scholarly journals Production of a B cell growth-promoting activity, (DL)BCGF, from a cloned T cell line and its assay on the BCL1 B cell tumor.

1982 ◽  
Vol 156 (6) ◽  
pp. 1821-1834 ◽  
Author(s):  
S L Swain ◽  
R W Dutton
Keyword(s):  
T Cell ◽  
B Cells ◽  
Cell Growth ◽  
B Cell ◽  
Cell Line ◽  
Growth Promoting ◽  
Alloreactive T Cell ◽  
Culture Supernatants

Culture supernatants from a long-term alloreactive T cell line, the Dennert line C.C3.11.75 (DL) contain a B cell-growth-promoting activity. This activity can be assayed on normal B cells or on the in vivo BCL1 tumor line. We have called this activity (DL)BCGF. This activity can be distinguished from the T cell-replacing factor activity we had earlier found in DL supernates [(DL)TRF], which is required together with IL2 for the B cell plaque-forming cell response to erythrocyte antigens. The (DL)BCGF can be absorbed on untreated or glutaraldehyde-fixed BCL1. This absorption does not remove (DL)TRF activity. The production of (DL)BCGF is greatly enhanced when DL is cultured with IL2-containing supernatants. Sublines or clones of DL (DL.B10 and DL.A4) have been obtained that make large amounts of (DL)BCGF in the absence of any stimulator cells or IL2. B cells from the Xid-deficient male (DBA/2 X CBA/N)F1 mice do not respond to (DL)BCGF.

scholarly journals A monoclonal T cell-replacing activity can act directly on B cells to enhance clonal expansion.

1982 ◽  
Vol 156 (1) ◽  
pp. 306-311 ◽  
Author(s):  
G D Wetzel ◽  
S L Swain ◽  
R W Dutton
Keyword(s):  
T Cell ◽  
B Cells ◽  
B Cell ◽  
B Lymphocyte ◽  
Interleukin 1 ◽  
Clonal Expansion ◽  
Factor Activity ◽  
Alloreactive T Cell ◽  
Culture Supernatants

We have used a B cell cloning system in which the response of a single isolated B cell to lipopolysaccharide and dextran sulfide can be followed. We have shown that culture supernatants from the Dennert long-term alloreactive T cell line C.C3.11.75 increase the frequency of B cells stimulated to clonal expansion by mitogens. These culture supernatants are devoid of interleukin 1 and 2 but contain the T cell-replacing factor activity (DL)TRF. These experiments provide unequivocal proof that a T cell-derived factor or factors can act directly on a B lymphocyte in the absence of any other cell.

Sustained Functional T Cell Persistence and B Cell Aplasia Following CD19-Targeting Adoptive T Cell Immunotherapy for Relapsed, Refractory CD19+ Malignacy

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 756-756
Author(s):  
Michael Kalos ◽  
Bruce L. Levine ◽  
Timothy L Macatee ◽  
Irina Kulikovskaya ◽  
Erica Suppa ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Surface Expression ◽  
University Of Pennsylvania ◽  
Cell And Gene Therapy

Abstract Abstract 756 Background Advances in ex vivo T cell engineering have facilitated clinical trials to evaluate the potential for adoptive T cell transfer to target malignancy. Gene-modified T cells have the potential to expand, functionally persist and mediate long-term anti-tumor activity. Most clinical studies have shown only limited persistence of infused cells, with modest and often temporary anti-tumor activity. We reported initial clinical data on CAR T cells targeting CD19 expressed on normal and malignant B cells (CART19 cells) (Porter, et al NEJM 2011; Kalos et al Sci Trans Med 2011). The CAR included signaling domains from CD3zeta and CD137 (4-1BB) that mediated effector and proliferation/persistence signals. Here we report functional persistence of CART19 cells from the initial cohort at approximately 2 years post-infusion, and data from a more recently treated cohort of CLL patients and a pediatric patient with advanced, treatment refractory ALL. Methods Persistence of gene-modified T cells was assessed by quantitative ABI-Taqman based PCR and a qualified assay that detects the CD137-TcR-zeta junction in the anti-CD19 CAR. CAR19 surface expression was detected by flow cytometry with an anti-CAR-19 idiotype specific antibody. B cell aplasia was evaluated by multiparametric flow cytometry. Multiplex cytokine analyses were performed using Luminex™assays. Results 10 patients with relapsed, refractory disease have been treated to date: 9 adults w/CLL and one child w/pre- B cell ALL. Each had been extensively pre-treated and had active disease at the time of CART19 infusion. All CLL patients received lymphodepleting chemotherapy 4–6 days before infusions, while the ALL patient did not get further lymphodepletion. Patients were infused with an average total of 1.7–50 × 108 total T cells which corresponded to 0.14–5.9 x108CART-19 cells. 9/10 treated patients were evaluable on 8.14.12. Detailed clinical outcomes will be reported separately at this meeting (Porter, D.L., Grupp S. et al). 4 pts (3 CLL, 1 ALL) had achieved CR at the primary endpoint (30 days post infusion) which is sustained and ongoing in all patients (range 1–24 months). Two CLL patients had a partial response (PR) lasting 3 and 5 months, while 3 patients did not respond (NR). In all patients with CR, robust in vivo expansion of CART19 cells was observed. By molecular analysis, CART19 cells demonstrated in vivo expansion, followed by contraction and an ongoing stable persistence at all evaluated timepoints. Expansion kinetics were unique for each patient; in all cases maximal expansion was observed by day +30 post CART-19 infusion. In patients with CR, observed peak marking for CART-19 ranged from 1 × 102-1 × 103 CART-19 cells/uL blood. Patients with PR demonstrated less robust in vivo expansion, with peak observed marking ∼1 × 101 CART-19 cells/uL blood. In NR patients, peak marking was <1 × 101CART-19 cells/uL blood. Long term peripheral blood persistence of CART19 cells and CAR19 surface expression was observed in all patients with CR in both CD3+/CD8+ and CD3+/CD4+ subsets. In patients with CR, elimination of peripheral B cells was observed at the time of CART19 in vivo expansion. Ongoing B cell aplasia has been documented in each CR patient in both peripheral blood and marrow by flow cytometry. Patients with PR showed transient elimination of malignant and normal B cells. Multiplex-cytokine analysis of serum samples from CR patients revealed a broad pro-inflammatory signature with significant elevation in a subset of soluble immune modulators including IL-6, IL-8, IFN-g, MIP1b, and IL2ra. In contrast, NR patients did not have elevated serum cytokines. In CR patients, elevation of cytokines tracked with expansion of CART19 cells and elimination of B cells, suggesting the potential for a cytokine-based diagnostic signature to monitor CART19 treatment efficacy. Conclusions Adoptive transfer of CART19 cells engineered to express CD137 and TCR-zeta signaling domains can result in in vivo expansion, homing to disease sites, and long-term functional persistence of CART19 cells, accompanied by ongoing complete clinical responses and long-term B cell aplasia in a substantial fraction of patients with advanced, refractory and high risk CLL and relapsed refractory ALL. A detailed cytokine profile and persistent B cell aplasia has been identified that may correlate with treatment efficacy. Disclosures: Kalos: University of Pennsylvania: Patents & Royalties. Levine:TxCell: Consultancy, Membership on an entity's Board of Directors or advisory committees; University of Pennsylvania: financial interest due to intellectual property and patents in the field of cell and gene therapy. Conflict of interest is managed in accordance with University of Pennsylvania policy and oversight, financial interest due to intellectual property and patents in the field of cell and gene therapy. Conflict of interest is managed in accordance with University of Pennsylvania policy and oversight Patents & Royalties. June:Novartis: Research Funding, institution owned patents have been licensed by Novartis, institution owned patents have been licensed by Novartis Patents & Royalties.

scholarly journals Development of a human T-T cell hybridoma secreting B cell growth factor.

1983 ◽  
Vol 157 (1) ◽  
pp. 60-68 ◽  
Author(s):  
J L Butler ◽  
A Muraguchi ◽  
H C Lane ◽  
A S Fauci
Keyword(s):  
T Cell ◽  
B Cells ◽  
Growth Factor ◽  
Cell Growth ◽  
B Cell ◽  
Interleukin 2 ◽  
Lymphocyte Function ◽  
B Cell Growth Factor ◽  
Normal Human

The success of long-term culture of normal human and murine B cells has been hampered by the limited availability of soluble factors capable of maintaining proliferation of activated B lymphocytes. Previous experiments using various culture-derived supernatants in a human system were unable to separate the activities of B cell growth factor (BCGF) and interleukin 2 (IL-2) by immunochemical means. Thus, purified factors with BCGF activity in the absence of IL-2 activity have not been available for study. In the present study, normal human peripheral blood T cells were fused with the hypoxanthine/aminopterin/thymidine-sensitive human T-leukemic cell line, CEM-6. Supernatants from the resulting hybrid cells were tested for the ability to maintain proliferation of normal human B cells in a recently described assay system for human BCGF. Hybrids demonstrating BCGF activity were cloned by limiting dilution. One hybrid clone, 2B11, continued to support proliferation of B cells in both long-term cultures and 6-d assays at a level significantly above that seen with conventionally produced growth factors. No IL-2 activity was found in the supernatant from hybrid 2B11. The hybridoma supernatant was fractionated by gel filtration, and maximum proliferation of B cells was supported by the 18-20,000 mol wt protein fraction. Thus, a human T-T cell hybridoma that has BCGF activity in the absence of any demonstrable IL-2 activity has been developed. Human T-T cell hybridomas secreting discrete immunoregulatory factors should prove to be powerful tools in dissecting the mechanisms of immunoregulation of human lymphocyte function.

Dual Inhibition of PI3K-Delta and Gamma By Duvelisib (IPI-145) Impairs CLL B- and T-Cell Migration, Survival and Proliferation in a Murine Xenograft Model Using Primary Chronic Lymphocytic Leukemia Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1753-1753 ◽  
Author(s):  
Shih-Shih Chen ◽  
Steven Ham ◽  
Kanti R. Rai ◽  
Karen McGovern ◽  
Jeffery L. Kutok ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
Cell Migration ◽  
T Cell ◽  
B Cells ◽  
Cell Growth ◽  
B Cell ◽  
Cell Survival ◽  
T Cell Migration

Abstract Duvelisib (IPI-145), a dual inhibitor of phosphoinositide 3-kinase (PI3K)-δ and -γ, has shown clinical activity in treatment-naïve and relapsed/refractory chronic lymphocytic leukemia (CLL) patients. Clinically, duvelisib results in a redistribution of malignant B cells and concomitant reduction in nodal enlargement. These effects are believed to be due to important roles of PI3K- δ and -γ in CXCL12-mediated CLL cell migration (Peluso 2014), cytokine-induced CLL B-cell proliferation, and BCR-stimulated B-cell survival (Balakrishnan 2015). Additional data suggest an effect of duvelisib on the tumor supporting cells of the CLL microenvironment. This includes preclinical studies demonstrating that PI3K-γ inhibition blocks normal T cell migration toward tumor chemokines and prevents murine bone marrow-derived M2 macrophage polarization (Peluso 2014), as well as clinical data in CLL patients receiving duvelisib showing reduced serum levels of myeloid and T cell-secreted cytokines and chemokines (Douglas 2015). To further characterize duvelisib's effect on CLL cells and the tumor microenvironment (TME), a murine xenograft model using primary human CLL cells was employed. We first studied duvelisib's effect on CLL B- and T-cell migration in vivo. CLL PBMCs (n=2; 1 IGHV unmutated (U)-CLL, 1 IGHV mutated (M)-CLL) pre-treated with duvelisib for 48 hours were injected retro-orbitally into NOD-scid IL2Rgammanull (NSG) mice. B- and T-cell localization in tissues and circulation was studied 1 and 24 hours post-injection. Duvelisib treatment (1000 nM) prevented the egress of CLL B and T cells from the circulation into the spleen, indicating impaired homing of CLL B and T cells. To better define the effect of duvelisib on T-cell migration, T cells from CLL patients (n=3; 2 U-CLL, 1 M-CLL) treated ex vivo with duvelisib at 1, 10, 100 and 1000 nM were injected into mice and analyzed for their trafficking 24 hours later. Inhibition of T-cell homing to spleen was dose dependent, with only 100 and 1000 nM having significant effects. Given duvelisib's cellular IC50s for PI3K isoforms, these results suggest that impaired T-cell migration is due to PI3K-γ inhibition, and studies with isoform-selective PI3K-δ and PI3K-γ inhibitors are currently underway to examine this possibility. The effect of duvelisib on CLL T-cell proliferation was evaluated after in vitro activation with anti-CD3/28 Dynabeads plus IL2 (n=6; 3 U-CLL, 3M-CLL). In duvelisib treated cells, CD4+, but not CD8+, T-cell proliferation was inhibited at doses of 100 and 1000 nM, suggesting a role for PI3K-γ. The effects of duvelisib on CLL B- and T-cell growth in vivo (n=4; 2 U-CLL, 2 M-CLL) were then studied. Autologous CLL T cells were stimulated as above and injected with CLL PBMCs into NSG mice. Animals treated orally with duvelisib for 3 weeks at 100 mg/kg/day had preferentially reduced CD4+ T-cell recovery from spleens, thereby decreasing the CD4 to CD8 ratio. In each case, duvelisib treatment reduced the number of splenic CLL B cells. This reduction reflected inhibition of both CLL cell proliferation and survival, since duvelisib treatment decreased the percentage of cycling CLL cells and increased the percentage of apoptotic B cells. Thus, duvelisib may target CLL B-cell growth directly, or indirectly by inhibiting the support of CD4+ T cells in the TME. The potential effect of duvelisib on the tumor-supporting myeloid compartment was also tested. Because of limited human myeloid-cell engraftment in our NSG model, we studied the effect of duvelisib on murine macrophages. Mice receiving duvelisib had reduced numbers of splenic CD11b+ GR-1low LY-6Clow LY-6Gneg macrophages compared to controls, suggesting duvelisib altered macrophage development. Prior in vitro studies demonstrated inhibition of CLL B-cell survival and proliferation by duvelisib, as well as blockade of T-cell migration and M2 macrophage polarization (Balakrishnan 2015; Peluso 2014). Our current in vivo studies further support duvelisib's effect on CLL B-cell growth and survival through inhibition of cellular homing to supportive tissue niches and alterations in the TME. The latter, in part, is through suppression of T-cell support and alterations in the macrophage compartment. Overall, these preclinical results suggest that inhibition of PI3K-δ and PI3K-γ by duvelisib affects CLL cell survival through direct and indirect mechanisms. Disclosures McGovern: Infinity Pharmaceuticals, Inc.: Employment. Kutok:Infinity Pharmaceuticals, Inc.: Employment.

scholarly journals B cell-stimulatory factor 1 (BSF-1) promotes growth of helper T cell lines.

1986 ◽  
Vol 164 (2) ◽  
pp. 580-593 ◽  
Author(s):  
R Fernandez-Botran ◽  
P H Krammer ◽  
T Diamantstein ◽  
J W Uhr ◽  
E S Vitetta
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Activated T Cells ◽  
Growth Promoting ◽  
T Cell Lines ◽  
Stimulatory Factor

T cell-derived supernatants (SN) that contain B cell-stimulatory factor 1 (BSF-1) and lack IL-2 promote the growth of the IL-2-dependent T cell line, HT-2, as well as three other clones or lines of T cells that can provide help to B cells. The BSF-1 purified from these SNs promotes growth of HT-2 cells approximately 50% as effectively as purified IL-2. A potential involvement for contaminating IL-2 in the BSF-1 preparations was excluded by the demonstration that anti-BSF-1 mAbs blocked the BSF-1-induced growth of HT-2 cells; in contrast, these antibodies did not block the IL-2-induced proliferation of the HT-2 cells. In addition, anti-IL-2 mAbs or anti-IL-2-R antibodies blocked the HT-2 growth-promoting activity of purified IL-2, but not BSF-1. Finally, BSF-1 promoted only a very modest growth of Con A-induced T cell blasts, and failed to induce significant growth in seven other cytotoxic, alloreactive, and long-term T cell lines. Taken together, these results indicate that in addition to its known effects on resting and LPS-stimulated B cells, BSF-1 can promote growth of certain subsets of activated T cells, in particular, those that provide help to B cells.

scholarly journals Transformation-associated alterations in interactions between pre-B cells and fibronectin

Blood ◽  
1990 ◽  
Vol 76 (11) ◽  
pp. 2311-2320 ◽  
Author(s):  
FM Lemoine ◽  
S Dedhar ◽  
GM Lima ◽  
CJ Eaves
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Cell Attachment ◽  
Attachment Site ◽  
Receptor Antibodies

Abstract Marrow stromal elements produce as yet uncharacterized soluble growth factors that can stimulate the proliferation of murine pre-B cells, although close contact between these two cell types appears to ensure a better pre-B cell response. We have now shown that freshly isolated normal pre-B cells (ie, the B220+, surface mu- fraction of adult mouse bone marrow) adhere to fibronectin (FN) via an RGD cell-attachment site, as shown in a serum-free adherence assay, and they lose this functional ability on differentiation in vivo into B cells (ie, the B220+, surface mu+ fraction). Similarly, cells from an immortalized but stromal cell-dependent and nontumorigenic murine pre-B cell line originally derived from a Whitlock-Witte culture were also found to adhere to fibronectin (FN) via an RGD cell-attachment site. Moreover, in the presence of anti-FN receptor antibodies, the ability of this immortalized pre-B cell line to proliferate when co-cultured with a supportive stromal cell line (M2–10B4 cells) was markedly reduced (down to 30% of control). This suggests that pre-B cell attachment to FN on stromal cells may be an important component of the mechanism by which stromal cells stimulate normal pre-B cell proliferation and one that is no longer operative to control their more differentiated progeny. Two differently transformed pre-B cell lines, both of which are autocrine, stromal-independent, tumorigenic in vivo, and partially or completely differentiation-arrested at a very early stage of pre-B cell development, did not bind to FN. In addition, anti-FN receptor antibodies were much less effective in diminishing the ability of these tumorigenic pre-B cells to respond to M2–10B4 cell stimulation, which could still be demonstrated when the tumorigenic pre-B cells were co- cultured with M2–10B4 cells at a sufficiently low cell density. Analysis of cell surface molecules immunoprecipitated from both the nontumorigenic and tumorigenic pre-B cell lines by an anti-FN receptor antibody showed an increase in very late antigen (VLA) alpha chain(s) in both tumorigenic pre-B cell lines and a decrease in the beta 1 chain in one. Interestingly, all of the pre-B cell lines expressed similar amounts of messenger RNA for the beta 1 chain of the FN receptor. These results suggest that alteration of FN receptor expression on pre-B cells may represent a mechanism contributing to the outgrowth of leukemic pre-B cells with an autocrine phenotype and capable of stromal cell-independent, autonomous growth.

Pax5 determines B- versus T-cell fate and does not block early myeloid-lineage development

Blood ◽  
2003 ◽  
Vol 101 (11) ◽  
pp. 4342-4346 ◽  
Author(s):  
Claudiu V. Cotta ◽  
Zheng Zhang ◽  
Hyung-Gyoon Kim ◽  
Christopher A. Klug
Keyword(s):  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cell Fate ◽  
Nk Cell ◽  
Cell Lineage ◽  
Blood Analysis ◽  
Hematopoietic Stem ◽  
Peripheral Blood Analysis

Abstract Progenitor B cells deficient in Pax5 are developmentally multipotent, suggesting that Pax5 is necessary to maintain commitment to the B-cell lineage. Commitment may be mediated, in part, by Pax5 repression of myeloid-specific genes. To determine whether Pax5 expression in multipotential cells is sufficient to restrict development to the B-cell lineage in vivo, we enforced expression of Pax5 in hematopoietic stem cells using a retroviral vector. Peripheral blood analysis of all animals reconstituted with Pax5-expressing cells indicated that more than 90% of Pax5-expressing cells were B220+ mature B cells that were not malignant. Further analysis showed that Pax5 completely blocked T-lineage development in the thymus but did not inhibit myelopoiesis or natural killer (NK) cell development in bone marrow. These results implicate Pax5 as a critical regulator of B- versus T-cell developmental fate and suggest that Pax5 may promote commitment to the B-cell lineage by mechanisms that are independent of myeloid gene repression.

scholarly journals B cell–intrinsic TLR signals amplify but are not required for humoral immunity

2007 ◽  
Vol 204 (13) ◽  
pp. 3095-3101 ◽  
Author(s):  
Almut Meyer-Bahlburg ◽  
Socheath Khim ◽  
David J. Rawlings
Keyword(s):  
B Cells ◽  
B Cell ◽  
Long Term Memory ◽  
Altered Expression ◽  
Intrinsic Signals ◽  
Antibody Titers ◽  
Specific Igg ◽  
B Cell Subsets

Although innate signals driven by Toll-like receptors (TLRs) play a crucial role in T-dependent immune responses and serological memory, the precise cellular and time-dependent requirements for such signals remain poorly defined. To directly address the role for B cell–intrinsic TLR signals in these events, we compared the TLR response profile of germinal center (GC) versus naive mature B cell subsets. TLR responsiveness was markedly up-regulated during the GC reaction, and this change correlated with altered expression of the key adaptors MyD88, Mal, and IRAK-M. To assess the role for B cell–intrinsic signals in vivo, we transferred MyD88 wild-type or knockout B cells into B cell–deficient μMT mice and immunized recipient animals with 4-hydroxy-3-nitrophenylacetyl (NP) chicken gamma globulin. All recipients exhibited similar increases in NP-specific antibody titers during primary, secondary, and long-term memory responses. The addition of lipopolysaccharide to the immunogen enhanced B cell-intrinsic, MyD88-dependent NP-specific immunoglobulin (Ig)M production, whereas NP-specific IgG increased independently of TLR signaling in B cells. Our data demonstrate that B cell–intrinsic TLR responses are up-regulated during the GC reaction, and that this change significantly promotes antigen-specific IgM production in association with TLR ligands. However, B cell–intrinsic TLR signals are not required for antibody production or maintenance.

scholarly journals Early Reconstitution of Antibody Secreting Cells after Allogeneic Stem Cell Transplantation

2022 ◽  
Vol 11 (1) ◽  
pp. 270
Author(s):  
Martina Hinterleitner ◽  
Clemens Hinterleitner ◽  
Elke Malenke ◽  
Birgit Federmann ◽  
Ursula Holzer ◽  
...  
Keyword(s):  
Innate Immunity ◽  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
B Cells ◽  
Stem Cell Transplantation ◽  
B Cell ◽  
Cell Transplantation ◽  
Antibody Secreting Cells

Immune cell reconstitution after stem cell transplantation is allocated over several stages. Whereas cells mediating innate immunity recover rapidly, adaptive immune cells, including T and B cells, recover slowly over several months. In this study we investigated kinetics and reconstitution of de novo B cell formation in patients receiving CD3 and CD19 depleted haploidentical stem cell transplantation with additional in vivo T cell depletion with monoclonal anti-CD3 antibody. This model enables a detailed in vivo evaluation of hierarchy and attribution of defined lymphocyte populations without skewing by mTOR- or NFAT-inhibitors. As expected CD3+ T cells and their subsets had delayed reconstitution (<100 cells/μL at day +90). Well defined CD19+ B lymphocytes of naïve and memory phenotype were detected at day +60. Remarkably, we observed a very early reconstitution of antibody-secreting cells (ASC) at day +14. These ASC carried the HLA-haplotype of the donor and secreted the isotypes IgM and IgA more prevalent than IgG. They correlated with a population of CD19− CD27− CD38low/+ CD138− cells. Of note, reconstitution of this ASC occurred without detectable circulating T cells and before increase of BAFF or other B cell stimulating factors. In summary, we describe a rapid reconstitution of peripheral blood ASC after CD3 and CD19 depleted haploidentical stem cell transplantation, far preceding detection of naïve and memory type B cells. Incidence before T cell reconstitution and spontaneous secretion of immunoglobulins allocate these early ASC to innate immunity, eventually maintaining natural antibody levels.

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