tested a 10× Sepax reduction using human bone marrow supplemented with human liquid plasma and RBCs. RESULTS: RBC reduction increased as the Sepax reduction rate increased, with an average of 86.06% (range of 70.85-96.39%) in the 10×, 98.80% (range of 98.1-99.5%) in the 12× and 98.89% (range of 98.80-98.89%) in the 15×. The reduction rate on the COBE ranged an average of 69.0-93.15%. However, white blood cell (WBC) recovery decreased as the Sepax reduction rate increased, with an average of 47.65% (range of 38.9-62.35%) in the 10×, 14.56% (range of 14.34-14.78%) in the 12× and 27.97% (range of 24.7-31.23%) in the 15×. COBE WBC recovery ranged an average of 53.17-76.12%. Testing a supplemented human bone marrow sample using a 10× Sepax reduction resulted in an average RBC reduction of 84.22% (range of 84.0-84.36%) and WBC recovery of 43.37% (range of 37.48-49.26%). Flow cytometry analysis also showed that 10× Sepax reduction resulted in higher purity and better recovery of CD34+, CD3+ and CD19+ cells compared with 12× and 15× reduction. Therefore, a 10× reduction rate was selected for the Sepax process. CONCLUSIONS: The fully automated and closed SmartRedux program on the Sepax was shown to be effective at reducing RBCs from "bone marrow-like" products and a supplemented bone marrow product using a 10× reduction rate.

Association of T-box gene polymorphisms with the risk of Wolff-Parkinson-White syndrome in a Han Chinese population.

Abnormal development of the atrioventricular ring can lead to the formation of a bypass pathway and the occurrence of Wolff-Parkinson-White (WPW) syndrome. The genetic mechanism underlying the sporadic form of WPW syndrome remains unclear. Existing evidence suggests that both T-box transcription factor 3 (TBX3) and T-box transcription factor 2 (TBX2) genes participate in regulating annulus fibrosus formation and atrioventricular canal development. Thus, we aimed to examine whether single-nucleotide polymorphisms (SNPs) in the TBX3 and TBX2 genes confer susceptibility to WPW syndrome in a Han Chinese Population. We applied a SNaPshot SNP assay to analyze 5 selected tagSNPs of TBX3 and TBX2 in 230 patients with sporadic WPW syndrome and 231 sex- and age-matched controls. Haplotype analysis was performed using Haploview software. Allele C of TBX3 rs1061657 was associated with a higher risk of WPW syndrome (odds ratio [OR] = 1.41, 95% confidence interval [CI]: 1.08-1.83, P = .011) and left-sided accessory pathways (OR = 1.40, 95% CI: 1.07-1.84, P = .016). However, allele C of TBX3 rs8853 was likely to reduce these risks (OR = 0.71, 95% CI: 0.54-0.92, P = .011; OR = 0.70, 95% CI: 0.53-0.92, P = .011, respectively). The data revealed no association between TBX3 rs77412687, TBX3 rs2242442, or TBX2 rs75743672 and WPW syndrome. TBX3 rs1061657 and rs8853 are significantly associated with sporadic WPW syndrome among a Han Chinese population. To verify our results, larger sample sizes are required in future studies.

Establishment and validation of in-house cryopreserved CAR/TCR-T cell flow cytometry quality control.

BACKGROUND: Chimeric antigen receptor (CAR) or T-cell receptor (TCR) engineered T-cell therapy has recently emerged as a promising adoptive immunotherapy approach for the treatment of hematologic malignancies and solid tumors. Multiparametric flow cytometry-based assays play a critical role in monitoring cellular manufacturing steps. Since manufacturing CAR/TCR T-cell products must be in compliance with current good manufacturing practices (cGMP), a standard or quality control for flow cytometry assays should be used to ensure the accuracy of flow cytometry results, but none is currently commercially available. Therefore, we established a procedure to generate an in-house cryopreserved CAR/TCR T-cell products for use as a flow cytometry quality control and validated their use. METHODS: Two CAR T-cell products: CD19/CD22 bispecific CAR T-cells and FGFR4 CAR T-cells and one TCR-engineered T-cell product: KK-LC-1 TCR T-cells were manufactured in Center for Cellular Engineering (CCE), NIH Clinical Center. The products were divided in aliquots, cryopreserved and stored in the liquid nitrogen. The cryopreserved flow cytometry quality controls were tested in flow cytometry assays which measured post-thaw viability, CD3, CD4 and CD8 frequencies as well as the transduction efficiency and vector identity. The long-term stability and shelf-life of cryopreserved quality control cells were evaluated. In addition, the sensitivity as well as the precision assay were also assessed on the cryopreserved quality control cells. RESULTS: After thawing, the viability of the cryopreserved CAR/TCR T-cell controls was found to be greater than 50%. The expression of transduction efficiency and vector identity markers by the cryopreserved control cells were stable for at least 1 year; with post-thaw values falling within ± 20% range of the values measured at time of cryopreservation. After thawing and storage at room temperature, the stability of these cryopreserved cells lasted at least 6 h. In addition, our cryopreserved CAR/TCR-T cell quality controls showed a strong correlation between transduction efficiency expression and dilution factors. Furthermore, the results of flow cytometric analysis of the cryopreserved cells among different laboratory technicians and different flow cytometry instruments were comparable, highlighting the reproducibility and reliability of these quality control cells. CONCLUSION: We developed and validated a feasible and reliable procedure to establish a bank of cryopreserved CAR/TCR T-cells for use as flow cytometry quality controls, which can serve as a quality control standard for in-process and lot-release testing of CAR/TCR T-cell products.

High efficiency closed-system gene transfer using automated spinoculation.

BACKGROUND: Gene transfer is an important tool for cellular therapies. Lentiviral vectors are most effectively transferred into lymphocytes or hematopoietic progenitor cells using spinoculation. To enable cGMP (current Good Manufacturing Practice)-compliant cell therapy production, we developed and compared a closed-system spinoculation method that uses cell culture bags, and an automated closed system spinoculation method to decrease technician hands on time and reduce the likelihood for microbial contamination. METHODS: Sepax spinoculation, bag spinoculation, and static bag transduction without spinoculation were compared for lentiviral gene transfer in lymphocytes collected by apheresis. The lymphocytes were transduced once and cultured for 9 days. The lentiviral vectors tested encoded a CD19/CD22 Bispecific Chimeric Antigen Receptor (CAR), a FGFR4-CAR, or a CD22-CAR. Sepax spinoculation times were evaluated by testing against bag spinoculation and static transduction to optimize the Sepax spin time. The Sepax spinoculation was then used to test the transduction of different CAR vectors. The performance of the process using healthy donor and a patient sample was evaluated. Functional assessment was performed of the CD19/22 and CD22 CAR T-cells using killing assays against the NALM6 tumor cell line and cytokine secretion analysis. Finally, gene expression of the transduced T-cells was examined to determine if there were any major changes that may have occurred as a result of the spinoculation process. RESULTS: The process of spinoculation lead to significant enhancement in gene transfer. Sepax spinoculation using a 1-h spin time showed comparable transduction efficiency to the bag spinoculation, and much greater than the static bag transduction method (83.4%, 72.8%, 35.7% n = 3). The performance of three different methods were consistent for all lentiviral vectors tested and no significant difference was observed when using starting cells from healthy donor versus a patient sample. Sepax spinoculation does not affect the function of the CAR T-cells against tumor cells, as these cells appeared to kill target cells equally well. Spinoculation also does not appear to affect gene expression patterns that are necessary for imparting function on the cell. CONCLUSIONS: Closed system-bag spinoculation resulted in more efficient lymphocyte gene transfer than standard bag transductions without spinoculation. This method is effective for both retroviral and lentiviral vector gene transfer in lymphocytes and may be a feasible approach for gene transfer into other cell types including hematopoietic and myeloid progenitors. Sepax spinoculation further improved upon the process by offering an automated, closed system approach that significantly decreased hands-on time while also decreasing the risk of culture bag tears and microbial contamination.

Characterization of HLH-like manifestations as a CRS variant in patients receiving CD22 CAR T cells.

Chimeric antigen receptor (CAR) T-cell toxicities resembling hemophagocytic lymphohistiocytosis (HLH) occur in a subset of patients with cytokine release syndrome (CRS). As a variant of conventional CRS, a comprehensive characterization of CAR T-cell-associated HLH (carHLH) and investigations into associated risk factors are lacking. In the context of 59 patients infused with CD22 CAR T cells where a substantial proportion developed carHLH, we comprehensively describe the manifestations and timing of carHLH as a CRS variant and explore factors associated with this clinical profile. Among 52 subjects with CRS, 21 (40.4%) developed carHLH. Clinical features of carHLH included hyperferritinemia, hypertriglyceridemia, hypofibrinogenemia, coagulopathy, hepatic transaminitis, hyperbilirubinemia, severe neutropenia, elevated lactate dehydrogenase, and occasionally hemophagocytosis. Development of carHLH was associated with preinfusion natural killer(NK) cell lymphopenia and higher bone marrow T-cell:NK cell ratio, which was further amplified with CAR T-cell expansion. Following CRS, more robust CAR T-cell and CD8 T-cell expansion in concert with pronounced NK cell lymphopenia amplified preinfusion differences in those with carHLH without evidence for defects in NK cell mediated cytotoxicity. CarHLH was further characterized by persistent elevation of HLH-associated inflammatory cytokines, which contrasted with declining levels in those without carHLH. In the setting of CAR T-cell mediated expansion, clinical manifestations and immunophenotypic profiling in those with carHLH overlap with features of secondary HLH, prompting consideration of an alternative framework for identification and management of this toxicity profile to optimize outcomes following CAR T-cell infusion.

CD4/CD8 T-Cell Selection Affects Chimeric Antigen Receptor (CAR) T-Cell Potency and Toxicity: Updated Results From a Phase I Anti-CD22 CAR T-Cell Trial.

PURPOSE: Patients with B-cell acute lymphoblastic leukemia who experience relapse after or are resistant to CD19-targeted immunotherapies have limited treatment options. Targeting CD22, an alternative B-cell antigen, represents an alternate strategy. We report outcomes on the largest patient cohort treated with CD22 chimeric antigen receptor (CAR) T cells. PATIENTS AND METHODS: We conducted a single-center, phase I, 3 + 3 dose-escalation trial with a large expansion cohort that tested CD22-targeted CAR T cells for children and young adults with relapsed/refractory CD22+ malignancies. Primary objectives were to assess the safety, toxicity, and feasibility. Secondary objectives included efficacy, CD22 CAR T-cell persistence, and cytokine profiling. RESULTS: Fifty-eight participants were infused; 51 (87.9%) after prior CD19-targeted therapy. Cytokine release syndrome occurred in 50 participants (86.2%) and was grade 1-2 in 45 (90%). Symptoms of neurotoxicity were minimal and transient. Hemophagocytic lymphohistiocytosis-like manifestations were seen in 19/58 (32.8%) of subjects, prompting utilization of anakinra. CD4/CD8 T-cell selection of the apheresis product improved CAR T-cell manufacturing feasibility as well as heightened inflammatory toxicities, leading to dose de-escalation. The complete remission rate was 70%. The median overall survival was 13.4 months (95% CI, 7.7 to 20.3 months). Among those who achieved a complete response, the median relapse-free survival was 6.0 months (95% CI, 4.1 to 6.5 months). Thirteen participants proceeded to stem-cell transplantation. CONCLUSION: In the largest experience of CD22 CAR T-cells to our knowledge, we provide novel information on the impact of manufacturing changes on clinical outcomes and report on unique CD22 CAR T-cell toxicities and toxicity mitigation strategies. The remission induction rate supports further development of CD22 CAR T cells as a therapeutic option in patients resistant to CD19-targeted immunotherapy.

Production of a cellular product consisting of monocytes stimulated with Sylatron® (Peginterferon alfa-2b) and Actimmune® (Interferon gamma-1b) for human use.

BACKGROUND: Monocytes are myeloid cells that reside in the blood and bone marrow and respond to inflammation. At the site of inflammation, monocytes express cytokines and chemokines. Monocytes have been shown to be cytotoxic to tumor cells in the presence of pro-inflammatory cytokines such as Interferon Alpha, Interferon Gamma, and IL-6. We have previously shown that monocytes stimulated with both interferons (IFNs) results in synergistic killing of ovarian cancer cells. We translated these observations to an ongoing clinical trial using adoptive cell transfer of autologous monocytes stimulated ex vivo with IFNs and infused into the peritoneal cavity of patients with advanced, chemotherapy resistant, ovarian cancer. Here we describe the optimization of the monocyte elutriation protocol and a cryopreservation protocol of the monocytes isolated from peripheral blood. METHODS: Counter flow elutriation was performed on healthy donors or women with ovarian cancer. The monocyte-containing, RO-fraction was assessed for total monocyte number, purity, viability, and cytotoxicity with and without a cryopreservation step. All five fractions obtained from the elutriation procedure were also assessed by flow cytometry to measure the percent of immune cell subsets in each fraction. RESULTS: Both iterative monocyte isolation using counter flow elutriation or cryopreservation following counter flow elutriation can yield over 2 billion monocytes for each donor with high purity. We also show that the monocytes are stable, viable, and retain cytotoxic functions when cultured with IFNs. CONCLUSION: Large scale isolation of monocytes from both healthy donors and patients with advanced, chemotherapy resistant ovarian cancer, can be achieved with high total number of monocytes. These monocytes can be cryopreserved and maintain viability and cytotoxic function. All of the elutriated cell fractions contain ample immune cells which could be used for other cell therapy-based applications.

Enhanced clinical-scale manufacturing of TCR transduced T-cells using closed culture system modules.

BACKGROUND: Genetic engineering of T-cells to express specific T cell receptors (TCR) has emerged as a novel strategy to treat various malignancies. More widespread utilization of these types of therapies has been somewhat constrained by the lack of closed culture processes capable of expanding sufficient numbers of T-cells for clinical application. Here, we evaluate a process for robust clinical grade manufacturing of TCR gene engineered T-cells. METHODS: TCRs that target human papillomavirus E6 and E7 were independently tested. A 21 day process was divided into a transduction phase (7 days) and a rapid expansion phase (14 days). This process was evaluated using two healthy donor samples and four samples obtained from patients with epithelial cancers. RESULTS: The process resulted in ~ 2000-fold increase in viable nucleated cells and high transduction efficiencies (64-92%). At the end of culture, functional assays demonstrated that these cells were potent and specific in their ability to kill tumor cells bearing target and secrete large quantities of interferon and tumor necrosis factor. Both phases of culture were contained within closed or semi-closed modules, which include automated density gradient separation and cell culture bags for the first phase and closed GREX culture devices and wash/concentrate systems for the second phase. CONCLUSION: Large-scale manufacturing using modular systems and semi-automated devices resulted in highly functional clinical-grade TCR transduced T-cells. This process is now in use in actively accruing clinical trials and the NIH Clinical Center and can be utilized at other cell therapy manufacturing sites that wish to scale-up and optimize their processing using closed systems.

Interferon-γ and Tumor Necrosis Factor-α Polarize Bone Marrow Stromal Cells Uniformly to a Th1 Phenotype.

Activated T cells polarize mesenchymal stromal cells (MSCs) to a proinflammatory Th1 phenotype which likely has an important role in amplifying the immune response in the tumor microenvironment. We investigated the role of interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α), two factors produced by activated T cells, in MSC polarization. Gene expression and culture supernatant analysis showed that TNF-α and IFN-γ stimulated MSCs expressed distinct sets of proinflammatory factors. The combination of IFN-γ and TNF-α was synergistic and induced a transcriptome most similar to that found in MSCs stimulated with activated T cells and similar to that found in the inflamed tumor microenvironment; a Th1 phenotype with the expression of the immunosuppressive factors IL-4, IL-10, CD274/PD-L1 and indoleamine 2,3 dioxygenase (IDO). Single cell qRT-PCR analysis showed that the combination of IFN-γ and TNF-α polarized uniformly to this phenotype. The combination of IFN-γ and TNF-α results in the synergist uniform polarization of MSCs toward a primarily Th1 phenotype. The stimulation of MSCs by IFN-γ and TNF-α released from activated tumor infiltrating T cells is likely responsible for the production of many factors that characterize the tumor microenvironment.

Leukemia cells induce changes in human bone marrow stromal cells.

BACKGROUND: Bone marrow stromal cells (BMSCs) are multipotent cells that support angiogenesis, wound healing, and immunomodulation. In the hematopoietic niche, they nurture hematopoietic cells, leukemia, tumors and metastasis. BMSCs secrete of a wide range of cytokines, growth factors and matrix proteins which contribute to the pro-tumorigenic marrow microenvironment. The inflammatory cytokines IFN-γ and TNF-α change the BMSC secretome and we hypothesized that factors produced by tumors or leukemia would also affect the BMSC secretome and investigated the interaction of leukemia cells with BMSCs. METHODS: BMSCs from healthy subjects were co-cultured with three myeloid leukemia cell lines (TF-1, TF-1α and K562) using a trans-well system. Following co-culture, the BMSCs and leukemia cells were analyzed by global gene expression analysis and culture supernatants were analyzed for protein expression. As a control, CD34+ cells were also cocultured with BMSCs. RESULTS: Co-culture induced leukemia cell gene expression changes in stem cell pluripotency, TGF-ß signaling and carcinoma signaling pathways. BMSCs co-cultured with leukemia cells up-regulated a number of proinflammatory genes including IL-17 signaling-related genes and IL-8 and CCL2 levels were increased in co-culture supernatants. In contrast, purine metabolism, mTOR signaling and EIF2 signaling pathways genes were up-regulated in BMSCs co-cultured with CD34+ cells. CONCLUSIONS: BMSCs react to the presence of leukemia cells undergoing changes in the cytokine and chemokine secretion profiles. Thus, BMSCs and leukemia cells both contribute to the creation of a competitive niche more favorable for leukemia stem cells.

Simplified method of the growth of human tumor infiltrating lymphocytes in gas-permeable flasks to numbers needed for patient treatment.

Adoptive cell therapy of metastatic melanoma with autologous tumor infiltrating lymphocytes (TIL) is clinically effective, but TIL production can be challenging. Here we describe a simplified method for initial TIL culture and rapid expansion in gas-permeable flasks. TIL were initially cultured from tumor digests and fragments in 40 mL capacity flasks with a 10 cm² gas-permeable silicone bottom, G-Rex10. A TIL rapid expansion protocol (REP) was developed using 500 mL capacity flasks with a 100 cm² gas-permeable silicone bottom, G-Rex100. TIL growth was successfully initiated in G-Rex10 flasks from tumor digests from 13 of 14 patients and from tumor fragments in all 11 tumor samples tested. TIL could then be expanded to 8-10×109 cells in a 2-step REP that began by seeding 5×106 TIL into a G-Rex100 flask, followed by expansion at day 7 into 3 G-Rex100 flasks. To obtain the 30-60×109 cells used for patient treatment, we seeded 6 G-Rex100 flasks with 5×106 cells and expanded into 18 G-Rex100 flasks. Large-scale TIL REP in gas-permeable flasks requires approximately 9-10 L of media, about 3-4 times less than other methods. In conclusion, TIL initiation and REP in gas-permeable G-Rex flasks require fewer total vessels, less media, less incubator space, and less labor than initiation and REP in 24-well plates, tissue culture flasks, and bags. TIL culture in G-Rex flasks will facilitate the production of TIL at the numbers required for patient treatment at most cell processing laboratories.