Activation Of CTLs Research Articles

The multiple roles of the CD8 coreceptor in T cell biology: opportunities for the selective modulation of self-reactive cytotoxic T cells.

Short peptide fragments generated by intracellular protein cleavage are presented on the surface of most nucleated cells bound to highly polymorphic MHCI molecules. These pMHCI complexes constitute an interface that allows the immune system to identify and eradicate anomalous cells, such as those that harbor infectious agents, through the activation of CTLs. Molecular recognition of pMHCI complexes is mediated primarily by clonally distributed TCRs expressed on the surface of CTLs. The coreceptor CD8 contributes to this antigen-recognition process by binding to a largely invariant region of the MHCI molecule and by promoting intracellular signaling, the effects of which serve to enhance TCR stimuli triggered by cognate ligands. Recent investigations have shed light on the role of CD8 in the activation of MHCI-restricted, antigen-experienced T cells and in the processes of T cell selection and lineage commitment in the thymus. Here, we review these data and discuss their implications for the development of potential therapeutic strategies that selectively target pathogenic CTL responses erroneously directed against self-derived antigens.

Construction of recombinant plasmid pIRES-GM-CSF-IL-21 and its therapeutic effect on orthotopically transplanted liver cancer in mice

To construct a recombinant plasmid pIRES-GM-CSF-IL-21, and to investigate its antitumor effect on tumors in the mice. Fifty Bal b/c mice were included in this study. Cultured hepatoma H22 cells were inoculated in the left lobe of the liver to develop orthotopically transplanted liver tumor models. The mice with orthotopically transplanted liver tumor were randomly divided into 5 groups (n = 10): (1) Each mouse received injection of recombinant plasmid pIRES-GM-CSF-IL-21; (2) Received injection of plasmid pIRES-GM-CSF; (3) pIRES-IL-21; (4) Received injection of ampty plasmid pIRES (H22/neo group); (5) Received injection of PBS (H22 group) via the tail vein, respectively. Therefore, the anti-tumor effect was induced by both GM-CSF and IL-21, or by either of them alone. The serum levels of IFN-γ and IL-2 were detected by ELISA, and the cytotoxicity of spleen NK and CTL cells were tested by MTT colorimetry. Comparing with the H22 and H22/Neo groups, the tumor weight in the mice of H22/GM-CSF group was (0.603 ± 0.223) g, H22/IL21-treated group (0.583 ± 0.290) g and H22/GM-CSF-IL21-treated group (0.303 ± 0.323) g, significantly lower than that in the H22 group [(1.591 ± 0.280) g] and H22/Neo group [(1.489 ± 0.155) g]. Among them the tumor growth was most significantly inhibited in the H22/GM-CSF-IL-21 group (0.303 ± 0.323) g, compared with that of H22 and H22/neo groups (P < 0.01). But there was no significant difference between the tumor weights of the H22/GM-CSF group and H22/IL-21 group, and between the tumor weights of the H22 and H22/Neo groups (P > 0.05). The levels of IFN-γ and IL-2 in peripheral blood of mouse models treated with H22/GM-CSF-IL-21 were significantly increased than that in the H22/GM-CSF group and H22/IL-21 group (all P < 0.01), but significantly decreased in the H22group and H22/Neo group (P < 0.01). The anti-tumor activity of splenic NK cells and CTLs in the H22/GM-CSF-IL21 group was significantly enhanced (P < 0.01), compared with the significantly decreased in the H22 and H22/Neo groups. Our results demonstrate apparent antitumor effect of the plasmid pIRES-GM-CSF-IL-21 on the orthotopically transplanted liver tumor in mice. The combination of both pIRES-GM-CSF and IL-21 is more effective than that of pIRES/IL21 or pIRES/GM-CSF treatment alone. In addition, the plasmid pIRES-GM-CSF-IL-21 can also promote the secretion of IFN-γ and IL-2 in vivo, and enhance the cytotoxic activity of splenic NK and CTLs against the transplanted liver tumor.

503. Improved Expansion and Anti-Tumor Activity of Tumor-Specific CTLs Using a Transgenic Chimeric Cytokine Receptor

503. Improved Expansion and Anti-Tumor Activity of Tumor-Specific CTLs Using a Transgenic Chimeric Cytokine Receptor

Abstract 761: Comparison of different classes of CpG-ODNs to augment generation, proliferation and specific cytotoxicity of human epitope-peptide specific CTLs in vitro

Abstract Three distinct classes of CpG- oligonucleotides (ODN) (CpG-A, CpG-B and CpG-C) have been identified based on differences in their structures and immune effect. So far, CpG-B is only applied for clinical treatments; however, it is still unknown which of the different CpG-ODN classes is most useful as adjuvant of human cancer vaccine therapy. In the present study, we examined the activity of these 3 types of CpG-ODNs to enhance the induction of human peptide-specific CTLs. In addition, we elucidated the effect of CpG-ODNs on tumor antigen epitope peptide-specific CTL clones to investigate the adjuvant mechanism of CpG-ODNs on peptide vaccine therapy. Our data showed that PDC activated by CpG-ODNs augmented induction of peptide specific CTLs. Importantly, CpG-A is superior to CpG-B and CpG-C in augmenting the generation, proliferation and cytotoxic activity of human peptide specific CTLs in vitro. Moreover, type-1 IFNs produced by PBMCs that were stimulated with CpG-ODNs accelerated the proliferation of peptide-specific CTL clones, and also directly augmented the peptide specific cytotoxicity of CTL clones through the up regulation of Granzyme-B. These data showed that IFN-α might play an important role for CpG-ODNs to induce the adjuvant effects in peptide vaccine therapy. In addition, because CpG-A induced high amount of type-1 IFNs, CpG-A might be the superior candidate for vaccine adjuvant than CpG-B or CpG-C. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 761. doi:10.1158/1538-7445.AM2011-761

Cytotoxic T Lymphocytes and Vaccine Development 2011

CTLs play a critical role in fighting chronic virus infection and cancers. Despite the tremendous efforts spent so far, generating potent and highly effective CTLs remain a major roadblock for disease control (prevention and therapies). Activation of CTLs requires different stimuli and is influenced by different disease conditions. In this special issue, several papers investigated functions of cytokines, such as IL-15 and IL-6, in activation of CTLs. Several papers are focusing on the therapeutic effects of CTLs in different cancers using novel approaches targeting tumor-specific CTLs. In addition, potential markers and mechanisms for rapid progression of AIDS are suggested using SIV model. In the first paper entitled “IL-6 production by dendritic cells is dispensable for CD8+ memory T-cell generation,” Daudelin et al. describe a new observation that IL-6 is not required in APC vaccination. In the second paper entitled “What is recent in pancreatic cancer immunotherapy?” Niccolai et al. summarize the current immunotherapy of pancreatic cancer targeting cancer-associated antigens. In the third paper entitled “MUC1-specific cytotoxic T lymphocytes in cancer therapy: induction and challenge,” Roulois et al. review current knowledge regarding MUC1 as a potential target tumor therapeutic vaccines. In the fourth paper entitled “Characterization of CD8+ T cell responses in the peripheral blood and skin injection sites of melanoma patients treated with mRNA electroporated autologous dendritic cells (TriMixDC-MEL),” Benteyn et al. report that functional TAA-specific CD8+ T cells are detected in both the skin and the peripheral blood after TriMixDC-MEL therapy. In the fifth paper entitled “CpG and interleukin-15 synergize to enhance IFN-production by activated CD8+ T cells,” Cobb et al. present evidence that IL-15 synergizes with CpG in the induction of IFN-γ in activated CD8+ T cells, expanding our understanding about the function of IL-15. In the sixth paper entitled “Increased Toll-like receptor signaling pathways characterize CD8+ cells in rapidly progressive SIV infection,” Marcondes et al. suggest that TLR overexpression may indicate ineffective T-cell response in rapid progression in HIV infection. We thank all of the authors for their great contributions to this special issue and appreciate their patience in processing their manuscripts. We also want to thank all of the reviewers who went through the manuscripts multiple times, providing insightful suggestions. We really hope this special issue will continue to thrive as a timely communication platform for basic and translational research on CTLs. Zhengguo Xiao Kim Klonowski Hanchun Yang Julie Curtsinger

Monocyte Chemoattractant Protein–1 Blockade Inhibits Lung Cancer Tumor Growth by Altering Macrophage Phenotype and Activating CD8+ Cells

The role of chemokines in the pathogenesis of lung cancer has been increasingly appreciated. Monocyte chemoattractant protein-1 (MCP-1, also known as CCL2) is secreted from tumor cells and associated tumor stromal cells. The blockade of CCL2, as mediated by neutralizing antibodies, was shown to reduce tumorigenesis in several solid tumors, but the role of CCL2 in lung cancer remains controversial, with evidence of both protumorigenic and antitumorigenic effects. We evaluated the effects and mechanisms of CCL2 blockade in several animal models of non-small-cell lung cancer (NSCLC). Anti-murine-CCL2 monoclonal antibodies were administered in syngeneic flank and orthotopic models of NSCLC. CCL2 blockade significantly slowed the growth of primary tumors in all models studied, and inhibited lung metastases in a model of spontaneous lung metastases of NSCLC. In contrast to expectations, no significant effect of treatment was evident in the number of tumor-associated macrophages recruited into the tumor after CCL2 blockade. However, a change occurred in the polarization of tumor-associated macrophages to a more antitumor phenotype after CCL2 blockade. This was associated with the activation of cytotoxic CD8(+) T lymphocytes (CTLs). The antitumor effects of CCL2 blockade were completely lost in CB-17 severe combined immunodeficient mice or after CD8 T-cell depletion. Our data from NSCLC models show that CCL2 blockade can inhibit the tumor growth of primary and metastatic disease. The mechanisms of CCL2 blockade include an alteration of the tumor macrophage phenotype and the activation of CTLs. Our work supports further evaluation of CCL2 blockade in thoracic malignancies.

Abstract 1911: Influence of gap-junction formation on melanoma specific-CD8 T cells function

Abstract Gap-junction channels (GJ) constitute a new potential pathway involved in the regulation of the tumor immunity. We previously showed, in a 3D construct model, that connexin43-based-GJ permitted the transfer of antigenic peptide from melanoma cell line to autologous endothelial cells. This antigenic peptide was cross-presented by endothelial cells leading to their specific recognition and lysis by specific autologous CD8+ CTL. We further investigate the potential role of GJ in the interaction between endothelial and melanoma cells. Using histological sections staining, our results indicate that gap-junction formation can occur in vivo from melanoma patients. Morever, we found that GJ are colocalized in the immunological synapse between CD8+ CTL and melanoma or melanoma-derived endothelial cells. Analysis of intracellular traffic of connexin43-GFP, using confocal live imaging, corroborates this observation since a translocation of intracellular connexin43-GFP to the contact area between CTL and target cells was observed. Our data demonstrate that CTL and melanoma cell lines exchange their intracytoplasmic content through GJ formation during the formation of the immunological synapse. The characterization of the GJ role and the influence of transferred molecules between cells will be discussed. These data open a new perspective on gap-junction importance in the anti-tumor activity of CTLs. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1911.

A Novel Haplo-Identical Adoptive CTL Therapy As Treatment For EBV-Associated Lymphoma After Stem Cell Transplantation

Epstein-Barr virus (EBV) related malignancies such as post-transplant lymphoproliferative disease (PTLD) are severe complications after allogeneic stem cell transplantation (SCT) and solid organ transplantation. In immune suppressed transplant recipients the activity of EBV-specific CTLs are often decreased or absent which leads to an increased risk of developing PTLD. If primary treatment modalities of PTLD fail, the most efficient way of treating the malignancy is adopting EBV-specific CTLs from the donor or, more recently, third-party donors. This is however both time-consuming and expensive and often it is too late to administer cells to the patient. We have for the first time, using a rapid isolation protocol of EBV-specific Tcells, treated and cured a patient suffering from PTLD with multiple associated tissue lesions, using her haplo-identical mother as a donor. This treatment approach paves way for a new possibility to within days treat patients with lifethreatening EBV-associated malignancies.

Cytotoxic T Lymphocytes and Vaccine Development

CTLs play a critical role in fighting chronic virus infection and cancers. Despite the tremendous efforts spent so far, generating potent and highly effective CTLs remain a major roadblock for disease control (prevention and therapies). Activation of CTLs requires different stimuli and is influenced by different disease conditions. In this special issue, several papers investigated functions of cytokines, such as IL-15 and IL-6, in activation of CTLs. Several papers are focusing on the therapeutic effects of CTLs in different cancers using novel approaches targeting tumor-specific CTLs. In addition, potential markers and mechanisms for rapid progression of AIDS are suggested using SIV model. In the first paper entitled “IL-6 production by dendritic cells is dispensable for CD8+ memory T-cell generation,” Daudelin et al. describe a new observation that IL-6 is not required in APC vaccination. In the second paper entitled “What is recent in pancreatic cancer immunotherapy?” Niccolai et al. summarize the current immunotherapy of pancreatic cancer targeting cancer-associated antigens. In the third paper entitled “MUC1-specific cytotoxic T lymphocytes in cancer therapy: induction and challenge,” Roulois et al. review current knowledge regarding MUC1 as a potential target tumor therapeutic vaccines. In the fourth paper entitled “Characterization of CD8+ T cell responses in the peripheral blood and skin injection sites of melanoma patients treated with mRNA electroporated autologous dendritic cells (TriMixDC-MEL),” Benteyn et al. report that functional TAA-specific CD8+ T cells are detected in both the skin and the peripheral blood after TriMixDC-MEL therapy. In the fifth paper entitled “CpG and interleukin-15 synergize to enhance IFN-production by activated CD8+ T cells,” Cobb et al. present evidence that IL-15 synergizes with CpG in the induction of IFN-γ in activated CD8+ T cells, expanding our understanding about the function of IL-15. In the sixth paper entitled “Increased Toll-like receptor signaling pathways characterize CD8+ cells in rapidly progressive SIV infection,” Marcondes et al. suggest that TLR overexpression may indicate ineffective T-cell response in rapid progression in HIV infection. We thank all of the authors for their great contributions to this special issue and appreciate their patience in processing their manuscripts. We also want to thank all of the reviewers who went through the manuscripts multiple times, providing insightful suggestions. We really hope this special issue will continue to thrive as a timely communication platform for basic and translational research on CTLs. Zhengguo Xiao Kim Klonowski Hanchun Yang Julie Curtsinger

In vivo Administration of Artificial Antigen-Presenting Cells Activates Low-Avidity T Cells for Treatment of Cancer

The development of effective antitumor immune responses is normally constrained by low-avidity, tumor-specific CTLs that are unable to eradicate the tumor. Strategies to rescue antitumor activity of low-avidity melanoma-specific CTLs in vivo may improve immunotherapy efficacy. To boost the in vivo effectiveness of low-avidity CTLs, we immunized mice bearing lung melanoma metastases with artificial antigen-presenting cells (aAPC), made by covalently coupling (pep)MHC-Ig dimers and B7.1-Ig molecules to magnetic beads. aAPC treatment induced significant tumor reduction in a mouse telomerase antigen system, and complete tumor eradication in a mouse TRP-2 antigen system, when low-avidity CTLs specific for these antigens were adoptively transferred. In addition, in an in vivo treatment model of subcutaneous melanoma, aAPC injection also augmented the activity of adoptively transferred CTLs and significantly delayed tumor growth. In vivo tumor clearance due to aAPC administration correlated with in situ proliferation of the transferred CTL. In vitro studies showed that aAPC effectively stimulated cytokine release, enhanced CTL-mediated lysis, and TCR downregulation in low-avidity CTLs. Therefore, in vivo aAPC administration represents a potentially novel approach to improve cancer immunotherapy.

Characterization and Antitumor Effect of a Novel Polysaccharide from Grifola frondosa

A novel polysaccharide, MZF, with a molecular mass of 23 kDa was isolated from Grifola frondosa . Results from methylation and (1)H NMR led to the conclusion that MZF is a heteropolysaccharide consisting of -->6)-alpha-D-Galp-(1--> (36.2%), -->3)-alpha-L-Fucp-(1--> (14.5%), -->6)-alpha-D-Manp-(1--> (9.4%), -->3)-beta-D-Glcp-(1--> (10.1%), alpha-D-Manp-(1--> (23.2%), and -->3,6)-beta-D-Glcp-(1--> (6.5%). Although MZF did not affect the proliferation of colon-26 cells in vitro, it significantly inhibited tumor growth in BALB/cA mice inoculated with colon-26 cancer cells. Moreover, MZF significantly induced the proliferation of splenocytes and peritoneal macrophages. The mRNA expression of IL-12p40, IL-2 and IFN-gamma were increased significantly in MZF-treated spleen. Furthermore, MZF augmented the percentage of IFN-gamma-producing cells in both splenic CD4(+) and CD8(+) T cells and tumor infiltrating CD4(+) and CD8(+) T cells and enhanced the cytotoxic activity of NK cells and CTLs. These results indicate that MZF is a novel effective immunomodulator that has antitumor activity associated with induced cell-mediated immunity.

Cancer vaccine with mimotopes of tumor-associated carbohydrate antigens

The GD2 ganglioside, displayed by five carbohydrate Neu5Acalpha2-8Neu5Acalpha2-3(GalNAcbeta1-4)Galbeta1-4Glcbeta residues attached to a ceramide chain that anchors the ganglioside in the cell membrane, is expressed on neuroectodermally derived tumors. GD2 has been used as a target for passive and active immunotherapy in patients with malignant melanoma and neuroblastoma. We have generated 47-LDA mimotope of GD2 by screening a phage display peptide library with anti-GD2 mAb 14G2a and reported that vaccination with the 47-LDA mimotope elicited GD2 cross-reactive IgG antibody responses as well as MHC class I-restricted CD8(+) T cells to syngeneic neuroblastoma tumor cells. The cytotoxic activity of the vaccine-induced CTLs was independent of GD2 expression, suggesting recognition of a novel tumor-associated antigen cross-reacting with 47-LDA. Immunoblotting studies using 14G2a mAb demonstrated that this antibody cross-reacts with a 105 kDa glycoprotein expressed by GD2(+) and GD2(-) neuroblastoma and melanoma cells. Functional studies of tumor cells grown in three-dimensional (3D) collagen cultures with 14G2a mAb showed decreases in matrix metalloproteinase-2 activation, a process regulated by 105 kDa activated leukocyte cell adhesion molecules (ALCAM/CD166). The CD166 glycoprotein was shown to be recognized by 14G2a antibody, and inhibition of CD166 expression by RNA interference ablated the cell sensitivity to lysis by 47-LDA-induced CD8(+) T cells in vitro and in vivo. These results suggest that the vaccine-induced CTLs recognize a 47-LDA cross-reactive epitope expressed by CD166 and reveal a novel mechanism of induction of potent tumor-specific cellular responses by mimotopes of tumor-associated carbohydrate antigens.

Specific antitumor effects of tumor vaccine produced by autologous dendritic cells transfected of allogeneic osteosarcoma total RNA through electroporation in rats.

Objective To investigate the antitumor effects of tumor vaccine produced by autologous dendritic cells transfected of allogeneic osteosarcoma total RNA through electroporation in tumor-bearing rats model. Methods In the present study, the tumor vaccine was got by transfecting UMRI08 cells total RNA to SD rat bone marrow-derived DCs through electroporation, and was applied to in tumor-bearing rats model and the specific antitumor effects of the tumor vaccine were observed. Then CTL activity was evaluated one week after the first immunization of SD rats with electroporated DCs. Results The immunization using au-tologous DCs electrotransfected with allogeneic osteosarcoma total RNA induced UMRlO6-specific CTL re-sponses which were statistically significant. In in vivo experiments, 80% of the rats immunized with allo-geneic osteosarcoma RNA transfected to DCs were typically able to reject tumor challenge and remained tu-mor-free. Vaccinated survivors developed long immunological memory and were able to reject a subsequent rechallenge with the same tumor cells but not a syngeneic unrelated tumor line. Conclusion The present study provides valid evidence of integration of autologous rat DCs electroporated with allogeneie tumor cell derived total RNA and reveales the effective potential of activation of CTLs. The research also confirmes the therapeutic effectiveness of positive immunization through corresponding animal experiment. This technique and its products may thus represent a promising strategy for DC-based immunotherapy of patients with os-teosarcoma. Key words: Dendritic cells; Osteosarcoma; Immunotherapy; T-lymphocytes, cytotoxic

EBAG9 tempers lymphocyte killing activity

The cytotoxic activity of lymphocytes is crucial for immune surveillance and homeostasis. Several independent, naturally occurring genetic models characterized by defects in granule trafficking or exocytosis have helped to decipher the multiple steps and molecules that regulate the cytotoxic process. The study by Rüder and colleagues in this issue of the JCI shows that an engineered absence of EBAG9, previously reported as a tumor-associated antigen, enhances cytotoxic activity of CTLs but not NK cells, likely acting on the endosomal-lysosomal trafficking of the cytotoxic effectors (see the related article beginning on page 2184). This finding adds a new piece to the puzzle of complex mechanisms that tightly regulate the capacity of the cytotoxic response and suggests a new target to negatively modulate CTL responsiveness.

Processing of Two Latent Membrane Protein 1 MHC Class I Epitopes Requires Tripeptidyl Peptidase II Involvement

The EBV Ag latent membrane protein 1 (LMP1) has been described as a potential target for T cell immunotherapy in EBV-related malignancies. However, only a few CD8(+) T cell epitopes are known, and the benefit of LMP1-specific T cell immunotherapy has not yet been proven. In this work, we studied the processing of the two LMP1 HLA-A02-restricted epitopes, YLLEMLRWL and YLQQNWWTL. We found that target cells endogenously expressing the native LMP1 are not recognized by CTLs specific for these epitopes because the N-terminal part of LMP1 limits the efficiency of epitope generation. We further observed that the proteasome is not required for the generation of both epitopes and that the YLLEMLRWL epitope seems to be destroyed by the proteasome, because blocking of proteasomal activities enhanced specific CTL activation. Activation of LMP1-specific CTLs could be significantly reduced after inhibition of the tripeptidyl peptidase II, suggesting a role for this peptidase in the processing of both epitopes. Taken together, our results demonstrate that the MHC class I-restricted LMP1 epitopes studied in this work are two of very few epitopes known to date to be processed proteasome independently by tripeptidyl peptidase II.

MHC Class I/Peptide Transfer between Dendritic Cells Overcomes Poor Cross-Presentation by Monocyte-Derived APCs That Engulf Dying Cells

In vivo data suggest that monocytes participate critically in cross-presentation, but other data suggest that lymph node resident dendritic cells (DCs) mainly cross-present. Here, we utilized a three-dimensional model of a blood vessel wall that endogenously supports DC development from human monocytes, and we incorporated dying autologous cells in the subendothelial matrix of the model. Flu-infected dying cells promoted monocytes to become mature DCs and cross-present cell-associated Ags for the activation of CTLs. Similar responses were induced by loading the dying cells with the TLR7/8 ligand ssRNA, whereas dying cells loaded with TLR3 ligand were less efficient. Monocyte-derived DCs that developed in this model cross-presented Ag to T cells efficiently regardless of whether they engulfed detectable amounts of labeled dying cells. Unexpectedly, the monocyte-derived cells that directly engulfed dying cells in vitro were not the major APCs stimulating CD8(+) lymphocytes. Instead, bystander DCs acquired more robust capacity to cross-prime through receipt of MHC class I/peptide from the phagocytic, monocyte-derived cells. In mice, lymph node-homing monocyte-derived DCs processed Ags from engulfed cells and then transferred MHC class I/peptide complexes to confer cross-priming capacity to MHC class I-deficient lymph node resident CD8alpha(+) DCs. Thus, natural or synthetic TLR7/8 agonists contained within dying cells promote the conversion of monocytes to DCs with capacity for cross-presentation and for "cross-dressing" other DCs. These data reveal a way in which migratory monocyte-derived DCs and other DCs, like lymph node resident DCs, both mediate cross-presentation.

Kinetics of primary and memory cytotoxic T lymphocyte responses to herpes simplex virus 1 infection: granzyme B mediated CTL activity.

Herpes simplex virus type 1 is one of the most common viruses among human population. Studies demonstrate the essential role of cell mediated immunity, especially CD8+ T cells, in prevention and clearance of HSV1. It is of great importance to improve our knowledge about the kinetics of CTL responses to primary and secondary HSV-1 infection. Using a sensitive technique for detection and analysis of CD8+ T cells, granzyme B ELISA, the CTL activity in the spleens of Balb/c mice at various time points after intraperitoneal administration of HSV1 (strain KOS) in primary and secondary infections were determined. During acute HSV-1 infection, virus specific cytotoxic T cells were detected at day 5 post virus inoculation and peaked at day 7. Six hours after secondary infection the activity of memory CD8+ T cells was detected and peaked at 12 hours post infection. The peak of CTL activity was found to be day 7 post infection in primary HSV-1 infections which decreased with time. In secondary infections, the activity of CTLs reached the highest level at 12 hours post infection.

Rapid Degranulation of NK Cells following Activation by HIV-Specific Antibodies

Ab-dependent cellular cytotoxicity (ADCC) Abs stimulate NK cell effector functions and play a role in protecting from and controlling viral infections. We characterized ADCC Abs in a cross-sectional cohort of 80 HIV-infected subjects not on antiretroviral therapy. We analyzed ADCC response by killing fluorescently labeled target cells, as well as expression of IFN-gamma and the degranulation marker CD107a from activated NK cells as measured by a novel intracellular cytokine assay. HIV-specific ADCC directed toward Envelope proteins were present in the majority of 80 untreated HIV-infected individuals measured by killing function. Similarly, most subjects had HIV-specific Abs that mediated degranulation or cytokine expression by NK cells. Interestingly, there was a poor correlation between ADCC-mediated killing of fluorescently labeled whole Envelope protein-pulsed cell lines and Ab-mediated expression of IFN-gamma by NK cells. However, in contrast to healthy donor NK cells, autologous patient NK cells more effectively degranulated granzyme B in response to ADCC activation. Activation of NK cells in response to stimulation by HIV-specific Abs occurs at least as rapidly as activation of Gag-specific CTLs. Our studies highlight the complexity of ab-mediated NK cell activation in HIV infection, and suggest new avenues toward studying the utility of ADCC in controlling HIV infection.

Inhibition of cellular alpha-glucosidases results in increased presentation of hepatitis B virus glycoprotein-derived peptides by MHC class I

Inhibitors of alpha glucosidases prevent the trimming of oligosaccharides on certain nascent glycoproteins, including the hepatitis B virus MHBs envelope glycoprotein. MHBs proteins with untrimmed oligosaccharides do not interact with calnexin, increasing protein misfolding and subsequent degradation by proteasomes. As peptides loaded onto newly synthesized MHC class I complexes are predominantly derived from proteasomes, the possibility that glucosidase inhibition could increase presentation by MHC class I was determined. Using either a model epitope, or a natural MHBs epitope, it was demonstrated that glucosidase inhibitors enhanced presentation by MHC class I and promoted activation of antigen-specific CTLs, suggesting a pharmacologic approach to immune modulation.

Immunization with a Mimotope of GD2 Ganglioside Induces CD8+ T Cells That Recognize Cell Adhesion Molecules on Tumor Cells

The GD2 ganglioside expressed on neuroectodermal tumor cells has been used as a target for passive and active immunotherapy in patients with malignant melanoma and neuroblastoma. We have reported that immunization of mice with a 47-LDA mimotope of GD2, isolated from a phage display peptide library with anti-GD2 mAb 14G2a, induces MHC class I-restricted CD8(+) T cell responses to syngeneic neuroblastoma tumor cells. The cytotoxic activity of the vaccine-induced CTLs was independent of GD2 expression, suggesting recognition of a novel tumor-associated Ag cross-reacting with 47-LDA. Glycan microarray and immunoblotting studies using 14G2a mAb demonstrated that this Ab is highly specific for the entire carbohydrate motif of GD2 but also cross-reacts with a 105 kDa glycoprotein expressed by GD2(+) and GD2(-) neuroblastoma and melanoma cells. Functional studies of tumor cells grown in three-dimensional collagen cultures with 14G2a mAb showed decreases in matrix metalloproteinase-2 activation, a process regulated by the 105 kDa-activated leukocyte cell adhesion molecule (ALCAM/CD166). A recombinant CD166 glycoprotein was shown to be recognized by 14G2a Ab and inhibition of CD166 expression by RNA interference ablated the cell sensitivity to lysis by 47-LDA-induced CD8(+) T cells in vitro and in vivo. The binding of 14G2a to CD166 was not disruptable by a variety of exo- and endo-glycosidases, implying recognition of a non-glycan epitope on CD166. These results suggest that the vaccine-induced CTLs recognize a 47-LDA cross-reactive epitope expressed by CD166, and reveal a novel mechanism of induction of potent tumor-specific cellular responses by mimotopes of tumor-associated carbohydrate Ags.