Regulatory perspective on in vitro potency assays for human dendritic cells used in anti-tumor immunotherapy

Dendritic cells (DCs) are key connectors between the innate and adaptive immune system and have an important role in modulating other immune cells. Therefore, their therapeutic application to steer immune responses is considered in various disorders, including cancer. Due to differences in the cell source and manufacturing process, each DC medicinal product is unique. Consequently, release tests to ensure consistent quality need to be product-specific.Although general guidance concerning quality control testing of cell-based therapies is available, cell type-specific regulation is still limited. Especially guidance related to potency testing is needed, because developing an in vitro assay measuring cell properties relevant for in vivo functionality is challenging. In this review, we provide DC-specific guidance for development of in vitro potency assays for characterisation and release. We present a broad overview of in vitro potency assays suggested for DC products to determine their anti-tumor functionality. Several advantages and limitations of these assays are discussed. Also, we provide some points to consider for selection and design of a potency test. The ideal functionality assay for anti-tumor products evaluates the capacity of DCs to stimulate antigen-specific T cells. Because this approach may not be feasible for release, use of surrogate potency markers could be considered, provided that these markers are sufficiently linked to the in vivo DC biological activity and clinical response. Further elucidation of the involvement of specific DC subsets in anti-tumor responses will result in improved manufacturing processes for DC-based products and should be considered during potency assay development.     

Gene gun application in the generation of effector T cells for adoptive immunotherapy

We utilized the gene gun to transfect subcutaneous D5 melanoma and MT-901 mammary carcinoma tumors in situ with a granulocyte/macrophage-colony-stimulating factor (GM-CSF) plasmid complexed to gold particles. There was diminished tumor growth following bombardment with GM-CSF plasmid, which was apparent only during the period of administration. Transgenic GM-CSF was produced by the skin overlying the tumors and not by the tumors themselves. GM-CSF plasmid bombardment resulted in increased cell yields within tumor-draining lymph nodes (TDLN) with at least a 12-fold increase in the percentage of dendritic cells (8.9%) compared to controls (0.7%). Secondarily activated TDLN cells from animals transfected with GM-CSF demonstrated enhanced cytokine release (interferon γ, GM-CSF and interleukin-10) in response to tumor stimulator cells compared to controls, and had an increased capacity to mediate tumor regression in adoptive immunotherapy. There was a small, but detectable, non-specific immune adjuvant effect observed with gold particle bombardment alone, which was less than with GM-CSF plasmid. The adjuvant effect of GM-CSF plasmid required peri-tumoral transgene expression since gene bombardment away from the tumor was ineffective. Received: 27 April 1999 / Accepted: 27 August 1999     

Closed loop bioreactor system for the ex vivo expansion of human T cells

Background aim

Translation of therapeutic cell therapies to clinical-scale products is critical to realizing widespread success. Currently, however, there are limited tools that are accessible at the research level and readily scalable to clinical-scale needs.

Replicative senescence of CD8 T cells: potential effects on cancer immune surveillance and immunotherapy

The process of replicative senescence, which stringently limits the proliferative potential of normal T cells, constitutes a potential problem for cancer immunotherapy. The ability of CD8 T cells to recognize and destroy tumor cells has been well-established, but the requirement for massive, prolonged proliferative T-cell expansion and maintenance of functional integrity poses a significant obstacle to the success of cancer immunotherapy. Cancer immune surveillance may also be compromised by the long-term exposure of T cells to tumor antigens, particularly those of latent viruses, which could drive certain T cells to replicative senescence. This review summarizes the major characteristics of T-cell replicative senescence and raises the possibility that this process has the potential to affect both cancer development and treatment. Experimental strategies aimed at preventing T-cell replicative senescence are discussed in the context of cancer immunotherapy and vaccines.This article forms part of the Symposium in Writing Tumor escape from the immune response, published in Vol. 53.     

In vitro mammalian mutagenesis as a model for genetic lesions in human cancer

Recently in vitro assays of mutagenesis have been criticized as being poorly predictive of long-term in vivo rodent assays of carcinogenicity. Questions have also been raised concerning the relevance of rodent assays to human risk. In vitro assays using mammalian cells can detect most types of genetic lesions thought to be important in human malignant disease. Molecular and cytogenetic analyses of mutations induced by a variety of genotoxic compounds at the heterozygous thymidine kinase locus in mouse lymphoma cells indicate that this in vitro assay does indeed register the range of genetic lesions recently found in a wide variety of human tumors. The types and complexity of the induced lesions are reflected in mutant colony phenotype in a compound-specific fashion. These studies point to the use of appropriate in vitro mammalian mutagenesis assays as new model systems for dissecting the genetic lesions important in human carcinogenesis, and as a means of determining the potential for compounds to induce such lesions.     

Regulatory T cells in colorectal cancer patients suppress anti-tumor immune activity in a COX-2 dependent manner

Objective Naturally occurring regulatory T (T_R) cells suppress autoreactive T cells whereas adaptive T_R cells, induced in the periphery, play an important role in chronic viral diseases and cancer. Several studies indicate that cyclooxygenase (COX) inhibitors prevent cancer development of colon adenomas and delay disease progression in patients with colorectal cancer (CRC). We have shown that adaptive T_R cells express COX-2 and produce PGE₂ that suppress effector T cells in a manner that is reversed by COX-inhibitors. Methods and results Here we demonstrate that CRC patients have elevated levels of PGE₂ in peripheral blood, and CRC tissue samples and draining lymph nodes display increased numbers of FOXP3+ T_R cells. Depletion of T_R cells from PBMC enhanced anti-tumor T-cell responses to peptides from carcinoembryonic antigen. Furthermore, the COX inhibitor indomethacin and the PKA type I antagonist Rp-8-Br-cAMPS significantly improved the anti-tumor immune activity. Conclusion We suggest that adaptive T_R cells contribute to an immunosuppressive microenvironment in CRC and inhibit effector T cells by a COX-2–PGE₂-dependent mechanism and thereby facilitate tumor growth. Therapeutic strategies targeting T_R cells and the PGE₂–cAMP pathway may be interesting to pursue to enhance anti-tumor immune activity in CRC patients.     

A novel system of artificial antigen-presenting cells efficiently stimulates Flu peptide-specific cytotoxic T cells in vitro

Therapeutic numbers of antigen-specific cytotoxic T lymphocytes (CTLs) are key effectors in successful adoptive immunotherapy. However, efficient and reproducible methods to meet the qualification remain poor. To address this issue, we designed the artificial antigen-presenting cell (aAPC) system based on poly(lactic-co-glycolic acid) (PLGA). A modified emulsion method was used for the preparation of PLGA particles encapsulating interleukin-2 (IL-2). Biotinylated molecular ligands for recognition and co-stimulation of T cells were attached to the particle surface through the binding of avidin–biotin. These formed the aAPC system. The function of aAPCs in the proliferation of specific CTLs against human Flu antigen was detected by enzyme-linked immunospot assay (ELISPOT) and MTT staining methods. Finally, we successfully prepared this suitable aAPC system. The results show that IL-2 is released from aAPCs in a sustained manner over 30 days. This dramatically improves the stimulatory capacity of this system as compared to the effect of exogenous addition of cytokine. In addition, our aAPCs promote the proliferation of Flu antigen-specific CTLs more effectively than the autologous cellular APCs. Here, this aAPC platform is proved to be suitable for expansion of human antigen-specific T cells.     

Characterization of human cells transformed by chemical and physical carcinogens in vitro

Summary Several different classes of chemical carcinogens induced the transformation of human fibroblasts grown in vitro. Characteristics of the events that occur from time of treatment through the expression of neoplastic transformation are presented. The S-phase appeared to be the portion of the cell cycle most vulnerable to insult. Staging of the cells by blocking them in G₁ before releasing them to proceed through scheduled DNA synthesis (S) was required to induce reproducible transformation. Compounds such as insulin were added to the cells upon release from the block to sensitize the cells to the carcinogen that was added during S. Growth of the transformed cells as distinct from nontransformed cells was promoted by growth in medium supplemented with 8X nonessential amino acids. Carcinogen-treated cells in the early stage of transformation exhibited abnormal colony morphology and were able to grow at 41°C, in air atmosphere, and in medium supplemented with only 1% serum. In addition, the transformed cells were insensitive to KB cell lysate and exhibited density independent, as well as anchorage independent, growth (i.e., growth in 0.33% agar). Cells that grew in soft agar also produced undifferentiated mesenchymal tumors in preirradiated nude mice. This work was supported in part by National Cancer Institute Grant ROI-CA-25907 and Air Force Office of Scientific Research Grant F49620-77-C0110 and EPA-R806638. The hydrazine compounds were furnished by Ms. Marilyn George and Dr. Kenneth Back, AFSOR Toxicology Division, Wright Patterson Air Force Base, Dayton, OH. The hydroxylate and phenyl napthylamines were furnished by Dr. Fred Kadlubar, Division of Chemical Carcinogenesis at the National Center for Toxicological Research, Jefferson, AR.     

On the role of APC-activation for in vitro versus in vivo T cell priming

Professional antigen-presenting cells take up antigens for processing and presentation in association with MHC class I and II molecules. When APCs receive the right stimuli, they undergo a maturation process and migrate to secondary lymphoid organs to trigger T cell activation. In this study, we compared side-by-side in vivo and in vitro activation of T cells. Transgenic CD8(+) T cells specific for the p33 epitope, derived from the lymphocytic choriomeningitis virus glycoprotein, were labeled with CFSE and injected into syngeneic mice or alternatively, co-cultured in vitro with APCs. The p33 epitope was delivered as free peptide or genetically fused to virus-like particles. Whereas proliferation of specific T cells was comparable in both systems, the production of IFN-gamma and the expression of CD25 showed important differences. Induction of effector function and expression of activation markers were strongly enhanced in vitro by both the free peptide and VLPs. Surprisingly, addition of CpG-containing immune-stimulating DNA for activation of APCs dramatically increased effector T cell differentiation in vitro, whereas no enhancement could be observed in vitro. Thus, activation of professional APCs was mandatory for induction of effector CD8(+) T cell responses in vivo, while this step was largely dispensable in vitro.     

Expression and function of the B and T lymphocyte attenuator (BTLA/CD272) on human T cells

Co-signal receptors provide crucial activating or attenuating signals for T cells. The B and T lymphocyte attenuator (BTLA/CD272) is a third member of co-inhibitory receptors, which belongs to the CD28 immunoglobulin-superfamily. Using monoclonal antibodies (mAbs) against human BTLA, we show that BTLA is constitutively expressed on most CD4+ and CD8+ T cells and its expression progressively decreases upon T cell activation. Polarized Th1 and Th2 cells contained both BTLA-positive and BTLA-negative populations, but the extended culture diminished BTLA expression. Cross-linking BTLA with an agonistic mAb inhibited T cell proliferation and the production of the cytokines IFN-gamma and IL-10 in response to anti-CD3 stimulation. BTLA-mediated inhibition of T cell activation occurred during both primary CD4+ T cell responses and secondary CD4+ and CD8+ T cell responses, suggesting that BTLA ligation sends a constitutive "off" signal to T cells and thus might play an important role in the maintenance of T cell tolerance.     

A simple filtration device for separating nonadherent microorganisms from mammalian cells in in vitro studies on microbial adherence

In adherence studies, the removal of nonadherent microorganisms is essential for the valid enumeration of microorganisms that adhere to host cells. Although filtration devices are available commercially for the removal of nonadherent microorganisms, these are expensive and not reusable. In this article, we describe a simple, inexpensive, and reusable filtration device composed of two chambers of nylon, a nylon membrane of desired pore size, a rubber washer, and supporting stainless steel mesh. The device was effective in in vitro adherence assays for removing nonadherent endospores of Rhinosporidium seeberi from human buccal epithelial cells, providing valid counts of adherent microorganisms.     

Interaction of PD-L1 on tumor cells with PD-1 on tumor-specific T cells as a mechanism of immune evasion: implications for tumor immunotherapy

Programmed death receptor ligand 1 (PD-L1, also called B7-H1) is a recently described B7 family member. In contrast to B7-1 and B7-2, PD-L1 does not interact with either CD28 or CTLA-4. To date, one specific receptor has been identified that can be ligated by PD-L1. This receptor, programmed death receptor 1 (PD-1), has been shown to negatively regulate T-cell receptor (TCR) signaling. Upon ligating its receptor, PD-L1 has been reported to decrease TCR-mediated proliferation and cytokine production. PD-1 gene–deficient mice developed autoimmune diseases, which early led to the hypothesis of PD-L1 regulating peripheral tolerance. In contrast to normal tissues, which show minimal surface expression of PD-L1 protein, PD-L1 expression was found to be abundant on many murine and human cancers and could be further up-regulated upon IFN- stimulation. Thus, PD-L1 might play an important role in tumor immune evasion. This review discusses the currently available data concerning negative T-cell regulation via PD-1, the blockade of PD-L1/PD-1 interactions, and the implications for adoptive T-cell therapies.     

Maintenance of testosterone production by purified adult rat leydig cells for 3 days in vitro

Summary Using a preparation of highly purified, adult rat Leydig cells and conditions of culture which we found to optimize testosterone production during 24 h, we sought to maintain optimal testosterone production for 3 d. Leydig cells cultured on Cytodex 3 beads at 19% O₂ in Dulbecco's modified Eagle's medium-Ham's nutrient mixture F12 (1:1; vol/vol) containing 0.5 mg/ml, total bovine lipoproteins (<1.222 g/ml) with maximal luteinizing hormone (LH) stimulation failed to maintain a constant amount of testosterone for 3 d. These cells did however secrete a similar amount of total delta 4-3-ketosteroids on each of the 3 culture d, indicating that their viability was preserved. The predominance of progesterone and 170H-progesterone relative to the amount of androstenedione found on Days 2 and 3 suggested that the activity of the cytochrome P₄₅₀ C₁₇-hydroxylase-C_{17, 20}-lyase enzyme in the smooth endoplasmic reticulum was diminished when Leydig cells were maintained in our primary culture for longer than 24 h. Decreasing the oxygen tension of the cultures from 19 to 5%, and decreasing the concentration of LH used to stimulate the Leydig cells from 100 to 0.1 ng/ml, were necessary to achieve maintenance of testosterone secretion without accumulation of other delta 4-3-ketosteroids during a 3-d period. Cells cultured in this fashion were still able to respond to maximal LH stimulation during Day 3, producing as much testosterone as if cultured for 24 h on Day 1 at 19% O₂ with 100 ng/ml LH stimulation. This research was supported in part by grant HD-07204 from the National Institutes of Health, Bethesda, MD, The Population Center (grant HD-06268), an EPA cooperative agreement (CR81-2765), an NSF equipment grant, and a Mellon Foundation Postdoctoral Fellowship for Gary Klinefelter. Although the research described herein has been funded in part by the U.S. Environmental Protection Agency through cooperative agreement (CR81-2765) to the Division of Reproductive Biology at Johns Hopkins University, it has not been subjected to the agency's peer and policy review; therefore, it does not necessarily reflect the views of the agency and no official endorsement should de inferred.     

Combination of Epstein-Barr virus nuclear antigen 1, 3 and lytic antigen BZLF1 peptide pools allows fast and efficient stimulation of Epstein-Barr virus-specific T cells for adoptive immunotherapy

BackgroundEpstein-Barr virus (EBV) infection is a major cause of morbidity following hematopoietic stem cell transplantation. EBV-infected B cells may not respond to rituximab treatment and may lead to a life-threatening post-transplantation lymphoproliferative disorder. Adoptive cellular immunotherapy using EBV-lymphoblastoid cell lines (LCL) as stimulating antigen has proved effective in restoring specific immunity. However, EBV presents several immunodominant antigens, and developing a swift and effective clinical-grade immunotherapy relies on the definition of a Good Manufacturing Practices (GMP) universal stimulating antigen.MethodsPeripheral blood mononuclear cells (PBMCs) from six donors with a cellular immune response against EBV were immunoselected after stimulation with a new EBV antigen associated with an EBNA3 peptide pool.ResultsAfter immunoselection, a mean of 0.53 ± 0.25 × 10⁶ cells was recovered consisting of a mean of 24.77 ± 18.01% CD4⁺-secreting interferon (IFN)-γ and 51.42 ± 26.92% CD8⁺-secreting IFN-γ. The T memory stem cell sub-population was identified. EBV-specific T cells were expanded in vitro, and their ability to secrete IFN-γ and to proliferate after re-stimulation with EBV antigen was confirmed. A specific lysis was observed against autologous target cells pulsed with EBV peptide pools (57.6 ± 11.5%) and against autologous EBV-LCL (18.3 ± 7.3%). A mean decrease of 94.7 ± 3.3% in alloreactivity against third-party donor mononuclear cells with EBV-specific T cells was observed compared with PBMCs before selection.ConclusionsOur results show that a combination of peptide pools including EBNA3 is needed to generate EBV-specific T cells with good specific cytotoxicity and devoid of alloreactivity, but as yet GMP grade is not fully achieved.     

Effect of fluvastatin on apoptosis in human CD4+ T cells

Statins are lipid-lowering agents with pleiotropic effects. We investigated the apoptotic effects of fluvastatin on peripheral CD4+ T cells from healthy subjects. Fluvastatin induced apoptosis in resting CD4+ T cells but not in CD4+ T cells strongly activated with a high concentration of PMA plus ionomycin (PMA/I) analyzed with annexin V and propidium iodide staining. However, CD4+ T cells activated with a low concentration of PMA/I or with anti-CD3 antibodies were apoptotic after treatment with fluvastatin. Activities of caspases-8, -9, and -3 were increased in resting CD4+ T cells treated with fluvastatin (10 microM). In strongly activated CD4+ T cells, fluvastatin inhibited the activation of caspase-8 induced by PMA/I and increased caspase-9 activity. The caspase-3 activity did not differ between untreated and fluvastatin-treated strongly activated CD4+ T cells. Treatment with fluvastatin (10 microM) enhanced cytochrome c release and increased the Bax/Bcl-2 ratio in both resting and strongly activated CD4+ T cells. Although the in vitro concentration of fluvastatin used in this study is higher than in vivo, other factors may sensitize apoptotic cell death of CD4+ T cells in vivo. In conclusion, fluvastatin induces apoptosis in resting T cells but not in strongly activated T cells, a difference that might be due to the interaction between caspase-8 and caspase-9.     

Biocompatibility testing of an experimental fluoride releasing resin using human gingival epithelial cells in vitro

Summary Cell culture is a valuable method of evaluating the biocompatibility of new dental materials. The purpose of this study was to compare the in vitro biocompatibility of an experimental fluoride composite resin with fluoride and non-fluoride-releasing materials currently available. The dental materials tested were: MQ Silicate (silicate cement), KETAC-CEM and FUJI (type II glass ionomer cements), VISIO DISPERS (a light-cured, nonfluoridated, microfilled composite resin), and FR-17 (an experimental fluoride-releasing composite resin). The Smulow-Glickman (S-G) human, gingival epithelial cell line, which exhibits semidifferentiated characteristics, was used in the study as a test system. Biocompatibility was quantified by counting the viable cells per unit area remaining after 24 and 48 h at two radial distances from cured specimens immersed in the cell culture medium. The test materials were observed to be most toxic to cells nearest the materials. A Time-Distance Cytotoxicity Index (TDCI) was calculated to relate the percentage of dead cells to viable cells at each diffusion distance for each exposure time compared to a nontoxic control. The relative toxicity ranking of the materials tested based on the TDCI was VISIO DISPERS (91%), FUJI (82%), FR-17 (30%), MQ Silicate (23%), and KETAC-CEM (10%), which exhibited the least toxicity. The cytotoxicity of the experimental resin FR-17 was within the range of cytotoxicity of currently accepted restorative materials. this study was supported in part by grant R01-DE04749 from the National Institutes of Health, Bethesda, MD, to H. R. R., and by grant no. S07-RR05704-13 from the Biomedical Research Grant Program, Division of Research Resources, National Institutes of Health, awarded to the Louisiana State University School of Dentistry.     

A new human pancreatic carcinoma cell line developed for adoptive immunotherapy studies with lymphokine-activated killer cells in nude mice

Summary A human tumor cell line designated SU.86 has been established from a moderate-to-poorly differentiated pancreatic carcinoma of ductal origin specifically for adoptive immunotherapy studies. This line was characterized as to its ability to be lysed in vitro by autologous and allogeneic lymphokine-activated killer (LAK) and natural killer cells and to grow in nude mice. SU.86 has been growing continuously in cell culture for more than 100 passages since 22 September 1986. Transplantation orthotopically and heterotopically into athymic Swiss nude mice showed that tumor take was 100% in the orthotopic position when young (4 to 6 wk old) mice were used and 0% when adult (8 wk old) mice were used (P=0.004). In the heterotopic position (subcutaneous), tumor take was 100% in neonate (2 to 3 wk old) and young mice and 50% in adults. The rate of tumor growth was inversely correlated with age (P<0.001). The histologic pattern is similar to that observed in most human pancreatic carcinomas with pseudoglandular structures and frequent mitotic figures. SU.86 has a doubling time of 77 h in vitro and produces carcinoembryonic antigen, 594 ng/10⁶ cells in 3 d. Chromosomal analysis shows heterogeneity with two notable cell subpopulations. The cell line is moderately sensitive to lysis by LAK cells in a standard, 4-h chromium-51 release assay (35.4±4.0%). When grown together with LAK cells in vitro, it is lysed completely in culture in 8 to 15 d, depending on the serum concentration.     

Effect of fetal bovine serum on 3-methylcholanthrene-induced transformation of hamster cells in vitro

Summary Eighteen lots of fetal bovine serum were tested for their ability to support clonal growth and 3-methylcholanthrene-induced morphological transformation of hamster embryo cells in vitro. Most of them supported cloning efficiencies of over 11%. However, cloning efficiency alone was an inadequate criterion for selecting serum for transformation studies, since no transformation was observed with some lots, even though their cloning efficiencies were over 16%. This shows the importance of pretesting serum for its ability to support morphological transformation before it is used in mammalian cell carcinogenesis tests. Research sponsored by the National Cancer Institute under Contract No. N01-CO-75380 with Litton Bionetics, Inc.     

Direct and indirect effects of recombinant human granulocyte-colony stimulating factor on in vitro colony formation of human bladder cancer cells

Although the present experimental use of recombinant human granulocyte-colony-stimulating factor (rG-CSF) has been proven to alleviate the myelosuppression induced by antitumor chemotherapy, it is also believed to stimulate growth of some nonhematopoietic tumor cells. We investigated both the direct and indirect effects of rG-CSF on in vitro colony formation of human bladder cancer cell lines using a modified human tumor clonogenic assay. Peripheral blood mononuclear cells (PBMC) were used as feeder cells (a mixture of 5×10⁴ monocytes/dish and 5×10⁵ lymphocytes/dish obtained from healthy donors). Human bladder cancer cell lines KK-47, TCCSUP and T24, all derived from human transitional-cell carcinomas, were incubated continuously with various concentrations of rG-CSF ranging from 0.01 ng/ml to 10 ng/ml both with and without PBMC for 7–21 days. The concentrations of rG-CSF used were chosen as being in the range of achievable serum concentrations in patients treated with rG-CSF. At the end of incubation, colonies were counted under an inverted phase-contrast microscope, and an increase in the number of colonies in comparison with the control was used to evaluate the effects of rG-CSF. Results were expressed as a percentage of controls. rG-CSF in the upper layer at concentrations ranging from 0.1 ng/ml to 10 ng/ml stimulated the colony formation of all the cancer cell lines tested in the absence of PBMC in the feeder layer, whereas cells with PBMC in the feeder layer were significantly stimulated more than those without PBMC in the feeder layer (P<0.05) up to a certain concentration, which varied from cell line to cell line. At higher concentrations of rG-CSF, no further stimulation but, on the contrary, a decrease in colony formation was observed in cells with PBMC in the feeder layer in all the cell lines tested. Colony formation in KK-47 and T24 cell lines was significantly inhibited at 5 ng/ml and/or 10 ng/ml rG-CSF compared with cells without PBMC in the feeder layer. Our results suggest that rG-CSF may have both direct and indirect stimulatory effects on the growth of human bladder cancer cell lines in vitro. The results obtained also raise the possibility of adverse effects of rG-CSF in bladder cancer patients whose malignant cells may be directly and indirectly stimulated by this factor while it is being used clinically to alleviate the myelosuppression induced by antitumor chemotherapy.