MHC Class I-restricted Cytotoxic T Lymphocytes Research Articles

The live-attenuated yellow fever vaccine 17D induces broad and potent T cell responses against several viral proteins in Indian rhesus macaques--implications for recombinant vaccine design.

The yellow fever vaccine 17D (YF17D) is one of the most effective vaccines. Its wide use and favorable safety profile make it a prime candidate for recombinant vaccines. It is believed that neutralizing antibodies account for a large measure of the protection afforded to YF17D-vaccinated individuals, however cytotoxic T lymphocyte (CTL) responses have been described in the setting of YF17D vaccination. YF17D is an ssRNA flavivirus that is translated as a full-length polyprotein, several domains of which pass into the lumen of the endoplasmic reticulum (ER). The processing and presentation machinery for MHC class I-restricted CTL responses favor cytoplasmic peptides that are transported into the ER by the transporter associated with antigen presentation proteins. In order to inform recombinant vaccine design, we sought to determine if YF17D-induced CTL responses preferentially targeted viral domains that remain within the cytoplasm. We performed whole YF17D proteome mapping of CTL responses in six Indian rhesus macaques vaccinated with YF17D using overlapping YF17D peptides. We found that the ER luminal E protein was the most immunogenic viral protein followed closely by the cytoplasmic NS3 and NS5 proteins. These results suggest that antigen processing and presentation in this model system is not preferentially affected by the subcellular location of the viral proteins that are the source of CTL epitopes. The data also suggest potential immunogenic regions of YF17D that could serve as the focus of recombinant T cell vaccine development.

Enhanced protective immunity against H5N1 influenza virus challenge by vaccination with DNA expressing a chimeric hemagglutinin in combination with an MHC class I-restricted epitope of nucleoprotein in mice

DNA vaccination is an effective means of eliciting both humoral and cellular immune responses. The hemagglutinin (HA) surface protein of influenza A virus is a major target of protective antibody responses induced by virus infection or by vaccination and is widely considered to be the antigen of choice for an influenza vaccine. Cytotoxic T lymphocyte (CTL) responses directed against the conserved nucleoprotein (NP) are thought to play an important role in clearing virus and promoting survival and recovery from influenza. In this study, we developed a novel DNA vaccine approach using a chimeric plasmid consisting of the HA of H5N1 influenza virus in which an MHC class I-restricted NP-specific CTL epitope (NP147-155) was inserted. Immunogenicity and antiviral efficacy of this vaccine was assessed in mouse models. A similar level of HA expression was achieved in 293T cells transfected with pHA/NP(147-155) compared to that with pHA. Besides eliciting the specific anti-HA antibody responses, vaccination using pHA/NP(147-155) in mice induced NP epitope-specific CD8(+) T cell responses, which are generally not inducible by vaccination with pHA alone. After H5N1 influenza virus challenge, BALB/c mice vaccinated with pHA/NP(147-155) exhibited reduced inflammation severity and lung viral titers compared to those vaccinated with pHA. Our work may contribute to improvement of HA-based influenza DNA vaccines.

Generation of robust cytotoxic T lymphocytes against prostate specific antigen by transduction of dendritic cells using protein and recombinant adeno-associated virus.

Prostate cancer is the most common male cancer and there is an urgent need for adjuvant therapy such as immunotherapy. Recombinant adeno-associated virus type 2 (rAAV) vectors are useful for antigen gene-loading of human dendritic cells (DC) and for the rapid generation of cytotoxic T lymphocytes (CTL). In this study, we report a protocol for AAV-loading of DC with the AAV-loading of self-antigen prostate specific antigen (PSA) resulting in generation of CTL. PSA and cytokine expression, Cell surface marker analysis of DC and CTL cells were done using a FACScalibur flow cytometer. Chromium-51 release assay was used to analyze the killing activity of CTL. It was found that AAV-loading of DC with the PSA gene is superior to PSA protein loading of the same antigen for generating effective CTL. AAV/PSA-loading of DC was found to result in: (1) strong, rapid PSA-specific, MHC Class I-restricted CTL, (2) PSA expression in DC, (3) high CD80, CD83, and CD86 expression on DC, (4) high level of IL-12 and low level of IL-10 in DC, (5) T cell populations with significant interferon gamma (IFNgamma) expression, but low IL-4 expression, (6) high proliferation of T cell populations, (7) high CD8:CD4 and CD8:CD56 T cell ratios. The reason for generation of robust CTL is partly explained by the characteristics of DC and CTL described. This protocol may be useful for adoptive immunotherapy against self antigens such as PSA for prostate cancer.

Defining MHC class II T helper epitopes for WT1 tumor antigen.

The product of Wilms' tumor gene 1 (WT1) is overexpressed in diverse human tumors, including leukemia, lung and breast cancer, and is often recognized by antibodies in the sera of patients with leukemia. Since WT1 encodes MHC class I-restricted peptides recognized by cytotoxic T lymphocytes (CTL), WT1 has been considered as a promising tumor-associated antigen (TAA) for developing anticancer immunotherapy. In order to carry out an effective peptide-based cancer immunotherapy, MHC class II-restricted epitope peptides that elicit anti-tumor CD4(+) helper T lymphocytes (HTL) will be needed. In this study, we analyzed HTL responses against WT1 antigen using HTL lines elicited by in vitro immunization of human lymphocytes with synthetic peptides predicted to serve as HTL epitopes derived from the sequence of WT1. Two peptides, WT1(124-138) and WT1(247-261), were shown to induce peptide-specific HTL, which were restricted by frequently expressed HLA class II alleles. Here, we also demonstrate that both peptides-reactive HTL lines were capable of recognizing naturally processed antigens presented by dendritic cells pulsed with tumor lysates or directly by WT1+ tumor cells that express MHC class II molecules. Interestingly, the two WT1 HTL epitopes described here are closely situated to known MHC class I-restricted CTL epitopes, raising the possibility of stimulating CTL and HTL responses using a relatively small synthetic peptide vaccine. Because HTL responses to TAA are known to be important for promoting long-lasting anti-tumor CTL responses, the newly described WT1 T-helper epitopes could provide a useful tool for designing powerful vaccines against WT1-expressing tumors.

Use and specificity of breast cancer antigen/milk protein BA46 for generating anti-self-cytotoxic T lymphocytes by recombinant adeno-associated virus-based gene loading of dendritic cells

Antigen-targeted immunotherapy is an emerging treatment for breast cancer. However, useful breast cancer antigens are only found in a subset of cancer patients. BA46, also known as lactadherin, is a membrane-associated glycoprotein that is expressed in most breast cancer cells but not in general hematopoietic cell populations. Moreover, it is much more difficult to generate CTLs against self-antigens. We wished to determine if the use of recombinant adeno-associated virus (rAAV) type 2 vectors for gene-loading of dendritic cells (DCs) could generate rapid, effective cytotoxic T lymphocytes (CTLs) against BA46. We were able to demonstrate that AAV/BA46/Neo-loading of DCs resulted in: (1) BA46 expression in DCs, (2) chromosomal integration of the AAV/BA46/Neo vector within DCs, (3) strong, rapid BA46-specific, MHC class I-restricted CTLs in only 1 week, (4) T-cell populations with significant interferon-gamma (IFN-gamma) expression but low IL-4 expression, (5) high CD80 and CD86 expression in DCs, and (6) high CD8:CD4 and CD8:CD56 T cell ratios. These data suggest that rAAV-loading of DCs may be useful for immunotherapeutic protocols against self-antigens in addition to viral antigens and that the BA46 antigen is potentially appropriate for cell-mediated immunotherapeutic protocols addressing ductal breast cancer.

Generation of cytotoxic T lymphocytes by MHC class I ligands fused to heat shock cognate protein 70.

Immunization with gp96 and heat shock cognate protein 70 (hsc70) purified with in vivo bound naturally occurring peptides or bound to synthetic peptides by in vitro reconstitution has been shown to induce peptide-specific cytotoxic T lymphocytes (CTL). In addition, mycobacterial heat shock protein 70 covalently fused to ovalbumin (OVA)-derived fragments has been shown to generate MHC class I-restricted CTL responses. Here, we genetically fused five different CTL epitopes, including peptides derived from Plasmodium yoelii circumsporozoite protein, tumor antigens, HY antigen and OVA, to either the N- or C-terminus of murine hsc70 and expressed the resulting proteins in Escherichia coli. Vaccination with all five fusion proteins induced peptide-specific CTL, indicating that no cognate flanking regions of CTL epitopes are necessary for the immune response. The point of injection was crucial for CTL induction. CD4(+) T cells were not required for the priming of CD8(+) T cells and vaccination with bone marrow-derived dendritic cells pulsed with hsc70 fusion proteins also elicited CTL responses. Furthermore, by using deletion mutants of hsc70, we identified amino acid residues 280-385 of hsc70 as the region most critical for inducing the CTL response.

Induction of Hepatitis C Virus-Specific Cytotoxic T Lymphocytes in Mice by an Intrahepatic Inoculation with an Expression Plasmid

We assessed the possibility of intrahepatic inoculation with a plasmid encoding hepatitis C virus (HCV) proteins to elicit HCV-specific cytotoxic T lymphocytes (CTL) in mice as a conventional animal model of HCV infection. BALB/c mice were intrahepatically or intramuscularly inoculated with an expression plasmid DNA encoding HCV structural proteins under the control of the elongation factor 1-α promoter. Expressions of HCV-core protein and envelope proteins (E1 and E2) in hepatocytes were detected immunohistochemically 6 days after inoculation. CTL responses were examined using target cells either pulsed with a specific peptide or infected with a recombinant vaccinia virus expressing HCV structural protein. Both intrahepatically and intramuscularly DNA-inoculated mice developed CD8+, MHC class I-restricted CTL responses that recognized the peptide pulsed as well as HCV proteins expressing target cells. These studies demonstrated the usefulness of a murine model of HCV infection induced by direct intrahepatic DNA inoculation for understanding the immunopathogenic mechanisms in HCV infection.

Fusogenic liposomes efficiently deliver exogenous antigen through the cytoplasm into the MHC class I processing pathway

Exogenous soluble proteins enter the endosomal pathway by endocytosis and are presented in association with MHC class II rather than class I. In contrast, the delivery of exogenous protein antigens (Ag) into the cytosol generates MHC class I-restricted cytotoxic T lymphocytes (CTL) responses. Although several immunization approaches, such as the utilization of liposomes, have induced the in vivo priming of MHC class I-restricted CTL responses to protein Ag, it remains unclear whether this priming results from the direct delivery of protein Ag to the cytosol. Here we report that fusogenic liposomes (FL), which are prepared by fusing simple liposomes with Sendai virus particles, can deliver the encapsulated soluble protein directly into the cytosol of cells cultured concurrently and introduce it into the conventional MHC class I Ag presentation pathway. Moreover, a single immunization with ovalbumin (OVA) encapsulated in FL but not in simple liposomes results in the potent priming of OVA-specific CTL. Thus, FL function as an efficient tool for the delivery of CTL vaccines.

Helper T cell determinant peptide contributes to induction of cellular immune responses by peptide vaccines against hepatitis C virus.

The capacity of novel subunit vaccines to generate cytotoxic T lymphocytes (CTLs) against hepatitis C virus (HCV) was assessed. BALB/c mice were immunized with peptides based on the CTL and helper T cell (Th) epitopes of the HCV core, with a mixture of CTL and Th peptides (CTL+Th) or with a conjugated Th-CTL peptide. Mice immunized with CTL, CTL+Th and Th-CTL peptides, but not those immunized with Th peptide, developed HCV core CTL epitope-specific effector cells. Cytotoxic activity induced by immunization with Th-CTL was much higher than that induced by immunization with CTL+Th or CTL alone. However, rapid and high cytotoxic activities against HCV core were not only detected after immunization with peptides containing the CTL epitope but also as a result of infection with recombinant vaccinia virus carrying the HCV core gene after immunization with the Th epitope alone. Immunization with peptides containing the Th epitope also elicited spleen cell proliferation. This study demonstrates the capacity of both Th and CTL activated peptide vaccines to elicit CD8+, MHC class I-restricted CTLs. The capacity of such CTLs to contribute towards a protective and/or pathogenic immune response against HCV can now be assessed in mouse models.

Tumor-specific killer cells in paraneoplastic cerebellar degeneration.

Models for immune-mediated tumor regression in mice have defined an essential role for cytotoxic T lymphocytes (CTLs); however, naturally occurring tumor immunity in humans is poorly understood. Patients with paraneoplastic cerebellar degeneration (PCD) provide an opportunity to explore the mechanisms underlying tumor immunity to breast and ovarian cancer. Although tumor immunity and autoimmune neuronal degeneration in PCD correlates with a specific antibody response to the tumor and brain antigen cdr2, this humoral response has not been shown to be pathogenic. Here we present evidence for a specific cellular immune response in PCD patients. We have detected expanded populations of MHC class I-restricted cdr2-specific CTLs in the blood of 3/3 HLA-A2.1+ PCD patients, providing the first description, to our knowledge, of tumor-specific CTLs using primary human cells in a simple recall assay. Cross-presentation of apoptotic cells by dendritic cells also led to a potent CTL response. These results indicate a model whereby immature dendritic cells that engulf apoptotic tumor cells can mature and migrate to draining lymph organs where they could induce a CTL response to tissue-restricted antigens. In PCD, peripheral activation of cdr2-specific CTLs is likely to contribute to the subsequent development of the autoimmune neuronal degeneration.

Induction of MHC class I-restricted CTL response by DNA immunization with ubiquitin-influenza virus nucleoprotein fusion antigens

DNA vaccines have been shown to be an effective means of inducing cytotoxic T-lymphocyte (CTL) responses in both young and aged mice. Better understanding of the pathways by which antigens encoded by DNA vaccines are processed and presented to CTL may allow for improvements in CTL responses in older animals. Since CTL recognize short peptides presented by MHC class I molecules, and since ubiquitin-dependent proteolysis is widely believed to be responsible for degradation of endogenously synthesized antigens and generation of these peptide ligands, we sought to use ubiquitin (Ub) conjugation to target influenza virus nucleoprotein (NP) antigen into the Ub-proteasome degradation pathway for MHC class I-restricted antigen processing and presentation. However, the addition of the Ub moiety did not affect the half-life of Ub-NP protein in transiently transfected human rhabdomyosarcoma (RD) cells. Moreover, the modifications of NP DNA vaccine with Ub conjugation did not affect their ability to induce a CTL response specific for the H-2K d-restricted NP147–155 epitope, as assessed by both percent cytolysis in bulk CTL culture and by CTL precursor (CTLp) frequency in limiting dilution analysis (LDA). In contrast, the anti-NP antibody (Ab) responses were dramatically reduced in mice immunized with low doses (1 μg) of Ub-NP constructs, compared with mice immunized with wild-type NP DNA. These results demonstrate that Ub conjugation alone does not guarantee targeting of endogenously synthesized antigens for rapid degradation by proteasomes. Furthermore, the ability of ubiquitination to reduce Ab responses to NP without affecting CTL responses suggests that the Ub modifications result in a lower availability of full-length NP from transfected cells in vivo. The implications of these data on antigen presentation and cross-priming are discussed.

Major histocompatibility complex class I presentation of exogenous soluble tumor antigen fused to the B-fragment of Shiga toxin.

Targeting exogenous antigen into the MHC class I-restricted presentation pathway is a prerequisite for the induction of cytotoxic T lymphocytes (CTL) which have been shown to represent an important component of the protective and therapeutic immune response to viral infections and tumors. In this study, we produced recombinant proteins composed of the receptor-binding non-toxic B-fragment of bacterial Shiga toxin derived from Shigella dysenteriae associated with an epitope from a model tumor antigen, Mage 1. We show that Shiga B-Mage 1 fusion proteins carrying an active or inactive endoplasmic reticulum retrieval signal (the C-terminal peptides KDEL or KDELGL, respectively) could be presented by peripheral blood mononuclear cells in an MHC class I-restricted manner to Mage 1-specific CTL. After pulsing B lymphoblastoid cells or dendritic cells with Shiga B-Mage 1 fusion protein, activation of the MHC class I-restricted Mage 1-specific CTL was also demonstrated. In further analysis, we showed that treatment with brefeldin A or paraformaldehyde fixation of Epstein-Barr virus-transformed B cells prevented the presentation of the Mage 1 T cell epitope, which excluded extracellular processing of the antigen. Immunofluorescence analysis also revealed that the Shiga B-Mage 1 fusion protein was largely excluded from Lamp-2-positive lysosomal structures. Therefore, the ability of Shiga toxin B-fragment to target dendritic cells and B cells and to direct antigen into the exogenous class I-restricted pathway makes it an attractive non-living and non-toxic vaccine vector.

CD8+ cytotoxic T lymphocytes of a cynomolgus macaque infected with simian immunodeficiency virus (SIV) mac32H-J5 recognize a nine amino acid epitope in SIV Gag p26.

A detailed analysis of simian immunodeficiency virus (SIV)-specific cytotoxic T lymphocyte (CTL) responses and the identification of the proteins and epitopes they target may improve the design of immunotherapeutic interventions and provide insights into AIDS pathogenesis. Here, we identified a new CTL epitope in the SIV Gag protein, recognized by CD8+ and MHC class I-restricted CTL clones from a long-term asymptomatic cynomolgus macaque (Macaca fascicularis) infected with SIVmac32H-J5. Using overlapping synthetic peptides, the optimal minimal epitope was characterized as a nine amino acid peptide representing amino acids 242-250 of p26 (SVDEQIQWM). CTL recognition was shown to be abolished by amino acid substitutions observed within homologous human immunodeficiency virus (HIV)-1 and HIV-2 sequences.

The mechanism of human autologous gastric signet ring cell tumor rejection by cytotoxic T lymphocytes in the possible context of HLA-A31 molecule.

Tumor rejection antigens in human melanomas, which are recognized by cytotoxic T lymphocytes (CTLs), have recently been identified. To elucidate the cytotoxic mechanism in tumors other than melanoma, several pairs of CTLs and tumor lines were established. The authors report that HLA-A31 may present a tumor rejection antigen that is recognized by the human autologous gastric signet ring cell carcinoma-specific CTL. They also briefly describe the in vitro enhancing effect of interferon-gamma (INF-gamma) on the lysis of tumor cells by autologous CTL. The MHC Class I-restricted CTL clone, TcHST-2, and autologous gastric signet ring cell carcinoma line, HST-2, were established. Cytotoxicity blocking assays of antibodies reacting against the MHC Class I nonpolymorphic determinant and HLA-A, B, and C haplotype elements, which are expressed on the HST-2 cells, were performed. Lysis of the autologous tumor cells (HST-2) by the CTL clone (TcHST-2) was enhanced when the tumor cells were pretreated with IFN-gamma. This lysis was selectively inhibited by the anti-nonpolymorphic MHC Class I determinant monoclonal antibody (MoAb) and anti-HLA-A31 haplotype-specific MoAb. However, TcHST-2 clone was not cytotoxic to HLA-A31+ allogeneic leukemia lines. Pretreatment of target cells with IFN-gamma may be a necessary procedure for the efficient lysis of HST-2 cells by autologous TcHST-2 CTL. The data indicate that TcHST-2 was MHC Class I-restricted HST-2 tumor-specific CTL and suggest that the HLA-A31 haplotype element is an antigen-presenting molecule. Also discussed is the nature of the antigenic peptides in gastric signet ring cell carcinoma.

Down-regulation of major histocompatibility complex class I synthesis by murine cytomegalovirus early gene expression

Murine cytomegalovirus (MCMV) infection of C57BL/6 (H-2b) mice prevents priming of antigen-specific helper and cytotoxic T lymphocytes (CTL) (J. S. Slater, W. S. Futch, V. J. Cavanaugh, and A. E. Campbell, Virology 185:132-139, 1991). In vitro, MCMV infection alters presentation of antigens to antigen-specific, MHC class I-restricted CTL (A. E. Campbell, J. S. Slater, V. J. Cavanaugh, and R. M. Stenberg, J. Virol. 66:3011-3017, 1992). This is accompanied by a significant decrease in surface expression of H-2Kb and H-2Db. We therefore examined the effects of MCMV infection on the intracellular expression of H-2Kb and H-2Db by immunoprecipitation with conformation-independent antibodies. MCMV early-gene products severely repressed synthesis of H-2Kb and H-2Db. This down-regulation was not restored by gamma interferon treatment. The MCMV-induced suppression of MHC class I protein synthesis resulted in a retarded rate of accumulation of these molecules within the cell. In addition, MCMV infection prevented maturation of class I heavy chains to the fully glycosylated forms and inhibited transport of H-2Db from the endoplasmic reticulum-cis Golgi. Virus infection had no effect on the rate of degradation of the class I molecules. These results demonstrate that MCMV early-gene products down-regulate synthesis and posttranslational events in MHC class I expression, thereby limiting the number of antigen-presenting molecules within infected cells. This limitation defines a potential mechanism for regulation of MHC class I-restricted antigen presentation by MCMV.

Can one predict antigenic peptides for MHC class I-restricted cytotoxic T lymphocytes useful for vaccination?

The cytotoxic T-lymphocyte (CTL) response can be crucial for efficient immunological control of intracellular pathogens and the MHC class I-restricted CTL have a major role to play in this process. They recognize complexes associating antigen-derived peptides with MHC class I molecules expressed on infected target cells. The characterization of these antigenic peptides is thus a key issue for developing vaccines efficient in inducing specific CTL. Recently, by sequencing the whole set of self-peptides eluted from a given MHC class I molecule, Falk and colleagues have found that they have a homogeneous 8–10 residue length and contain allele-specific peptidic motifs with two conservative dominant anchor residues. The existence of consensus motifs opens the way for a strategy to predict the MHC class I-restricted T-cell epitopes and here we discuss such an approach using hen egg lysozyme (HEL) as an antigenic model. Two HEL peptides corresponding to allele-specific motifs were found, HEL(49–56) and HEL(70–78) peptides, which can associate with MHC class I H-2K b and H-2D b molecules, respectively. The HEL peptide HEL(70–78) was found to be able to induce HEL-specific CTL in H-2 b mice also. Moreover, using an empirical approach, we have also characterized the N-terminal HEL(1–17) peptide as an immunodominant antigenic peptide in the H-2 k haplotype. This peptide presented by H-2K k molecules neither contained the corresponding allele-specific binding motif nor fitted the expected 8–10 residue length. These and other recently reported data illustrate the validity and limitation of predicting antigenic peptides that can bind to MHC class I by looking for allele-specific peptidie motifs in an antigen peptide sequence.

Induction of syngeneic cytotoxic T lymphocytes against a B cell tumor: III. MHC class I-Restricted CTL recognizes the processed form(s) of idiotype

The purpose of this work was to determine the molecular nature of the idiotypic Ig of a B cell tumor, 2C3, involved in the induction of anti-idiotypic cytotoxic T lymphocytes (CTL). We previously reported that hyperimmunization of mice with irradiated 2C3 cells provides effective tumor protection by inducing MHC class I-restricted CTL. Due to the enormous heterogeneity of the splenic CTL further study could not be undertaken on the idiotype (Id)-CTL interaction. Subsequently an anti-idiotypic CTL line, A102, and a highly Id-specific CTL clone, 102.F5, have been developed. In the present investigation we report that the processed forms of idiotypic determinants are responsible for induction and activation of these specific effector CTL. Inhibition studies using anti-TcR and anti-MHC class I mAbs showed that the TcR-CD3 complex of the anti-idiotypic CTL recognized 2C3 Id in the context of MHC class I antigens. The cytotoxicity of these CTL could not be inhibited with affinity-purified 2C3 Ig used as such or after pulsing with splenic antigen-presenting cells (APC). Furthermore, using brefeldin A (BFA) and chloroquine (CLQ), which are specific inhibitors of cytosolic and endosomal antigen processing pathways, respectively, it has also been observed that exposure of 2C3 to BFA but not CLQ prevents its cytolysis by both anti-idiotypic CTL line and clone. These results clearly indicate that endogenously produced idiotypic determinants of 2C3 Ig are processed in pre-Golgi vesicle, possibly in the ER, along with MHC class I antigens and then are transported to the membrane. Treatment of 2C3 with BFA, however, did not exert any effect on the expression of membrane-associated Ig of 2C3 cells. Therefore, it is the processed form rather than the bona fide receptor Ig on the cell surface that is recognized by the Id-specific CTL.