scholarly journals Syncytins enable novel possibilities to transduce human or mouse primary B cells and to achieve well-tolerated in vivo gene transfer

2019 ◽  
Author(s):  
Y. Coquin ◽  
M. Ferrand ◽  
A. Seye ◽  
L. Menu ◽  
A. Galy
Keyword(s):  
T Cells ◽  
B Cells ◽  
Gene Transfer ◽  
Gene Delivery ◽  
B Cell ◽  
Stable Gene ◽  
Murine Bone Marrow ◽  
High Level

AbstractSyncytins are cellular transmembrane glycoproteins with fusogenic and immunosuppressive properties that are encoded by endogenous retroviral envelope sequences in mammalian genomes. Based on their properties, syncytins may be useful to pseudotype lentiviral gene transfer vectors (LV) and to obtain well-tolerated in vivo gene delivery but their cellular targets are unknown in this context. We pseudotyped LV with human or murine syncytins. Such LV-Syn particles were infectious in vitro but required a transduction additive, as do other retroviral envelope LV pseudotypes. In these conditions, LV-Syn remarkably transduced quiescent human or murine primary B cells at high level in vitro including naïve blood B cells or B cell precursors from murine bone marrow. Transduced human B cells could be expanded in culture and were functional. Human or murine T cells were transduced less efficiently than B cells, in agreement with lower levels of syncytin receptors on T cells compared to B cells. Well-tolerated in vivo gene transfer was possible without additive, as demonstrated with murine syncytin A-mediated gene delivery in C57BL/6 mice. A single intravenous injection of LV-SynA vector to mice led to stable gene transfer into spleen germinal center B cells. LV-SynA were also intrinsically less immunogenic than LV-VSVG, leading to low antibody responses against the vector capsid. This is the first evidence of interactions between syncytins and B cells, providing novel opportunities for B cell genetic engineering and for well-tolerated gene transfer in vivo. The findings also suggest that some immunosuppressive properties of syncytins could be mediated by B cells.One Sentence SummarySyncytins are fusogenic cellular proteins that can pseudotype lentiviral gene transfer vector particles, achieving efficient gene transfer into primary quiescent B cells and reducing the in vivo immunogenicity of the particles following systemic administration.

scholarly journals Growth- and differentiation-associated expression of bcl-2 in B-chronic lymphocytic leukemia cells

Blood ◽  
1992 ◽  
Vol 79 (11) ◽  
pp. 2981-2989 ◽  
Author(s):  
M Schena ◽  
LG Larsson ◽  
D Gottardi ◽  
G Gaidano ◽  
M Carlsson ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Messenger Rna ◽  
Phorbol Esters ◽  
Lymphocytic Leukemia ◽  
Mantle Zone ◽  
Cell Stage ◽  
High Level

Abstract The bcl-2 gene is translocated into the Ig loci in about 80% of human follicular lymphomas and in 10% of B-type chronic lymphocytic leukemias (B-CLL), resulting in a high level of expression. We have compared the expression of bcl-2 transcripts and protein in B-CLL cells in their normal equivalent CD5+ B cells and in normal B-cell populations representative of different in vivo and in vitro stages of activation and proliferation. We report here that bcl-2 was expressed in 11 of 11 cases of CD5+ B-CLL clones, contrasting with the absent expression in normal CD5+ B cells. Activation of 173 and 183 B-CLL cells by phorbol esters (12-O-tetradecanoylphorbol-13-acetate [TPA]) to IgM secretion without concomitant DNA synthesis resulted in a rapid but transient downregulation of bcl-2 expression. In contrast, the reduction of bcl-2 at both the messenger RNA and protein levels was sustained after mitogenic stimulation, suggesting that bcl-2 expression and proliferation are inversely related in these cells. This notion was further supported by immunocytochemical analysis showing that bcl-2 was primarily expressed in small resting lymphocytes and in cells differentiating to the plasma cell stage, but less expressed in Ki67- positive proliferating B blasts. Moreover, it was also supported by the low level of bcl-2 in exponentially growing Epstein-Barr virus-carrying lymphoblastoid and B-CLL cell lines. The regulation of bcl-2 expression in B-CLL resembled that of normal tonsillar follicular B cells, in which a high level of expression was found in resting mantle zone B cells but not in the proliferating germinal center B cells. Based on these findings and the role of bcl-2 in maintaining B-cell memory, we propose that the phenotype of B-CLL cells corresponds to a mantle zone memory-type B cell.

In Vitro and in Vivo Imaging of Initial T-B Cell Interactions in the Setting of B Cell-Based Cancer Immunotherapy

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2740-2740
Author(s):  
Kerstin Wennhold ◽  
Nela Klein-Gonzalez ◽  
Michael von Bergwelt-Baildon ◽  
Alexander Shimabukuro-Vornhagen
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cancer Immunotherapy ◽  
Cell Interactions ◽  
Migration Behavior ◽  
Cell Zone

Abstract In recent years, there has been a growing interest in the use of B cells for cellular immunotherapy, since B cell-based cancer vaccines have yielded promising results in preclinical animal models. Contrary to dendritic cells (DCs), we know little about the migration behavior of B cells in vivo. Therefore, we investigated the interactions between CD40-activated (CD40) B cells and cytotoxic T cells in vitro and the migration behavior of CD40B cells in vivo. The dynamic interactions of human antigen-presenting cells and antigen-specific T cells were observed by time-lapse videomicroscopy. The migratory and chemoattractant potential of CD40B cells was analyzed by flow cytometry and standard transwell migration assays. GFP+ CD40B cells or CD40B cells isolated from Luciferase+mice were used for subsequent in vivo studies. Murine CD40B cells show similar migratory and chemotactic characteristics compared to human CD40B cells. Upon CD40-activation, B cells upregulate the important molecules involved in lymh node homing (CD62L, CCR7/CDCR4), which are functional and induce chemotaxis of T cells in vitro. Striking differences were observed for interactions of human CD40B cells or DCs with T cells. Antigen-loaded CD40B cells differ from immature and mature DCs by displaying a rapid migratory pattern undergoing highly dynamic, short-lived (7.5 min) and sequential interactions with cognate T cells. In vivo, CD40B cells migrate to the spleen and the lymph nodes, where they enrich in the B cell zone before traveling to B cell/ T cell boundary close to the T cell zone. CD40B cell interactions with T cells are dynamic and short-lived and thereby differ from DCs. Taken together, the migration behavior of CD40B cells and their interaction with T cells underline their potential as cellular adjuvant for cancer immunotherapy. Disclosures No relevant conflicts of interest to declare.

scholarly journals Engineered FVIII-expressing cytotoxic T cells target and kill FVIII-specific B cells in vitro and in vivo

2018 ◽  
Vol 2 (18) ◽  
pp. 2332-2340 ◽  
Author(s):  
Kalpana Parvathaneni ◽  
David W. Scott
Keyword(s):  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Cd8 T Cells ◽  
Hemophilia A ◽  
Cytotoxic T Cells ◽  
Cell Antibody ◽  
Engineered T Cells

Abstract Hemophilia A is an X-linked bleeding disorder caused by mutations in the factor VIII (FVIII) gene (F8). Treatment with recombinant or plasma-derived FVIII replacement therapy is standard therapy. A major problem in treating hemophilia A patients with therapeutic FVIII is that 20% to 30% of these patients produce neutralizing anti-FVIII antibodies (inhibitors) because they are not immunologically tolerant to this human protein. Hence, there is a need to establish tolerogenic protocols to FVIII epitopes. To specifically target FVIII-specific B cells, we engineered immunodominant FVIII domains (A2 and C2) as a chimeric antigen receptor expressed by both human and murine cytotoxic T cells. This FVIII domain engineered B-cell antibody receptor (BAR) that expresses T cells was capable of killing FVIII-reactive B-cell hybridomas in vitro and in vivo. Moreover, FVIII BAR CD8 T cells blocked the development of specific antibody from unimmunized spleen cells stimulated polyclonally with lipopolysaccharide in vitro. In addition, adoptive transfer of FVIII A2- and C2-BAR CD8 T cells significantly reduced the anti-FVIII antibody formation in hemophilic mice. These data suggest that BAR-engineered T cells are a promising approach for future prophylactic treatment for patients with severe hemophilia A who are at high risk of developing inhibitors.

scholarly journals The collaborative phenotype of secondary B cells is determined by T lymphocytes during in vivo immunization.

1982 ◽  
Vol 155 (2) ◽  
pp. 574-586 ◽  
Author(s):  
N A Speck ◽  
S K Pierce
Keyword(s):  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Cell Populations ◽  
Antibody Synthesis ◽  
Humoral Immune ◽  
Igg1 Antibody ◽  
Humoral Immune Responses

Previous studies have demonstrated that the B cells in immune and nonimmune mice manifest different major histocompatibility complex (MHC) collaborative phenotypes with antigen-specific T cells. Immune, or secondary B cells require syngeneic-like MHC recognition by collaborating T cells, and in its absence fail to be stimulated. Primary B cells manifest a much less stringent requisite for MHC recognition by T cells, and under conditions in which secondary B cells fail to be stimulated, primary B cells are stimulated to secrete IgM antibody. Experiments were conducted to determine whether the acquisition of the secondary B cells' MHC collaborative phenotype was dependent on the presence of T cells during in vivo immunization. B cell populations from T dependently and T independently immunized conventional BALB/c and athymic BALB/c nu/nu mice were compared in their ability to collaborate with allogeneic T cells. Although antigen alone promotes the differentiation of several secondary B cell characteristics, including an increase in the frequency of antigen-specific B cells and a preference for IgG1 antibody synthesis in vitro, the acquisition of the secondary B cells' MHC collaborative phenotype was dependent on the presence of T cells during in vivo immunization. B cell populations from T dependently and T independently immunized conventional BALB/c and athymic BALB/c nu/nu mice were compared in their ability to collaborate with allogeneic T cells. Although antigen alone promotes the differentiation of several secondary B cell characteristics, including an increase in the frequency of antigen-specific B cells and a preference of IgG1 antibody synthesis in vitro, the acquisition of the secondary B cells' MHC collaborative phenotype was found to be dependent on the presence of T cells during in vivo immunization. Thus, the restriction imposed on T cell-B-cell-collaborative interactions in secondary humoral immune responses appears to be the result of T dependent antigen-driven events.

scholarly journals Role of accessory cells in B cell activation. III. Cellular analysis of primary immune response deficits in CBA/N mice: presence of an accessory cell-B cell interaction defect.

1980 ◽  
Vol 152 (5) ◽  
pp. 1194-1309 ◽  
Author(s):  
H S Boswell ◽  
M I Nerenberg ◽  
I Scher ◽  
A Singer
Keyword(s):  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Genetic Defect ◽  
Accessory Cell ◽  
Male Mice ◽  
Accessory Cells ◽  
Antigen Presenting

The effect of the X-linked CBA/N genetic defect on the ability of mice to generate primary responses to thymic-dependent and thymic-independent antigens was assessed by comparing the ability of abnormal (CBA/N x DBA/2)F1 male mice and normal (DBA/2 x CBA/N)F1 male mice to generate 2,4,6-trinitrophenyl (TNP)-specific plaque-forming cell responses to TNP-keyhole limpet hemocyanin (KLH), TNP-conjugated Ficoll (TNP-Ficoll), TNP-Brucella abortus (BA), and TNP-lipopolysaccharide (LPS). The reciprocal F1 combinations used in this study differ genetically only in the origin of their X chromosome, but differ immunologically in that (CBA/N x DBA/2)F1 male mice express all the CBA/N immune abnormalities, whereas (DBA/2 x CBA/N)F1 male mice are immunologically normal. Analysis of thymic-dependent responses to TNP-KLH revealed that abnormal F1 mice were capable of generating primary responses in vivo to high doses of TNP-KLH, but failed to generate responses to suboptimal doses of TNP-KLH that were still immunogenic for normal F1 mice. Furthermore, under limiting in vitro micro-culture conditions, the abnormal F1 mice failed to generate primary thymic-dependent responses to any dose of TNP-KLH, even though under the identical conditions normal F1 mice consistently responded to a wide antigen dose range. The cellular basis of the failure of abnormal F1 mice to respond in vitro to TNP-KLH was investigated by assaying the ability of purified populations of accessory cells, T cells, and B cells from these mice to function in responses to TNP-KLH. The results of these experiments demonstrated that helper T cells and antigen-presenting accessory cells from abnormal F1 mice were competent and functioned as well as the equivalent cell populations from normal F1 mice. Instead, the failure of CBA/N mice to generate primary in vitro responses to TNP-KLH was solely the result of a defect in their B cell population such that B cells from these mice failed to be triggered by competent helper T cells and/or competent accessory cells. Similarly, the failure of abnormal F1 mice to respond either in vivo or in vitro to TNP-Ficoll was not the result of defective accessory cell presentation of TNP-Ficoll, but was the result of the failure of B cells from these mice to be activated by competent TNP-Ficoll-presenting accessory cells. In contrast to the failure of B cells from abnormal F1 mice to be activated in vitro in response to either TNP-KLH or TNP-Ficoll, B cells from abnormal F1 mice were triggered to respond to TNP-BA and TNP-LPS, antigens that did not require accessory cell presentation. The specific failure of B cells fron abnormal F1 mice to be activated in responses that required antigen-presentation by accessory cells suggested the possibility that the X-linked CBA/N genetic defect resulted in B cell populations that might be deficient in their ability to interact with antigen-presenting accessory cells...

scholarly journals CLL B Cells Develop Resistance to Ibrutinib By Reinvigorating the IL-4R - IL-4 Axis Blocked By Bruton's Tyrosine Kinase Inhibitors Including Acalabrutinib and Zanubrutinib

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 477-477
Author(s):  
Shih-Shih Chen ◽  
Constantine S. Tam ◽  
Alan G. Ramsay ◽  
Priyadarshini Ravichandran ◽  
Natalia C. Couto-Francisco ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Cell Subset ◽  
Advisory Committees

Bruton's tyrosine kinases inhibitors (BTKis) represent major advances in CLL therapy. However resistance to this form of therapy is emerging, and such patients often progress more rapidly. Hence there is an important need for therapies that address resistance. Microenvironmental input like IL-4 is critical for CLL disease progression. Compared with normal B cells, CLL cells exhibit significantly higher levels of surface membrane (sm) IL-4 receptor (IL4-R) and contain increased amounts of pSTAT6, a downstream mediator of IL-4R signaling. IL-4 stimulation of CLL B cells suppresses smCXCR4 and increases smIgM, thus promotes CLL cell retention and expansion. In this study, we aimed to examine if smIL-4R expression, IL4R signaling, and IL-4-producing cells are altered in patients sensitive or resistant to BTKis. To do so, T and B cell subset changes were studied overtime in 12 acalabrutinib-treated CLL patients, 6 zanubrutinib-treated CLL patients, 30 ibrutinib-sensitive and 5 ibrutinib-resistant CLL patients, 4 of which exhibited BTK mutations. Consistent with only ibrutinib inhibiting T-cell kinase (ITK), T-cell subset analyses revealed no changes in Th1, Th2, Th17, Th9, and Th22 cells after zanubrutinib or acalabrutinib treatment. In contrast, a Th1-biased T-cell immunity was observed in patients responsive to ibrutinib. In patients progressing on ibrutinib, significantly reduced Th2 T cells were found during the resistant as well as sensitive periods. In an in vitro T-cell function assay using T cells collected before and after the treatment with each BTKi, only ibrutinib treated patients exhibited a reduced ability of T cells to support CLL B cell survival. We next studied changes in CLL B cells, including numbers of IL-4, -10 and -13 producing B cells after BTKi treatment. IL-13 producing CLL B cells were not changed. IL-10 producing CLL B cells were reduced in both ibrutinib sensitive and resistant patients, but not in zanubrutinib or acalabrutinib treated patients. Importantly, IL-4 producing CLL B cells were significantly decreased in patients treated with all 3 BTKi. Significantly reduced smIL-4R levels, impaired IL-4R signaling, decreased smIgM and increased smCXCR4 were also seen in patients treated with each BTKi. To understand the mechanism responsible for inhibition of IL-4 production in CLL cells treated with BTKis, we stimulated CLL cells through IgM, Toll-like receptor and CD40L, finding that only anti-IgM stimulation significantly increased IL-4 production and p-STAT6 induction. We then explored the function of IL-4. IL-4 enhanced CLL B cell survival in vitro and this action was blocked by all 3 BTKis. Moreover, adhesion of CLL B cells to smIL-4R expressing stromal cells was decreased by IL-4 and IL-4R neutralizing antibodies, especially in M-CLL cases. In in vivo studies transferring autologous T cells and CLL PBMCs into alymphoid mice, we found less CLL B cells in mouse spleens post ibrutinib than zanubrutinib or acalabrutinib treatment. This might be due to the suppressed Th2 cells found only in ibrutinib, while IL-4 producing B cells were reduced in all 3 BTKi treated mice. These results support the idea that IL-4 promotes CLL B cell adhesion and growth in tissues. Finally, we investigated the IL-4/IL-4R axis in ibrutinib-resistant patients. Although IL-4 producing T cells remain reduced during the sensitive and resistant phases, CLL B cell production of IL-4 and expression of and signaling through smIL-4R returned when patients developed ibrutinib-resistance. When comparing paired ibrutinib-sensitive and -resistant CLL B cells collected from 3 patients in a xenograft model that requires T cell help, we found ibrutinib-resistant CLL B cells grew in vivo with only minimal (~15%) numbers of autologous T cells compared to B cells collected from ibrutinib-sensitive phase; this suggested a reduced requirement for T-cell help for growth of ibrutinib-resistant CLL cells. In summary, we found IL-4 is a key survival factor in the CLL microenvironment that also improves leukemia cell adhesion to stromal cells expressing smIL-4R. IL-4 production and signaling can be stimulated in CLL B cells through the B-cell receptor, and are consistently blocked by BTKis. Moreover, the recovered ability of ibrutinib-resistant CLL B cells to produce and respond to IL-4 leads to disease progression, suggesting blocking the IL-4/IL-4R axis is a potential treatment for ibrutinib-resistant CLL patients. Disclosures Chen: Pharmacyclics: Research Funding; Beigene: Research Funding; Verastem: Research Funding; ArgenX: Research Funding. Tam:Abbvie, Janssen: Research Funding; Abbvie, Janssen, Beigene, Roche, Novartis: Honoraria. Ramsay:Celgene Corporation: Research Funding; Roche Glycart AG: Research Funding. Kolitz:Boeringer-Ingelheim: Research Funding; Roche: Research Funding; Astellas: Research Funding. Zhou:BeiGene: Employment. Barrientos:Genentech: Consultancy; Gilead: Consultancy; Janssen: Consultancy; Abbvie: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding. Rai:Pharmacyctics: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees; Cellectis: Membership on an entity's Board of Directors or advisory committees; Genentech/Roche: Membership on an entity's Board of Directors or advisory committees.

Low Incidence of Post-Transplant EBV-Related Disease After Alemtuzumab-Mediated T Cell Depletion Is Explained by the Differential Susceptibility to Alemtuzumab of B Cells and Protective CD8 and CD4 T Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2330-2330
Author(s):  
Constantijn J.M. Halkes ◽  
Inge Jedema ◽  
Judith Olde Wolbers ◽  
Esther M van Egmond ◽  
Peter A. Von Dem Borne ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Cell Depletion ◽  
T Cell Depletion

Abstract Abstract 2330 In vivo T cell depletion with anti-thymocyte globulin (ATG) or alemtuzumab (anti-CD52) before reduced intensity allogeneic stem cell transplantation (alloSCT) in combination with in vitro T cell depletion with alemtuzumab reduces the risk of GVHD. Detectable levels of circulating antibodies are present up to several months after the alloSCT, leading to a delayed immune reconstitution which is associated with an increased incidence of opportunistic infections and early relapses. Prior to 2007, combined in vitro (Alemtuzumab 20 mg added “to the bag”) and in vivo T cell depletion with horse-derived ATG (h-ATG) resulted in good engraftment without GVHD in the absence of GVHD prophylaxis after reduced intensity alloSCT using conditioning with fludarabine and busulphan. Due to the unavailability of h-ATG, rabbit-derived ATG (r-ATG) 10–14 mg/kg was introduced in the conditioning regimen in 2007. Strikingly, in this cohort of patients, early EBV reactivation and EBV-associated post-transplantation lymphoproliferative disease (PTLD) was observed in 10 out of 18 patients at a median time of 6 weeks after alloSCT (range 5 to 11 weeks) in the absence of GVHD or immunosuppressive treatment. Analysis of T and B cell recovery early after transplantation revealed preferential depletion of T cells as compared to B cells, thereby allowing unrestricted proliferation of EBV infected B cells. Due to this unacceptable high incidence of EBV-related complications, in the conditioning regimen r-ATG was replaced by low dose alemtuzumab (15 mg i.v. day -4 and -3) in 2008. In this cohort of 60 patients, only 2 patients experienced transient EBV reactivation during the first 3 months after alloSCT and one patient developed an EBV-associated lymphoma 4 weeks after alloSCT. To investigate the mechanisms underlying the low incidence of EBV reactivation using alemtuzumab for T cell depletion, we studied the in vivo and in vitro effects of alemtuzumab on different lymphocyte subsets. First, lineage-specific reconstitution was studied in 20 patients from the alemtuzumab cohort with known CD52 negative diseases (11 AML and 9 multiple myeloma) to exclude the confounding effect of antibody absorption by malignant cells. Whereas at 3 weeks after alloSCT detectable numbers of circulating NK cells and T cells were observed (medians 71 (range 6–378), and 12 (range 1–1164)E6/L, respectively), no circulating B cells could be detected (median 0, range 0–1 E6/L). At 6 weeks after alloSCT, NK and T cell numbers further increased (medians 212 (52-813), and 130 (range 25–1509)E6/L, respectively), whereas B cell numbers still remained low in the majority of patients (median 15, range 0–813E6/L). In all patients, T cells were detectable before the appearance of circulating B cells. Furthermore, the expression of CD52 and the sensitivity to alemtuzumab-mediated complement-dependent cell lysis (CDC) of B cells, T cells and NK cells was measured in vitro. The highest CD52 expression was observed on B cells (mean fluorescence intensity (MFI) 120), resulting in 95% lysis after incubation with 10ug/mL alemtuzumab and rabbit complement. NK cells showed a significantly lower CD52 expression (MFI 41), which was also reflected by a lower susceptibility to alemtuzumab-mediated CDC (62% lysis). Interestingly, differential expression of CD52 was observed on CD4 and CD8 T cells (MFI 120 and 101, respectively). Cytotoxicity analysis revealed relative protection of CD8 compared to CD4 T cells against alemtuzumab-mediated CDC, resulting in 52% and 90% lysis, respectively. Based on these results, we investigated in detail the presence and phenotype of the CD4 and CD8 subsets and EBV-specific CD8 T cells using tetramer staining at 6 weeks after alloSCT. In accordance with the in-vitro expression and susceptibility data, circulating CD52+ CD8 T cells including EBV-specific T cells were detectable. Interestingly, the majority of circulating CD4 T cells (64-93%, n=4) lacked CD52 expression, explaining their capacity to persist in the presence of alemtuzumab. We conclude that in vivo and in vitro T cell depletion with alemtuzumab is associated with a relatively low risk of EBV-associated PTLD because of efficient B cell depletion and persistent EBV immunity allowed by the relative insusceptibility for alemtuzumab of CD8 T cells and the development of CD52 negative escape variants of CD4 T cells. Disclosures: No relevant conflicts of interest to declare.

Suppression of EBV-induced LCLs using CAR T cells redirected against HLA-DR.

2017 ◽  
Vol 35 (7_suppl) ◽  
pp. 146-146
Author(s):  
Chungyong Han ◽  
Rohit Singh ◽  
Seon-Hee Kim ◽  
Beom K. Choi ◽  
Byoung S. Kwon
Keyword(s):  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Side Effect ◽  
B Cell Malignancies ◽  
Car T Cells ◽  
Hla Dr ◽  
Car T

146 Background: Recent studies demonstrated a therapeutic potential of T cells with chimeric antigen receptor (CAR) targeting CD19 in refractory B cell malignancies. However, CD19-CAR T cells frequently caused on-target off-tumor side effect, i.e. B cell aplasia, and led to the recurrence of CD19-negative leukemic cells. Alternative target antigen for B cell malignancies has to be excavated. Methods: We developed antibody clone, MVR, which specifically bound to HLA-DR that is highly expressed on malignant B cells. In particular, MVR recognized polymorphic region of HLA-DR, and indicated different binding affinity against various HLA-DR alleles. Based on MVR binding strength, PBMCs from high binder (MVRHigh) and low binder (MVRLow) were tested to generate MVR-CAR T cells. To evaluate the anti-tumor efficacy on B cell malignancies, MVR-CAR T cells were assessed for immune responses against Epstein-Barr virus (EBV)-induced lymphoblastoid cell line (LCL) in vitro and in vivo. Results: Final yield of MVR-CAR T cells generated from MVRHigh PBMCs was 10-fold lower than that of CD19-CAR T cells, presumably caused by "fratricide" among HLA-DR-upregulated MVR-CAR T cells. In contrast, fratricidal effect was ameliorated in MVR-CAR T cells generated from MVRLow PBMCs indicating that the interaction between MVR-CAR and MVRLow-HLA-DR was weak enough to achieve tolerance to fratricide. Of note, in spite of such low binding, MVRLow-LCLs were killed efficiently by the CAR T cells. Further quantitative analysis revealed that HLA-DR was far more upregulated on LCLs compared with normal T and B cells which did not undergo EBV-transformation. In accordance with this observation, MVR-CAR T cells successfully induced LCL-specific cytotoxicity without causing normal B cell damage in vitro and efficiently suppressed the outgrowth of metastasized tumors in LCL-xenografted immune-deficient mice. Conclusions: MVR-CAR T cells redirected against HLA-DR for B cell malignancies were evaluated for the cytotoxic efficacy in vitro and in vivo. Considering the alleviated on-target off-tumor side effect and the feasibility of targeting HLA-DR for CD19-deficient malignant B cells, MVR-CAR T cells can be an alternative option for B cell malignancies.

scholarly journals Successful T cell priming in B cell-deficient mice.

1995 ◽  
Vol 182 (4) ◽  
pp. 915-922 ◽  
Author(s):  
M M Epstein ◽  
F Di Rosa ◽  
D Jankovic ◽  
A Sher ◽  
P Matzinger
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Protein Antigens ◽  
Capture Process ◽  
T Cell Priming ◽  
Deficient Mice

B cells are an abundant population of lymphocytes that can efficiently capture, process, and present antigen for recognition by activated or memory T cells. Controversial experiments and arguments exist, however, as to whether B cells are or should be involved in the priming of virgin T cells in vivo. Using B cell-deficient mice, we have studied the role of B cells as antigen-presenting cells in a wide variety of tests, including assays of T cell proliferation and cytokine production in responses to protein antigens, T cell killing to minor and major histocompatibility antigens, skin graft rejection, and the in vitro and in vivo responses to shistosome eggs. We found that B cells are not critical for either CD4 or CD8 T cell priming in any of these systems. This finding lends support to the notion that the priming of T cells is reserved for specialized cells such as dendritic cells and that antigen presentation by B cells serves distinct immunological functions.

scholarly journals Factor VIII BAR T-Cell Recognition By Antibodies to Different Domains

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1171-1171
Author(s):  
Kalpana Parvathaneni ◽  
Katherine Pohida ◽  
David W. Scott
Keyword(s):  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Conflicts Of Interest ◽  
Chimeric Receptors ◽  
Flow Cytometry Data ◽  
Cell Antibody ◽  
Engineered T Cells

Abstract Up to 30% of patients with hemophilia A develop inhibitory antibodies to therapeutic FVIII and these antibodies block the efficacy of this critical protein. To target the B cells specific for FVIII, we previously generated cytotoxic cells expressing the FVIII C2 or A2 immunodominant domains as chimeric receptors. We termed these antigen-expressing engineered T cells, "BARs", for B-cell Antibody Receptor. These could directly interact and kill with A2 or C2 FVIII-specific B cells as well as FVIII-specific hybridomas in vitro and in vivo. To increase the number of B-cell targets that could be killed, we recently engineered human and murine BAR CD8 T cells to express the FVIII light chain (LC), which includes A3 and C1, as well as C2 domains. In addition, since C2-expressing BAR T cells showed tonic signaling, we hypothesized that LC expressing BARs might not. Our data suggest that LC-expressing BAR T cells do not display tonic signaling but they can be stimulated to proliferate by anti-LC monoclonal antibody (mAb 5G12), for example. Flow cytometry data showed that LC-BAR T cells are stained by anti-C2 mAb (3G6) but this mAb did not stimulate them to proliferate. These data reflect the accessibility of different FVIII domains in BAR T cells, and are important in terms of the utility of FVIII domain expressing BARs to target specific B cells in vivo in the presence of inhibitors. (Supported by NIH grant R01 HL126727) Disclosures No relevant conflicts of interest to declare.

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