Activation of p38 mitogen-activated protein kinase in vivo selectively induces apoptosis of CD8(+) but not CD4(+) T cells

CD4(+) and CD8(+) T cells play specific roles during an immune response. Different molecular mechanisms could regulate the proliferation, death, and effector functions of these two subsets of T cells. The p38 mitogen-activated protein (MAP) kinase pathway is induced by cytokines and environmental stress and has been associated with cell death and cytokine expression. Here we report that activation of the p38 MAP kinase pathway in vivo causes a selective loss of CD8(+) T cells due to the induction of apoptosis. In contrast, activation of p38 MAP kinase does not induce CD4(+) T-cell death. The apoptosis of CD8(+) T cells is associated with decreased expression of the antiapoptotic protein Bcl-2. Regulation of the p38 MAP kinase pathway in T cells is therefore essential for the maintenance of CD4/CD8 homeostasis in the peripheral immune system. Unlike cell death, gamma interferon production is regulated by the p38 MAP kinase pathway in both CD4(+) and CD8(+) T cells. Thus, specific aspects of CD4(+) and CD8(+) T-cell function are differentially controlled by the p38 MAP kinase signaling pathway.     

Molecular analysis of sunitinib resistant renal cell carcinoma cells after sequential treatment with RAD001 (everolimus) or sorafenib

Sequential application of target drugs is standard procedure after renal cell carcinoma (RCC) patients develop resistance. To optimize the sequence, antitumour effects of the mTOR inhibitor RAD001 or the tyrosine kinase inhibitor (TKI) sorafenib on RCC cells with acquired resistance to the TKI sunitinib was evaluated. RCC cells were exposed to 1 μM sunitinib for 24 hrs (as control) and for 8 weeks (to induce resistance) and then switched to RAD001 (5 nM) or sorafenib (5 μM) for a further 8 weeks. Tumour cell growth, cell cycle progression, cell cycle regulating proteins and intracellular signalling were then investigated. Short‐term application of sunitinib (24 hrs) induced cell growth blockade with accumulation in the G2/M phase. RCC cells became resistant to sunitinib after 8 weeks, demonstrated by accelerated cell growth along with enhanced cdk1, cdk2, loss of p27, activation of Akt, Rictor and Raptor. Switching to sorafenib only slightly reduced growth of the sunitinib resistant RCC cells and molecular analysis indicated distinct cross‐resistance. In contrast, full response was achieved when the cancer cells were treated with RAD001. p19 and p27 strongly increased, phosphorylated Akt, Rictor and Raptor decreased and the tumour cells accumulated in G0/G1. It is concluded that an mTOR‐inhibitor for second‐line therapy could be the strategy of choice after first‐line sunitinib failure.     

Exposure of normal monocyte-derived dendritic cells to human immunodeficiency virus type-1 particles leads to the induction of apoptosis in co-cultured CD4+ as well as CD8+ T cells

The depletion of immune T cells by human immunodeficiency virus type-1 (HIV-1) infection is a major mechanism involved in the pathogenesis of AIDS. Here, we examined a possible effector function of blood monocyte-derived dendritic cells (DCs) to induce apoptosis in bystander CD4+ and CD8+ T cells. The DCs were generated by culturing monocytes in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4. The DCs exposed to HIV-1 particles were co-cultured with healthy donor-derived blood T cells at a ratio of 1:20. Analyses by percent cell mortality, staining with propidium iodide and reactivity with Annexin V revealed the induction of apoptosis in both CD4+ and CD8+ target T cells. Further, this apoptosis occurred without stimulation with mitogens when the cell cycle of target T cells shifted from G0 to G1, probably due to the mitogenic effect of the DCs. Thus, induction of apoptosis in both CD4+ and CD8+ T cells occurred via interaction with DCs adsorbed with HIV-1 particles.     

Constitutive caspase activation and impaired death-inducing signaling complex formation in CD95-resistant,long-term activated,antigen-specific T cells

Elimination of T cells during an immune response is mediated by activation-induced cell death (AICD) and CD95-mediated apoptosis. Chronic graft-vs-host disease and T cell-mediated autoimmune diseases are caused by the persistence of activated T cells that escaped tolerance induction by deletion or silencing. To mimic the in vivo situation of long-term activated T cells, we generated an in vitro system using HLA-A1-specific T cells, weekly restimulated by Ag. While short-term activated T cells (two to five rounds of stimulation) were CD95 sensitive and susceptible to AICD, T cells stimulated more than eight times acquired constitutive CD95 resistance and exhibited reduced AICD. Phenotypically, these long-term activated T cells could be identified as effector/memory T cells. The expression of the proforms of the CD95 receptor initiator caspases, caspase-8 and -10, and the effector caspase-3 was strongly decreased in these cells, and only active caspase fragments were detected. In contrast to short-term activated T cells, constitutive CD95 receptor clustering was observed on the cell surface, and caspase-8 was bound to the CD95 receptor in the absence of receptor triggering. After further cross-linking of CD95, additional formation of the death-inducing signaling complex (DISC) was strongly impaired. Reduced DISC formation in long-term activated T cells was associated with the loss of PTEN expression and the increased phosphorylation of protein kinase B. Inhibitors of phosphoinositol 3-kinase restored CD95 sensitivity and DISC formation in long-term activated T cells. These data suggest that defective CD95 signaling in effector/memory T cells may contribute to the apoptosis resistance toward physiological stimuli in T cells mediating tissue destruction in vivo.     

Low‐dose angiostatic tyrosine kinase inhibitors improve photodynamic therapy for cancer: lack of vascular normalization

Photodynamic therapy (PDT) is an effective clinical treatment for a number of different cancers. PDT can induce hypoxia and inflammation, pro‐angiogenic side effects, which may counteract its angio‐occlusive mechanism. The combination of PDT with anti‐angiogenic drugs offers a possibility for improved anti‐tumour outcome. We used two tumour models to test the effects of the clinically approved angiostatic tyrosine kinase inhibitors sunitinib, sorafenib and axitinib in combination with PDT, and compared these results with the effects of bevacizumab, the anti‐VEGF antibody, for the improvement of PDT. Best results were obtained from the combination of PDT and low‐dose axitinib or sorafenib. Molecular analysis by PCR revealed that PDT in combination with axitinib suppressed VEGFR‐2 expression in tumour vasculature. Treatment with bevacizumab, although effective as monotherapy, did not improve PDT outcome. In order to test for tumour vessel normalization effects, axitinib was also applied prior to PDT. The absence of improved PDT outcome in these experiments, as well as the lack of increased oxygenation in axitinib‐treated tumours, suggests that vascular normalization did not occur. The current data imply that there is a future for certain anti‐angiogenic agents to further improve the efficacy of photodynamic anti‐cancer therapy.     

Specific elimination of effector memory CD4+ T cells due to enhanced Fas signaling complex formation and association with lipid raft microdomains

Elimination of autoreactive CD4⁺ T cells through the death receptor Fas/CD95 is an important mechanism of immunological self-tolerance. Fas deficiency results in systemic autoimmunity, yet does not affect the kinetics of T-cell responses to acute antigen exposure or infection. Here we show that Fas and TCR-induced apoptosis are largely restricted to CD4⁺ T cells with an effector memory phenotype (effector memory T cells (T_EM)), whereas central memory and activated naïve CD4⁺ T cells are relatively resistant to both. Sensitivity of T_EM to Fas-induced apoptosis depends on enrichment of Fas in lipid raft microdomains, and is linked to more efficient formation of the Fas death-inducing signaling complex. These results explain how Fas can cull T cells reactive against self-antigens without affecting acute immune responses. This work also identifies Fas-induced apoptosis as a possible immunotherapeutic strategy to eliminate T_EM linked to the pathogenesis of a number of autoimmune diseases.     

Gene expression in antigen-specific CD8+ T cells during viral infection

Following infection with intracellular pathogens, Ag-specific CD8(+) T cells become activated and begin to proliferate. As these cells become activated, they elaborate effector functions including cytokine production and cytolysis. After the infection has been cleared, the immune system returns to homeostasis through apoptosis of the majority of the Ag-specific effector cells. The surviving memory cells can persist for extended periods and provide protection against reinfection. Little is known about the changes in gene expression as Ag-specific cells progress through these stages of development, i.e., naive to effector to memory. Using recombinant MHC class I tetramers, we isolated Ag-specific CD8(+) T cells from mice infected with lymphocytic choriomeningitis virus at various time points and performed semiquantitative RT-PCR. We examined expression of: 1) genes involved in cell cycle control, 2) effector and regulatory functions, and 3) susceptibility to apoptosis. We found that Ag-specific CD8(+) memory T cells contain high steady-state levels of Bcl-2, BAX:, IFN-gamma, and lung Kruppel-like factor (LKLF), and decreased levels of p21 and p27 mRNA. Moreover, the pattern of gene expression between naive and memory cells is distinct and suggests that these two cell types control susceptibility to apoptosis through different mechanisms.     

Immune modulation of effector CD4+ and regulatory T cell function by sorafenib in patients with hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a difficult to treat cancer characterized by poor tumor immunity with only one approved systemic drug, sorafenib. If novel combination treatments are to be developed with immunological agents, the effects of sorafenib on tumor immunity are important to understand. In this study, we investigate the impact of sorafenib on the CD4+CD25? effector T cells (Teff) and CD4+CD25+ regulatory T cells (Tregs) from patients with HCC. We isolated Teff and Treg from peripheral mononuclear cells of HCC patients to determine immune reactivity by thymidine incorporation, ELISA and flow cytometry. Teff cultured alone or with Treg were supplemented with different concentrations of sorafenib. The effects of sorafenib on Teff responses were dose-dependent. Pharmacologic doses of sorafenib decreased Teff activation by down regulating CD25 surface expression. In contrast, sub-pharmacologic concentrations of sorafenib resulted in Teff activation. These low doses of sorafenib in the Teff cultures led to a significant increase in Teff proliferation, IL2 secretion and up-regulation of CD25 expression on the cell surface. In addition, low doses of sorafenib in the suppression Teff/Treg cocultures restored Teff responses by eliminating Treg suppression. The loss of Treg suppressive function correlated with an increase in IL2 and IL6 secretion. Our findings show that sub-pharmacologic doses of sorafenib impact subsets of T cells differently, selectively increasing Teff activation while blocking Treg function. In conclusion, this study describes novel immune activating properties of low doses of sorafenib by promoting immune responsiveness in patients with HCC.     

Genetic Deletion of Mst1 Alters T Cell Function and Protects against Autoimmunity

Mammalian sterile 20-like kinase 1 (Mst1) is a MAPK kinase kinase kinase which is involved in a wide range of cellular responses, including apoptosis, lymphocyte adhesion and trafficking. The contribution of Mst1 to Ag-specific immune responses and autoimmunity has not been well defined. In this study, we provide evidence for the essential role of Mst1 in T cell differentiation and autoimmunity, using both genetic and pharmacologic approaches. Absence of Mst1 in mice reduced T cell proliferation and IL-2 production in vitro, blocked cell cycle progression, and elevated activation-induced cell death in Th1 cells. Mst1 deficiency led to a CD4⁺ T cell development path that was biased toward Th2 and immunoregulatory cytokine production with suppressed Th1 responses. In addition, Mst1^−/− B cells showed decreased stimulation to B cell mitogens in vitro and deficient Ag-specific Ig production in vivo. Consistent with altered lymphocyte function, deletion of Mst1 reduced the severity of experimental autoimmune encephalomyelitis (EAE) and protected against collagen-induced arthritis development. Mst1^−/− CD4⁺ T cells displayed an intrinsic defect in their ability to respond to encephalitogenic antigens and deletion of Mst1 in the CD4⁺ T cell compartment was sufficient to alleviate CNS inflammation during EAE. These findings have prompted the discovery of novel compounds that are potent inhibitors of Mst1 and exhibit desirable pharmacokinetic properties. In conclusion, this report implicates Mst1 as a critical regulator of adaptive immune responses, Th1/Th2-dependent cytokine production, and as a potential therapeutic target for immune disorders.     

Distinct mechanisms control human naive and antigen-experienced CD8+ T lymphocyte proliferation

Human Ag-specific CD8(+) T lymphocytes are heterogeneous and include functionally distinct populations. In this study, we report that at least two distinct mechanisms control the expansion of circulating naive, memory, and effector CD8(+) T lymphocytes when exposed to mitogen or Ag stimulation. The first one leads to apoptosis and occurs shortly after in vitro stimulation. Susceptibility to cell death is prominent among primed T cell subsets, and it is inversely correlated with the size of the ex vivo Bcl-2(high) population within these subsets. Importantly, the Bcl-2(high) phenotype is associated to the proportion of responsive CD8(+) T cells, independently of their differentiation stage. The second one depends on the expression of newly synthesized cyclin-dependent kinase inhibitor p16(INK4a) that occurs in a significant fraction of T cells that had been actively cycling, leading to their cell cycle arrest upon stimulation. Strikingly, accumulation of p16(INK4a) protein preferentially occurs in naive as opposed to primed derived T lymphocytes and is not related to apoptosis. Significant levels of p16 are readily detectable in a small number of ex vivo CD8(+) T cells. Our observations reveal that activation-induced p16 expression represents an alternative process to apoptosis, limiting the proliferation potential of activated naive derived T lymphocytes.     

Tailored Immune Responses: Novel Effector Helper T Cell Subsets in Protective Immunity

Differentiation of naïve CD4⁺ cells into functionally distinct effector helper T cell subsets, characterised by distinct “cytokine signatures,” is a cardinal strategy employed by the mammalian immune system to efficiently deal with the rapidly evolving array of pathogenic microorganisms encountered by the host. Since the T_H1/T_H2 paradigm was first described by Mosmann and Coffman, research in the field of helper T cell biology has grown exponentially with seven functionally unique subsets having now been described. In this review, recent insights into the molecular mechanisms that govern differentiation and function of effector helper T cell subsets will be discussed in the context of microbial infections, with a focus on how these different helper T cell subsets orchestrate immune responses tailored to combat the nature of the pathogenic threat encountered.     

Immune response to pre-erythrocytic stages of malaria parasites

Immunization with radiation-attenuated Plasmodium spp. sporozoites induces sterile protective immunity against parasite challenge. This immunity is targeted primarily against the intrahepatic parasite and appears to be sustained long term even in the absence of sporozoite exposure. It is mediated by multifactorial mechanisms, including T cells directed against parasite antigens expressed in the liver stage of the parasite life cycle and antibodies directed against sporozoite surface proteins. In rodent models, CD8+ T cells have been implicated as the principal effector cells, and IFN-gamma as a critical effector molecule. IL-4 secreting CD4+ T cells are required for induction of the CD8+ T cell responses, and Th1 CD4+ T cells provide help for optimal CD8+ T cell effector activity. Components of the innate immune system, including gamma-delta T cells, natural killer cells and natural killer T cells, also play a role. The precise nature of pre-erythrocytic stage immunity in humans, including the contribution of these immune responses to the age-dependent immunity naturally acquired by residents of malaria endemic areas, is still poorly defined. The importance of immune effector targets at the pre-erythrocytic stage of the parasite life cycle is highlighted by the fact that infection-blocking immunity in humans rarely, if ever, occurs under natural conditions. Herein, we review our current understanding of the molecular and cellular aspects of pre-erythrocytic stage immunity.     

Potentiating the Efficacy of Molecular Targeted Therapy for Hepatocellular Carcinoma by Inhibiting the Insulin-Like Growth Factor Pathway

Insulin-like growth factor (IGF) signaling pathway is an important regulatory mechanism of tumorigenesis and drug resistance in many cancers. The present study explored the potential synergistic effects between IGF receptor (IGFR) inhibition and other molecular targeted agents (MTA) in HCC cells. HCC cell lines (Hep3B, PLC5, and SK-Hep1) and HUVECs were tested. The MTA tested included sorafenib, sunitinib, and the IGFR kinase inhibitor NVP-AEW541. The potential synergistic antitumor effects were tested by median dose effect analysis and apoptosis assay in vitro and by xenograft models in vivo. The activity and functional significance of pertinent signaling pathways and expression of apoptosis-related proteins were measured by RNA interference and Western blotting. We found that IGF can activate IGFR and downstream AKT signaling activities in all the HCC cells tested, but the growth-stimulating effect of IGF was most prominent in Hep3B cells. NVP-AEW541 can abrogate IGF-induced activation of IGFR and AKT signaling in HCC cells. IGF can increase the resistance of HCC cells to sunitinib. The apoptosis-inducing effects of sunitinib, but not sorafenib, were enhanced when IGFR signaling activity was inhibited by NVP-AEW541 or IGFR knockdown. Chk2 kinase activation was found contributory to the synergistic anti-tumor effects between sunitinib and IGFR inhibition. Our data indicate that the apoptosis-potentiating effects of IGFR inhibition for HCC may be drug-specific. Combination therapy of IGFR inhibitors with other MTA may improve the therapeutic efficacy in HCC.     

CD7 is a differentiation marker that identifies multiple CD8 T cell effector subsets

The adaptive immune response of human CD8 T cells to invading pathogens involves the differentiation of naive cells into memory and effector cells. However, the lineage relationship between memory and effector cells and the differentiation of CD8 T cells into distinct subsets of effector cell subpopulations are subjects of considerable debate. CD7 identifies three populations of CD8 T cells: CD7 high (CD7(high)), low (CD7(low)), and negative (CD7(neg)) that translate into subsets with distinct functional properties. The CD7(high) subset contains naive and memory cells and the CD7(low) and CD7(neg) subsets contain effector cells. The effector cells can functionally be divided into cytokine-secreting effector CD8 T cells and lytic effector CD8 T cells. These data provide a model of human CD8 T cell differentiation in which specialized distinct subpopulations can be identified by expression of CD7.     

Distinct subsets of accessory cells activate Thy-1+ triple negative (CD3-, CD4-, CD8-) cells and Th-1 delayed-type hypersensitivity effector T cells.

The SJL strain of mice possess a unique developmental delay in the ability to exhibit delayed-type hypersensitivity (DTH) responses after immunization with a wide variety of Ag. Similar to other models of DTH, the adoptive transfer of syngeneic Ag-pulsed macrophages from DTH-responsive mice into these DTH-unresponsive mice results in the activation of Ag-specific, CD4+ DTH effector Th1 T cells. The absence of other defects in APC-dependent immune responses indicate that the macrophages is the sole APC required for the induction of DTH effector T cells in SJL mice. The defect occurs during the sensitization phase of the DTH response; however, it has not been determined whether a Th cell, which is required for the induction of CD4+ DTH effector T cells, was present in the DTH unresponsive SJL mice. In this study, we have determined that the Thy-1+ helper cell is induced upon Ag stimulation of nonresponder mice and present evidence for the existence of an accessory cell distinct from the macrophage that induces CD4+ DTH effector T cells. Our data indicate that CD4+ DTH effector T cells are induced in an Ag-specific and MHC-restricted manner by an adherent macrophage that expresses the Mac-1+, Mac-2-, Mac-3+, I-A+ phenotype. Adoptive transfer of as few as 100 of the Mac-1+, Mac-2-, or Mac-3+ subsets from DTH responsive donors to DTH unresponsive recipients is able to overcome the DTH deficit. The activation of CD4+ DTH effector T cells in the SJL mouse cells also requires a Thy-1+, Lyt-1+, CD3-, CD4-, CD8-, helper cell. In contrast to the Mac-1+, Mac-3+, I-A+ accessory cell, this helper cell requires an adherent, irradiation resistant, accessory cell that expresses the Mac-1+, Mac-2-, Mac-3-, I-A- surface phenotype for activation. Further, the interaction between this accessory cell and the Thy-1+ helper cell is neither Ag-specific nor MHC restricted. This is the first demonstration of an accessory cell requirement for the Thy-1+, Lyt-1+, B220-, CD4-, CD8-, CD3- DTH Th cell. These data indicate that the activation of the triple negative helper cells and subsequent activation of the CD4+ effector T cells are regulated by two distinct macrophage subpopulations.     

Profiling calcium signals of in vitro polarized human effector CD4+ T cells

Differentiation of naïve CD4⁺ T cells into effector subtypes with distinct cytokine profiles and physiological roles is a tightly regulated process, the imbalance of which can lead to an inadequate immune response or autoimmune disease. The crucial role of Ca²⁺ signals, mainly mediated by the store operated Ca²⁺ entry (SOCE) in shaping the immune response is well described. However, it is unclear if human effector CD4⁺ T cell subsets show differential Ca²⁺ signatures in response to different stimulation methods. Herein, we provide optimized in vitro culture conditions for polarization of human CD4⁺ effector T cells and characterize their SOCE following both pharmacological store depletion and direct T-cell receptor (TCR) activation. Moreover, we measured whole cell Ca²⁺ release activated Ca²⁺ currents (I_CRAC) and investigated whether the observed differences correlate to the expression of CRAC genes. Our results show that Ca²⁺ profiles of helper CD4⁺ Th1, Th2 and Th17 are distinct and in part shaped by the intensity of stimulation. Regulatory T cells (Treg) are unique being the subtype with the most prominent SOCE response. Analysis of in vivo differentiated Treg unraveled the role of differential expression of ORAI2 in fine-tuning signals in Treg vs. conventional CD4⁺ T cells.