Targeting T cell activation in immuno-oncology.

The years since 2009 have seen tremendous progress in unlocking the curative potential of the immune system for the treatment of cancer. Much of that revolution in immuno-oncology has been fueled by the clinical success of immune checkpoint inhibitors, particularly those targeting the PD-1 axis. Unfortunately, many patients still fail to benefit from checkpoint blockade or other immunotherapies. An inability to fully activate antitumour T cells contributes in part to the failure of those therapies. Here, we review the basic biology of T cell activation, with particular emphasis on the essential role of the dendritic cell and the innate immune system in T cell activation. The current understanding of the multiple factors that govern T cell activation and how they impinge on tumour immunotherapy are also discussed. Lastly, treatment strategies to potentially overcome barriers to T cell activation and to enhance the efficacy of immunotherapy are addressed.

Efficacy of adoptive therapy with tumor-infiltrating lymphocytes and recombinant interleukin-2 in advanced cutaneous melanoma: a systematic review and meta-analysis.

Adoptive cell therapy (ACT) using autologous tumor-infiltrating lymphocytes (TIL) has been tested in advanced melanoma patients at various centers. We conducted a systematic review and meta-analysis to assess its efficacy on previously treated advanced metastatic cutaneous melanoma. The PubMed electronic database was searched from inception to 17 December 2018 to identify studies administering TIL-ACT and recombinant interleukin-2 (IL-2) following non-myeloablative chemotherapy in previously treated metastatic melanoma patients. Objective response rate (ORR) was the primary end point. Secondary end points were complete response rate (CRR), overall survival (OS), duration of response (DOR) and toxicity. Pooled estimates were derived from fixed or random effect models, depending on the amount of heterogeneity detected. Analysis was carried out separately for high dose (HD) and low dose (LD) IL-2. Sensitivity analyses were carried out. Among 1211 records screened, 13 studies (published 1988 - 2016) were eligible for meta-analysis. Among 410 heavily pretreated patients (some with brain metastasis), 332 received HD-IL-2 and 78 LD-IL-2. The pooled overall ORR estimate was 41% [95% confidence interval (CI) 35% to 48%], and the overall CRR was 12% (95% CI 7% to 16%). For the HD-IL-2 group, the ORR was 43% (95% CI 36% to 50%), while for the LD-IL-2 it was 35% (95% CI 25% to 45%). Corresponding pooled estimates for CRR were 14% (95% CI 7% to 20%) and 7% (95% CI 1% to 12%). The majority of HD-IL-2 complete responders (27/28) remained in remission during the extent of follow-up after CR (median 40 months). Sensitivity analyses yielded similar results. Higher number of infused cells was associated with a favorable response. The ORR for HD-IL-2 compared favorably with the nivolumab/ipilimumab combination following anti-PD-1 failure. TIL-ACT therapy, especially when combined with HD-IL-2, achieves durable clinical benefit and warrants further investigation. We discuss the current position of TIL-ACT in the therapy of advanced melanoma, particularly in the era of immune checkpoint blockade therapy, and review future opportunities for improvement of this approach.

Molecularly targeted therapies in cancer: a guide for the nuclear medicine physician.

Molecular imaging continues to influence every aspect of cancer care including detection, diagnosis, staging and therapy response assessment. Recent advances in the understanding of cancer biology have prompted the introduction of new targeted therapy approaches. Precision medicine in oncology has led to rapid advances and novel approaches optimizing the use of imaging modalities in cancer care, research and development. This article focuses on the concept of targeted therapy in cancer and the challenges that exist for molecular imaging in cancer care.

Idh1 protects murine hepatocytes from endotoxin-induced oxidative stress by regulating the intracellular NADP(+)/NADPH ratio.

Isocitrate dehydrogenase-1 (Idh1) is an important metabolic enzyme that produces NADPH by converting isocitrate to α-ketoglutarate. Idh1 is known to reduce reactive oxygen species (ROS) induced in cells by treatment with lipopolysaccharide (LPS) in vitro. Here, we used Idh1-deficient knockout (Idh1 KO) mice to investigate the role of Idh1 in antioxidant defense in vivo. Idh1 KO mice showed heightened susceptibility to death induced by LPS and exhibited increased serum levels of inflammatory cytokines such as tumor necrosis factor-α and interleukin-6. The serum of LPS-injected Idh1 KO mice also contained elevated levels of AST, a marker of inflammatory liver damage. Furthermore, after LPS injection, livers of Idh1 KO mice showed histological evidence of elevated oxidative DNA damage compared with livers of wild-type (WT) mice. Idh1 KO livers showed a faster and more pronounced oxidative stress than WT livers. In line with that, Idh1 KO hepatocytes showed higher ROS levels and an increase in the NADP(+)/NADPH ratio when compared with hepatocytes isolated from WT mice. These results suggest that Idh1 has a physiological function in protecting cells from oxidative stress by regulating the intracellular NADP(+)/NADPH ratio. Our findings suggest that stimulation of Idh1 activity may be an effective therapeutic strategy for reducing oxidative stress during inflammatory responses, including the early stages of septic shock.

Toso regulates differentiation and activation of inflammatory dendritic cells during persistence-prone virus infection.

During virus infection and autoimmune disease, inflammatory dendritic cells (iDCs) differentiate from blood monocytes and infiltrate infected tissue. Following acute infection with hepatotropic viruses, iDCs are essential for re-stimulating virus-specific CD8(+) T cells and therefore contribute to virus control. Here we used the lymphocytic choriomeningitis virus (LCMV) model system to identify novel signals, which influence the recruitment and activation of iDCs in the liver. We observed that intrinsic expression of Toso (Faim3, FcµR) influenced the differentiation and activation of iDCs in vivo and DCs in vitro. Lack of iDCs in Toso-deficient (Toso(-/-)) mice reduced CD8(+) T-cell function in the liver and resulted in virus persistence. Furthermore, Toso(-/-) DCs failed to induce autoimmune diabetes in the rat insulin promoter-glycoprotein (RIP-GP) autoimmune diabetes model. In conclusion, we found that Toso has an essential role in the differentiation and maturation of iDCs, a process that is required for the control of persistence-prone virus infection.

IRF4 and BATF are critical for CD8⁺ T-cell function following infection with LCMV.

CD8(+) T-cell functions are critical for preventing chronic viral infections by eliminating infected cells. For healthy immune responses, beneficial destruction of infected cells must be balanced against immunopathology resulting from collateral damage to tissues. These processes are regulated by factors controlling CD8(+) T-cell function, which are still incompletely understood. Here, we show that the interferon regulatory factor 4 (IRF4) and its cooperating binding partner B-cell-activating transcription factor (BATF) are necessary for sustained CD8(+) T-cell effector function. Although Irf4(-/-) CD8(+) T cells were initially capable of proliferation, IRF4 deficiency resulted in limited CD8(+) T-cell responses after infection with the lymphocytic choriomeningitis virus. Consequently, Irf4(-/-) mice established chronic infections, but were protected from fatal immunopathology. Absence of BATF also resulted in reduced CD8(+) T-cell function, limited immunopathology, and promotion of viral persistence. These data identify the transcription factors IRF4 and BATF as major regulators of antiviral cytotoxic T-cell immunity.

Reactive oxygen species delay control of lymphocytic choriomeningitis virus.

Cluster of differentiation (CD)8(+) T cells are like a double edged sword during chronic viral infections because they not only promote virus elimination but also induce virus-mediated immunopathology. Elevated levels of reactive oxygen species (ROS) have been reported during virus infections. However, the role of ROS in T-cell-mediated immunopathology remains unclear. Here we used the murine lymphocytic choriomeningitis virus to explore the role of ROS during the processes of virus elimination and induction of immunopathology. We found that virus infection led to elevated levels of ROS producing granulocytes and macrophages in virus-infected liver and spleen tissues that were triggered by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Lack of the regulatory subunit p47phox of the NADPH oxidase diminished ROS production in these cells. While CD8(+) T cells exhibited ROS production that was independent of NADPH oxidase expression, survival and T-cell function was elevated in p47phox-deficient (Ncf1(-/-)) mice. In the absence of p47phox, enhanced T-cell immunity promoted virus elimination and blunted corresponding immunopathology. In conclusion, we find that NADPH-mediated production of ROS critically impairs the immune response, impacting elimination of virus and outcome of liver cell damage.

The interaction between caveolin-1 and Rho-GTPases promotes metastasis by controlling the expression of alpha5-integrin and the activation of Src, Ras and Erk.

Proteins containing a caveolin-binding domain (CBD), such as the Rho-GTPases, can interact with caveolin-1 (Cav1) through its caveolin scaffold domain. Rho-GTPases are important regulators of p130(Cas), which is crucial for both normal cell migration and Src kinase-mediated metastasis of cancer cells. However, although Rho-GTPases (particularly RhoC) and Cav1 have been linked to cancer progression and metastasis, the underlying molecular mechanisms are largely unknown. To investigate the function of Cav1-Rho-GTPase interaction in metastasis, we disrupted Cav1-Rho-GTPase binding in melanoma and mammary epithelial tumor cells by overexpressing CBD, and examined the loss-of-function of RhoC in metastatic cancer cells. Cancer cells overexpressing CBD or lacking RhoC had reduced p130(Cas) phosphorylation and Rac1 activation, resulting in an inhibition of migration and invasion in vitro. The activity of Src and the activation of its downstream targets FAK, Pyk2, Ras and extracellular signal-regulated kinase (Erk)1/2 were also impaired. A reduction in α5-integrin expression, which is required for binding to fibronectin and thus cell migration and survival, was observed in CBD-expressing cells and cells lacking RhoC. As a result of these defects, CBD-expressing melanoma cells had a reduced ability to metastasize in recipient mice, and impaired extravasation and survival in secondary sites in chicken embryos. Our data indicate that interaction between Cav1 and Rho-GTPases (most likely RhoC but not RhoA) promotes metastasis by stimulating α5-integrin expression and regulating the Src-dependent activation of p130(Cas)/Rac1, FAK/Pyk2 and Ras/Erk1/2 signaling cascades.

Duration and strength of extracellular signal-regulated kinase signals are altered during positive versus negative thymocyte selection.

During thymocyte development, high-affinity/avidity TCR engagement leads to the induction of negative selection and apoptosis, while lower TCR affinity-avidity interactions lead to positive selection and survival. To elucidate how these extracellular interactions are translated into intracellular signals that distinguish between positive and negative selection, we developed a culture system in which naive double-positive thymocytes were either induced to differentiate along the CD8(+) lineage pathway or were triggered for clonal deletion. Using this system, we show that sustained low level activation of extracellular signal-regulated kinases (ERKs) promotes positive selection, whereas strong but transient ERK activation is coupled with negatively selecting stimuli. Importantly, similar ERK activation profiles were demonstrated during positive selection for strong agonist ligands presented at low concentrations or weak agonist ligands. This is consistent with the affinity/avidity model and a role for strong or weak agonists during positive selection. Surprisingly, the addition of a pharmacological inhibitor which blocks ERK activation prevented the induction of negative selection. These data suggest that the duration and strength of the TCR signal is involved in discriminating between positive and negative selection.

Positive regulation of T cell activation and integrin adhesion by the adapter Fyb/Slap.

The molecular adapter Fyb/Slap regulates signaling downstream of the T cell receptor (TCR), but whether it plays a positive or negative role is controversial. We demonstrate that Fyb/Slap-deficient T cells exhibit defective proliferation and cytokine production in response to TCR stimulation. Fyb/Slap is also required in vivo for T cell-dependent immune responses. Functionally, Fyb/Slap has no apparent role in the activation of known TCR signaling pathways, F-actin polymerization, or TCR clustering. Rather, Fyb/Slap regulates TCR-induced integrin clustering and adhesion. Thus, Fyb/Slap is the first molecular adapter to be identified that couples TCR stimulation to the avidity modulation of integrins governing T cell adhesion.

Expression of active protein kinase B in T cells perturbs both T and B cell homeostasis and promotes inflammation.

The molecular mechanisms that contribute to autoimmunity remain poorly defined. While inflammation is considered to be one of the major checkpoints in autoimmune disease progression, very little is known about the initiating events that trigger inflammation. We have studied transgenic mice expressing the prosurvival molecule protein kinase B/Akt under control of a T cell-specific CD2 promoter. In this study, we demonstrate that aged mice develop lymphadenopathy and splenomegaly that result from an accumulation of CD4, CD8, and unexpectedly B cells. An increased proportion of T cells express activation markers, while T cell proliferative responses remain normal. B cells are hyperproliferative in response to anti-IgM F(ab')(2) and anti-CD40, and increased IgA and IgG2a were found in the sera. In addition, a profound multiorgan lymphocytic infiltration is observed, and T cells from these mice display a defect in Fas-mediated apoptosis, which may be the mechanism underlying this phenotype. Therefore, T cell expression of active protein kinase B can alter T cell homeostasis, indirectly influence B cell homeostasis, and promote inflammation in vivo.

ICOS is essential for effective T-helper-cell responses.

The outcome of T-cell responses after T-cell encounter with specific antigens is modulated by co-stimulatory signals, which are required for both lymphocyte activation and development of adaptive immunity. ICOS, an inducible co-stimulator with homology to CD28, is expressed on activated, but not resting T cells, and shows T-cell co-stimulatory function in vitro. ICOS binds specifically to its counter-receptor B7RP-1 (refs 5,6,7), but not to B7-1 or B7-2. Here we provide in vivo genetic evidence that ICOS delivers a co-stimulatory signal that is essential both for efficient interaction between T and B cells and for normal antibody responses to T-cell-dependent antigens. To determine the physiological function of ICOS, we generated and characterized gene-targeted ICOS-deficient mice. In vivo, a lack of ICOS results in severely deficient T-cell-dependent B-cell responses. Germinal centre formation is impaired and immunoglobulin class switching, including production of allergy-mediating IgE, is defective. ICOS-deficient T cells primed in in vivo and restimulated in vitro with specific antigen produce only low levels of interleukin-4, but remain fully competent to produce interferon-gamma.

T cell-specific loss of Pten leads to defects in central and peripheral tolerance.

PTEN, a tumor suppressor gene, is essential for embryogenesis. We used the Cre-loxP system to generate a T cell-specific deletion of the Pten gene (Pten(flox/-) mice). All Pten(flox/-) mice develop CD4+ T cell lymphomas by 17 weeks. Pten(flox/-) mice show increased thymic cellularity due in part to a defect in thymic negative selection. Pten(flox/-) mice exhibit elevated levels of B cells and CD4+ T cells in the periphery, spontaneous activation of CD4+ T cells, autoantibody production, and hypergammaglobulinemia. Pten(flox/-) T cells hyperproliferate, are autoreactive, secrete increased levels of Th1/Th2 cytokines, resist apoptosis, and show increased phosphorylation of PKB/Akt and ERK. Peripheral tolerance to SEB is also impaired in Pten(flox/-) mice. PTEN is thus an important regulator of T cell homeostasis and self-tolerance.

A point mutation in CD28 distinguishes proliferative signals from survival signals.

Upon interaction with its ligand, B7, CD28 becomes phosphorylated on tyrosines. One tyrosine in particular (Y170 in mouse CD28, Y173 in human CD28) has received much attention. This is because it permits CD28 to recruit SH2-containing signaling molecules, including phosphoinositide 3 kinase, Grb2 and Gads. Using mice we employed a transgenic approach to express a tyrosine-->phenylalanine mutant form of CD28 that uncouples these SH2-mediated interactions from CD28. The CD28 mutant is unable to up-regulate expression of the prosurvival protein Bcl-xL, rendering the T cells more susceptible to radiation-induced death. Nonetheless, this mutated form of CD28 still prevents the induction of anergy and promotes T cell proliferation, interleukin 2 secretion and B cell help. Thus, we describe a single point mutation within the CD28 cytoplasmic domain that uncouples signals required for proliferation and survival.

Bcl10 is a positive regulator of antigen receptor-induced activation of NF-kappaB and neural tube closure.

Bcl10, a CARD-containing protein identified from the t(1;14)(p22;q32) breakpoint in MALT lymphomas, has been shown to induce apoptosis and activate NF-kappaB in vitro. We show that one-third of bcl10-/- embryos developed exencephaly, leading to embryonic lethality. Surprisingly, bcl10-/- cells retained susceptibility to various apoptotic stimuli in vivo and in vitro. However, surviving bcl10-/- mice were severely immunodeficient and bcl10-/- lymphocytes are defective in antigen receptor or PMA/Ionomycin-induced activation. Early tyrosine phosphorylation, MAPK and AP-1 activation, and Ca2+ signaling were normal in mutant lymphocytes, but antigen receptor-induced NF-kappaB activation was absent. Thus, Bcl10 functions as a positive regulator of lymphocyte proliferation that specifically connects antigen receptor signaling in B and T cells to NF-kappaB activation.

Knockout mice: a paradigm shift in modern immunology.

In the past decade, advances in genetic engineering and mouse knockout technology have transformed our understanding of the immune system. In particular, new perspectives on T-cell development, co-stimulation and activation have emerged from the study of single and multiple gene-knockout animals, as well as from conditional knockout and 'knock-in' mutants. Analysis of these animals has clarified important intracellular signalling pathways and has shed light on the regulatory mechanisms that govern normal immune responses and autoimmunity.

Cbl-b is a negative regulator of receptor clustering and raft aggregation in T cells.

Stimulation of T cells via the antigen and costimulatory receptors leads to the organization of a supramolecular activation cluster called the immune synapse. We report that loss of the molecular adaptor Cbl-b in T cells frees antigen receptor-triggered receptor clustering, lipid raft aggregation, and sustained tyrosine phosphorylation from the requirement for CD28 costimulation. Introduction of the cbl-b mutation into a vav1-/- background relieved the functional defects of vav1-/- T cells and caused spontaneous autoimmunity. Wiscott Aldrich Syndrome protein (WASP) was found to be essential for deregulated proliferation and membrane receptor reorganization of cbl-b mutant T cells. Antigen receptor-triggered Ca2+ mobilization, cytokine production, and receptor clustering can be genetically uncoupled in cbl-b mutant T cells. Thus, Cbl-b functions as a negative regulator of receptor clustering and raft aggregation in T cells.

The quantity of TCR signal determines positive selection and lineage commitment of T cells.

It is generally accepted that the avidity of TCR for self Ag/MHC determines the fate of immature thymocytes. However, the contribution of the quantity of TCR signal to T cell selection has not been well established, particularly in vivo. To address this issue, we analyzed DO-TCR transgenic CD3zeta-deficient (DO-Tg/zetaKO) mice in which T cells have a reduced TCR on the cell surface. In DO-Tg/zetaKO mice, very few CD4 single positive (SP) thymocytes developed, indicating that the decrease in TCR signaling resulted in a failure of positive selection of DO-Tg thymocytes. Administration of the peptide Ag to DO-Tg/zetaKO mice resulted in the generation of functional CD4 SP mature thymocytes in a dose-dependent manner, and, unexpectedly, DO-Tg CD8 SP cells emerged at lower doses of Ag. TCR signal-dependent, sequential commitment from CD8(+) SP to CD4(+) SP was also shown in a class I-restricted TCR-Tg system. These in vivo analyses demonstrate that the quantity of TCR signal directly determines positive and negative selection, and further suggest that weak signal directs positively selected T cells to CD8 lineage and stronger signal to CD4 lineage.