scholarly journals Single-Cell Analysis of Target Antigens of CAR-T Reveals a Potential Landscape of “On-Target, Off-Tumor Toxicity”

2021 ◽  
Vol 12 ◽  
Author(s):  
Yinyin Zhang ◽  
Yingmei Li ◽  
Weijie Cao ◽  
Fang Wang ◽  
Xinsheng Xie ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Expression Patterns ◽  
Antigen Expression ◽  
Cellular Immunotherapy ◽  
Target Antigen ◽  
Aortic Tissue ◽  
Great Success ◽  
Healthy Human ◽  
Peripheral Blood Mononuclear ◽  
Car T

Cellular immunotherapy represented by CD19-directed chimeric antigen receptor T (CAR-T) cells has achieved great success in recent years. An increasing number of CAR-T therapies are being developed for cancer treatment, but the frequent and varied adverse events, such as “on-target, off-tumor toxicity”, limit CAR-T application. Here, we identify the target antigen expression patterns of CAR therapies in 18 tissues and organs (peripheral blood mononuclear cells, bone marrow, lymph nodes, spleen, heart, ascending aortic tissue, trachea, lung, skin, kidney, bladder, esophagus, stomach, small intestine, rectum, liver, common bile duct, and pancreas) from healthy human samples. The atlas determines target antigens expressed on some normal cell types, which facilitates elucidating the cause of “on-target, off-tumor toxicity” in special tissues and organs by targeting some antigens, but not others. Moreover, we describe the target antigen expression patterns of B-lineage-derived malignant cells, acute myeloid leukemia (AML), and solid tumors. Overall, the present study indicates the pathogenesis of “on-target, off-tumor toxicity” during CAR therapies and provides guidance on taking preventive measures during CAR treatment.

scholarly journals P855 High-resolution maps of heterogeneous antigen expression in glioblastoma and implications for immunotherapy

2020 ◽  
Vol 8 (Suppl 1) ◽  
pp. A6.1-A6
Author(s):  
Michael Barish ◽  
Lihong Weng ◽  
Dina Awabdeh ◽  
Blake Brewster ◽  
Massimo D’Apuzzo ◽  
...  
Keyword(s):  
Tumor Heterogeneity ◽  
Diversity Index ◽  
Expression Patterns ◽  
Antigen Expression ◽  
Target Antigen ◽  
Spatial Distributions ◽  
Combination Therapies ◽  
Cell Immunotherapy ◽  
Color Deconvolution ◽  
Car T

BackgroundGlioblastoma (GBM) remains an almost universally fatal brain tumor. While CAR T cell immunotherapy has shown promising clinical efficacy, therapeutic failure may reflect our incomplete understanding of target antigen expression. We previously examined variations in antigen expression at the level of individual patients (inter-patient or inter-tumor heterogeneity), focusing on immunotherapy targets IL13Rα2, HER2 and EGFR. We concluded that antigen expression diverged from expectations from random expression. Because antigen escape may arise from GBM cell heterogeneity, we have mapped target antigen expression within individual tumors (intra-tumor heterogeneity).MethodsSerial sections from a 43 patient cohort were immunostained (DAB with hematoxylin counterstain) for target antigens IL13Rα2, HER2 and EGFR. Each section was annotated directly from the slide by a neuropathologist. Sections were scanned (0.46 µm/pixel; Hamamatsu), and then working within Fiji/ImageJ, images were segmented by color deconvolution into hematoxylin (nuclei) and DAB layers. Images of nuclear layers were aligned, and used to align the DAB layers. Two schemes were used to examine the spatial distributions of the three target antigens. When tumor domains could be identified, we determined expression of each antigen as optical density (OD). In the second scheme, a 10 µm grid was superimposed on each section, and OD was determined for each position and assembled into spreadsheets (Origin v2019b). Maps for expression were generated from the OD in each position.ResultsApproaching these maps from the perspective of antigen escape, we examined the extent to which expression of target antigens was spatially mixed, how rapidly antigen dominance could shift (spatial frequency), and whether spatial distributions were arrayed in a coordinated manner.When tumor domains could be identified, we calculated the Shannon diversity index (H) for each domain within a section. While values of H clustered within some tumors, usually values of H varied widely.The superimposed grid was used to examine heterogeneity within entire tumor sections. Expression was intermixed, and EGFR and IL13Rα2/HER2 displayeds complementary expression patterns. In tumors with large EGFR+ areas, IL13Rα2+/HER2+ areas could overlap, while when EGFR+ areas were smaller, IL13Rα2+ and HER2+ areas were more distinct. Borders could be quite diffuse, or quite sharp (a few cell diameters).ConclusionsOur results indicate that expression of IL13Rα2, HER2 and EGFR is highly heterogeneous and not always spatially distinct. Because GBM tumors adapt to the selection pressures of immunotherapies, we suggest that combination therapies should be designed accordingly, and immunotherapies targeting IL13Rα2/HER2 could benefit from inclusion of EGFR.

scholarly journals Investigation of the PD-1 and PD-L1 Immune Checkpoint Molecules Throughout Healthy Human Pregnancy and in Nonpregnant Women

2020 ◽  
Vol 9 (8) ◽  
pp. 2536
Author(s):  
Matyas Meggyes ◽  
David U. Nagy ◽  
Laszlo Szereday
Keyword(s):  
T Cells ◽  
Cd8 T Cells ◽  
Mononuclear Cells ◽  
Expression Patterns ◽  
Treg Cells ◽  
Control Group ◽  
Limited Information ◽  
Healthy Human ◽  
Peripheral Blood Mononuclear ◽  
Healthy Pregnancy

Background: A growing body of evidence supports the importance of PD-1 and PD-L1, especially in the materno-fetal interface, although limited information is available about the peripheral expression of these molecules during the trimesters of pregnancy. Methods: 13 healthy women were enrolled from the 1st, 10 from the 2nd and 12 from the 3rd trimester of pregnancy at the same time, 10 healthy, age-matched nonpregnant women formed the control group. From peripheral blood, mononuclear cells were separated and stored at –80 °C. From freshly thawed samples, surface and intracellular staining were performed for flow cytometric analyses. CD107a degranulation assay was used to evaluate the cytotoxicity. Results: significant alternation was detected in PD-1 expression by CD8+T cells and in PD-L1 expression by CD8+T, CD4+T and Treg cells. An interesting relationship was revealed between the PD-1 and PD-L1 expression by the investigated subpopulations in 2nd trimester of pregnancy. Different expression patterns of an activation receptor NKG2D by the PD-1+ CD8+T cells was observed during pregnancy. The notable relationship was further determined in cytotoxicity between PD-1+ and NKG2D+ CD8+T cells throughout pregnancy. Conclusions: the different PD-1 presence and the relationship with NKG2D could contribute to the dynamic changes of the Th1 and Th2 predominance throughout the three trimesters of a healthy pregnancy.

scholarly journals Phytochemical characterization and biological activities of Plectranthus barbatus Andrews

2022 ◽  
Vol 82 ◽  
Author(s):  
M. F. Cordeiro ◽  
T. R. S. Nunes ◽  
F. G. Bezerra ◽  
P. K. M. Damasco ◽  
W. A. V. Silva ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Trichophyton Rubrum ◽  
Biological Activities ◽  
Sandwich Elisa ◽  
Immunomodulatory Activity ◽  
Potential Candidate ◽  
Leaf Extracts ◽  
Healthy Human ◽  
Peripheral Blood Mononuclear ◽  
Normal Peripheral Blood

Abstract Plectranthus barbatus Andrews (Lamiaceae) is widely distributed in the world and has a range of popular therapeutic indications. This work aimed to evaluate the phytochemical characterization of two leaf extracts of P. barbatus, and their antimicrobial, antineoplastic and immunomodulatory potential. After collection, herborization and obtainment of the P. barbatus aqueous extract (PBA) and acetone:water 7:3 P. barbatus organic extract (PBO), the phytochemical characterization was performed by high-performance liquid chromatography (HPLC). The antimicrobial activity was performed to determine the minimum inhibitory concentration (MIC) against eight bacterial strains using the microdilution test and the fungus Trichophyton rubrum by disc diffusion assay and microdilution test. Cytotoxicity was assessed by MTT and trypan blue methods in normal peripheral blood mononuclear cells (PBMCs) at concentrations ranged between 0.1 to 100 µg.mL-1 and in neoplastic cell lines Toledo, K562, DU-145 and PANC-1 at 1, 10 and 100 µg.mL-1 . Immunomodulatory activity, was evaluated by sandwich ELISA of proinflammatory cytokines at BALB/c mice splenocytes cultures supernatant. Both extracts presented flavonoids, cinnamic derivatives, steroids and ellagic acid. PBO showed bacteriostatic activity against Acinetobacter baumannii (MIC = 250 µg.mL-1) clinical isolate and PBA fungistatic activity against Trichophyton rubrum (MIC = 800 µg.mL-1). The extracts did not exhibit toxicity to PBMCs and neoplastic cells (IC50 > 100 µg.mL-1). Additionally, PBO at 100 µg.mL-1 significantly inhibited IFN-γ and IL-17A cytokines (p = 0.03). Plectranthus barbatus is a potential candidate for therapeutic use due to its low toxicity in healthy human cells and exhibits biological activities of medical interest as bacteriostatic, fungistatic and immunomodulatory.

scholarly journals A 70-Kilodalton Recombinant Heat Shock Protein ofCandida albicans Is Highly Immunogenic and Enhances Systemic Murine Candidiasis

1998 ◽  
Vol 66 (5) ◽  
pp. 2154-2162 ◽  
Author(s):  
Carla Bromuro ◽  
Roberto La Valle ◽  
Silvia Sandini ◽  
Francesca Urbani ◽  
Clara M. Ausiello ◽  
...  
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Mononuclear Cells ◽  
Cell Mediated Immunity ◽  
Healthy Human ◽  
Peripheral Blood Mononuclear ◽  
Necrosis Factor Alpha ◽  
Ifn Γ

ABSTRACT The 70-kDa recombinant Candida albicans heat shock protein (CaHsp70) and its 21-kDa C-terminal and 28-kDa N-terminal fragments (CaHsp70-Cter and CaHsp70-Nter, respectively) were studied for their immunogenicity, including proinflammatory cytokine induction in vitro and in vivo, and protection in a murine model of hematogenous candidiasis. The whole protein and its two fragments were strong inducers of both antibody (Ab; immunoglobulin G1 [IgG1] and IgG2b were the prevalent isotypes) and cell-mediated immunity (CMI) responses in mice. CaHsp70 preparations were also recognized as CMI targets by peripheral blood mononuclear cells of healthy human subjects. Inoculation of CaHsp70 preparations into immunized mice induced rapid production of interleukin-6 (IL-6) and tumor necrosis factor alpha, peaking at 2 to 5 h and declining within 24 h. CaHsp70 and CaHsp70-Cter also induced gamma interferon (IFN-γ), IL-12, and IL-10 but not IL-4 production by CD4+ lymphocytes cocultured with splenic accessory cells from nonimmunized mice. In particular, the production of IFN-γ was equal if not superior to that induced in the same cells by whole, heat-inactivated fungal cells or the mitogenic lectin concanavalin A. In immunized mice, however, IL-4 but not IL-12 was produced in addition to IFN-γ upon in vitro stimulation of CD4+ cells with CaHsp70 and CaHsp70-Cter. These animals showed a decreased median survival time compared to nonimmunized mice, and their mortality was strictly associated with organ invasion by fungal hyphae. Their enhanced susceptibility was attributable to the immunization state, as it did not occur in congenitally athymic nude mice, which were unable to raise either Ab or CMI responses to CaHsp70 preparations. Together, our data demonstrate the elevated immunogenicity of CaHsp70, with which, however, no protection against but rather some enhancement of Candida infection seemed to occur in the mouse model used.

scholarly journals Chloroquine and Rapamycin Augment Interleukin-37 Expression via the LC3, ERK, and AP-1 Axis in the Presence of Lipopolysaccharides

2020 ◽  
Vol 2020 ◽  
pp. 1-14
Author(s):  
Xiaoyi Shi ◽  
Chunhui Lai ◽  
Lianyu Zhao ◽  
Mingying Zhang ◽  
Xi Liu ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Therapeutic Potential ◽  
Inflammatory Diseases ◽  
Human Peripheral Blood ◽  
Signal Pathways ◽  
U937 Cells ◽  
Healthy Human ◽  
Peripheral Blood Mononuclear ◽  
Phosphorylated Proteins

IL-37 is a cytokine that plays critical protective roles in many metabolic inflammatory diseases, and its therapeutic potential has been confirmed by exogenous IL-37 administration. However, its regulatory mechanisms remain unclear. U937 cells were treated with autophagy-modifying reagents (3-MA, chloroquine, and rapamycin) with or without LPS stimulation. Thereafter, IL-37 expression and autophagic markers (Beclin1, P62/SQSTM1, and LC3) were determined. For regulatory signal pathways, phosphorylated proteins of NF-κB (p65 and IκBα), AP-1 (c-Fos/c-Jun), and MAPK signal pathways (Erk1/2 and p38 MAPK) were quantified, and the agonists and antagonists of MAPK and NF-κB pathways were also used. Healthy human peripheral blood mononuclear cells were treated similarly to confirm our results. Four rhesus monkeys were also administered chloroquine to evaluate IL-37 induction in vivo and its bioactivity on CD4 proliferation and activation. IL-37 was upregulated by rapamycin and chloroquine in both U937 cells and human PBMCs in the presence of LPS. IL-37 was preferentially induced in autophagic cells associated with LC3 conversion. AP-1 and p65 binding motifs could be deduced in the sequence of the IL-37 promoter. Inductive IL-37 expression was accompanied with increased phosphorylated Erk1/2 and AP-1 and could be completely abolished by an Erk1/2 inhibitor or augmented by Erk1/2 agonists. In monkeys, chloroquine increased IL-37 expression, which was inversely correlated with CD4 proliferation and phosphorylated STAT3. IL-37 levels were induced by rapamycin and chloroquine through the LC3, Erk1/2, and NF-κB/AP-1 pathways. Functional IL-37 could also be induced in vivo.

scholarly journals Identification of IFITM1 and IFITM3 in Goose: Gene Structure, Expression Patterns, and Immune Reponses against Tembusu Virus Infection

2017 ◽  
Vol 2017 ◽  
pp. 1-13 ◽  
Author(s):  
Anqi Wang ◽  
Lipei Sun ◽  
Mingshu Wang ◽  
Renyong Jia ◽  
Dekang Zhu ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Expression Patterns ◽  
Harderian Gland ◽  
Bursa Of Fabricius ◽  
Poly I:C ◽  
Peripheral Blood Mononuclear ◽  
Interferon Stimulated Genes ◽  
Cecal Tonsil ◽  
Tembusu Virus ◽  
First Time

As interferon-stimulated genes (ISGs), interferon-inducible transmembrane proteins 1 and 3 (IFITM1 and IFITM3) can effectively inhibit the replication of multiple viruses. Here, goose IFITM1 and IFITM3 were cloned and identified for the first time. The two proteins share the same topological structure and several important sites critical for the antiviral functions in other species are conserved in the goose. Goose IFITM1 and IFITM3 are most closely related to their respective orthologs in ducks; these proteins exhibited high mRNA transcript levels in immune-related tissues, including the thymus, bursa of Fabricius, and Harderian gland, compared to other tissues. Moreover, goose IFITM1 was highly constitutively expressed in gastrointestinal tract tissues, while goose IFITM3 was expressed in respiratory organs. Furthermore, goose IFITM3 was activated in goose peripheral blood mononuclear cells (PBMCs) infected with Tembusu virus (TMUV) or treated with Toll-like receptors (TLRs) agonists, while only the R848 and Poly (I:C) agonists induced significant upregulation of goose IFITM1. Furthermore, goose IFITM1 and IFITM3 were upregulated in the sampled tissues, to some extent, after TMUV infection. Notably, significant upregulation of goose IFITM1 and IFITM3 was detected in the cecum and cecal tonsil, where TMUV was primarily distributed. These data provide new insights into the immune effectors in geese and promote our understanding of the role of IFITM1 and IFITM3 in the defense against TMUV.

scholarly journals Cytokine influence on killing of fresh chronic lymphocytic leukemia cells by human leukocytes

Blood ◽  
1991 ◽  
Vol 77 (12) ◽  
pp. 2707-2715 ◽  
Author(s):  
D Cemerlic ◽  
B Dadey ◽  
T Han ◽  
L Vaickus
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Mononuclear Cells ◽  
Protein A ◽  
Human Leukocyte ◽  
Lymphocytic Leukemia ◽  
Target Antigen ◽  
Peripheral Blood Mononuclear ◽  
Ifn Gamma ◽  
Hla Dr

Abstract The feasibility of combining the Lym-1 monoclonal antibody (MoAb) with interferon-gamma (IFN-gamma) in the treatment of chronic lymphocytic leukemia (CLL) was evaluated. We used an in vitro tumor lysis model that incorporated fresh CLL cells from 21 different patients as targets for two distinct normal human leukocyte effector subsets, neutrophils, and peripheral blood mononuclear cells (PBMCs). Lym-1 antigen (Lym-1- Ag) expression varied greatly and did not correlate with the expression of other CLL-associated antigens such as CD5, CD19, or HLA-DR. CLL cells were not lysed by neutrophils alone or with IFN-gamma in the absence of Lym-1. Neutrophil Lym-1-dependent cytotoxicity (ADCC) in the absence of IFN-gamma was weak and inconsistent. IFN-gamma exposure induced MoAb-dependent lysis of 80% of 21 CLL targets and resulted in an eightfold augmentation of neutrophil ADCC against the remainder. Cytotoxicity correlated directly and positively with Lym-1-Ag expression. Confirmation of the need for interaction between neutrophil IgG Fc receptors (Fc gamma Rs) and the Fc portion of the Lym-1 MoAb was obtained by demonstrating that purified Staphylococcus aureus Protein A (SpA) inhibited ADCC. IFN-gamma exposure caused no consistent alternations in Lym-1-Ag expression on CLL cells so that target antigen upregulation was unlikely to account for augmentation of neutrophil ADCC. PBMCs alone, exposed to interkeukin-2 (IL-2) or IFN-gamma, or with Lym-1 in the presence or absence of IL-2 or IFN-gamma were unable to lyse CLL targets. PBMCs were able to kill Raji Burkitt lymphoma cells in conjunction with Lym-1, so their ability to interact with Lym- 1-coated targets and their lytic functions appeared intact. These results emphasize the importance of examining fresh tumor cells with different leukocyte effector subsets before designing a clinical trial that combines a therapeutic MoAb with a cytokine.

Anti-CD20 Chimeric Antigen Receptor Transduced T Cells Can Recognize Very Low Antigen Expression: Determination Of The Lower Threshold Required To Activate The CAR-T Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4493-4493 ◽  
Author(s):  
Keisuke Watanabe ◽  
Seitaro Terakura ◽  
Tatsunori Goto ◽  
Ryo Hanajiri ◽  
Nobuhiko Imahashi ◽  
...  
Keyword(s):  
T Cells ◽  
Antigen Expression ◽  
Expression Level ◽  
Target Antigen ◽  
Ifn Gamma ◽  
Cd20 Antigen ◽  
Car T Cells ◽  
Car T ◽  
Cd20 Expression ◽  
Anti Cd20

Background In chimeric antigen receptor transduced T-cell (CAR-T cell) therapy, setting of the target antigen is critical in terms of both the efficacy and the possible adverse effects. However, how low expressing antigen CAR-T cells can recognize and present cytotoxicity, and how low expressing antigen would be a candidate for the target or should be avoided for the concern of off-organ effect is still unclear. Thus we developed novel anti-CD20 CAR-T cells, and investigated the threshold antigen expression level required to activate CAR-T cells. Methods In this study, we generated a retrovirus vector that encodes a novel CAR consisting of anti-CD20 single chain variable fragments linked to a CD3-zeta chain, a CD28 costimulatory domain, and a truncated epidermal growth factor receptor (tEGFR) as a transduction and selection marker. CD3 positive T cells from a healthy donor were activated with anti-CD3/28 beads, transduced with the CAR on day 3, enriched by selection with anti-EGFR mAb, and expanded by stimulation with gamma-irradiated B-lymphoblastoid cells. CD20 expression level of the target cells was evaluated with CD20 mean fluorescence intensity (CD20-MFI) and was quantified as CD20 specific antibody biding capacity (CD20-ABC). Results To determine the threshold expression level of CD20 antigen to induce cytotoxicity, we performed 51Cr release assay by anti-CD20 CAR-T cells against 30 clones of CD20 transduced CCRF-CEM (CD20-CEM) expressing variable levels of CD20 (CD20-MFI: 126-6,924/ CD20-ABC: 240-230,546 molecules) (kindly provided by Dr. A.C.M. Martens, University Medical Center Utrecht). CAR-T cells lysed CD20-CEMs equally well from low to high level of CD20 (CD20MFI: 157/ CD20ABC: 5,172 molecules or more, 40-60% of lysis at E:T ratio of 10:1), and lysed the clone expressing the lowest level of CD20 (126/ 240 molecules, 22.8±2% of lysis). However, in rituximab-induced CDC assay against the same 30 clones, a minimum CD20 MFI of 600 (ABC of 65,000 molecule) was required to induce CDC. Next, cytokine production and proliferation upon stimulation were investigated by using four representative CD20-CEM clones out of the 30 clones that cover a wide range of CD20 expression levels; Very low (VL-CEM) (MFI: 126/ ABC:240 molecules), Low (L-CEM)(252/ 26,990 molecules), Mid (M-CEM)(826/ 91,567 molecules), High (H-CEM)(6,924/ 142,722 molecules). In intracellular IFN-gamma assay, CAR-T cells produced IFN-gamma equally well (L-CEM, 45.9%, M-CEM, 35.4% and H-CEM, 38.0%, respectively), except against VL-CEM (0.2%). In CFSE division assay, CAR-T cells proliferated upon stimulation with L-CEM, M-CEM, H-CEM (31.4%, %36.1, 37.3% at 72h and, 88.9%, 90.2%, 91.1% at 96h), except with LV-CEM (0% at 72h/ 96h). In intracellular signaling assay, phosphorylation of downstream signaling molecule, CD3-zeta and ERK were evaluated. Whereas only minimal phosphorylations of CD3-zeta and ERK were observed upon VL-CEM stimulation, other 3 CEMs induced similar strength of signals in the majority of CAR-T cells, and induced phosphorylation of proximal signaling molecule, CD3-zeta according to the CD20 expression level. Finally, we evaluated cytotoxicity against low-CD20 expressing primary tumor cells and cell lines. CAR-T cells lysed RRBL1 which is a cell line established from a patient who exhibited relapse of B-cell lymphoma with very weak CD20 expression and became resistant to rituximab (47.8±3% of lysis at E:T ratio 10:1) and primary DLBCL cells isolated from pleural effusion with low CD20 expression (32.6±2.9% of lysis). Conclusions We observed that CAR-T cells recognize CD20 antigen with considerably low expression. A minimum number of antigen molecules required to activate CAR-T cells is very low; the threshold is a few hundreds of target antigen. For the future search of a novel target of CAR-T therapy, antigens with expression above the threshold, even if that is below the range of mAb therapy, could be also considered. Conversely, antigens with off-organ expression above the threshold may induce off-organ effect, thus should be avoided. CD20-CAR therapy may be also effective for the patients with CD20 down-regulated lymphoma and refractory to CD20 mAb therapy. Disclosures: No relevant conflicts of interest to declare.

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