Autologous Peripheral Blood Mononuclear Cells Research Articles

Modulatory Impact of Adipose-Derived Mesenchymal Stem Cells of Ankylosing Spondylitis Patients on T Helper Cell Differentiation.

The domination of pro-inflammatory Th subsets (Th1, Th17) is characteristic of ankylosing spondylitis (AS). Mesenchymal stem cells (MSC) were reported to normalize Th imbalance, but whether MSCs from AS adipose tissue (AS/ASCs) possess such properties is unknown. We examined AS/ASCs’ impact on Th-cell differentiation, using healthy donors ASCs (HD/ASCs) as a control. The assessment of the expression of transcription factors defining Th1 (T-bet), Th2 (GATA3), Th17 (RORc), and Treg (FoxP3) subsets by quantitative RT-PCR, the concentrations of subset-specific cytokines by ELISA, and Treg (CD4+CD25highFoxP3+) formation by flow cytometry, were performed in the co-cultures of ASCs with activated CD4+ T cells or peripheral blood mononuclear cells (PBMCs). AS/ASCs and HD/ASCs exerted similar immunomodulatory effects. Acting directly on CD4+ T cells, ASCs decreased the T-bet/GATA3 and RORc/FoxP3 ratios, diminished Treg formation, but increase IFNγ and IL-17AF production, while ASCs co-cultured with PBMCs enhanced Treg generation and reduced IFNγ release. ASCs failed to up-regulate the anti-inflammatory IL-10 and TGFβ. AS/ASCs’ impact on allogeneic and autologous PBMCs was similar. In conclusion, to shift Th differentiation to a functional anti-inflammatory direction, ASCs require accessory cell support, whereas their direct effect may be pro-inflammatory. Because ASCs neither inhibit IL-17AF nor up-regulate anti-inflammatory cytokines, their usefulness for AS patients’ treatment remains uncertain.

Application of Autologous Peripheral Blood Mononuclear Cells into the Area of Spinal Cord Injury in a Subacute Period: A Feasibility Study in Pigs.

Simple SummarySpinal cord injury is a medical and social issue causing severe disability. The potential to overcome the consequences of spinal cord injury is related to cell therapy. Peripheral blood is a prospective and available source of cells for further clinical use. In our study, we have evaluated the therapeutic potential of peripheral blood mononuclear cells (PBMCs) on the model of spinal cord injury in pigs. In the subacute period (6 weeks after injury), PBMCs enclosed in fibrin glue were applied into the dorsal area of the injured spinal cord. In this study, we observed that the tissue integrity increased in the area adjacent to the epicenter of injury, and conduction along spinal axons was partially restored after cell therapy in pigs.Peripheral blood presents an available source of cells for both fundamental research and clinical use. In our study, we have evaluated the therapeutic potential of peripheral blood mononuclear cells (PBMCs) excluding the preliminary sorting or mobilization of peripheral blood stem cells. We have evaluated the regenerative potential of PBMCs embedded into a fibrin matrix (FM) in a model of pig spinal cord injury. The distribution of transplanted PBMCs in the injured spinal cord was evaluated; PBMCs were shown to penetrate into the deep layers of the spinal cord and concentrate mainly in the grey matter. The results of the current study revealed an increase in the tissue integrity in the area adjacent to the epicenter of injury and the partially restored conduction along posterior columns of the spinal cord in animals after FM+PBMC application. The multiplex analysis of blood serum and cerebrospinal fluid showed the cytokine imbalance to occur without significantly shifting toward pro-inflammatory or anti-inflammatory cytokine cascades.

Autologous Bone-Marrow vs. Peripheral Blood Mononuclear Cells Therapy for Peripheral Artery Disease in Diabetic Patients.

Diabetes mellitus (DM) remains one of the most important risk factors for peripheral artery disease (PAD), with approximately 20% of DM patients older than 40 years old are affected with PAD. The current standard management for severe PAD is endovascular intervention with or without surgical bypass. Unfortunately, up to 40% of patients are unable to undergo these revascularization therapies due to excessive surgical risk or adverse vascular side effects. Stem cell therapy has emerged as a novel therapeutic strategy for these ‘no-option’ patients. Several types of stem cells are utilized for PAD therapy, including bone marrow mononuclear cells (BMMNC) and peripheral blood mononuclear cells (PBMNC). Many studies have reported the safety of BMMNC and PBMNC, as well as its efficacy in reducing ischemic pain, ulcer size, pain-free walking distance, ankle-brachial index (ABI), and transcutaneous oxygen pressure (TcPO2). However, the capacity to establish the efficacy of reducing major amputation rates, amputation free survival, and all-cause mortality is limited, as shown by several randomized placebo-controlled trials. The present literature review will focus on comparing safety and efficacy between BMMNC and PBMNC as cell-based management in diabetic patients with PAD who are not suitable for revascularization therapy.

IL-7 During Antigenic Stimulation Using Allogeneic Dendritic Cells Promotes Expansion of CD45RA-CD62L+CD4+ Invariant NKT Cells With Th-2 Biased Cytokine Production Profile.

Invariant natural killer T (iNKT) cells are innate-like T lymphocytes cells that recognize glycolipid antigens associated with CD1d, non-classical antigen presenting proteins. They can drive either pro-inflammatory (Th-1) or anti-inflammatory (Th-2) immune microenvironment through the production of both Th-1 and Th-2 type cytokines upon activation, thus play a vital role in cancer, infection, and autoimmune diseases. Adoptive cell therapy using ex vivo expanded iNKT cells is a promising approach to enhance anti-tumor immunity or immunosuppression. However, overcoming phenotypic and functional heterogeneity and promoting in vivo persistency of iNKT cells remains to be a challenge. Here, we compared various methods for ex vivo expansion of human iNKT cells and assessed the quality of expansion, phenotype, and cytokine production profile of expanded iNKT cells. While a direct stimulation of iNKT cells in peripheral blood mononuclear cells with agonist glycolipid led to the expansion of iNKT cells in varying degrees, stimulation of enriched iNKT cells by irradiated autologous peripheral blood mononuclear cells or allogeneic dendritic cells resulted in consistent expansion of highly pure iNKT cells. Interestingly, the mode of antigenic stimulation influenced the dominant subtype of expanded iNKT cells. Further, we evaluated whether additional IL-7 or IL-15 during antigenic stimulation with allogeneic dendritic cells can improve the phenotypic heterogeneity and modify cytokine production profile of iNKT cells expanded from 18 consecutive donors. The presence of IL-7 or IL-15 during antigenic stimulation did not affect the fold of expansion or purity of expanded iNKT cells. However, IL-7, but not IL-15, led to a better expansion of CD4+ iNKT cells, enhanced Th-2 type cytokine production of CD4+ iNKT cells, and maintained the expansion of central memory (CD45RA-CD62L+) CD4+ iNKT cells. Our results suggest the addition of IL-7 during antigenic stimulation with allogeneic dendritic cells can promote the expansion of CD62L+Th-2+CD4+ human iNKT cells that can be used as novel immunotherapeutic to control excessive inflammation to treat various autoimmune diseases.

Fidelity of human ovarian cancer patient-derived xenografts in a partially humanized mouse model for preclinical testing of immunotherapies

BackgroundImmune checkpoint blockers (ICBs) have been approved by the Food and Drug Administration to be used alone in front-line therapies or in combination with other regimens for certain advanced cancers....

Low Level Donor Chimerism of CD19 CAR-T Cells Returned to Complete Donor Chimerism in Patients with Relapse After Allo-Hematopoietic Stem Cell Transplant.

PurposeTo investigate the donor chimerism changes and curative effects associated with the use of autologous anti-CD19 chimeric antigen receptor (CAR) T cells with B-cell acute lymphoblastic leukemia (B-ALL) presenting with a low donor chimerism level and relapse after allogeneic hematopoietic stem cell transplant (allo-HSCT).MethodsNine patients with B-ALL showing low donor chimerism level and relapse after allo-HSCT were enrolled. Patients 1–3 received CD19 CAR-T cell therapy using cells derived from autologous peripheral blood mononuclear cells (PBMCs) (comprising a mixture of patient and original donor cells) as their donors could not provide PBMCs. Samples from the other six patients (Patients A–F) were investigated only in vitro. The changes in the degree of donor chimerism, function of the CD19 CAR-T cells and T cells in all nine patients were analyzed in vitro. The therapeutic effects and adverse events (AEs) were also evaluated in Patients 1–3.ResultsThe CAR-T cells and T cells in all nine patients showed complete donor chimerism restoration following a 12-day culture period in vitro. These CD19 CAR-T cells demonstrated strong cytotoxicity towards Nalm 6 cells in vitro except in patients 3 and D. In the latter patients, the absolute numbers of all subsets, especially the CD8 + T-cell absolute numbers in peripheral blood were very low. Patients 3 and D showed relatively short durations from transplant to recurrence and received chemotherapy after relapse. In the patients receiving CD19 CAR-T cell therapy, the most commonly observed AE was grade 1 to 2 cytokine release syndrome. None of the cases showed acute graft-versus-host disease during treatment. Patients 1 and 2 achieved complete response with complete restoration of donor chimerism. Patient 3, who received the same CD19 CAR-T cell therapy, did not respond to this therapy.ConclusionCD19 CAR-T cells derived from patients relapsed after allo-HSCT with a low level of donor chimerism were effective for salvage therapy and could restore to complete donor chimerism after 12 days’ culture in vitro.Trial RegistrationHumanized CD19 CAR-T cell therapy for relapse or refractory B-cell lymphoma or acute B lymphocytic leukemia, ChiCTR1800019622, Registered 24 November 2018, http://www.chictr.org.cn/index.aspx.

Adoptive transfer of zoledronate-expanded autologous Vγ9Vδ2 T-cells in patients with treatment-refractory non-small-cell lung cancer: a multicenter, open-label, single-arm, phase 2 study

BackgroundNot all non-small cell lung cancer (NSCLC) patients possess drug-targetable driver mutations, and response rates to immune checkpoint blockade therapies also remain unsatisfactory. Therefore, more effective treatments are still needed....

Feasibility and preliminary safety and efficacy of first-in-human intraperitoneal delivery of MCY-M11, anti-human-mesothelin CAR mRNA transfected into peripheral blood mononuclear cells, for ovarian cancer and malignant peritoneal mesothelioma.

3014 Background: MCY-M11 is a mesothelin-targeting chimeric antigen receptor (CAR) therapy made by a non-viral, mRNA-based platform, for rapid ( < 1 day) CAR manufacturing. We are conducting a phase I dose escalation trial in ovarian cancer and malignant peritoneal mesothelioma (MPM) (NCT03608618). Methods: MCY-M11 are fresh, non-expanded, autologous peripheral blood mononuclear cells (PBMCs) transfected by flow electroporation with mRNA encoding a human anti-mesothelin CAR. Following a 3+3 design, patients are treated in dose level (DL) escalating cohorts (DL1 1.0 x 107, DL2 5.0 x 107, DL3 1.0 x 108, DL4 5.0 x 108 cells/dose), in one cycle of weekly x 3 doses, intraperitoneal (ip) without preconditioning chemotherapy. Results: By January 2020, CP-M11-101 study successfully completed DL1 and DL2 without safety concerns. Based on 11 patients treated in DL1, DL2 and DL3, ip infusion of MCY-M11 is safe and well tolerated. No infusion-related adverse events and no dose limiting toxicities (DLTs) have occurred. No neurotoxicity has been observed. Most reported treatment-related adverse events have been Grades 1-2 per NCI CTCAE. One patient in DL3 presented with G2 pericarditis, fever and transient neutropenia clinically assessed as related SAEs, that resolved without further complications. These events were assessed as on-target off-tumor effects and possibly G1 cytokine release syndrome (CRS). Two unrelated SAEs (G2 confusion in a patient in DL2; G3 enterocutaneous fistula in a patient in DL3) were reported. These 2 patients have been replaced as they did not complete the evaluation period (3 weekly infusions and the DLT 43 day follow up). There have been no treatment-related discontinuations or deaths. Three patients in DL2 showed stable disease (SD) by RECIST 1.1 at the end of the DLT period. Of them, 1 completed the study and did not participate in additional follow up, 1 remained in SD 6 months, and 1 remained in SD 2 months. In DL3, 1 patient remains in SD at 2 months, and evaluation is pending for the other 2 patients. Enrollment is ongoing. Conclusions: Feasibility of 1-day manufacturing of MCY-M11 for ip delivery is demonstrated. Treatment has been safe. Initial SD observed in DL2 and DL3 with one-cycle infusions is encouraging and supports exploration of additional strategies such as the addition of preconditioning chemotherapy and multiple cycles to increase efficacy. Clinical trial information: NCT03608618 .

Generation of high-affinity CMV-specific T cells for adoptive immunotherapy using IL-2, IL-15, and IL-21

Cytomegalovirus (CMV) infection remains a life-threatening condition in individuals with a suppressed immune system. CMV may also represent a clinically relevant target for immune responses in CMV-positive malignancies. We established a protocol to expand CMV-specific T cells (CMV-T) using peripheral blood mononuclear cells (PBMCs). PBMCs from 16 HLA-A*0201 donors were cultured with a cytokine cocktail comprising IL-2/IL-15/IL-21 along with overlapping peptides from CMV-pp65. Ten days later, T cells were stimulated with anti-CD3 (OKT3) and irradiated autologous PBMCs. CMV-T were detected by HLA-A*0201 CMV-pp65NLVPMVATV wild type and q226a mutant tetramers (for high-affinity T cells), intracellular cytokine staining, a CD107a mobilization assays as well as IFN-γ and TNF-α production in cell culture supernatants. We reliably obtained 50.25 ± 27.27% of CD8+ and 22.08 ± 21.83% of CD4+ T cells post-CMV-pp65 stimulation of PBMCs with a Th1-polarized phenotype and decreased Th2/Th17 responses. Most CD3 + CD8 + tetramer+ T cells were effector-memory cells, particularly among high-affinity CMV-T (q226a CMV-tetramer+). High-affinity CMV-T cells, compared to WT-tetramer+ cells, expressed higher IL-21R and lower FasL post-stimulation with CMV-pp65. The IL-2/IL-15/IL-21 cocktail also promoted CCR6 and CXCR3 expression necessary for T-cell migration into tissues. We have optimized methods for generating high-affinity CMV-specific T cells that can be used for adoptive cellular therapy in clinical practice.

Identification and generation of tumor antigen-specific T cells directed against AML

Background & Aim Acute myeloid leukemia (AML) is an aggressive hematological malignancy characterized by aberrant expression of leukemia associated antigens (LAA) that can be presented in context of HLA molecules, to elicit anti-tumor T cell response and improve clinical outcomes. This study aimed to 1) identify targets of endogenous LAA-specific T cells in AML patients post allogeneic hematopoietic stem cell transplant (allo-HSCT) and whose detection was associated with favorable clinical outcomes and 2) assess the feasibility of generating LAA-specific T cells targeting these antigens for adoptive immunotherapy. Methods, Results & Conclusion We obtained blood from patients (n=8) with relapsed and/or refractory AML who were in remission post allo-HCST, and measured the frequency of T cells reactive against known LAAs (e.g. WT1, CCNA1, survivin, PRAME). These patients were followed for 1 year post-sampling. Of the tested antigens, we identified that the best predictor of those who would eventually relapse was the magnitude CCNA1-directed T cell activity (clinical remission vs relapse: 171 vs 7.13 mean spot forming colonies/1 × 10e5; p=0.03). We next hypothesized that outcomes for AML could be improved by the ex vivo preparation and adoptive transfer of stem cell donor-derived CCNA1-specific T cells to patients at risk of relapse post allo-HSCT. Thus, we exposed autologous peripheral blood mononuclear cells from healthy donors (n=14) with CCNA1 pepmix and were able to induce specific responses in 13 of 14 donors tested. The cultures expressed both central (30±3%) and memory markers (30±6%) and were polyclonal though the dominant CCNA1-directed response was CD8-mediated. Reactive cells were polyfunctional (dual IFNg+/TNFa+ secretion), and capable of lysing HLA-matched CCNA1+ myeloid leukemia cell lines and primary AML blasts, while CCNA1 negative and/or HLA mismatched targets were spared (mean±SEM %lysis =42.6±10 vs 4.3±1.7; p=0.03) Thus, by measuring endogenous immune activity and correlating this data with clinical outcomes we identify clinically relevant target antigens that may be suitable for future T cell therapy studies in AML. As a proof of principle, this study demonstrated the importance of CCNA1-targeted T cells and established the preclinical feasibility of expanding Th1-polarized reactive cells from healthy donors that are suitable for adoptive transfer. Our future studies involve identification of other novel targets in AML including neoantigens and clinical translation of these findings.

Humanized Rag2 mice as patient-derived orthotopic mouse models for immune checkpoint blockade and conventional chemotherapeutic drug treatment for human solid tumors

Abstract To evaluate therapeutic efficacy of immune checkpoint blockade (ICB) and conventional chemotherapy in pre-clinical models, we developed humanized patient-derived orthotopic xenograft (PDOX) models for colorectal cancer (CRC) and renal cell carcinoma (RCC). Rag2 mice were humanized by donor peripheral blood mononuclear cells (PBMC) transfer. Luciferase-tagged CRC and RCC tumor cells were injected intra-rectally and intra renal subcapsular, respectively. Groups of mice (n=5–10) received ICB, anti-PD-1 and anti-PD-L1 antibodies (Nivolumab and Atezolizumab, 200 μg each/mouse) for 4 weeks, and conventional therapy of 5FU (200 mg/kg, for CRC) alone or in combination. Tumor growth was measured by weekly bioluminescent image (BLI) and tumor weight at necropsy. The presence of human immune cells and tumor infiltrating lymphocytes were confirmed by flow cytometry and immunohistochemistry staining. Humanization was evidenced by >45% HLA-ABC+CD45+ circulating human cells in both CRC and RCC models. CRC tumor weight showed significant reduction in group treated with combination of ICB and 5FU compared with untreated controls or 5FU monotherapy group (p<0.05) using patient tumor cell CoCa302. The lung/liver metastasis detected by ex vivo BLI were reduced from 50% of mice in control group to 33% and 11%, respectively. For RCC, tumor size was reduced by >30% from controls in ICB treated group using patient tumor cell KiCa118 and cell line SN12K1. The later had 20% reduction of liver metastasis. The combination of ICB and conventional chemotherapies for CRC and RCC can be tested in our humanized PDOX mouse models. The effect of the combination therapy using MHC matched donors or patient autologous PBMC will be further investigated.

Ayu (Plecoglossus altivelis) CD46 isoforms protect the cells from autologous complement attack

CD46 is an important immune regulatory receptor with multiple functions. However, studies on the function of teleost CD46, especially the different CD46 isoforms are limited. In this study, we identified three membrane cofactor protein (MCP, CD46) gene isoforms from ayu (Plecoglossus altivelis) and tentatively named as PaCD46 isoforms. PaCD46 isoforms were generated by alternative splicing and all consisted of four conserved short consensus repeats (SCRs), and the variable serine-threonine-proline-rich domain, transmembrane hydrophobic domain, and cytoplasmic tail. Phylogenetic analysis showed that the isoforms clustered together with other fish CD46 and then with higher animal CD46. Western blotting analysis of peripheral blood mononuclear cells (PBMC) revealed three bands, all of which had much larger molecular weights than the theoretical values of the three PaCD46 isoforms. Moreover, three PaCD46 isoforms were individually expressed on HEK293 cells, and Western blotting showed the similar band profile to that of PBMC. The recombinant extracellular domain of the PaCD46 isoforms, obtained by expression in Pichia pastoris, significantly reduced hemolysis activity of ayu sera. Furthermore, each of the three PaCD46 isoforms respectively protected the HEK293 cells expressing the isoform. The isoforms were also identified for their protection of autologous PBMC from complement activation. These results provided the first evidence that PaCD46 isoforms may be complement regulatory proteins to prevent complement-induced damage to self-tissue.

The Role of Autologous Mesenchymal Stem Cell Recellularization in Rescuing the Xenoreactive Immune Response

Emerging evidence suggests that current xenograft valve replacements provoke an intense cell-mediated and humoral immune response that is a major regulator of time-dependent structural valve deterioration (SVD). Autologous recellularization may improve valvular durability by masking the xenograft scaffold from immune recognition. The purpose of this study was to directly address the role of autologous recellularization of xenograft scaffolds, in the regulation of the xenoreactive immune response in an in-vitro human model. To study the human xenoreactive immune response, we collected bone marrow, human pericardium and isolated peripheral blood mononuclear cells (PBMCs) from adult patients undergoing elective cardiac surgery. Decellularized bovine pericardium underwent recellularization with human mesenchymal stem cells (MSCs). Grafts were tested as either non-decellularized (fresh) bovine pericardium, decellularized bovine pericardium and recellularized bovine pericardium. We show that when decellularized bovine pericardium was recellularized with human MSCs and exposed to autologous human PBMCs in-vitro, there was a significant reduction in TNF-α and IFN-γ expression, along with a significant decrease in T-cell proliferation when compared to both wild-type and decellularized bovine pericardium (p<0.01). Importantly, recellularized bovine pericardium had an equivalent expression of TNF-α, IFN-γ and T-cell proliferation when compared to human pericardium exposed to autologous human PBMCs. Our data suggest that autologous MSC recellularization of xenogenic tissue abrogates the xenoreactive immune response with subsequent reduction in pro-inflammatory cytokine production and T-cell proliferation in an in-vitro human model. As such, autologous MSC recellularization of an acellular xenogenic scaffolds may be an effective approach to decrease the progression of bioprosthetic SVD.

Effect of autologous transplant of peripheral blood mononuclear cells in combination with proangiogenic factors during experimental revascularization of lower limb ischemia.

Peripheral blood mononuclear cells (PBMCs) contain a cell fraction of mononuclear progenitor cells (MPCs), which own significant angiogenic potential. Autologous transplant of PBMC and/or platelet-rich plasma (PRP) promotes endothelial cells differentiation in experimental lower limb ischemia, which is considered a safe and effective strategy to support revascularization, either in animal models or clinical trials. In addition, thrombin has been proposed to enrich biological scaffolds, hence increasing MPC viability after intramuscular administration, whereas proangiogenic mediators such as vascular endothelial growth factor (VEGF), tumor necrosis factor alpha (TNF-α), inhibitor of the plasminogen activator-1 (PAI-1), and chemokine (CXCL1; GRO-α) participate in the endothelial response to ischemia, through their proangiogenic effects over endothelial cells proliferation, survival, migration, endothelial integrity maintenance, and physiologic vascular response to injury. In the present study, we describe the effect of autologous PBMCs transplant and PRP, either with or without thrombin, over proangiogenic mediators (measured by enzyme-linked immunosorbent assay) and revascularization response (angiographic vascular pattern at 30 days after vascular occlusion) in a rat model of lower limb ischemia. The group treated with PBMC + PRP significantly induced PAI-1, an effect that was prevented by the addition of thrombin. Furthermore, treatment with PBMC + PRP + thrombin resulted in the induction of VEGF. GRO-α showed a sensitive induction of all proangiogenic mediators. All treatments significantly stimulated revascularization, according to angiographic assessment, whereas higher effect was observed with PBMC + PRP treatment (p < .0001). In conclusion, autologous PBMC transplant stimulates revascularization during experimental ischemia of the lower limb, whereas particular effects over proangiogenic and fibrinolytic mediators may be attributed to PBMCs and its combination with PRP and thrombin.

Abstract B85: Combination therapy using the programmed death receptor-1 and the programmed death-ligand 1 inhibitors and Fluorouracil in human colorectal carcinoma in a humanized patient-derived orthotopic mouse model

Abstract Background: One of the recently understood mechanisms associated with the progression of cancer is the immune checkpoint pathway. Many tumors can stimulate the expression of immune checkpoint molecules, resulting in a phenotype of exhausted T cells that cannot restrain tumor progression. One such inhibitory ligand and receptor pair in solid tumors are the programmed death-ligand 1 (PD-1) and programmed death receptor-1 (PD-L1). They prevent the killing of cancer cells by cytotoxic T-lymphocytes. PD-1 receptor is expressed by activated T cells among other cells, while PD-L1 is overexpressed on many tumor types including colorectal carcinoma (CRC). We have developed a humanized patient-derived orthotopic xenograft (hPDOX) model for CRC. Here, we investigate the potential efficacy of combination of immune checkpoint inhibitors (ICIs) and fluorouracil (5FU) in this CRC hPDOX model. Methods: Humanized mice were established by IP injection of donor peripheral blood mononuclear cells (PBMCs) into recombinase activating gene 2 (Rag2) and common cytokine receptor gamma chain gene (IL2Rγ) double knockout (Rag2-/-/IL2Rγ-/-) Rag2 mice. Luciferase-tagged patient tumor CoCaPt302 cells were injected intrarectally into Rag2 mice. Groups of mice (n=7-10) received ICIs, anti-PD-1 and anti-PD-L1 antibodies (nivolumab, 200 μg/mouse and atezolizumab, 200 μg/mouse), and 5FU (200 mg/kg) once a week for 4 weeks, IV injection alone or in combination. Tumor growth was measured weekly by bioluminescent imaging (BLI). At necropsy, tumor weights were measured. Human HLA-ABC+CD45+ hematopoietic cells and CD4+ or CD8+ T cells were detected in peritoneal lavage, blood, and spleen by flow cytometry. The presence of human immune cells (humanization) and tumor-infiltrating lymphocytes was confirmed by immunohistochemistry staining. Results: Humanization was evidenced by mouse blood containing more than 45% HLA-ABC+CD45+ human cells when tested by flow cytometry. Tumor weight showed significant reduction in group treated with combination of ICIs and 5FU compared with untreated controls (p=.0038). Tumors were also significantly smaller than ICIs (p=.0123) or 5FU (p=.0427) monotherapy groups. In addition, the lung and liver metastasis detected by ex vivo BLI were reduced from 50% of mice in control group to 33% and 11%, respectively, and exhibited better antitumor response as well as less development of metastases. CD3+ T cells, especially CD4+, CD8+ T cells in blood and spleen, were reduced in the ICIs only and combination treatment groups, but not in 5FU group compared to the controls. Conclusion: Our study provides preclinical evidence of establishment of humanized Rag2 mice to be used as a hPDOX model for CRC; and treatment efficacy including inhibiting tumor growth and distant organ metastasis was significantly enhanced through ICI and 5FU combination therapy. The effect of the combination therapy using CRC patient-derived specimens and PBMCs from MHC matched donors or autologous PBMCs will be further investigated. Citation Format: Xin Zhang, Grace Maresh, Linh Hellmers, Henry Yip, Lara McKean Baste, Heather Green, David Margolin, Li Li. Combination therapy using the programmed death receptor-1 and the programmed death-ligand 1 inhibitors and Fluorouracil in human colorectal carcinoma in a humanized patient-derived orthotopic mouse model [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B85.

Analysis of the Application of Regulatory Dendritic Cells in Kidney Transplantation

Kidney transplantation (KT_x) is the optimum therapy for end-stage renal diseases. However, long-term usage of immunosuppressive agents results in various toxicities and side effects, which has been a major obstacle for recipients. How to reduce the dosages of immunosuppressive agents has become a problem that desperately needs to be solved. Among potential methods, cell therapy has great potential, and regulatory dendritic cells (DC_reg) have attracted special attention for their tolerogenic ability. Currently, some DC-based clinical trials are ongoing or have been completed at several research centers, including an immune tolerance trial in KT_x named “The One Study”, in which autologous peripheral blood mononuclear cells were isolated and stimulated with low doses of GM-CSF or immunosuppressive agents. DC_reg generated with different induction methods or from different cell sources may function in different ways in different organs.

Predicting combinations of immunomodulators to enhance dendritic cell-based vaccination based on a hybrid experimental and computational platform

Dendritic cell (DC)-based vaccines have been largely used in the adjuvant setting for the treatment of cancer, however, despite their proven safety, clinical outcomes still remain modest. In order to improve their efficacy, DC-based vaccines are often combined with one or multiple immunomodulatory agents. However, the selection of the most promising combinations is hampered by the plethora of agents available and the unknown interplay between these different agents. To address this point, we developed a hybrid experimental and computational platform to predict the effects and immunogenicity of dual combinations of stimuli once combined with DC vaccination, based on the experimental data of a variety of assays to monitor different aspects of the immune response after a single stimulus. To assess the stimuli behavior when used as single agents, we first developed an in vitro co-culture system of T cell priming using monocyte-derived DCs loaded with whole tumor lysate to prime autologous peripheral blood mononuclear cells in the presence of the chosen stimuli, as single adjuvants, and characterized the elicited response assessing 18 different phenotypic and functional traits important for an efficient anti-cancer response. We then developed and applied a prediction algorithm, generating a ranking for all possible dual combinations of the different single stimuli considered here. The ranking generated by the prediction tool was then validated with experimental data showing a strong correlation with the predicted scores, confirming that the top ranked conditions globally significantly outperformed the worst conditions. Thus, the method developed here constitutes an innovative tool for the selection of the best immunomodulatory agents to implement in future DC-based vaccines.

Modulation of T-Cell Activation Markers Expression by the Adipose Tissue–Derived Mesenchymal Stem Cells of Patients with Rheumatic Diseases

Background: Activated T lymphocytes play an important role in the pathogenesis of rheumatic diseases (RD). Mesenchymal stem cells (MSCs) possess immunoregulatory activities but such functions of MSCs from bone marrow of systemic lupus erythematosus (SLE), systemic sclerosis (SSc), and ankylosing spondylitis (AS) patients are impaired. Adipose tissue–derived MSCs (ASCs) are an optional pool of therapeutically useful MSCs, but biology of these cells in RD is poorly known. This study aimed at investigating the effect of ASCs from RD patients and healthy donors (HD) on the expression of the key T-cell activation markers. Methods: ASCs were isolated from subcutaneous abdominal fat from SLE (n = 16), SSc (n = 18), and AS (n = 16) patients, while five human ASCs lines from HD were used as a control. Untreated and cytokine (tumor necrosis factor α + interferon γ)-treated ASCs were co-cultured with allogenic, mitogen (phytohemagglutinin)-stimulated peripheral blood mononuclear cells (PBMCs) or purified anti-CD3/CD28-activated CD4+ T lymphocytes. Contacting and noncontacting ASCs-PBMCs co-cultures were performed. RD/ASCs were analyzed in co-cultures with both allogeneic and autologous PBMCs. Flow cytometry analysis was used to evaluate expression of CD25, HLA-DR, and CD69 molecules on CD4+ and CD8+ cells. Results: In co-cultures with allogeneic, activated CD4+ T cells and PBMCs, HD/ASCs and RD/ASCs downregulated CD25 and HLA-DR, while upregulated CD69 molecules expression on both CD4+ and CD8+ cells with comparable potency. This modulatory effect was similar in contacting and noncontacting co-cultures. RD/ASCs exerted weaker inhibitory effect on CD25 expression on autologous than allogeneic CD4+ and CD8+ T cells. Conclusion: RD/ASCs retain normal capability to regulate expression of activation markers on allogeneic T cells. Both HD/ASCs and RD/ASCs exert this effect independently of their activation status, mostly through the indirect pathway and soluble factors. However, autologous CD4+ and CD8+ T cells are partially resistant to RD/ASCs inhibition of CD25 expression, suggesting weaker control of T-cell activation in vivo.

Natural Killer Cell Expansion with Autologous Feeder Layer and Anti-CD3 Antibody for Immune Cell Therapy of Hepatocellular Carcinoma.

Background:one of the promising approaches for treatment of some cancers is adoptive cell therapy using natural killer (NK) cells. Various methods have been investigated for ex vivo expansion of NK cells in large-scale, but most of them involved cancer or genetically modified cells as feeder layer and also some of them have the risk of T cell contamination and graft-versus-host disease (GVHD). Method:In this study, irradiated autologous peripheral blood mononuclear cells (PBMCs) as feeder layer with an anti-CD3 monoclonal antibody (mAb) were used. For activation and expansion of NK cells, human recombinant IL2 and IL15 were used. After co-culturing of expanded NK cells (eNKC) and isolated NK cells (iNKC) with hepatocellular carcinoma (HCC) cells, the viability of eNKC in compared to iNKC were analyzed by CCK-8 assay and degranulation of NK cells after co-culturing was assayed by measuring CD107a expression. Enzyme-Linked Immunosorbent Assay (ELISA) assay was used for the ability of NK cells to secretion of IFN-γ (interferon-γ) and TNF-α (Tumor Necrosis Factor-α) after co-culture with HCC cells. Real Time PCR analysis was used for expression of human Perforin and Granzyme B genes in the NK cells exposed to target HepG2 cells. Result:This method strongly expanded highly purified NK cells with powerful cytotoxicity against HCC cells. The expanded NK cells showed high level of expression of degranulation marker and human Perforin and Granzyme B genes, and also was secreted larger amounts of TNF-α and IFN-γ compared with fresh isolated NK cells. Conclusion:we proposed an effective method for expansion of cytotoxic NK cells using irradiated autologous PBMC as feeder layer for more successful transfer of allogeneic NK cell in immuno cell therapy of HCC.

Sequential CD19- and Bcma-Specific Chimeric Antigen Receptor T Cell Treatment for RRMM: Report from a Single Center Study

Background: Relapsed and/or refractory multiple myeloma (RRMM) has a very poor prognosis, especially for chinese patients with limited therapeutic options. Chimeric antigen receptor transduced T cells (CARTs) had been approved to be one of the rescue strategies for malignant B cell hematological tumors. CARTs targeting B-Cell Maturation Antigen (BCMA) were effective against RRMM in recent published clinical studies. CD19 is expressed by B cells before terminal differentiation into plasma cells and is associated with the enhancement of tumor-propagating and drug-resistance properties of myeloma. Thus, CD19 is also a potential therapeutic target. To our knowledge, we firstly designed the clinical study to evaluate the safety and efficacy of sequential infusion of CD19 and BCMA-Specific CARTs for RRMM patients (pts) (NCT 03196414). Our initial results showed this stragety had substantial anti-myeloma activity and well tolerated toxicities, which had been released at the 59th ASH meeting. Here, we will report the latest clinical data of a total of 28 RRMM patients receiving combined autologous CARTs infusion. Methods: Human T cells were collected from autologous peripheral blood mononuclear cells (PBSC). CART production was performed by the Unicar-Therapy Bio-medicine Technology Company (Shanghai, China). In this trial, RRMM pts firstly receive FC regimen as lymphodepleting conditioning , then were infused into CART-19 (1×107/kg on day 0) and CART-BCMA (total dose range: 2-6.8×107/kg) cells as split-dose infusions (40% on day 1 and 60% on day 2). After one month from CARTs infusion, some pts were adminstrated IMiDs for maintence therapy after hematopoietic recovery, including oral thalidomide 50mg/d or lenalidomide 10mg/d. This trial design and observation was seen in Blood,2017,130(Suppl.):506.The median follow-up time was 16 (3-28) months. Results: By January 2019, 28 pts were enrolled in this clinical trial. Among them, 23 (82.1%) pts were males and 5 (17.9%) pts were females. The median age was 57.5 (range: 42-69) years old. All pts were resistant to proteasome inhibitors (PIs), immunomodulatory drugs (IMiDs), or both. These pts had received an average of 3 (2-8) lines of therapy prior to enrollment. The most common adverse event of CART therapy was acute cytokine release syndrome (CRS), which occurred in 28 pts (100%). 19 pts (67.9%) were evaluated grade 1-2 CRS. 7 pts (25.0%) were evaluated grade 3 CRS, and 2 pts (7.1%) were grade 4. High-grade CRS was associated with elevated levels of IL-6, IFNγ, IL-10 and CRP, especially IL-6 upgradation to thousands of times the baseline. Other toxicities within two weeks of CARTs infusion included fatigue (n=28; 100%), cytopenia (n=28; 100%), anemia (n=28; 100%), prolonged APTT (n=23; 82.1%), elevated creatinine (n=13; 46.4%), gastrointestinal reaction(n=9; 32.1%), elevated transaminase (n=9; 32.1%). Notably, one patient (CRS grade 4) developed grade 4 CRES who exhibited generalized seizure. But this life-threatening complication was quickly relieved after corticosteroids and sodium valproate administration. Two pts (7.1%) had HLH/MAS. No fatal or severe adverse events emerged after two weeks following the infusion. Chronic side effects included chronic diarrhea, hematocytopenia, mild infection, grade ≤2 bilirubin and/or transaminase elevation, hypogammaglobulinemia, etc. No treatment related mortality occurred in this group of pts. Among the 28 pts who completed the CARTs infusion, 27 pts were monitored at predetermined time points and one patient lost follow-up. The overall response rate was 92.6%, and 88.9% of the pts were above PR. 11 pts (40.7%) were CR or sCR, 8 pts (29.6%) were VGPR, 5 pts (18.5%) were PR and one (3.7%) was MR. The remaining 2 pts, one (3.7%) was evaluated as SD and another (3.7%) was PD. 4 pts died from myeloma progression during the follow-up. Of those pts, the median PFS was 8 months. The median OS was 16 months. Conclusions: Combined sequential administration of CART-19 and CART-BCMA cells can be manufactured from heavily-treated MM pts, and could elicit sustained remission for RRMM pts. Toxicities can be well tolerated. CARTs early expansion and persistence in vivo post infusion may be predictive of clinical outcome. How to prolong the survival time of CARTs in vivo is one of the subjects worth studying in the future. Disclosures No relevant conflicts of interest to declare.