Cytotoxic T Lymphocytes Killing Research Articles

Autocrine, not paracrine, interferon-gamma gene delivery enhances ex vivo antigen-specific cytotoxic T lymphocyte stimulation and killing.

The adoptive transfer of antigen-specific cytotoxic T lymphocytes (CTL) shows promise in the treatment of cancer and infectious diseases. We utilize adeno-associated virus-(AAV-) based antigen gene-loaded dendritic cells (DCs) to stimulate such antigen-specific CTL. Yet further improvements in CTL stimulation and killing may result by gene delivery of various Th1-response interferons/cytokines, such as interferon γ (IFN-γ), as the delivered gene can continuously produce that interferon. However which immune cell type should optimally express IFN-γ is unclear as the phenotypes of both DC and T cells are enhanced by it. Here, we used AAV to compare and contrast IFN-γ gene delivery into DC or T cells, and versus the addition of exogenous IFN-γ, for stimulating carcinoembryonic antigen-(CEA-) specific CTL. It was found that AAV/IFN-γ delivery into T cells (autocrine) resulted in T cell populations with the highest CD8(+)/CD4(+) ratio, highest IFN-γ(+)/IL-4(+) ratio, highest CD69(+),CD8(+) levels, and lowest CD4(+)/CD25(+) levels, all consistent with the strongest Th1 response. Most importantly, AAV/IFN-γ transduction of T cells resulted in antigen-specific T cell populations with the highest killing capabilities, 49% above other treatments. These data strongly suggest that AAV/IFN-γ autocrine gene delivery into T cells is worthy of further study towards maximizing the generation of antigen-specific anticancer CTL killers.

Investigating CTL Mediated Killing with a 3D Cellular Automaton

Cytotoxic T lymphocytes (CTLs) are important immune effectors against intra-cellular pathogens. These cells search for infected cells and kill them. Recently developed experimental methods in combination with mathematical models allow for the quantification of the efficacy of CTL killing in vivo and, hence, for the estimation of parameters that characterize the effect of CTL killing on the target cell populations. It is not known how these population-level parameters relate to single-cell properties. To address this question, we developed a three-dimensional cellular automaton model of the region of the spleen where CTL killing takes place. The cellular automaton model describes the movement of different cell populations and their interactions. Cell movement patterns in our cellular automaton model agree with observations from two-photon microscopy. We find that, despite the strong spatial nature of the kinetics in our cellular automaton model, the killing of target cells by CTLs can be described by a term which is linear in the target cell frequency and saturates with respect to the CTL levels. Further, we find that the parameters describing CTL killing on the population level are most strongly impacted by the time a CTL needs to kill a target cell. This suggests that the killing of target cells, rather than their localization, is the limiting step in CTL killing dynamics given reasonable frequencies of CTL. Our analysis identifies additional experimental directions which are of particular importance to interpret estimates of killing rates and could advance our quantitative understanding of CTL killing.

Triglyceride lipids can enhance the cytotoxicity of induced cytotoxic T lymphocytes (CTLs) (83.8)

Abstract IL-4 induces pancreatic lipase related protein 2 (PLRP2) in cytotoxic T lymphocytes (CTLs). Because PLRP2 in semen mediates lipid-dependent toxicity to sperm, we questioned whether CTL-derived PLRP2 could support similar cytotoxicity towards tumor cells. First we tested recombinant PLRP2. Microgram/ml concentrations were toxic to P815 cells in 48 hr assays when lipid and co-lipase were present. Next we found that CTLs secrete triglyceride lipase but that most IL-2 or IL-4 induced CTLs were unable to mediate lipid-dependent cytotoxicity. A single, exceptional lot of IL-4 reproducibly induced lipid-dependent cytotoxicity that was also perforin-independent. In efforts to induce the lipid-dependent CTL killing again, attempts were made to block type 1-polarizing cytokines, including biasing the T cells cultures with more IL-4 or with IL-12 blocking antibodies, and using T cells without IFN-ã receptors. These attempts were unsuccessful at inducing triglyceride-enhanced cytotoxicity. The CTLs induced with IL-4 consistently had mRNA for PLRP2 but lacked mRNA for co-lipase. Therefore, we added exogenous co-lipase to the cytotoxicity assays. Cytotoxicity was unaltered. We conclude (1) that lipid-dependent cytotoxicity by PLRP needs co-lipase and (2) that more than PLRP2 is required for lipid-dependent cytotoxicity by CTLs. Supported by NIH R01CA38942 and T32CA09563.

Enhancement of dendritic cell-based vaccine potency by anti-apoptotic siRNAs targeting key pro-apoptotic proteins in cytotoxic CD8 + T cell-mediated cell death

Dendritic cells (DCs) have become an important measure for the treatment of malignancies. Current DC preparations, however, generate short-lived DCs because they are subject to cell death from various apoptotic pressures. Antigen-specific CD8 + cytotoxic T lymphocytes (CTLs) is one of the main obstacles to limit the DC-mediated immune priming since CTLs can recognize the target antigen expressing DCs as target cells and kill the DCs. CTLs secret perforin and serine protease granzymes during CTL killing. Perforin and serine protease granzymes induce the release of a number of mitochondrial pro-apoptotic factors, which are controlled by members of the BCL-2 family, such as BAK, BAX and BIM. FasL linking to Fas on DCs triggers the activation of caspase-8, which eventually leads to mitochondria-mediated apoptosis via truncation of BID. In this study, we tried to enhance the DC priming capacity by prolonging DC survival using anti-apoptotic siRNA targeting these key pro-apoptotic molecules in CTL killing. Human papillomavirus (HPV)-16 E7 antigen presenting DCs that were transfected with these anti-apoptotic siRNAs showed increased resistance to T cell-mediated death, leading to enhanced E7-specific CD8 + T cell activation in vitro and in vivo. Among them, siRNA targeting BIM (siBIM) generated strongest E7-specific E7-specific CD8 + T cell immunity. More importantly, vaccination with E7 presenting DCs transfected with siBIM was capable of generating a marked therapeutic effect in vaccinated mice. Our data indicate that ex vivo manipulation of DCs with siBIM may represent a plausible strategy for enhancing dendritic cell-based vaccine potency.

Intracellular and Extracellular Remodeling Effectively Prevents Human CD8 +Cytotoxic T Lymphocyte-Mediated Xenocytotoxicity by Coexpression of Membrane-Bound Human FasL and Pig c-FLIP L in Pig Endothelial Cells

Human CD8 + cytotoxic T lymphocyte (CTL)–mediated cytotoxicity, which participates in xenograft rejection, is mediated mainly by the Fas/FasL apoptotic pathway. We previously developed methods to inhibit human CTL xenocytotoxicity by extracellular remodeling using overexpression of membrane-bound human FasL on pig xenograft cells, and by intracellular blockade of death receptor-mediated apoptotic signals, such as the Fas/FasL pathway using the pig c-FLIP L molecule. To investigate the cooperative effects of both membrane-bound FasL and pig c-FLIP L, we cotransfected both genes into pig endothelial cells (PEC). The double remodeling with these molecules effectively prevented CD8 + CTL killing. Although double transfectants and single high transfectants of either membrane-bound FasL or c-FLIP L gene displayed similar inhibition of CTL cytotoxicity, the expression levels of these 2 molecules in double transfectants were almost half the expression levels of single transfectants. Furthermore, to show in vivo prolongation of xenograft survival, we transplanted PEC transfectants under the rat kidney capsule. Prolonged survival was displayed by PEC double transfectant xenografts whereas those from either parental PEC or MOCK (vehicle control) were completely rejected by day 5 posttransplantation. These data suggested that intracellular and extracellular remodeling by coexpression of membrane-bound FasL and pig c-FLIP L in xenograft cells may prevent an innate cellular response to xenografts. The gene compatibility of these molecules to generate transgenic pigs may be sufficient to create a window of opportunity to facilitate long-term xenograft survival.

Adenovirus-Mediated Gene Expression of the Human c-FLIPL Gene Protects Pig Islets Against Human CD8 + Cytotoxic T Lymphocyte-Mediated Cytotoxicity

Cell-mediated immunity, especially of human CD8+ cytotoxic T lymphocytes (CTLs) is believed to have an important role in the long-term survival of pig islet xenografts. Protection against human CD8+ CTL cytotoxicity may reduce the direct damage to pig islets and enable long-term xenograft survival in pig-to-human islet xenotransplantation. We have previously reported that c-FLIP S/L genes, which are potent inhibitors of death receptor–mediated proapoptotic signals through binding competition with caspase-8 for recruitment to the Fas-associated via death domain (FADD), markedly suppress human CD8+ CTL-mediated xenocytotoxicity. In addition, the cytoprotective effects of c-FLIP L seem to be significantly stronger than those of c-FLIP S . Accordingly, in the present study, expression of c-FLIP L was induced in intact pig islets by adenoviral transduction. Consequently, the cytoprotective capacity of the transgene in pig islets was examined in in vitro and in vivo exposure to human CD8+ CTLs. Cells from untransduced islets or mock islets were sensitive to CD8+ CTL-mediated lysis (59.3% ± 15.9% and 64.0% ± 8.9% cytotoxicity, respectively). In contrast, cells from pig islets transduced with the c-FLIP L gene were markedly protected from lysis (30.5% ± 3.5%). Furthermore, prolonged xenograft survival was elicited from pig islets transduced with this molecule as assessed using an islet transplant model using the rat kidney capsule. Thus, these data indicate that intact pig islets can be transduced to express c-FLIP L with adenovirus. Pig islets expressing c-FLIP L are significantly resistant to human CTL killing and further exhibit beneficial effects to prolong xenograft survival.

Predicting the Impact of Blocking Human Immunodeficiency Virus Type 1 Nef In Vivo

Human immunodeficiency virus type 1 (HIV-1) Nef is a multifunctional protein that confers an ability to evade killing by cytotoxic T lymphocytes (CTLs) as well as other advantages to the virus in vivo. Here we exploited mathematical modeling and related statistical methods to estimate the impact of Nef activity on viral replication in vivo in relation to CTLs. Our results indicate that downregulation of major histocompatibility complex class I (MHC-I) A and B by wild-type Nef confers an advantage to the virus of about 82% in decreased CTL killing efficiency on average, meaning that abolishing the MHC-I downregulation function of Nef would increase killing by more than fivefold. We incorporated this estimate, as well as prior estimates of replicative enhancement by Nef, into a previously published model of HIV-1 and CTLs in vivo (W. D. Wick, O. O. Yang, L. Corey, and S. G. Self, J. Virol. 79:13579-13586, 2005), generalized to permit CTL recognition of multiple epitopes. A sequence database analysis revealed that 92.9% of HIV-1 epitopes are A or B restricted, and a previous study found an average of about 19 epitopes recognized (M. M. Addo et al., J. Virol. 77:2081-2092, 2003). We combined these estimates in the model in order to predict the impact of inhibiting Nef function in the general (chronically infected) population by a drug. The predicted impact on viral load ranged from negligible to 2.4 orders of magnitude, depending on the effects of the drug and the CTL dynamical scenario assumed. We conclude that inhibiting Nef could make a substantial reduction in disease burden, lengthening the time before the necessity of undertaking combination therapy with other antiretroviral drugs.

Enhancement of cytotoxic T lymphocyte activity by dendritic cells loaded with Tat-protein transduction domain-fused hepatitis B virus core antigen

The protein transduction domain (PTD) of human immunodeficiency virus-1-Tat protein has a unique potency to penetrate the cellular membranes. To synthesize the sequence of Tat-PTD47-57 and hepatitis B virus core antigen (HBcAg), we spliced these sequences and linked a fusion gene into the pMAL-c2x vector. The fusion proteins were purified by affinity chromatography and pulsed with bone marrow-derived dendritic cells (DCs), and the transduction of recombinant protein was detected by immunofluorescence antibody assay. Results showed that recombinant PTD-HBcAg could penetrate into DC cytoplasm while recombinant HBcAg was detected on the surface of cells. The percentage of DC surface molecules, such as CD80, CD86 and major histocompatibility complex II, and production of cytokine (IL-12p70) induced by recombinant PTD-HBcAg were significantly higher than those induced by recombinant HBcAg or tumor necrosis factor-alpha. DCs treated with PTD-HBcAg induced T cells to differentiate into specific cytotoxic T lymphocytes (CTLs) and enhanced the CTL killing response. In conclusion, the expressed and purified PTD-HBcAg fusion protein could penetrate into cells through the plasma membrane, promote DC maturation, and enhance T cells response to generate HBcAg-specific CTLs efficiently.

RAAV/Her-2/neu Loading of Dendritic Cells for a Potent Cellular-Mediated MHC Class I Restricted Immune Response Against Ovarian Cancer

Recent studies demonstrate that recombinant adeno-associated virus (rAAV)-based antigen loading of dendritic cells (DCs) generates, in vitro, significant and rapid cytotoxic T-lymphocyte (CTL) responses against viral antigens. We used the rAAV system to induce specific CTLs against tumor antigens for the development of ovarian cancer (OC) gene therapy. As an extension of the versatility of the rAAV system, we incorporated a self-antigen, Her-2/neu, which is expressed in many cancers, including breast and ovarian. We analyzed two different vectors containing a short (157-612) and long domain (1-1197). Our rAAV vector induced strong stimulation of CTLs directed against the self tumor antigen, Her-2/neu. We then investigated the efficiency of the CTLs in killing Her-2/neu-targeted cells. A significant MHC class I-restricted, anti-Her-2/neu-specific CTL killing was demonstrated against Her-2/neu-positive OC cells after one in vitro stimulation. In summary, single peripheral blood mononuclear cell (PBMC) stimulation with rAAV/157-612- or rAAV/1-1197-pulsed DCs induces strong antigen-specific CTL generation. The CTLs were capable of lysing low doses of peptides pulsed into target cells or OC Her-2/neu(+) tumors. These data suggest that AAV-based antigen loading of DCs is highly effective for generating human CTL responses against OC antigens.

Modulation of Mesenchymal Stem Cell Immunogenicity through Forced Expression of Human Cytomegalovirus Proteins

Mesenchymal stem cells (MSC) are promising candidates for cell replacement therapy since they have the ability to differentiate, under appropriate conditions, into a broad range of specialized cell types. Furthermore, MSC have low inherent immunogenicity, immunomodulatory properties, and preferentially home to/engraft damaged tissues. However, they are not invisible to the immune system, and upon allogeneic transplantation, MSC can elicit an immune response, that results in activation of the recipient's cytotoxic T lymphocytes (CTL) and Natural Killer (NK) cells and hence rejection of the transplanted cells. Human cytomegalovirus (HCMV), a virus ubiquitously present in humans, has developed several strategies to evade CTL and NK cell recognition. HCMV avoids CTL attack by producing proteins coded for by the unique short region (US) of the genome that downregulate MHC-I surface expression. Moreover, expression of HCMV UL18 protein, which is an MHC-I homolog or decoy, renders CMV-infected cells resistant to NK lysis. Thus, we hypothesized that simultaneous expression of HCMV US protein US2, US3, US6, or US11 with UL18 would render MSC less susceptible to CTL and NK cell recognition and killing. To this end, we started by transducing MSC with the retroviral vectors encoding: US2; US3; US6; or US11, and tested the effect of each of these proteins on expression of MHC-I and CD59, a complement membrane attack complex inhibitor whose expression has been shown to prevent destruction of transplanted cells. MSC expressing US3, US2, US11 and US6 showed substantial MHC-I downregulation of 69%, 74%, 78 % and 95% respectively, when compared to untransduced MSC. Moreover, MSC expressing either US6 or US2 proteins displayed a significant up-regulation of CD59, with US6 causing a 2.5 and US2 a 3.9 fold increase, when compared to untransduced MSC (Mean Fluorescence Intensity: 869.8; 1331.19; 344.99, respectively). Each of the US transduced MSC were then tested for their capability to activate MHC-I mismatched CTL proliferation in both an allogeneic and a xenogeneic setting, using MSC transduced with an empty retrovirus as a control. MSC expressing US2 or US6 reduced CTL proliferation by the same degree, whether CTL were mismatched human- or sheep-derived. Using MSC-US2 we obtained a reduction in CTL proliferation by 18% and 23% in human-or sheep-CTL, respectively, while using MSC-US6 resulted in a reduction in human and sheep CTL proliferation of 60% and 53%. Expression of US11 on MSC was more efficient at abrogating the sheep CTL proliferation than that of mismatched human CTL (reduction in proliferation by 53% vs 31%, respectively). Finally, expression of US3 on MSC only reduced proliferation of human CTL by 21.5%, but had no effect on sheep CTL. In conclusion, engineering MSC to over-express US6 seems to be the most effective way to enable MSC to evade the host immune system, resulting in efficient downregulation of MHC-I surface expression, significant reduction in CTL proliferation, and a reduced formation of the membrane attack complex at the end of the complement cascade, which will reduce destruction of MSC by this system. In vivo studies using a large animal sheep model are underway to demonstrate whether the results obtained in vitro using US6 HCMV protein expression will translate into improved engraftment in vivo.

Resistance of Tumor Cells to Cytolytic T Lymphocytes Involves Rho-GTPases and Focal Adhesion Kinase Activation

Tumor cells evade adaptive immunity by a variety of mechanisms, including selection of variants that are resistant to specific cytotoxic T lymphocyte (CTL) pressure. Recently, we have reported that the reorganization of the actin cytoskeleton can be used by tumor cells as a strategy to promote their resistance to CTL-mediated lysis. In this study, we further examined the functional features of a CTL-resistant tumor variant and investigated the relationship between cytoskeleton alteration, the acquisition of tumor resistance to CTL-induced cell death, Rho-GTPases, and focal adhesion kinase (FAK) pathways. Our data indicate that although the resistant cells do not display an increased migratory potential, an alteration of adhesion to the extracellular matrix was observed. When Rho-GTPases were activated in cells by the bacterial CNF1 (cytotoxic necrotizing factor 1), striking changes in the cell morphology, including actin cytoskeleton, focal adhesions, and membrane extensions, were observed. More importantly, such activation also resulted in a significant attenuation of resistance to CTL-induced cell death. Furthermore, we demonstrate that FAK signaling pathways were constitutively defective in the resistant cells. Silencing of FAK in the sensitive target cells resulted in the inhibition of immune synapse formation with specific CTLs and their subsequent lysis. Expression of the FAK mutant (Y397F) resulted in an inhibition of IGR-Heu cell adhesion and of their susceptibility to specific lysis. These results suggest that FAK activation plays a role in the control of tumor cell susceptibility to CTL-mediated lysis.

EGF and HGF levels are increased during active HBV infection and enhance survival signaling through extracellular matrix interactions in primary human hepatocytes

The hepatitis B virus (HBV) is a major causative agent of chronic liver disease and subsequent liver cirrhosis worldwide. The reduced sensitivity of virus-infected liver cells to apoptosis may play a role in the failure to remove virus-infected cells and eventually promote viral chronicity. The purpose of our study was to investigate whether survival factors induced during compensatory liver regeneration may protect hepatocytes against apoptosis. We evaluated the serum levels of hepatocyte growth factor (HGF) and epidermal growth factor (EGF) in HBV-infected patients and found significant increases in HGF and EGF in patients with active virus infection. In primary human hepatocytes we show that HGF and EGF have a protective effect against CD95-mediated apoptosis and cytotoxic T-cell killing. Simultaneous treatment with both regeneration factors enhanced the cytoprotective effect. The PI 3-K/Akt kinase inhibitor, wortmannin, and the STAT3 pathway inhibitor, Tyrphostin AG490, both effectively attenuated the cytoprotective effect of HGF and EGF. Furthermore, we show an EGF/HGF-dependent upregulation of beta(1)-integrin chains, increased adhesion to extracellular matrix and an increase in focal adhesions, suggesting outside-in signaling from the extracellular matrix as an additional cytoprotective mechanism. Our study demonstrates that HGF and EGF can interfere with CD95-mediated apoptosis and the action of cytotoxic T-cells through multiple mechanisms in human hepatocytes. Together our results argue that a survival mileau generated by activation of liver regeneration factors may be a risk factor for establishing viral persistence.

Protective CD8+ T-cell responses to cytomegalovirus driven by rAAV/GFP/IE1 loading of dendritic cells

BackgroundRecent studies demonstrate that recombinant adeno-associated virus (rAAV)-based antigen loading of dendritic cells (DCs) generates in vitro, significant and rapid cytotoxic T-lymphocyte (CTL) responses against viral antigens.MethodsWe used the rAAV system to induce specific CTLs against CVM antigens for the development of cytomegalovirus HCMV) gene therapy. As an extension of the versatility of the rAAV system, we incorporated immediate-early 1 (IE1), expressed in HCMV. Our rAAV vector induced a strong stimulation of CTLs directed against the HCMV antigen IE1. We then investigated the efficiency of the CTLs in killing IE1 targeted cells.ResultsA significant MHC Class I-restricted, anti-IE1-specific CTL killing was demonstrated against IE1 positive peripheral blood mononuclear cells (PBMC) after one, in vitro, stimulation.ConclusionIn summary, single PBMC stimulation with rAAV/IE1 pulsed DCs induces strong antigen specific-CTL generation. CTLs were capable to lyse low doses of peptides pulsed into target cells. These data suggest that AAV-based antigen loading of DCs is highly effective for generating human CTL responses against HCMV antigens.

Prolonged survival of pig islets xenograft by adenovirus‐mediated expression of either the membrane‐bound human FasL or the human decoy Fas antigen gene

Pig islets are considered an attractive alternative treatment for patients with Type 1 diabetes. However, pig islet xenografts, transplanted into non-human primates, are directly rejected by cell-mediated processes. We have previously reported that cell-mediated xenograft-rejections, and especially human CD8(+) cytotoxic T lymphocytes (CTL)-mediated cytotoxicity, are highly detrimental to pig xenograft cells. Moreover, we have explored novel strategies for the prevention of CTL killing by overexpression of either human decoy Fas antigen or membrane-bound human FasL in pig endothelial cells. In this study, we assessed the cytoprotective effects of these molecules for pig islets both in vitro and in vivo. Pig islets were freshly isolated by modified Ricordi's methods. Subsequently, these islets were transfected with an adenoviral expression vector containing the DNA fragments of either membrane-bound human FasL or human decoy Fas. Transfected islets were transplanted into preimmunized diabetic rats under the kidney capsule. Control pig islets (i.e., MOCK), which were transfected with an adenoviral expression vector containing only the enhanced green fluorescent protein gene, were also transplanted. Efficiency of adenoviral expressions of these molecules in pig islets was approximately 80% at a multiplicity of infection of 100. In an in vitro assay, approximately 80% suppression of cytotoxicity was observed in membrane-bound human FasL-expressing pig islets and 60% inhibition of CTL killing was displayed in decoy Fas expression pig islets. In an in vivo transplant model, prolonged survival of pig islets xenografts, expressing either membrane-bound human FasL or human decoy Fas genes, was elicited in comparison with that of control islets xenografts. The extracellular remodeling of either death receptor or death ligand genes by adenoviral expression was effective for the prevention of CTL-mediated xenocytotoxicity in pig islets.

CD4 + Target Cell Availability Determines the Dynamics of Immune Escape and Reversion In Vivo

Infections with human immunodeficiency virus (HIV) and the closely related monkey viruses simian-human immunodeficiency virus (SHIV) and simian immunodeficiency virus (SIV) are characterized by progressive waves of immune responses, followed by viral mutation and "immune escape." However, escape mutation usually leads to lower replicative fitness, and in the absence of immune pressure, an escape mutant (EM) virus "reverts" to the wild-type phenotype. Analysis of the dynamics of immune escape and reversion has suggested it is a mechanism for identifying the immunogens best capable of controlling viremia. We have analyzed and modeled data of the dynamics of wild-type (WT) and EM viruses during SHIV infection of macaques. Modeling suggests that the dynamics of reversion and immune escape should be determined by the availability of target cells for infection. Consistent with this suggestion, we find that the rate of reversion of cytotoxic T-lymphocyte (CTL) EM virus strongly correlates with the number of CD4(+) T cells available for infection. This phenomenon also affects the rate of immune escape, since this rate is determined by the balance of CTL killing and the WT fitness advantage. This analysis predicts that the optimal timing for the selection of immune escape variants will be immediately after the peak of viremia and that the development of escape variants at later times will lead to slower selection. This has important implications for comparative studies of immune escape and reversion in different infections and for identifying epitopes with high fitness cost for use as vaccine targets.

MHC-Unrestricted Lysis of MUC1-Expressing Cells by Human Peripheral Blood Mononuclear Cells

Many human adenocarcinomas can be killed in vitro by targeted cytotoxic T-lymphocytes (CTL); however, major histocompatibility complex (MHC)-restrictions are typically required. The MUC1 antigen is common in many human adenocarcinomas, and is associated with a variable number of tandem repeats. It has been proposed that antigens with such repeated epitopes may be vulnerable to cytotoxic T-lymphocyte killing without MHC-restriction. Therefore, it is possible that MUC1-expressing malignant cells may be killed by targeted cytotoxic T-lymphocyte in the absence of MHC-restriction. In this study, a human MUC1-expressing murine mammary carcinoma cell line was used to determine if cytotoxic T-lymphocyte killing of MUC1-expressing adenocarcinoma cells requires MHC-restriction. Specifically, MUC1-stimulated human mononuclear cells (M1SMC) were observed to kill human MUC1-transfected, MUC1-expressing murine mammary carcinoma cells, but not the mock-transfected, non-MUC1-expressing murine mammary carcinoma cells. Furthermore, the killing was blocked by antibody to MUC1, indicating MUC1-specific killing. In conclusion, cytotoxic T-lymphocyte killing of MUC1-expressing adenocarcinoma cells can be MHC-unrestricted.

Revisiting Estimates of CTL Killing Rates In Vivo

Recent experimental advances have allowed the estimation of the in vivo rates of killing of infected target cells by cytotoxic T lymphocytes (CTL). We present several refinements to a method applied previously to quantify killing of targets in the spleen using a dynamical model. We reanalyse data previously used to estimate killing rates of CTL specific for two epitopes of lymphocytic choriomeningitis virus (LCMV) in mice and show that, contrary to previous estimates the “killing rate” of effector CTL is approximately twice that of memory CTL. Further, our method allows the fits to be visualized, and reveals one potentially interesting discrepancy between fits and data. We discuss extensions to the basic CTL killing model to explain this discrepancy and propose experimental tests to distinguish between them.

Aberrant Subcellular Localization of Azurophil Granule Proteins in Myeloid Leukemia Favors Peptide Antigen Presentation on MHC-I and Susceptibility to Killing by Cytotoxic T Lymphocytes.

The recognition of leukemia-associated antigens (LAA) by immune cells is essential for generating anti-leukemia responses. Whether elicited by vaccine or hematopoietic stem cell transplantation (HSCT), anti-leukemia immunity requires that cytotoxic T lymphocytes (CTL) recognize and mount an effective response against leukemia cells. PR1 is a nonomeric HLA-A2-restricted peptide (VLQELNVTV) derived from the myeloid-restricted serine proteases neutrophil elastase (ELA2) and proteinase-3 (PRTN3), recognized azurophil granule proteins. PR1-specific cytotoxic T lymphocytes (PR1-CTL) have been shown to preferentially recognize and kill myeloid leukemia cells. We hypothesized that aberrant protein expression in target leukemia cells predisposes to CTL killing. To determine the subcellular localization of ELA2 and PRTN3 in leukemia and in healthy donor (HD) neutrophils, high-speed ultracentrifugation of leukapheresed cells from patients with AML and CML in blast crises yielded 4 fractions: cytoplasmic, nuclear, membrane and golgi/endoplasmic reticulum (ER). Western blot analyses identified higher levels of ELA2 and PRTN3 in cytoplasmic, nuclear, and golgi/ER fractions in leukemia, compared to HD granulocytes. Confocal microscopy of leukapheresed myeloid blasts and HD granulocytes confirmed these findings. We further hypothesized that cross-presentation of PR1 is necessary for priming anti-leukemia immunity. Confocal microscopy of the human B-cell line HMy.CIR-A2, co-incubated with either purified or recombinant ELA2 and PRTN3 demonstrated co-localization of both proteins with LAMP-2, a marker of lysosomes and early endosomes, and small distribution in cytoplasm after 2 hours. Uptake of ELA2 and PRTN3 by HMy.CIR-A2 cells was additionally confirmed by Western blots. Priming of naive PR1-CTL by co-culture of HD lymphocytes with PRTN3 or ELA2-transfected HMy.CIR-A2 cells was confirmed by cell proliferation assays using BrDU. This could be abrogated by addition of lactacystin and cyclohexamide to cell culture. Together, these results demonstrate that ELA2 and PRTN3 are aberrantly localized preferentially in gogli/ER and cytoplasmic fractions in leukemia, rather than azurophil granules, which are often absent or deficient in myeloid leukemia. Similar forced subcellular localization of these proteins in B cells leads to their susceptibility to PR1-CTL killing. Therefore, we conclude that the natural mistrafficking of PRTN3 and ELA2 in myeloid leukemia is necessary to render cells susceptible to CTL attack and that cross-presentation of ELA2 and PRTN3 contributes to priming of a PR1-CTL response.

Mathematical models of cytotoxic T‐lymphocyte killing

By killing infected host cells, cytotoxic T lymphocytes (CTL) mediate an important defense mechanism against viruses and other intracellular pathogens. Quantitative aspects of this killing process have been studied for several decades in vitro. More recently, methods have been developed to measure the timescales of CTL killing in vivo. Here, we review the estimates of kinetic rates involved in CTL killing which were obtained in these studies, and elaborate on the differences between them.

Enhanced T-cell immunity induced by dendritic cells with phagocytosis of heat shock protein 70 gene-transfected tumor cells in early phase of apoptosis

The dual role of heat shock protein 70 (HSP70), as antigenic peptide chaperone and danger signal, makes it especially important in dendritic cell (DC)-based vaccination. In this study, we investigated the impacts of apoptotic transgenic MCA/HSP tumor cells expressing HSP70 on DC maturation, T-cell stimulation and vaccine efficacy. We found that DCs with phagocytosis of MCA/HSP in early phase of apoptosis expressed more pMHC I complexes, stimulated stronger cytotoxic T lymphocyte (CTL) responses (40% specific killing at an E:T cell ratio of 50) and induced immune protection in 90% of mice against MCA tumor cell challenge, compared with 25% specific CTL killing activity and 60% immune protection seen in mice immunized with DC with phagocytosis of MCA/HSP in late phase of apoptosis (P<0.05). Similar results were confirmed in another EG7 tumor model also expressing HSP70. Taken together, our data demonstrate that HSP70 on apoptotic tumor cells stimulate DC maturation, and DC with phagocytosis of apoptotic tumor cells expressing HSP70 in early phase of apoptosis more efficiently induced tumor-specific CTL responses and immunity than DCs with phagocytosis of apoptotic tumor cells in late phase of apoptosis. These results may have an important impact in designing DC-based antitumor vaccines.