Long-term functionality of TCR-transduced T cells in vivo

To broaden the applicability of adoptive T cell therapy to cancer types for which tumor-specific T cells cannot routinely be isolated, an effort has been made to develop the transfer of tumor-specific TCR genes into autologous T cells as a novel immunotherapeutic approach. Although such TCR-modified T cells have been shown to react to Ag encounter and can be used to break tolerance to defined self-Ags, the persistence and capacity for renewed expansion of TCR-modified T cells has not been analyzed. To establish whether TCR-transduced T cells can provide recipients with long-term Ag-specific immune protection, we analyzed long-term function of TCR transduced T cells in mouse model systems. We demonstrate that polyclonal populations of T cells transduced with a class I restricted OVA-specific TCR are able to persist in vivo and respond upon re-encounter of cognate Ag as assessed by both proliferation and cytolytic capacity. These experiments indicate that TCR gene transfer can be used to generate long-term Ag-specific T cell responses and provide a useful model system to assess the factors that can promote high-level persistence of TCR-modified T cells.     

CD4 cell priming and tolerization are differentially programmed by APCs upon initial engagement

Bone marrow-derived APCs present both parenchymal-self and pathogen-derived Ags in a manner that elicits either T cell tolerization or immunity, respectively. To study the parameters that confer tolerogenic vs immunogenic APC function we used an adoptive transfer system in which naive TCR transgenic hemagglutinin (HA)-specific CD4(+) T cells are either tolerized upon encountering HA expressed constitutively as a parenchymal self-Ag (self-HA) or primed to express effector function upon encountering transiently expressed vaccinia-derived HA (viral-HA). When the duration of viral-HA presentation was extended for the period required to elicit tolerization toward self-HA, CD4 cell tolerization to viral-HA did not occur. Furthermore, CD4 cells exhibited both phenotypic as well as functional differences during early stages of tolerization and priming, suggesting that these divergent differentiation processes are programmed soon after the initial APC-CD4 cell interaction. When mice expressing self-HA were infected with an irrelevant vaccinia, CD4 cell tolerization still occurred, indicating that priming vs tolerization cannot be explained by pathogen-induced third parties (i.e., non-APCs) that act directly on CD4 cells. Taken together, these results suggest that CD4 cell tolerization to parenchymal self-Ags and priming to pathogen-derived Ags are initiated by functionally distinct APCs.     

Dual T cell receptor expressing CD8+ T cells with tumor- and self-specificity can inhibit tumor growth without causing severe autoimmunity

The engineering of Ag-specific T cells by expression of TCR genes is a convenient method for adoptive T cell immunotherapy. A potential problem is the TCR gene transfer into self-reactive T cells that survived tolerance mechanisms. We have developed an experimental system with T cells that express two TCRs with defined Ag-specificities, one recognizing a tumor-specific Ag (LCMV-gp(33)), the other recognizing a self-Ag in the pancreas (OVA). By using tumor cells expressing high and low amounts of Ag and mice expressing high and low levels of self-Ag in the pancreas (RIP-OVA-Hi and RIP-OVA-Lo), we show that 1) tumor rejection requires high amount of tumor Ag, 2) severe autoimmunity requires high amount of self-Ag, and 3) if Ag expression on tumor cells is sufficient and low in the pancreas, successful adoptive T cell therapy can be obtained in the absence of severe autoimmunity. These results are shown with T cells from dual TCR transgenic mice or T cells that were redirected by TCR gene transfer. Our data demonstrate that the approach of adoptively transferring TCR redirected T cells can be effective without severe side effects, even when high numbers of T cells with self-reactivity were transferred.     

Low-avidity self-specific T cells display a pronounced expansion defect that can be overcome by altered peptide ligands

Thymic expression of self-Ags results in the deletion of high-avidity self-specific T cells, but, at least for certain Ags, a residual population of self-specific T cells with low-affinity TCRs remains after negative selection. Such self-specific T cells are thought to play a role in the induction of T cell-mediated autoimmunity, but may also be used for the induction of antitumor immunity against self-Ags. In this study, we examine the functional competence of a polyclonal population of self-specific CD8+ T cells. We show that low-affinity interactions between TCR and peptide are associated with selective loss of critical T cell functions. Triggering of low levels of IFN-gamma production and cytolytic activity through low-affinity TCRs readily occurs provided high Ag doses are used, but IL-2 production and clonal expansion are severely reduced at all Ag doses. Remarkably, a single peptide variant can form an improved ligand for the highly diverse population of low-avidity self-specific T cells and can improve their proliferative capacity. These data provide insight into the inherent limitations of self-specific T cell responses through low-avidity TCR signals and the effect of modified peptide ligands on self-specific T cell immunity.     

Effective adoptive therapy of tap-deficient lymphoma using diverse high avidity alloreactive T cells

High avidity for antigen and diversity of T cell receptor (TCR) repertoire are essential for effective immunity against cancer. We have previously created a transgenic mouse strain with increased TCR avidity in a diverse T cell population. In this report, we show that strong alloreactive responses of transgenic T cells against targets with low MHC class I expression can be used for effective adoptive transfer of tumor immunity in vivo. Alloreactive transgenic T cells could be an effective therapeutic approach counteracting tumor evasion of the immune system.     

Requirements for effective antitumor responses of TCR transduced T cells

Adoptive transfer of TCR gene-modified T cells has been proposed as an attractive approach to target tumors for which it is difficult or impossible to induce strong tumor-specific T cell responses by vaccination. Whereas the feasibility of generating tumor Ag-specific T cells by gene transfer has been demonstrated, the factors that determine the in vivo effectiveness of TCR-modified T cells are largely unknown. We have analyzed the value of a number of clinically feasible strategies to enhance the antitumor potential of TCR modified T cells. These experiments reveal three factors that contribute greatly to the in vivo potency of TCR-modified T cells. First, irradiation-induced host conditioning is superior to vaccine-induced activation of genetically modified T cells. Second, increasing TCR expression through genetic optimization of TCR sequences has a profound effect on in vivo antitumor activity. Third, a high precursor frequency of TCR modified T cells within the graft is essential. Tumors that ultimately progress in animals treated with this optimized regimen for TCR-based adoptive cell transfer invariably display a reduced expression of the target Ag. This suggests TCR gene therapy can achieve a sufficiently strong selective pressure to warrant the simultaneous targeting of multiple Ags. The strategies outlined in this study should be of value to enhance the antitumor activity of TCR-modified T cells in clinical trials.     

Alpha 3 domain mutants of peptide/MHC class I multimers allow the selective isolation of high avidity tumor-reactive CD8 T cells

The goal of adoptive T cell therapy in cancer is to provide effective antitumor immunity by transfer of selected populations of tumor Ag-specific T cells. Transfer of T cells with high TCR avidity is critical for in vivo efficacy. In this study, we demonstrate that fluorescent peptide/MHC class I multimeric complexes incorporating mutations in the alpha3 domain (D227K/T228A) that abrogate binding to the CD8 coreceptor can be used to selectively isolate tumor Ag-specific T cells of high functional avidity from both in vitro expanded and ex vivo T cell populations. Sorting, cloning, and expansion of alpha3 domain mutant multimer-positive CD8 T cells enabled rapid selection of high avidity tumor-reactive T cell clones. Our results are relevant for ex vivo identification and isolation of T cells with potent antitumor activity for adoptive T cell therapy.     

Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of human Type 1 diabetes (T1D). Cell transfer studies in irradiated recipient mice have established that T cells are pivotal in T1D pathogenesis in this model. We describe herein a simple method to rapidly induce T1D by adoptive transfer of purified, primary CD4+ T cells from pre-diabetic NOD mice transgenic for the islet-specific T cell receptor (TCR) BDC2.5 into NOD.SCID recipient mice. The major advantages of this technique are that isolation and adoptive transfer of diabetogenic T cells can be completed within the same day, irradiation of the recipients is not required, and a high incidence of T1D is elicited within 2 weeks after T cell transfer. Thus, studies of pathogenesis and therapeutic interventions in T1D can proceed at a faster rate than with methods that rely on heterogenous T cell populations or clones derived from diabetic NOD mice.     

Chronic immunodeficiency in mice lacking RasGRP1 results in CD4 T cell immune activation and exhaustion

The Ras-guanyl nucleotide exchange factor RasGRP1 is an important link between TCR-mediated signaling and the activation of Ras and its downstream effectors. RasGRP1 is especially critical for the survival and differentiation of developing thymocytes whereas negative selection of thymocytes bearing an autoreactive TCR appears to be RasGRP1 independent. Despite apparently normal central tolerance, RasGRP1(-/-) mice spontaneously acquire an acutely activated and proliferating CD4 T cell population that exhibits characteristics of T cell exhaustion, including strong expression of programmed cell death-1. To elucidate the basis for RasGRP1(-/-) CD4 T cell immune activation, we initiated a series of adoptive transfer experiments. Remarkably, the copious amounts of cytokines and self-Ags present in hosts made lymphopenic through irradiation failed to induce the majority of RasGRP1(-/-) CD4 T cells to enter cell cycle. However, their infusion into either congenitally T cell- or T/B cell-deficient recipients resulted in robust proliferation and L-selectin down-regulation. These findings imply that the activation and proliferation of RasGRP1(-/-) CD4 T cells may be dependent on their residence in a chronically immunocompromised environment. Accordingly, bacterial and viral challenge experiments revealed that RasGRP1(-/-) mice possess a weakened immune system, exhibiting a T cell-autonomous defect in generating pathogen-specific T cells and delayed pathogen clearance. Collectively, our study suggests that chronic T cell immunodeficiency in RasGRP1(-/-) mice may be responsible for CD4 T cell activation, proliferation, and exhaustion.     

Cutting edge: persistence of transferred lymphocyte clonotypes correlates with cancer regression in patients receiving cell transfer therapy

The lack of persistence of transferred autologous mature lymphocytes in humans has been a major limitation to the application of effective cell transfer therapies. The results of a pilot clinical trial in 13 patients with metastatic melanoma suggested that conditioning with nonmyeloablative chemotherapy before adoptive transfer of activated tumor-reactive T cells enhances tumor regression and increases the overall rates of objective clinical responses. The present report examines the relationship between T cell persistence and tumor regression through analysis of the TCR beta-chain V region gene products expressed in samples obtained from 25 patients treated with this protocol. Sequence analysis demonstrated that there was a significant correlation between tumor regression and the degree of persistence in peripheral blood of adoptively transferred T cell clones, suggesting that inadequate T cell persistence may represent a major factor limiting responses to adoptive immunotherapy.     

Redirecting T lymphocyte specificity by T cell receptor gene transfer--a new era for immunotherapy

The therapeutic efficacy of adoptively transferred cytotoxic T lymphocytes (CTL) has been demonstrated in clinical trials for the treatment of chronic myelogenous leukemia, cytomegalovirus-mediated disease, and Epstein-Barr virus-positive B cell lymphomas. It is however limited by the difficulty of generating sufficient amounts of CTLs in vitro, especially for the treatment of solid tumors. Recent gene therapy approaches, including two clinical trials, successfully apply genetic engineering of T cell specificity by T cell receptor (TCR) gene transfer. In this review we want to elucidate several principles of the redirection of T cell specificity. We cover basic aspects of retroviral gene transfer, regarding transduction efficacy and transgene expression levels. It was demonstrated that the number of TCR molecules on a T cell is important for its function. Therefore, an efficient transfer system that yields high transduction efficiency and strong and stable transgene expression is a prerequisite to achieve effector function by redirected T cells. Furthermore, we consider more recent aspects of T cell specificity engineering. These include the possibility of co-transferring coreceptors to create for example functional T helper cells by engrafting CD4(+) T cells with a MHC class I restricted TCR and the CD8 coreceptor and vice versa. Also, risks related to the adoptive transfer of TCR gene-modified T cells and possible safety mechanisms are discussed. Finally, we summarize recent findings describing transferred TCRs capable of displacing endogenous TCRs from the cell surface.     

Reconstitution of EBV-directed T cell immunity by adoptive transfer of peptide-stimulated T cells in a patient after allogeneic stem cell transplantation for AITL

Reconstitution of the T cell repertoire after allogeneic stem cell transplantation is a long and often incomplete process. As a result, reactivation of Epstein-Barr virus (EBV) is a frequent complication that may be treated by adoptive transfer of donor-derived EBV-specific T cells. We generated donor-derived EBV-specific T cells by stimulation with peptides representing defined epitopes covering multiple HLA restrictions. T cells were adoptively transferred to a patient who had developed persisting high titers of EBV after allogeneic stem cell transplantation for angioimmunoblastic T-cell lymphoma (AITL). T cell receptor beta (TCRβ) deep sequencing showed that the T cell repertoire of the patient early after transplantation (day 60) was strongly reduced and only very low numbers of EBV-specific T cells were detectable. Manufacturing and in vitro expansion of donor-derived EBV-specific T cells resulted in enrichment of EBV epitope-specific, HLA-restricted T cells. Monitoring of T cell clonotypes at a molecular level after adoptive transfer revealed that the dominant TCR sequences from peptide-stimulated T cells persisted long-term and established an EBV-specific TCR clonotype repertoire in the host, with many of the EBV-specific TCRs present in the donor. This reconstituted repertoire was associated with immunological control of EBV and with lack of further AITL relapse.     

Protective capacity of virus-specific T cell receptor-transduced CD8 T cells in vivo

The transfer of T cell receptor (TCR) genes by viral vectors represents a promising technique to generate antigen-specific T cells for adoptive immunotherapy. TCR-transduced T cells specific for infectious pathogens have been described, but their protective function in vivo has not yet been examined. Here, we demonstrate that CD8 T cells transduced with the P14 TCR specific for the gp33 epitope of lymphocytic choriomeningitis virus exhibit protective activities in both viral and bacterial infection models in mice.     

Optimization of TCR transgenic T cells for in vivo tracking of immune responses

T-cell receptor (TCR) transgenic mice have proven useful for the study of various immune parameters. Despite this, it has been suggested that transferred T cells respond differently to their endogenous counterparts at least in terms of conversion to antigen-experienced populations bearing memory cell markers. Here, we have compared the response of TCR transgenic T cells to endogenous populations within the context of infection with herpes simplex virus. We found that adoptive transfer at numbers approaching those of the endogenous virus-specific subset results in a response with similar kinetics, magnitude and memory subset conversion. This suggests that this form of optimized T-cell transfer remains a useful means of tracking antiviral immune responses.     

The immunopathology of acute experimental allergic encephalomyelitis induced with myelin proteolipid protein. T cell receptors in inflammatory lesions.

To determine whether there is predominance of T cells expressing a particular TCR V beta chain in the inflammatory lesions of an autoimmune disease model, TCR expression was analyzed in central nervous system (CNS) tissues of mice with experimental allergic encephalomyelitis (EAE). Acute EAE was induced in SJL/J mice either by sensitization with a synthetic peptide corresponding to myelin proteolipid protein residues 139-151 or by adoptive transfer of myelin proteolipid protein peptide 139-151-specific encephalitogenic T cell clones. Mice were killed when they showed clinical signs of EAE or by 40 days after sensitization or T cell transfer. Cryostat CNS and lymphoid tissue sections were immunostained with a panel of mAb to T cell markers and proportions of stained cells were counted in inflammatory foci. In mice with both actively induced and adoptively transferred EAE, infiltrates consisted of many CD3+, TCR alpha beta+, and CD4+ cells, fewer CD8+ cells, and small numbers of TCR gamma delta+ cells. Approximately 30% of CD45+ leukocytes in the inflammatory foci were T cells. Cells expressing TCR V beta 2, 3, 4, 6, 7 and 14 were detected in the infiltrates, whereas TCR V beta 8 and 11, which that are deleted in SJL mice, were absent. When EAE was induced by transfer of T cell clones that use either V beta 2, 6, 10, or 17, there was also a heterogeneous accumulation of T cells in the lesions. Similar proportions of TCR V beta+ and gamma delta+ cells were detected in EAE lesions and in the spleens of the mice. Thus, at the time that clinical signs are present in acute EAE, peripherally derived, heterogeneous TCR V beta+ cells are found in CNS lesions, even when the immune response is initiated to a short peptide Ag or by a T cell clone using a single TCR V beta.     

Steady state dendritic cells present parenchymal self-antigen and contribute to, but are not essential for, tolerization of naive and Th1 effector CD4 cells

Bone marrow-derived APC are critical for both priming effector/memory T cell responses to pathogens and inducing peripheral tolerance in self-reactive T cells. In particular, dendritic cells (DC) can acquire peripheral self-Ags under steady state conditions and are thought to present them to cognate T cells in a default tolerogenic manner, whereas exposure to pathogen-associated inflammatory mediators during the acquisition of pathogen-derived Ags appears to reprogram DCs to prime effector and memory T cell function. Recent studies have confirmed the critical role of DCs in priming CD8 cell effector responses to certain pathogens, although the necessity of steady state DCs in programming T cell tolerance to peripheral self-Ags has not been directly tested. In the current study, the role of steady state DCs in programming self-reactive CD4 cell peripheral tolerance was assessed by combining the CD11c-diphtheria toxin receptor transgenic system, in which DC can be depleted via treatment with diphtheria toxin, with a TCR-transgenic adoptive transfer system in which either naive or Th1 effector CD4 cells are induced to undergo tolerization after exposure to cognate parenchymally derived self-Ag. Although steady state DCs present parenchymal self-Ag and contribute to the tolerization of cognate naive and Th1 effector CD4 cells, they are not essential, indicating the involvement of a non-DC tolerogenic APC population(s). Tolerogenic APCs, however, do not require the cooperation of CD4(+)CD25(+) regulatory T cells. Similarly, DC were required for maximal priming of naive CD4 cells to vaccinia viral-Ag, but priming could still occur in the absence of DC.     

Impaired specific CD8 T cell response with aging is not due to decreased expression of CD90 on TCR transgenic T cells

Background

CD90 (Thy-1) is a small glycoprotein that is particularly abundant on the surface of mouse thymocytes and peripheral T cells, and is often used as a marker in adoptive transfer experiments to distinguish donor and recipient T cells with different CD90 subtypes. We have performed adoptive transfer experiments with T cell receptor transgenic (TCR Tg) mice to study the impaired CD8 T cell response with aging.

Findings

After stimulation with a CD8 T cell epitope, HA_518-524, the response of TCR Tg CD8 T cells from aged mice was decreased compared to the response of TCR Tg T cells from young mice. CD90 expression was also substantially decreased on the TCR Tg CD8 T cells of aged mice. However, the responses of CD90^hi and CD90^low CD8 T cells of the aged mice were similar in both early activation and proliferation, demonstrating that the impaired Tg T cell response with aging is not associated with the altered CD90 expression on CD8 T cells.

Conclusions

The impaired Tg CD8 T cell response in aged mice is not due to age-associated changes in CD90 expression on Tg CD8 T cells.

     

Clonal competition inhibits the proliferation and differentiation of adoptively transferred TCR transgenic CD4 T cells in response to infection

CD4 and CD8 T cells have been shown to proliferate and differentiate to different extents following antigenic stimulation. CD4 T cells form a heterogenous pool of effector cells in various stages of division and differentiation, while nearly all responding CD8 T cells divide and differentiate to the same extent. We examined CD4 and CD8 T cell responses during bacterial infection by adoptive transfer of CFSE-labeled monoclonal and polyclonal T cells. Monoclonal and polyclonal CD8 T cells both divided extensively, whereas monoclonal CD4 T cells underwent limited division in comparison with polyclonal CD4 T cells. Titration studies revealed that the limited proliferation of transferred monoclonal CD4 T cells was due to inhibition by a high precursor frequency of clonal T cells. This unusually high precursor frequency of clonal CD4 T cells also inhibited the differentiation of these cells. These results suggest that the adoptive transfer of TCR transgenic CD4 T cells significantly underestimates the extent of proliferation and differentiation of CD4 T cells following infection.     

Gamma delta T cells assist alpha beta T cells in adoptive transfer of contact sensitivity.

Cutaneous immune responses to contact sensitizers such as picryl chloride or oxazolone, are classical manifestations of T cell-mediated immunity in vivo. In fact, the first documentation of T cell-mediated immunity was the ability to adoptively transfer contact sensitivity (CS) responses. Although it is now clear that Ag/MHC-restricted alpha beta TCR positive effector T cells are responsible for 24 to 48 h CS responses, other subsets of Thy-1+ cells in mice also participate in the elicitation of CS. Thus, Thy-1+, CD5+, CD3-, B220+, hapten-specific, non-MHC-restricted early-acting cells are required to initiate CS responses by leading to local serotonin release, which allows for extravascular recruitment of the late-acting, alpha beta TCR+, CS effector T cells. This study describes another T cell population that is needed for the adoptive transfer of CS by alpha beta T cells. In vitro treatment of a mixture of CS effector cells with hamster mAb to gamma delta TCR, together with rabbit complement, or by panning on anti-hamster Ig-coated dishes, diminished substantially the subsequent transfer of CS reactivity without affecting either CS-initiating cells, or the later-acting, alpha beta TCR+ CS effector T cells. Immune cells treated with anti-alpha beta TCR mAb, or recovered as adherent cells from petri dishes after anti-gamma delta TCR panning (i.e., gamma delta TCR-enriched cells), reconstituted the ability of anti-gamma delta TCR-treated immune cells (i.e., alpha beta TCR-enriched cells) to transfer 24-h CS responsiveness. The phenotype of the gamma delta T cells that assisted CS effector alpha beta T cells was: CD3+, CD4-, and CD8+. The gamma delta T cells that assisted alpha beta T cells were not Ag-specific since anti-alpha beta-TCR-treated cells (gamma delta T-enriched) from picryl chloride immunized donors aided alpha beta T cells (anti-gamma delta TCR-treated) from oxazolone-immunized donors, and conversely gamma delta T cells from oxazolone-immunized donors aided alpha beta T cells from picryl chloride immunized donors. Furthermore, the CS-regulating gamma delta T cells were not MHC-restricted because gamma delta T cells from H2d or H2b donors could assist alpha beta T cells from H2k donors. It was concluded that a regulatory population of non-Ag specific, non-MHC-restricted gamma delta T cells was needed to assist immune effector, Ag/MHC-specific alpha beta T cells in the adoptive transfer of CS.     

Defective CD8+ T cell peripheral tolerance in nonobese diabetic mice.

Nonobese diabetic (NOD) mice develop spontaneous autoimmune diabetes that involves participation of both CD4+ and CD8+ T cells. Previous studies have demonstrated spontaneous reactivity to self-Ags within the CD4+ T cell compartment in this strain. Whether CD8+ T cells in NOD mice achieve and maintain tolerance to self-Ags has not previously been evaluated. To investigate this issue, we have assessed the extent of tolerance to a model pancreatic Ag, the hemagglutinin (HA) molecule of influenza virus, that is transgenically expressed by pancreatic islet beta cells in InsHA mice. Previous studies have demonstrated that BALB/c and B10.D2 mice that express this transgene exhibit tolerance of HA and retain only low-avidity CD8+ T cells specific for the dominant peptide epitope of HA. In this study, we present data that demonstrate a deficiency in peripheral tolerance within the CD8+ T cell repertoire of NOD-InsHA mice. CD8+ T cells can be obtained from NOD-InsHA mice that exhibit high avidity for HA, as measured by tetramer (K(d)HA) binding and dose titration analysis. Significantly, these autoreactive CD8+ T cells can cause diabetes very rapidly upon adoptive transfer into NOD-InsHA recipient mice. The data presented demonstrate a retention in the repertoire of CD8+ T cells with high avidity for islet Ags that could contribute to autoimmune diabetes in NOD mice.