Protein kinase C delta localizes to secretory lysosomes in CD8+ CTL and directly mediates TCR signals leading to granule exocytosis-mediated cytotoxicity

Lytic granule exocytosis is the major effector function used by CD8(+) CTL in response to intracellular pathogens and tumors. Despite recent progress in the field, two important aspects of this cytotoxic mechanism remain poorly understood. First, TCR-signaling pathway(s) that selectively induces granule exocytosis in CTL has not been defined to date. Second, it is unclear how Ag receptor-induced signals are converted into mobilization of lytic granules. We recently demonstrated that protein kinase C delta (PKC delta) selectively regulates TCR-induced lytic granule polarization in mouse CD8(+) CTL. To better understand how PKC delta facilitates granule movement, here we studied dynamics of intracellular localization of PKC delta in living CD8(+) CTL. Strikingly, we found that PKC delta localizes to the secretory lysosomes and polarizes toward immunological synapse during the process of target cell killing. Also, biochemical and structure-function studies demonstrated that upon TCR ligation, PKC delta becomes rapidly phosphorylated on the activation loop and regulates granule exocytosis in a kinase-dependent manner. Altogether, our current studies provide new insights concerning the regulation of TCR-induced lytic granule exocytosis by revealing novel intracellular localization of PKC delta, providing the first example of colocalization of a kinase with secretory lysosomes in CD8(+) CTL and demonstrating that PKC delta directly transduces TCR signals leading to polarized granule secretion.     

Protein kinase Cdelta is activated by Shiga toxin and regulates its transport

Protein kinase C (PKC) isozymes regulate different vesicular trafficking steps in the recycling or degradative pathways. However, a possible role of these kinases in the retrograde pathway from endosomes to the Golgi complex has previously not been investigated. We report here the involvement of a specific PKC isozyme, PKCdelta, in the intracellular transport of the glycolipid-binding Shiga toxin (Stx), which utilizes the retrograde pathway to intoxicate cells. Upon binding to cells, Stx was shown to specifically activate PKCdelta and not PKCalpha. The involvement of PKCdelta and PKCalpha in the retrograde transport of Stx was then monitored biochemically and by immunofluorescence after inhibition or depletion of the isozymes. PKCdelta, but not PKCalpha, was shown to selectively regulate the endosome-to-Golgi transport of StxB. Upon inhibition or knockdown of PKCdelta, StxB molecules colocalized less with giantin and more with EEA1, indicating that the molecules were accumulated in endosomes, unable to reach the Golgi complex. The inhibition of Golgi transport of Stx was reflected by a strong reduction in the toxic effect, demonstrating that transport of Stx to the cytosol is dependent on PKCdelta activity. These results are in agreement with our previous data, which show that Stx is able to stimulate its own transport.     

Human CD8+ CTL specific for the mycobacterial major secreted antigen 85A

The role of CD8(+) CTL in protection against tuberculosis in human disease is unclear. In this study, we stimulated the peripheral blood mononuclear cells of bacillus Calmette-Guérin (BCG)-vaccinated individuals with live Mycobacterium bovis BCG bacilli to establish short-term cell lines and then purified the CD8(+) T cells. A highly sensitive enzyme-linked immunospot (ELISPOT) assay for single cell IFN-gamma release was used to screen CD8(+) T cells with overlapping peptides spanning the mycobacterial major secreted protein, Ag85A. Three peptides consistently induced a high frequency of IFN-gamma responsive CD8(+) T cells, and two HLA-A*0201 binding motifs, P(48-56) and P(242-250), were revealed within the core sequences. CD8(+) T cells responding to the 9-mer epitopes were visualized within fresh blood by ELISPOT using free peptide or by binding of HLA-A*0201 tetrameric complexes. The class I-restricted CD8(+) T cells were potent CTL effector cells that efficiently lysed an HLA-A2-matched monocyte cell line pulsed with peptide as well as autologous macrophages infected with Mycobacterium tuberculosis or recombinant vaccinia virus expressing the whole Ag85A protein. Tetramer assays revealed a 6-fold higher frequency of peptide-specific T cells than IFN-gamma ELISPOT assays, indicating functional heterogeneity within the CD8(+) T cell population. These results demonstrate a previously unrecognized, MHC class I-restricted, CD8(+) CTL response to a major secreted Ag of mycobacteria and supports the use of Ag85A as a candidate vaccine against tuberculosis.     

Protein kinase C-delta mediates adenosine A3 receptor-induced delayed cardioprotection in mouse

We investigated the role of protein kinase C in adenosine A3 receptor (A3AR)-induced delayed cardioprotection in the mouse heart. Mice were treated with selective A3AR agonist N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA). Twenty-four hours later, hearts were perfused in the Langendorff mode and subjected to 30 min of global ischemia and 30 min of reperfusion. Infarct size was determined by computer morphometry of tetrazolium-stained sections, and ventricular function was monitored by inserting a fluid-filled balloon into the left ventricle (LV). Chelerythrine chloride (CHE, 5.0 mg/kg) and rottlerin (Rot, 0.3 mg/kg) were given 30 min before IB-MECA to block total and PKC-delta isoforms, respectively. IB-MECA caused postischemic reduction in necrosis and improvement in ventricular function, which was abolished by CHE. Western blot analysis demonstrated translocation of the PKC-delta isoform but not the alpha, epsilon, xi, eta isoform(s) from cytoplasm to the membrane fraction after 30 min of IB-MECA administration. A3AR antagonist MRS-1191 and CHE blocked the translocation of PKC-delta. Furthermore, IB-MECA-induced increase in nuclear factor-kappaB binding was diminished by CHE. These results provide direct evidence of an essential role of PKC, and more specifically, PKC-delta in A3AR-induced delayed cardioprotection.     

Cytotoxic CD4+ T cell responses to EBV contrast with CD8 responses in breadth of lytic cycle antigen choice and in lytic cycle recognition

EBV, a B lymphotropic herpesvirus, encodes two immediate early (IE)-, >30 early (E)-, and >30 late (L)-phase proteins during its replication (lytic) cycle. Despite this, lytic Ag-induced CD8 responses are strongly skewed toward IE and a few E proteins only, all expressed before HLA I presentation is blocked in lytically infected cells. For comparison, we examined CD4(+) T cell responses to eight IE, E, or L proteins, screening 14 virus-immune donors to overlapping peptide pools in IFN-γ ELISPOT assays, and established CD4(+) T cell clones against 12 defined epitopes for target-recognition assays. We found that the lytic Ag-specific CD4(+) T cell response differs radically from its CD8 counterpart in that it is widely distributed across IE, E, and L Ag targets, often with multiple reactivities detectable per donor and with IE, E, or L epitope responses being numerically dominant, and that all CD4(+) T cell clones, whether IE, E, or L epitope-specific, show strong recognition of EBV-transformed B cell lines, despite the lines containing only a small fraction of lytically infected cells. Efficient recognition occurs because lytic Ags are released into the culture and are acquired and processed by neighboring latently infected cells. These findings suggested that lytic Ag-specific CD4 responses are driven by a different route of Ag display than drives CD8 responses and that such CD4 effectors could be therapeutically useful against EBV-driven lymphoproliferative disease lesions, which contain similarly small fractions of EBV-transformed cells entering the lytic cycle.     

Imaging of lytic granule exocytosis in CD8+ cytotoxic T lymphocytes reveals a modified form of full fusion

Here we imaged the exocytosis of lytic granules from human CD8⁺ cytotoxic T lymphocytes using rapid total internal reflection microscopy, Lamp-1 tagged with mGFP to follow the fate of the lytic granule membrane, and granzyme A, granzyme B or serglycin tagged with mRFP to follow the fate of lytic granule cargo. Lytic granules were released by full fusion with the plasma membrane, such that the entire granule content for all three cargos visualized was released on a subsecond time scale. The behavior of GFP-Lamp-1 was, however, more complex. While it entered the plasma membrane in all cases, the extent to which it then diffused away from the site of exocytosis varied from nearly complete to highly restricted. Finally, the diffusion properties upon release of the three cargos examined put an upper limit on the size of the macromolecular complex of granzyme and serglycin that is presented to the target cell.     

Activation of dendritic cells that cross-present tumor-derived antigen licenses CD8+ CTL to cause tumor eradication

The fate of naive CD8(+) T cells is determined by the environment in which they encounter MHC class I presented peptide Ags. The manner in which tumor Ags are presented is a longstanding matter of debate. Ag presentation might be mediated by tumor cells in tumor draining lymph nodes or via cross-presentation by professional APC. Either pathway is insufficient to elicit protective antitumor immunity. We now demonstrate using a syngeneic mouse tumor model, expressing an Ag derived from the early region 1A of human adenovirus type 5, that the inadequate nature of the antitumor CTL response is not due to direct Ag presentation by the tumor cells, but results from presentation of tumor-derived Ag by nonactivated CD11c(+) APC. Although this event results in division of naive CTL in tumor draining lymph nodes, it does not establish a productive immune response. Treatment of tumor-bearing mice with dendritic cell-stimulating agonistic anti-CD40 mAb resulted in systemic efflux of CTL with robust effector function capable to eradicate established tumors. For efficacy of anti-CD40 treatment, CD40 ligation of host APC is required because adoptive transfer of CD40-proficient tumor-specific TCR transgenic CTL into CD40-deficient tumor-bearing mice did not lead to productive antitumor immunity after CD40 triggering in vivo. CpG and detoxified LPS (MPL) acted similarly as agonistic anti-CD40 mAb with respect to CD8(+) CTL efflux and tumor eradication. Together these results indicate that dendritic cells, depending on their activation state, orchestrate the outcome of CTL-mediated immunity against tumors, leading either to an ineffective immune response or potent antitumor immunity.     

Development of antigen-specific CD8+ CTL in MHC class I-deficient mice through CD4 to CD8 conversion

CD8+ CTL are the predominant tumoricidal effector cells. We find, however, that MHC class I-deficient mice depleted of CD8+ T cells are able to mount an effective antitumor immunity after immunization with fused dendritic/tumor cells. Such immunity appears to be mediated by the generation of phenotypic and functional CD8+ CTL through CD4+ to CD8+ conversion, which we have demonstrated at the single cell level. CD4+ to CD8+ conversion depends on effective in vivo activation and is promoted by CD4+ T cell proliferation. The effectiveness of this process is shown by the generation of antitumor immunity through adoptive transfer of primed CD4 T cells to provide protection against tumor cell challenge and to eliminate established pulmonary metastases.     

MHC recognition by hapten-specific HLA-A2-restricted CD8+ CTL

T cell recognition by peptide-specific alphabeta TCRs involves not only recognition of the peptide, but also recognition of multiple molecular features on the surface of the MHC molecule to which the peptide has been bound. We have previously shown that TCRs that are specific for five different peptides presented by HLA-A2 recognize similar molecular features on the surface of the alpha1 and alpha2 helices of the HLA-A2 molecule. We next asked whether these same molecular features of the HLA-A2 molecule would be recognized by hapten-specific HLA-A2-restricted TCRs, given that hapten-specific T cells frequently show reduced MHC dependence/restriction. The results show that a panel of CD8+ CTL that are specific for the hapten DNP bound to two different peptides presented by HLA-A2 do the following: 1) show stringent MHC restriction, and 2) are largely affected by the same mutations on the HLA-A2 molecule that affected recognition by peptide-specific CTL. A small subset of this panel of CD8+ CTL can recognize a mutant HLA-A2 molecule in the absence of hapten. These data suggest that TCR recognition of a divergent repertoire of ligands presented by HLA-A2 is largely dependent upon common structural elements in the central portion of the peptide-binding site.     

Soluble MHC-peptide complexes induce rapid death of CD8+ CTL

Soluble MHC-peptide (pMHC) complexes, commonly referred to as tetramers, are widely used to enumerate and to isolate Ag-specific CD8(+) CTL. It has been noted that such complexes, as well as microsphere- or cell-associated pMHC molecules compromise the functional integrity of CTL, e.g., by inducing apoptosis of CTL, which limits their usefulness for T cell sorting or cloning. By testing well-defined soluble pMHC complexes containing linkers of different length and valence, we find that complexes comprising short linkers (i.e., short pMHC-pMHC distances), but not those containing long linkers, induce rapid death of CTL. This cell death relies on CTL activation, the coreceptor CD8 and cytoskeleton integrity, but is not dependent on death receptors (i.e., Fas, TNFR1, and TRAILR2) or caspases. Within minutes of CTL exposure to pMHC complexes, reactive oxygen species emerged and mitochondrial membrane depolarized, which is reminiscent of caspase-independent T cell death. The morphological changes induced during this rapid CTL death are characteristic of programmed necrosis and not apoptosis. Thus, soluble pMHC complexes containing long linkers are recommended to prevent T cell death, whereas those containing short linkers can be used to eliminate Ag-specific CTL.     

Protein kinase Cdelta regulates endothelial nitric oxide synthase expression via Akt activation and nitric oxide generation

In this study, we explore the roles of the delta isoform of PKC (PKCdelta) in the regulation of endothelial nitric oxide synthase (eNOS) activity in pulmonary arterial endothelial cells isolated from fetal lambs (FPAECs). Pharmacological inhibition of PKCdelta with either rottlerin or with the peptide, deltaV1-1, acutely attenuated NO production, and this was associated with a decrease in phosphorylation of eNOS at Ser1177 (S1177). The chronic effects of PKCdelta inhibition using either rottlerin or the overexpression of a dominant negative PKCdelta mutant included the downregulation of eNOS gene expression that was manifested by a decrease in both eNOS promoter activity and protein expression after 24 h of treatment. We also found that PKCdelta inhibition blunted Akt activation as observed by a reduction in phosphorylated Akt at position Ser473. Thus, we conclude that PKCdelta is actively involved in the activation of Akt. To determine the effect of Akt on eNOS signaling, we overexpressed a dominant negative mutant of Akt and determined its effect of NO generation, eNOS expression, and phosphorylation of eNOS at S1177. Our results demonstrated that Akt inhibition was associated with decreased NO production that correlated with reduced phosphorylation of eNOS at S1177, and decreased eNOS promoter activity. We next evaluated the effect of endogenously produced NO on eNOS expression by incubating FPAECs with the eNOS inhibitor 2-ethyl-2-thiopseudourea (ETU). ETU significantly inhibited NO production, eNOS promoter activity, and eNOS protein levels. Together, our data indicate involvement of PKCdelta-mediated Akt activation and NO generation in maintaining eNOS expression.     

B cell receptor-induced cAMP-response element-binding protein activation in B lymphocytes requires novel protein kinase Cdelta

The cAMP-response element-binding protein (CREB) is activated by phosphorylation on Ser-133 and plays a key role in the proliferative and survival responses of mature B cells to B cell receptor (BCR) signaling. The signal link between the BCR and CREB activation depends on a phorbol ester (phorbol 12-myristate 13-acetate)-sensitive protein kinase C (PKC) activity and not protein kinase A or calmodulin kinase; however, the identity and role of the PKC(s) activity has not been elucidated. We found the novel PKCdelta (nPKCdelta) activator bistratene A is sufficient to induce CREB phosphorylation in murine splenic B cells. The pharmacological inhibitor G?6976, which targets conventional PKCs and PKCmu, has no effect on CREB phosphorylation, whereas the nPKCdelta inhibitor rottlerin blocks CREB phosphorylation following BCR cross-linking. Bryostatin 1 selectively prevents nPKCdelta depletion by phorbol 12-myristate 13-acetate when coapplied, coincident with protection of BCR-induced CREB phosphorylation. Ectopic expression of a kinase-inactive nPKCdelta blocks BCR-induced CREB phosphorylation in A20 B cells. In addition, BCR-induced CREB phosphorylation is significantly diminished in nPKCdelta-deficient splenic B cells in comparison with wild type mice. Consistent with the essential role for Bruton's tyrosine kinase and phospholipase Cgamma2 in mediating PKC activation, Bruton's tyrosine kinase- and phospholipase Cgamma2-deficient B cells display defective CREB phosphorylation by the BCR. We also found that p90 RSK directly phosphorylates CREB on Ser-133 following BCR cross-linking and is positioned downstream of nPKCdelta. Taken together, these results suggest a model in which BCR engagement leads to the phosphorylation of CREB via a signaling pathway that requires nPKCdelta and p90 RSK in mature B cells.     

PD-1 regulates self-reactive CD8+ T cell responses to antigen in lymph nodes and tissues

PD-1, an inhibitory receptor expressed on activated lymphocytes, regulates tolerance and autoimmunity. We tested the role of PD-1:PD-1 ligand (PD-L) interactions in cross-presentation and the generation and control of CD8(+) responses against self-Ag. Ag-naive PD-1(-/-) OVA-specific OT-I CD8(+) T cells exhibited exacerbated responses to cross-presented Ag in mice expressing soluble OVA under the control of the rat insulin promoter (RIP-ova(high)). Following adoptive transfer into RIP-ova(high) recipients, PD-1(-/-) OT-I T cells expanded in the pancreatic lymph node. In contrast to wild-type OT-I cells, PD-1(-/-) OT-I T cells secreted IFN-gamma and migrated into the pancreas, ultimately causing diabetes. Loss of PD-1 affected CD8(+) cells intrinsically, and did not significantly alter the responses of wild-type OT-I T cells adoptively transferred into the same RIP-ova(high) recipient mouse. PD-1:PD-L interactions also limited CD8(+) effector cells, and PD-L1 expression on parenchymal tissues protected against effector OT-I T cell attack. Finally, we found that the loss of PD-1 on effector OT-I cells lowers the threshold for Ag recognition in peripheral tissues. These findings indicate two checkpoints where PD-1 attenuates self-reactive T cell responses: presentation of self-Ag to naive self-reactive T cells by dendritic cells in the draining lymph node and reactivation of pathogenic self-reactive T cells in the target organ.     

Protein kinase C activity in mouse eggs regulates gamete membrane interaction

Gamete membrane interaction is critical to initiate the development of a new organism. The signaling pathways governing this event, however, are poorly understood. In this report, we provide the first evidence that protein kinase C activity in mouse eggs plays a crucial role in the regulation of this process. Stimulating PKC activity in mouse eggs by phorbol 12-myristate 13-acetate (PMA) drastically inhibited the egg's membrane ability to bind and fuse with sperm. Surprisingly, this significant reduction of gamete membrane interaction was also observed in eggs treated with the PKC inhibitors staurosporine and calphostin c. In further analysis, we found that while no change of egg actin cytoskeleton was detected after either PMA or calphostin c treatment, the structural morphology of egg surface microvilli was severely altered in the PMA-treated eggs, but not in the calphostin c-treated eggs. Moreover, sperm, which bound but did not fuse with the eggs treated with the anti-CD9 antibody KMC8, were liberated from the egg membrane after PMA, but not calphostin c, treatment. Taken together, these results suggest that egg PKC may be precisely balanced to regulate gamete membrane interaction in a biphasic mode, and this biphasic regulation is executed through two different mechanisms.     

MEK activity regulates negative selection of immature CD4+CD8+ thymocytes

CD4+CD8+ thymocytes are either positively selected and subsequently mature to CD4 single positive (SP) or CD8 SP T cells, or they die by apoptosis due to neglect or negative selection. This clonal selection is essential for establishing a functional self-restricted T cell repertoire. Intracellular signals through the three known mitogen-activated protein (MAP) kinase pathways have been shown to selectively guide positive or negative selection. Whereas the c-Jun N-terminal kinase and p38 MAP kinase regulate negative selection of thymocytes, the extracellular signal-regulated kinase (ERK) pathway is required for positive selection and T cell lineage commitment. In this paper, we show that the MAP/ERK kinase (MEK)-ERK pathway is also involved in negative selection. Thymocytes from newborn TCR transgenic mice were cultured with TCR/CD3epsilon-specific Abs or TCR-specific agonist peptides to induce negative selection. In the presence of the MEK-specific pharmacological inhibitors PD98059 or UO126, cell recovery was enhanced and deletion of DP thymocytes was drastically reduced. Furthermore, development of CD4 SP T cells was blocked, but differentiation of mature CD8 SP T cells proceeded in the presence of agonist peptides when MEK activity was blocked. Thus, our data indicate that the outcome between positively and negatively selecting signals is critically dependent on MEK activity.     

Memory CD8+ T cells protect dendritic cells from CTL killing

CD8(+) T cells have been shown to be capable of either suppressing or promoting immune responses. To reconcile these contrasting regulatory functions, we compared the ability of human effector and memory CD8(+) T cells to regulate survival and functions of dendritic cells (DC). We report that, in sharp contrast to the effector cells (CTLs) that kill DCs in a granzyme B- and perforin-dependent mechanism, memory CD8(+) T cells enhance the ability of DCs to produce IL-12 and to induce functional Th1 and CTL responses in naive CD4(+) and CD8(+) T cell populations. Moreover, memory CD8(+) T cells that release the DC-activating factor TNF-alpha before the release of cytotoxic granules induce DC expression of an endogenous granzyme B inhibitor PI-9 and protect DCs from CTL killing with similar efficacy as CD4(+) Th cells. The currently identified DC-protective function of memory CD8(+) T cells helps to explain the phenomenon of CD8(+) T cell memory, reduced dependence of recall responses on CD4(+) T cell help, and the importance of delayed administration of booster doses of vaccines for the optimal outcome of immunization.     

Defective proximal TCR signaling inhibits CD8+ tumor-infiltrating lymphocyte lytic function

CD8+ tumor-infiltrating lymphocytes (TIL) are severely deficient in cytolysis, a defect that may permit tumor escape from immune-mediated destruction. Because lytic function is dependent upon TCR signaling, we have tested the hypothesis that primary TIL have defective signaling by analysis of the localization and activation status of TIL proteins important in TCR-mediated signaling. Upon conjugate formation with cognate target cells in vitro, TIL do not recruit granzyme B+ granules, the microtubule-organizing center, F-actin, Wiskott-Aldrich syndrome protein, nor proline rich tyrosine kinase-2 to the target cell contact site. In addition, TIL do not flux calcium nor demonstrate proximal tyrosine kinase activity, deficiencies likely to underlie failure to fully activate the lytic machinery. Confocal microscopy and fluorescence resonance energy transfer analyses demonstrate that TIL are triggered by conjugate formation in that the TCR, p56lck, CD3zeta, LFA-1, lipid rafts, ZAP70, and linker for activation of T cells localize at the TIL:tumor cell contact site, and CD43 and CD45 are excluded. However, proximal TCR signaling is blocked upon conjugate formation because the inhibitory motif of p56lck is rapidly phosphorylated (Y505) and COOH-terminal Src kinase is recruited to the contact site, while Src homology 2 domain-containing protein phosphatase 2 is cytoplasmic. Our data support a novel mechanism explaining how tumor-induced inactivation of proximal TCR signaling regulates lytic function of antitumor T cells.