In Vivo Assessment of NK Cell-Mediated Cytotoxicity by Adoptively Transferred Splenocyte Rejection.

NK cells are innate lymphocytes that are vital to clearance of virally infected or malignantly transformed cells. Assessment of the cytotoxic response is an important component of NK cell research and investigation of human disease. Standard assays of NK cell-mediated cytotoxicity of CD107a degranulation or 51Cr release assay utilize cultured or freshly isolated NK cell populations in vitro. In addition to requirements to maintain multiple target cell lines and radioactivity precautions in the case of 51Cr, these are in vitro evaluations of a complex in vivo function. Here, we describe the in vivo assessment of NK cell-mediated cytotoxicity through the adoptive transfer of splenocytes and their subsequent rejection. This protocol offers rapid, quantitative, and concurrent assessment of NK cell-mediated cytotoxicity against the prototypic NK stimulations of "missing-self" and "nonself."

Assessment of Gene Function of Mouse Innate Lymphoid Cells for In Vivo Analysis Using Retroviral Transduction.

Retroviral transduction is commonly used to modulate gene expression and is a powerful approach to understand the role of a gene using gain- or loss-of-function strategies. Retroviral vectors can stably integrate non-viral genes into host genomes, providing long-term modulation of gene expression in infected cells and their progeny. Here we describe the generation of retroviral supernatants and the steps to efficiently transduce genes in innate lymphoid cell (ILC) progenitors for subsequent analysis of ILC populations in vivo.

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients.

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms requires reliable models. Here, we describe models of tolerance to cardiac allograft in rat, induced by blockade of costimulation signals or by upregulation of immunoregulatory molecules through gene transfer. Each of these models allowed in vivo generation of regulatory cells such as regulatory T cells (Tregs), regulatory B cells (Bregs) or regulatory myeloid cells (RegMCs). In this manuscript, we describe two complementary protocols that have been used to identify and define in vitro and in vivo regulatory cell activity to determine their responsibility in tolerance induction and maintenance. First, an in vitro suppressive assay allowed rapid identification of cells with suppressive capacity on effector immune responses in a dose dependent manner, and can be used for further analysis such as cytokine measurement or cytotoxicity. Second, the adoptive transfer of cells from a tolerant treated recipient to a newly irradiated grafted recipient, highlighted the tolerogenic properties of these cells in controlling graft directed immune responses and/or converting new regulatory cells (termed infectious tolerance). These methods are not restricted to cells with known phenotypic markers and can be extended to any cell population. Furthermore, donor directed allospecificity of regulatory cells (an important goal in the field) can be assessed by using third party donor cells or graft either in vitro or in vivo. Finally, to determine the specific tolerogenic capacity of these regulatory cells, we provide protocols to assess the humoral anti-donor antibody responses and the capacity of the recipient to develop humoral responses against new or former known antigens. The models of tolerance described can be used to further characterize regulatory cells, to identify new biomarkers, and immunoregulatory molecules, and are adaptable to other transplantation models or autoimmune diseases in rodent or human.

A Higher Activation Threshold of Memory CD8+ T Cells Has a Fitness Cost That Is Modified by TCR Affinity during Tuberculosis.

T cell vaccines against Mycobacterium tuberculosis (Mtb) and other pathogens are based on the principle that memory T cells rapidly generate effector responses upon challenge, leading to pathogen clearance. Despite eliciting a robust memory CD8+ T cell response to the immunodominant Mtb antigen TB10.4 (EsxH), we find the increased frequency of TB10.4-specific CD8+ T cells conferred by vaccination to be short-lived after Mtb challenge. To compare memory and naïve CD8+ T cell function during their response to Mtb, we track their expansions using TB10.4-specific retrogenic CD8+ T cells. We find that the primary (naïve) response outnumbers the secondary (memory) response during Mtb challenge, an effect moderated by increased TCR affinity. To determine whether the expansion of polyclonal memory T cells is restrained following Mtb challenge, we used TCRß deep sequencing to track TB10.4-specific CD8+ T cells after vaccination and subsequent challenge in intact mice. Successful memory T cells, defined by their clonal expansion after Mtb challenge, express similar CDR3ß sequences suggesting TCR selection by antigen. Thus, both TCR-dependent and -independent factors affect the fitness of memory CD8+ responses. The impaired expansion of the majority of memory T cell clonotypes may explain why some TB vaccines have not provided better protection.

Low-cost generation of Good Manufacturing Practice-grade CD19-specific chimeric antigen receptor-expressing T cells using piggyBac gene transfer and patient-derived materials.

BACKGROUND AIMS: Protocols for the production of CD19-specific chimeric antigen receptor (CAR19) T cells are often complex and expensive because of the use of retroviral and lentiviral vectors or the need for CAR19 T-cell enrichment. We aimed to simplify the generation of CAR19 T cells from the peripheral blood of normal donors and patients using the piggyBac transposon system of gene modification. METHODS: We varied electroporation voltage, cytokines and stimulation conditions for the generation and expansion of CAR19 T cells over a 3-week culture period. RESULTS: Using optimized electroporation voltage, interleukin-15 alone and co-culturing CAR T cells with peripheral blood mononuclear cells, we were able to expand CAR19 T-cell cultures by up to 765-fold over 3 weeks in normal donors and 180-fold in patients with B-cell malignancies. Final median CAR19 expression of 72% was seen in normal donors, and 81% was seen in patients with acute lymphoblastic leukaemia, chronic lymphocytic leukemia or non-Hodgkin lymphoma. CAR19 T cells produced interferon gamma on stimulation with CD19(+) cell lines and efficiently lysed both CD19(+) cell lines and primary leukemia cells. In addition, combining CAR expression with an inducible caspase safety switch allowed elimination of CAR19 T cells by the application of a small molecule dimerizer. DISCUSSION: We have produced a simple, inexpensive and easily adoptable protocol for the generation of CAR19 T cells suitable for use in clinical trials using the piggyBac transposon system. This provides a robust platform for further enhancing the T-cell product and testing new CAR technologies.

Mechanistic Assessment of PD-1H Coinhibitory Receptor-Induced T Cell Tolerance to Allogeneic Antigens.

PD-1H is a recently identified cell surface coinhibitory molecule of the B7/CD28 immune modulatory gene family. We showed previously that single injection of a PD-1H agonistic mAb protected mice from graft-versus-host disease (GVHD). In this study, we report two distinct mechanisms operate in PD-1H-induced T cell tolerance. First, signaling via PD-1H coinhibitory receptor potently arrests alloreactive donor T cells from activation and expansion in the initiation phase. Second, donor regulatory T cells are subsequently expanded to maintain long-term tolerance and GVHD suppression. Our study reveals the crucial function of PD-1H as a coinhibitory receptor on alloreactive T cells and its function in the regulation of T cell tolerance. Therefore, PD-1H may be a target for the modulation of alloreactive T cells in GVHD and transplantation.

Ex vivo assessment of cellular immune function - applications in patient care and clinical studies.

Cellular ex vivo assays have a broad range of applications in patient care and clinical studies, especially when they are standardized and highly sensitive. As compared to analyses by molecular genetics such as the single nucleotide polymorphism (SNP) testing, they are usually more global. These assays partly mimic the in vivo situation, relying on a complex interaction of various immune cells. For example, they can be used to determine modulation of alloresponses by treatment or underlying disease, diagnose and quantify primary and secondary cellular immunodeficiency, follow-up vaccination responses, measure adoptive transfer of virus-specific immunity via hematopoietic stem cell or liver transplantation, assess allergy, antimicrobial immunity and also rare effector/memory cells directed against tumor antigens. This review will first shortly describe various cellular in vitro methods and then present applications, summarizing some own studies performed within the last 18 years.

In vivo assessment of specific cytotoxic T lymphocyte killing.

The direct killing of target cells by cytotoxic T lymphocytes (CTLs) plays a fundamental role in protective immunity to viral, bacterial, protozoan and fungi infections, as well as to tumor cells. In vivo cytotoxic assays take into account the interaction of target and effector cells in the context of the proper microenvironment making the analysis biologically more relevant than in vitro cytotoxic assays. Thus, the development, improvement and validation of in vivo methods are necessary in view of the importance of the results they may provide. We describe and discuss in this manuscript a method to evaluate in vivo specific cytotoxic T lymphocyte killing. We used as model system mice immunized with human recombinant replication-deficient adenovirus 5 (HAd5) containing different transgenes as the trigger of a CTL-mediated immune response. To these mice, we adoptively transferred syngeneic cells labeled with different vital fluorescent dyes. Donor cells were pulsed (target) or not (control non-target) with distinct CD8 T-cell epitopes, mixed in a 1:1 ratio and injected i.v. into immunized or non-immunized recipient mice. After 18-24h, spleen cells are collected and analysed by flow cytometry. A deviation from the 1:1 ratio of control and target cell populations indicates antigen specific lysis of target cells.

Robust and cost effective expansion of human regulatory T cells highly functional in a xenograft model of graft-versus-host disease.

The low frequency of naturally occurring regulatory T cells (nTregs) in peripheral blood and the suboptimal protocols available for their ex vivo expansion limit the development of clinical trials based on the adoptive transfer of these cells. We have, therefore, generated a simplified, robust and cost-effective platform for the large-scale expansion of nTregs using a gas permeable static culture flask (G-Rex) in compliance with Good Manufacturing Practice. More than 10(9) putative Tregs co-expressing CD25 and CD4 molecules (92 ± 5%) and FoxP3 (69 ± 19%) were obtained within 21 days of culture. Expanded Tregs showed potent regulatory activity in vitro (80 ± 13% inhibition of CD8(+) cell division) and in vivo (suppression or delay of graft-versus-host disease in a xenograft mouse model) indicating that the cost-effective and simplified production of nTregs we propose will facilitate the implementation of clinical trials based on their adoptive transfer.

Novel anti-melanoma immunotherapies: disarming tumor escape mechanisms.

The immune system fights cancer and sometimes temporarily eliminates it or reaches an equilibrium stage of tumor growth. However, continuous immunological pressure also selects poorly immunogenic tumor variants that eventually escape the immune control system. Here, we focus on metastatic melanoma, a highly immunogenic tumor, and on anti-melanoma immunotherapies, which recently, especially following the FDA approval of Ipilimumab, gained interest from drug development companies. We describe new immunomodulatory approaches currently in the development pipeline, focus on the novel CEACAM1 immune checkpoint, and compare its potential to the extensively described targets, CTLA4 and PD1. This paper combines multi-disciplinary approaches and describes anti-melanoma immunotherapies from molecular, medical, and business angles.

Long-term outcome of EBV-specific T-cell infusions to prevent or treat EBV-related lymphoproliferative disease in transplant recipients.

T-cell immunotherapy that takes advantage of Epstein-Barr virus (EBV)-stimulated immunity has the potential to fill an important niche in targeted therapy for EBV-related cancers. To address questions of long-term efficacy, safety, and practicality, we studied 114 patients who had received infusions of EBV-specific cytotoxic T lymphocytes (CTLs) at 3 different centers to prevent or treat EBV(+) lymphoproliferative disease (LPD) arising after hematopoietic stem cell transplantation. Toxicity was minimal, consisting mainly of localized swelling at sites of responsive disease. None of the 101 patients who received CTL prophylaxis developed EBV(+) LPD, whereas 11 of 13 patients treated with CTLs for biopsy-proven or probable LPD achieved sustained complete remissions. The gene-marking component of this study enabled us to demonstrate the persistence of functional CTLs for up to 9 years. A preliminary analysis indicated that a patient-specific CTL line can be manufactured, tested, and infused for $6095, a cost that compares favorably with other modalities used in the treatment of LPD. We conclude that the CTL lines described here provide safe and effective prophylaxis or treatment for lymphoproliferative disease in transplantation recipients, and the manufacturing methodology is robust and can be transferred readily from one institution to another without loss of reproducibility.

Clinical development of combination strategies in immunotherapy: are we ready for more than one investigational product in an early clinical trial?

Stimulating the innate and adaptive immunity against cancer necessitates the tricking of a system evolved to fight microbial pathogens and directing its activity towards transformed self-tissue. Efficacious interventions to start and sustain the response will probably require a number of agents to tamper simultaneously or sequentially with several immune mechanisms. Although master switches controlling various functions may exist, the goal of a curative immune response will probably demand the combined actions of several therapeutic components. Synergy occurs when drugs interact in ways that enhance or magnify one or more effects or side effects. In cancer immunotherapy, two agents that have minor or no therapeutic effects as single agents can be powerful when combined. Mouse experimentation provides multiple examples of synergistic combinations. Elements to be combined include chiefly: tumor vaccines, adoptive T-cell therapies, cytokines, costimulatory molecules, molecular deactivation of immunosuppressive or tolerogenic pathways and immunostimulatory monoclonal antibodies. These novel therapies, even as single agents, are extremely complex products to be developed owing to the associated biomolecules, cell therapies or gene therapies. At present, drug-development programs are run individually for each immunotherapeutic agent and combinations are considered only at a later stage in clinical development, even in the absence of formal compulsory regulations to prevent clinical trials with combinations. As a result, instead of the search for maximal efficacy, ease of combination with standard treatments, intellectual property management, regulations and business-based decisions often guide the way. Even though the maximal effort must be made in order to prevent adverse effects in patients, it seems reasonable that combination pilot trials should be performed at an early stage, following safe completion of Phase I trials. These trials should be performed based on evidence for synergy in animal models and be simplified in terms of regulatory requirements. Such 'short-cut' combination immunotherapy trials can bring much needed efficacy earlier to the bedside.

Antigen-specific cytotoxic T lymphocytes protect against lethal West Nile virus encephalitis.

Infection with West Nile virus (WNV) causes fatal encephalitis in immunocompromised animals. Previous studies in mice have established that T cell protection is required for clearance of WNV infection from tissues and preventing viral persistence. The current study assessed whether specific WNV peptide epitopes could elicit a cytotoxic T lymphocyte (CTL) response capable of protecting against virus infection. Hidden Markov model analysis was used to identify WNV-encoded peptides that bound the MHC class I proteins K(b) or D(b). Of the 35 peptides predicted to bind MHC class I molecules, one immunodominant CTL recognition peptide was identified in each of the envelope and non-structural protein 4B genes. Addition of these but not control peptides to CD8(+) T cells from WNV-infected mice induced IFN-gamma production. CTL clones that were generated ex vivo lysed peptide-pulsed or WNV-infected target cells in an antigen-specific manner. Finally, adoptive transfer of a mixture of envelope- and non-structural protein 4B-specific CTL to recipient mice protected against lethal WNV challenge. Based on this, we conclude that CTL responses against immundominant WNV epitopes confer protective immunity and thus should be targets for inclusion in new vaccines.

Simultaneous assessment of antigen-stimulated cytokine production and memory subset composition of memory CD8 T cells.

The functional identification of antigen-specific CD8 T cell populations is critical to understanding host responses to infection by intracellular pathogens. Furthermore, assessing the properties of protective memory CD8 T cell populations generated by immunization is necessary in the rational design of vaccines. Recently, a classification scheme was proposed in which memory CD8 T cells were divided into one of two distinct subsets, based on CD62L expression, that have different functional properties and protective capacities. Intracellular cytokine staining functionally identifies antigen-specific CD8 T cell populations after short in vitro stimulation with cognate peptide. This short stimulation, however, results in the cleavage of CD62L from the cell surface of antigen-specific CD8 T cells and precludes distinguishing CD62L(hi)- and CD62L(lo)-expressing memory cell subsets within this population. Here, we describe a method of preserving CD62L expression by the antigen-specific CD8 T cell population during coculture with antigen. This methodology allows for the identification and functional assessment of antigen-specific memory CD8 T cell populations, while simultaneously characterizing the memory subset composition of that population. Using this method, we directly identify differences in IL-2 production capacity by CD62L(hi)- and CD62L(lo)-expressing antigen-specific memory CD8 T cell populations.

Quantitative assessment concerning the contribution of IL-2Rbeta for superantigen-mediated T cell responses in vivo.

IL-2- and IL-2R-deficient mice readily develop T cell-dependent immune responses in vivo, but the relevance of this finding is complicated by severe concurrent autoimmunity. Furthermore, the detection of such responses does not address whether under normal circumstances IL-2 dominates T cell immunity. In the present report, we investigated the extent IL-2-independent T cell growth is mediated by other cytokines in the IL-2 family and compared such responses to those generated by IL-2/IL-2R-sufficient T cells. T cell expansion and contraction to the superantigen staphylococcal enterotoxin A (SEA) by autoimmune-free IL-2Rbeta-/- CD4 and CD8 T cells were comparable to normal control mice, although their CD8+ T cells did not optimally develop into IFNgamma-producing effector cells. The proliferation by these IL-2Rbeta-deficient T cells in vivo was independent of IL-2, IL-4 and IL-15 and not blocked by mAbs to the common gamma chain. However, in co-adoptive transfer experiments, wild-type T cells exhibited somewhat more extensive proliferation than IL-2Rbeta-deficient T cells to SEA and this difference was almost entirely accounted for by CD8+ T cells. Collectively, these data indicate that substantial T cell proliferation occurs in the absence of responsiveness to cytokines in the IL-2 family, although maximal T cell proliferation and development of IFNgamma-producing effector CD8+ T cells depend upon IL-2Rbeta.

Effects of polyinosinic-polycytidylic acid and adoptive transfer of immune cells in the Lew.1AR1-iddm rat and in its coisogenic LEW.1AR1 background strain.

The importance of the cellular immune system for the development of T1DM in the LEW.1AR1-iddm rat was investigated by use of polyinosinic-polycytidylic acid (Poly I:C) and by adoptive transfer of concanavalin A (Con A) activated lymphocytes from diabetic LEW.1AR1-iddm rats and the coisogenic LEW.AR1 background strain. Poly I:C treatment induced diabetes, characterized morphologically by a diffuse infiltration of the pancreas, in up to 20% of the animals of the coisogenic LEW.1AR1 background strain. It did not increase the diabetes incidence of 30% of the LEW.1AR1-iddm strain. In contrast Poly I:C treatment induced diabetes in up to 80% of the animals of the Mhc congenic LEW.1WR1 strain. Adoptive transfer of lymphocytes activated by the T-cell mitogen Con A from diabetic donors doubled the incidence of diabetes, characterized morphologically by a focal insulitis, in diabetes prone LEW.1AR1-iddm recipients. In contrast, animals of the LEW.1AR1 background strain did not develop diabetes after adoptive transfer. Moreover, adoptive transfer of Con A activated lymphocytes from LEW.1AR1 rats to LEW.1AR1-iddm rats with 30 or 60% diabetes incidence, significantly decreased the incidence of diabetes in LEW.1AR1-iddm rats with 60% diabetes incidence. The results show that autoreactive lymphocytes induce beta cell destruction in the LEW.1AR1-iddm rat, while the LEW.AR1 background strain apparently contains regulatory potential, which is able to counteract the autoimmune response.