Localized expression of GITR-L in the tumor microenvironment promotes CD8<Superscript>+</Superscript> T cell dependent anti-tumor immunity

The systemic administration of an agonist antibody against glucocorticoid-induced tumor necrosis factor receptor related (GITR) protein has been shown to be effective in overcoming immune tolerance and promoting tumor rejection in a variety of murine tumor models. However, little is known regarding the functional consequence of ligation of GITR with its natural ligand (GITR-L) in the context of regulatory T cell (Treg) suppression in vivo. To determine the mechanism of GITR-L action in vivo, we generated a panel of tumor cell clones that express varying levels of GITR-L. The ectopic expression of GITR-L on the tumor cell surface was sufficient to enhance anti-tumor immunity and delay tumor growth in syngeneic BALB/c mice. Within the range examined, the extent of anti-tumor activity in vivo did not correlate with the level of GITR-L expression, as all clones tested exhibited a similar delay in tumor growth. The localized expression of GITR-L on tumor cells led to a significant increase in CD8⁺ T cell infiltration compared to the levels seen in control tumors. The increased proportion of CD8⁺ T cells was only observed locally at the tumor site and was not seen in the tumor draining lymph node. Depletion studies showed that CD8⁺ T cells, but not CD4⁺ T cells, were required for GITR-L mediated protection against tumor growth. These studies demonstrate that signaling between GITR-L and GITR in the tumor microenvironment promotes the infiltration of CD8⁺ T cells, which are essential for controlling tumor growth. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Specific tumor memory induced by polyethylene-glycol-modified interleukin-2 requires both helper and cytotoxic T cells

Local polyethylene-glycol (PEG)-modified interleukin-2 (IL-2) immunotherapy of the guinea pig Line 10 (L10) tumor was previously demonstrated to evoke long-lasting systemic immunity after cure of the tumor and metastases. T cells, most likely the helper T cell subpopulation, were demonstrated to be crucial to the antitumor effects. Here we show that systemic immunity is induced within 7 days after the start of PEG-IL-2 therapy, indicating a rapid systemic priming of L10-specific T cells. No in vitro cytotoxic activity was detected in cell suspensions obtained from the primary tumor site, the regional lymph node or the spleen when isolated during (days 21 and 28) intratumoral treatment with 200 000 IU PEG-IL-2. These data confirm our carlier results obtained with 60 000 IU PEG-IL-2. Moreover, no cytolytic activity was observed in the chromium-release assay after in vitro restimulation with irradiated tumor cells. Specific L10 immunity can be transferred using spleen cell suspensions. Depletion of such a suspension of helper T cells resulted in rejection of the primary tumor in two out of four animals, but all the guinea pigs developed lymph node metastases. Removal of the cytotoxic/suppressor phenotype caused rejection of the dermal tumor in four of eight guinea pigs, but the capacity to prevent lymph node metastases was retained in all animals. Thus, depletion of either subtype reduces, but does not abrogate, the capacity to transfer L10 immunity with spleen cells. In conclusion, our data suggest that tumor cell killing through direct T cell cytotoxicity is not the main mode of action in PEG-IL-2-induced L10 tumor regression, PEG-IL-2 therapy induces early systemic immunity, resulting in rejection of a distant tumor, and the transfer of this immunity depends mainly on the presence of helper T cells, although cytotoxic T cells may also play a role.     

Directed differentiation of induced pluripotent stem cells towards T lymphocytes

Adoptive cell transfer (ACT) of antigen-specific CD8(+) cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of malignancies (1). CTLs can recognize malignant cells by interacting tumor antigens with the T cell receptors (TCR), and release cytotoxins as well as cytokines to kill malignant cells. It is known that less-differentiated and central-memory-like (termed highly reactive) CTLs are the optimal population for ACT-based immunotherapy, because these CTLs have a high proliferative potential, are less prone to apoptosis than more differentiated cells and have a higher ability to respond to homeostatic cytokines (2-7). However, due to difficulties in obtaining a high number of such CTLs from patients, there is an urgent need to find a new approach to generate highly reactive Ag-specific CTLs for successful ACT-based therapies. TCR transduction of the self-renewable stem cells for immune reconstitution has a therapeutic potential for the treatment of diseases (8-10). However, the approach to obtain embryonic stem cells (ESCs) from patients is not feasible. Although the use of hematopoietic stem cells (HSCs) for therapeutic purposes has been widely applied in clinic (11-13), HSCs have reduced differentiation and proliferative capacities, and HSCs are difficult to expand in in vitro cell culture (14-16). Recent iPS cell technology and the development of an in vitro system for gene delivery are capable of generating iPS cells from patients without any surgical approach. In addition, like ESCs, iPS cells possess indefinite proliferative capacity in vitro, and have been shown to differentiate into hematopoietic cells. Thus, iPS cells have greater potential to be used in ACT-based immunotherapy compared to ESCs or HSCs. Here, we present methods for the generation of T lymphocytes from iPS cells in vitro, and in vivo programming of antigen-specific CTLs from iPS cells for promoting cancer immune surveillance. Stimulation in vitro with a Notch ligand drives T cell differentiation from iPS cells, and TCR gene transduction results in iPS cells differentiating into antigen-specific T cells in vivo, which prevents tumor growth. Thus, we demonstrate antigen-specific T cell differentiation from iPS cells. Our studies provide a potentially more efficient approach for generating antigen-specific CTLs for ACT-based therapies and facilitate the development of therapeutic strategies for diseases.     

Comparative analysis of cytotoxic T lymphocyte response induced by dendritic cells loaded with hepatocellular carcinoma -derived RNA or cell lysate

The choice of the tumor antigen preparation used for dendritic cell (DC) loading is important for optimizing DC vaccines. In the present study, we compared DCs pulsed with hepatocellular carcinoma (HCC) total RNA or cell lysates for their capacity to activate T cells. We showed here that HCC total RNA pulsed-DCs induced effector T lymphocyte responses which showed higher killing ability to HCC cell lines, as well as higher frequency of IFN-γ producing of CD4+ and CD8+ T cells when compared with lysate pulsed-DCs. Both of RNA and lysate loading did not influence the changes of mature DC phenotype and the capacity of inducing T cell proliferation. However, HCC lysate loading significantly inhibited the production of inflammatory cytokines IL-12p70, IFN-γ and enhanced the secretion of anti-inflammatory cytokines IL-10 of mature DCs. Our results indicated that DCs loaded with HCC RNA are superior to that loaded with lysate in priming anti-HCC CTL response, suggesting that total RNA may be a better choice for DCs-based HCC immunotherapy.     

Characterization of MHC class-I restricted TCRαβ<Superscript>+</Superscript> CD4<Superscript>−</Superscript> CD8<Superscript>−</Superscript> double negative T cells recognizing the gp100 antigen from a melanoma patient after gp100 vaccination

The immune attack against malignant tumors require the concerted action of CD8⁺ cytotoxic T lymphocytes (CTL) as well as CD4⁺ T helper cells. The contribution of T cell receptor (TCR) αβ⁺ CD4⁻ CD8⁻ double-negative (DN) T cells to anti-tumor immune responses is widely unknown. In previous studies, we have demonstrated that DN T cells with a broad TCR repertoire are present in humans in the peripheral blood and the lymph nodes of healthy individuals. Here, we characterize a human DN T cell clone (T4H2) recognizing an HLA-A2-restricted melanoma-associated antigenic gp100-peptide isolated from the peripheral blood of a melanoma patient. Antigen recognition by the T4H2 DN clone resulted in specific secretion of IFN-γ and TNF. Although lacking the CD8 molecule the gp100-specifc DN T cell clone was able to confer antigen-specific cytotoxicity against gp100-loaded target cells as well as HLA-A2⁺ gp100 expressing melanoma cells. The cytotoxic capacity was found to be perforin/granzymeB-dependent. Together, these data indicate that functionally active antigen-specific DN T cells recognizing MHC class I-restricted tumor-associated antigen (TAA) may contribute to anti-tumor immunity in vivo. A. Mackensen and K. Fischer contributed equally to this work and should be considered joint senior authors. This work was supported by the Deutsche Forschungsgemeinschaft (MA 1351/5-1, KFO 146) and NIH grants CA90873, CA102280, 104947 (MIN). Companion paper: “Relationship between CD8-dependent antigen recognition, T cell functional avidity, and tumor cell recognition” by Tamson V. Moore et al. doi: .     

Osteosarcoma cell line growth inhibition by zoledronate-stimulated effector cells

Bisphosphonates have a profound effect on bone resorption and are widely used in the treatment of osteoclast-mediated bone diseases. Zoledronic acid (ZA), a third-generation biphosphonate, has a potent antitumor activity and expands gammadelta (γδ) T cells endowed of major histocompatibility complex-unrestricted lytic activity. Many solid tumors express tumor-specific antigens on their surface, representing targets for immune effector T cells. Nevertheless, the immune surveillance against clinically manifested tumors is relatively inefficient. Therefore, we investigated the hitherto unknown effects of ZA activated γδ T cells of normal donors on osteosarcoma cell lines. γδ T cells were stimulated with ZA and low doses of interleukin-2, and then analyzed for proliferation and generation of effector activity against osteosarcoma cell lines. Our results show the potent anti-tumor activity of ZA-stimulated γδ T cells and the enhanced immunosensitivity of osteosarcoma cell lines to γδ T cells suggesting that osteosarcoma is another γδ T cell susceptible tumor type.     

Functional heterogeneity of cytotoxic T cells and tumor resistance to cytotoxic hits limit anti‐tumor activity in vivo

Cytotoxic T cells (CTLs) can eliminate tumor cells through the delivery of lethal hits, but the actual efficiency of this process in the tumor microenvironment is unclear. Here, we visualized the capacity of single CTLs to attack tumor cells in vitro and in vivo using genetically encoded reporters that monitor cell damage and apoptosis. Using two distinct malignant B‐cell lines, we found that the majority of cytotoxic hits delivered by CTLs in vitro were sublethal despite proper immunological synapse formation, and associated with reversible calcium elevation and membrane damage in the targets. Through intravital imaging in the bone marrow, we established that the majority of CTL interactions with lymphoma B cells were either unproductive or sublethal. Functional heterogeneity of CTLs contributed to diverse outcomes during CTL–tumor contacts in vivo. In the therapeutic settings of anti‐CD19 CAR T cells, the majority of CAR T cell–tumor interactions were also not associated with lethal hit delivery. Thus, differences in CTL lytic potential together with tumor cell resistance to cytotoxic hits represent two important bottlenecks for anti‐tumor responses in vivo.     

Genetic engineering of cytolytic T lymphocytes for adoptive T-cell therapy of neuroblastoma

BACKGROUND: Disease relapse is the leading cause of mortality for children diagnosed with disseminated neuroblastoma. The adoptive transfer of tumor-specific T cells is an attractive approach to target minimal residual disease following conventional therapies. We describe here the genetic engineering of human cytotoxic T lymphocytes (CTL) to express a chimeric immunoreceptor for re-directed HLA-independent recognition of neuroblastoma. METHODS: The CE7R chimeric immunoreceptor was constructed by PCR splice overlap extension and is composed of a single-chain antibody extracellular domain (scFv) derived from the L1-CAM-specific murine CE7 hybridoma fused to human IgG1 hinge-Fc, the transmembrane portion of human CD4, and the cytoplasmic tail of huCD3-zeta chain (scFvFc:zeta). Primary human T cells were genetically modified by naked DNA electrotransfer of plasmid expression vector CE7R-pMG then analyzed by Western blotting, flow cytometry for CE7R expression and cell surface trafficking, 4-h chromium release assay for re-directed neuroblastoma lysis, and ELISA for tumor-specific activation of cytokine production. RESULTS: CE7R is expressed as an intact chimeric protein that trafficks to the cell surface as a type I transmembrane protein. Primary human CE7R-expressing CD8(+) CTL clones specifically recognize human neuroblastoma tumor cells and are activated for tumor cell lysis and T(c)1 cytokine production. CONCLUSIONS: These data demonstrate the utility of CE7R for re-directing the effector function of CTL to neuroblastoma and have provided the rationale to initiate a FDA-authorized (BB-IND#9149) pilot clinical trial to establish the feasibility and safety of adoptive transfer of autologous CE7R(+)CD8(+) CTL clones to children with recurrent/refractory neuroblastoma.     

Therapeutic efficacy of antitumor dendritic cell vaccinations correlates with persistent Th1 responses,high intratumor CD8+ T cell recruitment and low relative regulatory T cell infiltration

Despite the increasing number of immunotherapeutic strategies for the treatment of cancer, most approaches have failed to correlate the induction of an anti-tumor immune response with therapeutic efficacy. We therefore took advantage of a successful vaccination strategy-combining dendritic cells and irradiated GM-CSF secreting tumor cells-to compare the immune response induced against 9L gliosarcoma tumors in cured rats versus those with progressively growing tumors. At the systemic level, the tumor specific cytotoxic responses were quite heterogeneous in uncured vaccinated rats, and were surprisingly often high in animals with rapidly-growing tumors. IFN-gamma secretion by activated splenic T cells was more discriminative as the CD4+ T cell-mediated production was weak in uncured rats whereas high in cured ones. At the tumor level, regressing tumors were strongly infiltrated by CD8+ T cells, which demonstrated lytic capacities as high as their splenic counterparts. In contrast, progressing tumors were weakly infiltrated by T cells showing impaired cytotoxic activities. Proportionately to the T cell infiltrate, the expression of Foxp3 was increased in progressive tumors suggesting inhibition by regulatory T cells. In conclusion, the main difference between cured and uncured vaccinated animals does not depend directly upon the induction of systemic cytotoxic responses. Rather the persistence of higher CD4+ Th1 responses, a high intratumoral recruitment of functional CD8+ T cells, and a low proportion of regulatory T cells correlate with tumor rejection.     

Cord blood and adult T cells show different responses to C1q-bearing immune complexes

We have previously shown that T cells can be activated through cell-surface C1q receptors, resulting in secretion of interferon-gamma (IFN-gamma) and tumor necrosis factor alpha (TNFalpha), further demonstrating the intimate linkage between innate and adaptive immunity. In this current report, we sought to determine whether: (1) T cell responses to C1q-bearing immune complexes are dependent on the maturational status of the T cells and (2) whether signaling through the C1q receptor on T cells modulates conventional activation mediated through the conventional T cell receptor (TCR)/CD3 signaling complex. We first examined the capacity of neonatal T cells to respond to C1q-bearing immune complexes using IFNgamma, IL-2, and MIF secretion as measures of activation (MIF was chosen because of its crucial role in coordinating innate and adaptive immunity). Neonatal T cells produced significantly less IFNgamma but not IL-2, when stimulated by C1q immune complexes compared with adult T cells. MIF levels did not exceed background levels in these experiments. Next, we examined the capacity of C1q-bearing immune complexes to regulate signaling through the conventional TCR/CD3 signaling complex. Pre-incubating adult T cells with C1q-bearing immune complexes significantly reduced IFNgamma secretion when those same cells were subsequently stimulated with anti-CD3 and anti-CD28 monoclonal antibodies. Pre-incubation of neonatal T-cells with C1q-bearing immune complexes had no effect on IFNgamma secretion, although IFNgamma secretion was lower than that found in adult T cells for each experimental condition. We speculate that reduced IFNgamma secretion after pre-incubation with C1q immune complexes may be due to IL-10 secretion, which was observed in C1q-stimulated adult (but not neonatal) T cells. Conclusions: C1q-bearing immune complexes exert complex effects on mature T cells that include both pro- and anti-inflammatory responses. Immunologic maturation is required for these effects, as cord blood T cells are relatively hyporesponsive to C1q-bearing immune complexes compared with adult T cells.     

Depressed Immune Surveillance Against Cancer: Role of Deficient T Cell: Extracellular Matrix Interactions

Although T cells infiltrate malignant tumors, the local immune response is usually inefficient and tumors escape destruction. While extracellular matrix proteins strongly costimulate T cell responses in normal individuals, our studies indicate that peripheral blood T cells from cancer patients and tumor infiltrating cells respond poorly or are resistant to stimulative signals mediated by collagen I and IV and fibronectin. Moreover, the adhesive properties of cancer T cells are markedly depressed. Those functional deficiencies are paralleled by variable deficits in integrin and non-integrin T cell receptors for extracellular matrix. Immunotherapy with BCG causes a dramatic but transient increase in T cell: ECM interactions.     

Depressed Immune Surveillance Against Cancer: Role of Deficient T Cell: Extracellular Matrix Interactions

Although T cells infiltrate malignant tumors, the local immune response is usually inefficient and tumors escape destruction. While extracellular matrix proteins strongly costimulate T cell responses in normal individuals, our studies indicate that peripheral blood T cells from cancer patients and tumor infiltrating cells respond poorly or are resistant to stimulative signals mediated by collagen I and IV and fibronectin. Moreover, the adhesive properties of cancer T cells are markedly depressed. Those functional deficiencies are paralleled by variable deficits in integrin and non-integrin T cell receptors for extracellular matrix. Immunotherapy with BCG causes a dramatic but transient increase in T cell: ECM interactions.     

Conventional, regulatory, and unconventional T cells in the immunologic response to Helicobacter pylori

Infection by the gastroduodenal pathogen Helicobacter pylori elicits a complex immunologic response in the mucosa involving neutrophils, plasma cells, eosinophils, and lymphocytes, of which T cells are the principal orchestrators of immunity. While so-called classical T cells (e.g. T-helper cells) that are activated by peptide fragments presented on antigen-presenting cells have received much attention in H. pylori infection, there exists a diverse array of other T cell populations that are potentially important for the outcome of the ensuing immune response, some of which have not been extensively studied in H. pylori infection. Pathogen-specific regulatory T cells that control and prevent the development of immunopathology associated with H. pylori infection have been investigated, but these cells can also benefit the bacterium in helping to prolong the chronicity of the infection by suppressing protective immune responses. An overlooked T cell population, the more recently described Th17 cells, may play a role in H. pylori-induced inflammation, due to triggering responses that ultimately lead to the recruitment of polymorphs, including neutrophils. The so-called innate or unconventional T cells, that include two conserved T cell subsets expressing invariant antigen-specific receptors, the CD1d-restricted natural killer T cells which are activated by glycolipids, and the mucosal-associated invariant T cells which play a role in defense against orally acquired pathogens in the intestinal mucosa, have only begun to receive attention. A greater knowledge of the range of T cell responses induced by H. pylori is required for a deeper understanding of the pathogenesis of this bacterium and its ability to perpetuate chronic infection, and could reveal new strategies for therapeutic exploitation.     

T cell exhaustion and Interleukin 2 downregulation

T cells reactive to tumor antigens and viral antigens lose their reactivity when exposed to the antigen-rich environment of a larger tumor bed or viral load. Such non-responsive T cells are termed exhausted. T cell exhaustion affects both CD8+ and CD4+ T cells. T cell exhaustion is attributed to the functional impairment of T cells to produce cytokines, of which the most important may be Interleukin 2 (IL2). IL2 performs functions critical for the elimination of cancer cells and virus infected cells. In one such function, IL2 promotes CD8+ T cell and natural killer (NK) cell cytolytic activities. Other functions include regulating naïve T cell differentiation into Th1 and Th2 subsets upon exposure to antigens. Thus, the signaling pathways contributing to T cell exhaustion could be linked to the signaling pathways contributing to IL2 loss. This review will discuss the process of T cell exhaustion and the signaling pathways that could be contributing to T cell exhaustion.     

Blocking CD38-driven fratricide among T cells enables effective antitumor activity by CD38-specific chimeric antigen receptor T cells

Chimeric antigen receptor T-cell(CAR T) therapy is a kind of effective cancer immunotherapy. However,designing CARs remains a challenge because many targetable antigens are shared by T cells and tumor cells. This shared expression of antigens can cause CAR T cell fratricide. CD38-targeting approaches(e.g.,daratumumab) have been used in clinical therapy and have shown promising results. CD38 is a kind of surface glycoprotein present in a variety of cells, such as T lymphocytes and tumor cells. It was previously reported that CD38-based CAR T cells may undergo apoptosis or T cell-mediated killing(fratricide) during cell manufacturing. In this study, a CAR containing a sequence targeting human CD38 was designed to be functional. To avoid fratricide driven by CD38 and ensure the production of CAR T cells, two distinct strategies based on antibodies(clone MM12 T or clone MM27) or proteins(H02 H or H08 H) were used to block CD38 or the CAR single-chain variable fragment(scFv) domain, respectively, on the T cell surface.The results indicated that the antibodies or proteins, especially the antibody MM27, could affect CAR T cells by inhibiting fratricide while promoting expansion and enrichment. Anti-CD38 CAR T cells exhibited robust and specific cytotoxicity to CD38~+ cell lines and tumor cells. Furthermore, the levels of the proinflammatory factors TNF-a, IFN-g and IL-2 were significantly upregulated in the supernatants of A549~(CD38~+) cells. Finally, significant control of disease progression was demonstrated in xenograft mouse models. In conclusion, these findings will help to further enhance the expansion, persistence and function of anti-CD38 CAR T cells in subsequent clinical trials.     

Immune tolerance: Are regulatory T cell subsets needed to explain suppression of autoimmunity?

The potential for self-reactive T cells to cause autoimmune disease is held in check by Foxp3(+) regulatory T cells (Tregs), essential mediators of peripheral immunological tolerance. Tregs have the capacity to suppress multiple branches of the immune system, tightly controlling the different subsets of effector T cells across multiple different tissue environments. Recent genetic experiments have found mutations that disrupt specific Treg: effector T cell relationships, leading to the possibility that subsets of Tregs are required to suppress each subset of effector T cells. Here we review the environmental factors and mechanisms that allow Tregs to suppress specific subsets of effector T cells, and find that a parsimonious explanation of the genetic data can be made without invoking Treg subsets. Instead, Tregs show a functional and chemotactic plasticity based on microenvironmental influences that allows the common pool of cells to suppress multiple distinct immune responses.