Support T-cell Proliferation Research Articles

Using serum-free media to streamline and optimize CAR T-cell manufacturing workflows

<!--StartFragment-->CAR T-cell therapies have led to breakthroughs in treating hematological malignancies, but the complexity and cost involved in their production limits their availability to patients. One challenge in CAR T-cell manufacturing outside of efficacy of your CAR-T product is achieving consistent expansion of T cells available after gene transfer. Optimizing the media used to support T-cell proliferation is, therefore, a key consideration—but it has proven difficult to develop media that enable consistent expansion while meeting regulatory quality requirements. However, recent advances in cell culture strategies have improved productivity and performance in CAR T-cell workflows using serum-free media, enabling the development of large-scale regulatory-compliant processes capable of producing billions of T cells within a short timeframe. This article will discuss how serum-free media can streamline CAR T-cell manufacturing workflows, highlighting how this can reduce time-to-market and make these treatments more widely available to patients.<!--EndFragment-->

Abstract B173: G protein coupling signaling as regulators of dendritic cell maintenance and function in immune responses

Abstract Dendritic cells (DCs) are well known as the professional antigen presenT-cells (APCs), which can scan peripheral tissues where they can recognize, uptake, process and present all kinds of antigens, including pathogens and tumor antigens, to antigen specific naïve T-cells within the lymphoid organs. In these processes, DCs form a remarkable bridge between innate and adaptive immunity by interacting with various lymphoid and myeloid cells. DCs originate from the bone marrow hematopoietic progenitor cells and DC precursors migrate from blood to tissues for developing into immature DCs. Upon activation, DCs migrate to stimulate T-cells and induce immune responses to protect our bodies. However, the mechanism for maintenance and activation of DCs on the local microenvironment is largely unknown. As GPCR signaling has important roles in many cellular process, including cell division, survival, migration and adhesion. Using competitive mixed BM chimaera, I checked some molecules involved in GPCR signal pathways, and found that geneA was required intrinsically in the maintenance of CD4+ conventional DCs (cDCs) in the spleen, as evidenced by observation that geneA-deficient CD4+ DCs were dramatically decreased in spleen but increased in blood. By in vivo 3min CD45-PE labeling assay, cells exposed to the vascular compartment in spleen can be selectively labeled. With this method, I found without geneA, CD4+ DCs had disadvantage to be maintained in the blood-exposed region within the spleens under the shear flow stress. I further showed that geneA-deficient CD4+ DCs had disadvantage to uptake blood-derived Sheep-blood cells (SRBCs). Besides this stimulation, this signal also controlled CD4+ DCs activation in response to different kinds of TLR stimulators. By OT-II adoptive transfer system, geneA-expressing DCs were required to support T-cell proliferation and differentiation efficiently. Therefore, these results demonstrate a key role of G protein-coupled receptor signaling in promoting the maintenance and activation of DCs, and reveal a mechanism in DC positioning in vivo. Thus, it will broaden our understanding of GPCR signaling in DC immunology, which is pivotal for modulating DCs for vaccines and therapies against pathogens, autoimmune diseases and tumors. Citation Format: Dan Liu, Jason G. Cyster. G protein coupling signaling as regulators of dendritic cell maintenance and function in immune responses [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B173.

Abstract B045: Arginase inhibitor CB-1158 elicits immune-mediated antitumor responses as a single agent and in combination with other immunotherapies

Abstract Myeloid derived suppressor cells (MDSCs) and polymorphonuclear cells (PMNs) are mediators of tumor immune evasion, however there are no approved clinical agents that directly antagonize the immunosuppressive activity of these cells. One of the mechanisms by which MDSCs and PMNs suppress anti-tumor immunity is expression of the enzyme arginase, which reduces L-arginine levels rendering T-cells and natural killer (NK)-cells unable to proliferate and mount an effective anti-tumor response. As a rationale to pursue arginase inhibition as a potential therapeutic strategy, we investigated the prevalence of arginase in tumors and peripheral blood from patients with various types of cancer. Our results confirm reports that multiple tumor types have arginase-expressing PMN infiltrates and that cancer patients have higher levels of plasma arginase and lower levels of plasma arginine compared to healthy volunteers. We also confirmed that in vitro-activated primary human T-cells and NK-cells require arginine to proliferate, secrete pro-inflammatory cytokines, and express the intracellular signaling molecule CD3ζ and cell surface activation marker PD-1. We developed CB-1158 to be a potent, selective, and orally-bioavailable small molecule inhibitor of arginase. CB-1158 potently inhibits recombinant human arginase 1 (IC50 = 98 nM), recombinant human arginase 2 (IC50 = 249 nM), and endogenous arginase from human neutrophils (IC50 = 160 nM). In a co-culture system, neutrophils strongly suppressed T-cell proliferation. The addition of CB-1158 blocked arginase activity, maintained arginine levels in the media, and allowed T-cells to proliferate in the presence of suppressive myeloid cells, suggesting that arginase is a major mechanism of myeloid cell suppression of lymphocyte proliferation and that CB-1158 can reverse the suppression. To extend these findings to MDSCs, we isolated peripheral monocytic MDSCs (M-MDSCs) or granulocytic MDSCs (G-MDSCs) from cancer patients and found that G-MDSCs expressed higher levels of arginase, depleted more arginine from cell-culture media, and were more suppressive to T-cell proliferation compared to M-MDSCs. Incubation of G-MDSCs with CB-1158 resulted in media arginine levels that could support T-cell proliferation. CB-1158 has high oral bioavailability in mice and rats. Twice-daily oral dosing of CB-1158 produced dose-dependent pharmacodynamic increases in plasma and tumor arginine levels and resulted in single-agent anti-tumor efficacy in the murine syngeneic tumor models Lewis Lung carcinoma (LLC1), Madison-109 lung carcinoma, and B16F10 melanoma. Antibody-mediated depletion of either CD8+ T-cells or NK-cells partially abrogated the anti-tumor effect of CB-1158 in the LLC1 and B16F10 models indicating that the mechanism of action of CB-1158 is immune cell-mediated through the actions of both T-cells and NK cells. Corroborating the immune-mediated anti-tumor efficacy in CB-1158-treated LLC1 tumors, following dosing with CB-1158 we observed increases in tumor infiltrating CD8+ T-cells; increased levels of tumor Th1 T-cell cytokines; increased expression of T-cell and NK-cell activation markers; and increased interferon-inducible genes. Based on the novel mechanism of action of CB-1158, there is potential for enhanced therapeutic benefit by combining CB-1158 with other immunotherapies. We observed improved anti-tumor activity by combining CB-1158 with either epacadostat or anti-PD-L1 in the B16F10 model, with low dose ionizing radiation in the Madison-109 model, and with gemcitabine in the LLC1 model. These results support the development of CB-1158, a first-in-class arginase inhibitor, as a novel immuno-oncology agent targeting the immunosuppressive effects of tumor-infiltrating myeloid cells. Citation Format: Susanne M. Steggerda, Mark Bennett, Jason Chen, Ethan Emberley, Matthew Gross, Tony Huang, Weiqun Li, Andy MacKinnon, Amani Makkouk, Gisele Marguier, Silinda Neou, Alison Pan, Tracy Wang, Melissa Works, Jing Zhang, Winter Zhang, Francesco Parlati. Arginase inhibitor CB-1158 elicits immune-mediated antitumor responses as a single agent and in combination with other immunotherapies [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B045.

Human Vascular Smooth Muscle Cells Lack Essential Costimulatory Molecules to Activate Allogeneic Memory T Cells

The arterial media, populated by vascular smooth muscle cells (VSMC), is an immunoprivileged compartment and, in contrast to the intima or adventitia containing endothelial cells, is generally spared by inflammatory processes, such as arteriosclerosis. To determine mechanisms of medial immunoprivilege, we investigated the ability of human VSMC versus endothelial cells to activate allogeneic T cells in vitro. Unlike cultured endothelial cells, cultured VSMC do not activate allogeneic memory CD4 or CD8 T cells and fail to effectively support T-cell proliferation to the polyclonal activator, phytohemagglutinin, consistent with a defect in costimulation function. Although many costimulators are comparably expressed on both cell types, endothelial cells but not VSMC basally express OX40 ligand and upregulate inducible costimulator ligand in response to proinflammatory cytokines. OX40 ligand-transduced, but not control- or inducible costimulator ligand-transduced, VSMC acquire the capacity to stimulate allogeneic memory CD4 T cells to produce cytokines and to proliferate in the presence of supplemental l-tryptophan. OX40 ligand overexpression, although not essential, also enhances allogeneic memory CD8 T-cell responses to VSMC after l-tryptophan supplementation. The inability of cultured VSMC to activate memory T cells results from a lack of essential costimulators, particularly OX40 ligand, in addition to indoleamine 2,3-dioxygenase-mediated tryptophan depletion.

Exovesicles from Human Activated Dendritic Cells Fuse with Resting Dendritic Cells, Allowing Them to Present Alloantigens

Dendritic cells (DCs) can release microvesicles, but the latter's numbers, size, and fate are unclear. Fluorescently labeled DCs were visualized by laser-scanning microscopy. Using a Surpass algorithm, we were able to identify and quantify per cell several hundred microvesicles released from the surface of stimulated DCs. We show that most of these microvesicles are not of endocytic origin but result from budding of the plasma membrane, hence their name, exovesicle. Using a double vital staining, we show that exovesicles isolated from activated DCs can fuse with the membrane of resting DCs, thereby allowing them to present alloantigens to lymphocytes. We concluded that, within a few hours from their release, exovesicles may amplify local or distant adaptive immunological response.

Murine cytomegalovirus abortively infects human dendritic cells, leading to expression and presentation of virally vectored genes.

Dendritic cells (DC) are potent antigen-presenting cells that play a crucial role in antigen-specific immune responses. Thus, the targeting of exogenous antigens to DC has become a popular approach for cancer immunotherapy and vaccine development. In this report, we studied the interplay between murine cytomegalovirus (MCMV) and human monocyte-derived DC. The results showed that an enhanced green fluorescent protein (EGFP)-encoding, replication-competent MCMV vector underwent abortive infection in human DC; this was accompanied by the efficient expression of EGFP. Infection of human DC by this vector resulted in a modest increase in the expression of cell surface proteins associated with DC maturation and has no significant effect on the immunostimulatory function of the cells, as reflected by their ability to support T-cell proliferation in a mixed-lymphocyte reaction. Finally, an MCMV vector encoding the human immunodeficiency virus type 1 (HIV-1) gp120 envelope glycoprotein was constructed and used to infect cultured human DC. The infected DC were shown to be capable of stimulating the expansion of autologous, gp120-specific, class I-restricted T lymphocytes from an HIV-1-negative donor, as determined by tetramer staining and enzyme-linked immunospot analysis. Taken together, these results suggest that MCMV may have potential utility as a vector for human vaccine development.

Disparate effects of phorbol esters, CD3 and the costimulatory receptors CD2 and CD28 on RANTES secretion by human T lymphocytes.

This study has examined the stimuli required for secretion of regulated upon activation, normal T-cell expressed, presumed secreted (RANTES) from T lymphocytes and found that stimuli such as phorbol 12-myristate 13-acetate (PMA), which are unable to support T-cell proliferation and interleukin-2 (IL-2) production, are nevertheless able to elicit strong secretion of RANTES. Conversely, stimuli such as CD2 and CD28 ligation, which are able to support T-cell proliferation, are unable to elicit RANTES secretion. Coligation of CD3 and CD28 drives T-cell proliferation to a similar degree as CD2 and CD28 coligation, yet also supports modest RANTES secretion. Furthermore, CD28 ligation enhances the secretion of RANTES stimulated by PMA and this costimulatory effect is abrogated by the phosphoinositide 3-kinase inhibitor wortmannin. Our data also indicate that the observed effects of PMA on RANTES secretion are probably due to activation of protein kinase C (PKC) isoenzymes, since RANTES secretion was unaffected by the non-PKC activating 4alpha-phorbol ester, whilst the general PKC inhibitor Ro-32-0432 inhibits PMA-stimulated RANTES secretion. Moreover, the effect of PMA appears to be chemokine-specific because PMA was unable to increase secretion of the related CC chemokine MIP-1alpha. Under stimulation conditions where increases in [Ca2+]i occur (e.g. PMA plus ionomycin or CD3 plus CD28 ligation) RANTES secretion can be severely reduced compared with the levels observed in response to the phorbol ester PMA. Hence, whilst PKC-dependent pathways are sufficient for strong RANTES secretion, a calcium-dependent factor is activated which negatively regulates RANTES secretion. This correlates well with the observation that ligation of cytolytic T lymphocyte-associated antigen-4 (CTLA-4) (expression of which has been reported to be dependent on a sustained calcium signal), inhibits RANTES secretion induced by CD3/CD28, but has no effect on PMA-stimulated RANTES secretion.

Accessory function of endothelial cells in anti-CD3-induced T-cell proliferation: synergism with monocytes.

Monoclonal antibodies to CD3 can induce proliferation of resting T cells. In vitro this effect is dependent on the presence of monocytes. They serve as accessory cells providing a co-stimulatory signal after cross-linking of the antibody-coated TcR/CD3 complex by the Fc receptor on the monocytes. We have studied whether endothelial cells can replace monocytes with regard to this function. Highly purified T-cell preparations were cultured in the presence of anti-CD3 antibody, purified monocytes, and human umbilical vein endothelial cells. Anti-CD3 and endothelial cells alone were unable to support T-cell proliferation, due to lack of FcR expression. Addition, however, of as few as 1000 FcR+ monocytes (0.8% of the number of T cells present) to a coculture of T cells and endothelial cells (EC) in the presence of soluble anti-CD3 resulted in a strong proliferation of T cells. When anti-CD3 was presented in an immobilized form (coated to the culture well or to Sepharose beads), or when phytohaemagglutinin was added to the culture as a cross-linking agent, EC could support T-cell proliferation in the absence of any monocytes. We conclude that EC by themselves cannot support the proliferation of pure T cells induced by soluble anti-CD3, but are potent generators of the co-stimulatory signal(s). They provide a suitable starting material to further define this co-stimulatory activity.

Antigen presentation by adherent cells from human peripheral blood. Correlation between T-cell activation and expression of HLA-DQ and -DR antigens

The ability of cells with different amounts of HLA-DO or -DR to support T-cell proliferation in response to foreign antigens was investigated. Adherent cells were stained with monoclonal antibodies and sorted in the fluorescence-activated cell sorter (FACS) into (a) DQ-positive and DQ-negative subsets, with monoclonal anti-DQ; or (b) subsets expressing different density of DR determinants. Expression of HLA-DQ correlated with increased density of DR. The subset of cells expressing detectable DQ and increased density of DR was found to be more efficient in presenting mumps or tetanus toxoid antigen to T cells than were the DQ-negative, low-DR density adherent cells. Similar results were obtained with primary cultures of T cells from blood and with cloned antigen-specific T-cell lines, restricted by a single DR-subregion specificity. Our results suggest that quantitative variation in DR/DQ molecules expressed on monocytes correlates with their ability to support T-cell response to nominal antigens. It is not clear whether this is due to only class II antigen density on the surface of the accessory cells or whether other factors are involved.

Role of Ia antigens and interleukin 1 in T-cell proliferation to phytohemagglutinin

Highly purified human T cells were obtained by a four-step purification procedure which included: removal of plastic adherent cells, rosetting with sheep red cells, passage over nylon-wood columns, and treatment with mouse monoclonal antibodies to human Ia antigens and complement. The resulting T cells did not proliferate to phytohemagglutinin (PHA). Purified human interleukin 1 (IL-1) could not substitute for accessory cells in supporting a PHA response. Reconstitution with as little as 0.03% adherent cells resulted in a proliferative response to PHA. T-Cell proliferation to PHA was supported by monocytes, by Ia + Epstein-Barr virus-transformed lymphoblastoid B-cells lines, and by Ia − cultured human dermal fibroblasts but not by Ia-containing liposomes. Addition of anti-Ia antibodies to monocyte-containing cultures did not inhibit the T-cell proliferative response to PHA. These results suggest that Ia antigen expression by accessory cells is neither necessary nor sufficient to support T-cell proliferation to PHA and that IL-1 is not sufficient to support the proliferation of T cells to PHA.

Heterogeneity in the response of T cells to antigens presented by B lymphoma cells

Proliferation of antigen-specific T-cell populations was induced in cultures stimulated with antigen and a suitable source of antigen-presenting cells. Soluble (keyhole limpet hemocyanin) and particulate (horse red blood cells) antigens were presented by irradiated spleen cells and by a variety of B-lymphoma-cell lines, providing support for antigen-specific H-2-restricted T-cell responses. A marked heterogeneity was demonstrated, however, in the capacity of T-cell lines to proliferate in response to antigen presented by the B-lymphoma cells. T-cell populations were prepared from the lymph nodes of antigen-primed mice and restimulated in vitro in the presence of antigen and irradiated spleen cells. During the first six in vitro restimulations, these T-cell populations maintained the capacity to respond to antigen presented either by irradiated spleen cells or by B-lymphoma cells. Continued growth of these T-cell populations, again in the presence of antigen and irradiated spleen cells, resulted in the generation of T-cell lines which had lost the ability to respond to antigen presented by B-lymphoma cells. These lines however, fully retained the capacity to proliferate in the presence of antigen and irradiated spleen cells. T-cell clones derived from one of these lines were also unable to respond to antigen presented by B-lymphoma cells but again proliferated in the presence of antigen and irradiated spleen cells. Supernatants containing high levels of IL-1, IL-2, or IL-3 activity failed to reconstitute the antigen-specific response of T-cell lines which had lost the capacity to respond to antigen presented by B-lymphoma cells. Furthermore, titrated numbers of irradiated spleen cells, while having the capacity to support T-cell proliferation themselves, failed to synergize with B-lymphoma cells in the support of antigen-specific T-cell proliferation. Thus we have defined populations of antigen-specific, H-2-restricted T cells which do not recognize antigen presented by B-lymphoma cells and can therefore discriminate between different antigen-presenting cell types.

Interleukin 1 secretion is not required for human macrophage support of T-cell proliferation

Interleukin 1 (IL-1) is a soluble factor secreted by stimulated monocytes (Mo) and animal macrophages (Mx). We have previously demonstrated that human Mo cultured in vitro for 1–6 days transform to Mx, and retain their ability to support concanavalin A (Con A)-driven T-cell proliferation. We have also shown that, paradoxically, these Mx do not secrete IL-1, when stimulated by endotoxin (LPS). In this study we examined two alternative hypotheses: T cells plus mitogen induce Mx IL-1 production, and human Mx deliver a second signal to T cells via a non-IL-1 mechanism. IL-1 was assayed in a mouse CD-1 thymocyte system without concanavalin A. Mo Mx were cultured with T cells at low (2 × 10 4/200 μl) or high (1 × 10 5/200 μl) concentrations for 2 or 4 days, in the presence of Con A. Six hours prior to quantitation of proliferation, 50 μl of supernatant was removed and assayed for IL-1. As expected both Mo and Mx enhanced T-cell proliferation eight- to tenfold. Mo secreted large amounts of IL-1; there was no demonstrable IL-1 activity present in supernatants from cultures containing either T cells and Mx, or Mx alone. Similar results were obtained by preincubating the cells (Mo, Mx, and T cells) with Con A for 12 hr and removing Con A prior to a 36-hr coculture. We examined the possibility that a small amount of IL-1 may be able to support Con A-stimulated T-cell proliferation and yet may not induce thymocyte proliferation. The highest dilutions of Mo supernatant (1:125) which supported T-cell proliferation also caused a fivefold increase in thymocyte proliferation. Supernatants from Mx failed to stimulate thymocyte proliferation or support Con A-driven T-cell proliferation. However, Mo and Mx lysates contain II-1 activity. We conclude that human Mx support Con A-induced T-cell proliferation in the absence of IL-1 secretion. Mx may support T-cell proliferation by cell-bound IL-1 or by a non-IL-1 mechanism.