Class I-restricted Cytotoxic T Lymphocytes Research Articles

Endogenous Immunoglobulin-Derived Neoepitopes Are Processed and Form a Sizeable Fraction of the HLA Class I Ligandome of Human Lymphoma Cells

T cells recognizing solid tumors and Hodgkin's lymphoma can be released from anergy by immune checkpoint inhibitors. Its therapeutic success seems to be associated with the abundance of neoepitopes within the tumor mutanome. In neoplastic B cells, VDJ recombination and somatic hypermutation (SHM) generate unique immunoglobulin (Ig) peptide sequences that predictably contribute to the lymphoma mutanome. Efficient presentation of a neoepitope by an individual's HLA complex is an essential requirement for neoepitope-directed T-cell immunity. Presentation of Ig-derived peptides in HLA class I has hitherto been demonstrated only indirectly, and there is as yet no direct evidence for presentation of Ig-derived neoepitopes of primary human lymphoma cells.We analyzed HLA-presented Ig-derived peptides in 9 clonal B-cell populations: 3 chronic lymphocytic leukemias (CLL), 1 hairy cell leukemia, 1 follicular lymphoma (FL), 3 EBV-transformed lymphoblastoid cell lines, and the U299 myeloma cell line. Full-length B-cell receptor (BCR) VDJ and VJ sequences were obtained by unbiased ARTISAN PCR and Pacific Biosciences next generation sequencing. 1067 unique Ig-derived nonamers were predicted to bind the HLA class I alleles expressed by the respective B-cell clone by the NetCTLpan bioinformatics tool. 650 candidate epitopes (60.9%) were derived from constant (C) regions; 417 (39.1%) from variable (V) regions. 146 predicted peptides from V regions (35.0%) contained at least one amino acid change due to SHM or at least one amino acid from CDR3 and were therefore considered potential neoepitopes.To investigate experimentally which epitopes are processed intracellularly and presented by HLA class I, HLA class I-peptide complexes were immunoaffinity purified by the monoclonal antibody W6.23 and analyzed by liquid chromatography and tandem mass spectrometry. In total, 53,663 unique peptides (8-15mers) were identified by Uniprot matching with a Mascot Ion Score of >20. HLA ligandome sizes of individual B-cell populations ranged from 600 to 14,091 unique peptides per case. Within any HLA ligandome, 6 to 81 peptides were annotated as BCR-derived epitopes if they matched the individual BCR sequences. Of the total of 276 eluted BCR peptides, 203 (73.5%) where derived from C regions and 73 (26.5%) from V regions. 25 eluted peptides (range 0-10 per case) were derived from CDR3 regions or contained SHM-induced amino acid changes, thus fulfilling the definition of neoepitopes. Neoepitopes were detected in all cases with more than 109 cells as input material. Up to date, 4 neoepitopes have been synthesized and their mass spectra confirmed. Confirmation of all remaining neoepitopes is ongoing. No BCR-derived neoepitopes could be detected in an IGHV-unmutated CLL and the FL. These two cases had the lowest number of input cells and small overall ligandome sizes of only 600 and 1015 unique UniProt-matched peptides, respectively.We demonstrate for the first time that primary neoplastic B cells process and present BCR-derived neoepitopes in the context of HLA class I. Despite their origin from only two polypeptides, BCR epitopes and neoepitopes represent app. 0.5% and 0.05% of the total HLA class I ligandome, respectively. The challenging identification of BCR neoepitopes was possible by unbiased identification of BCR transcripts combined with next generation sequencing and targeted search for HLA class I-bound peptides. Matching fragmentation patterns of native and synthetic peptides suggest high specificity of this strategy. With respect to sensitivity, the ability to identify low frequency peptides appears to be strongly dependent on the amount of input cells.Our data close a gap in the mechanism underlying the evidence that highly immunogenic formulations of an idiotype vaccine are able to induce MHC class I-restricted cytotoxic T-cell responses. While phase III trials of idiotype vaccination aiming to induce anti-Ig antibodies failed to demonstrate convincing prolongation of clinical remissions achieved by chemotherapy, our data lend support for exploring idiotype-specific T-cell immunity against B-cell lymphomas. With recent advances in peptide synthesis, adjuvant formulations, and the availability of check point inhibitors to surpass regulatory activity, active immunotherapy targeting the lymphoma idiotype may regain appeal as truly personalized immune therapy. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Crystal structure of the N-myristoylated lipopeptide-bound MHC class I complex.

The covalent conjugation of a 14-carbon saturated fatty acid (myristic acid) to the amino-terminal glycine residue is critical for some viral proteins to function. This protein lipidation modification, termed N-myristoylation, is targeted by host cytotoxic T lymphocytes (CTLs) that specifically recognize N-myristoylated short peptides; however, the molecular mechanisms underlying lipopeptide antigen (Ag) presentation remain elusive. Here we show that a primate major histocompatibility complex (MHC) class I-encoded protein is capable of binding N-myristoylated 5-mer peptides and presenting them to specific CTLs. A high-resolution X-ray crystallographic analysis of the MHC class I:lipopeptide complex reveals an Ag-binding groove that is elaborately constructed to bind N-myristoylated short peptides rather than prototypic 9-mer peptides. The identification of lipopeptide-specific, MHC class I-restricted CTLs indicates that the widely accepted concept of MHC class I-mediated presentation of long peptides to CTLs may need some modifications to incorporate a novel MHC class I function of lipopeptide Ag presentation.

Population-Level Immune-Mediated Adaptation in HIV-1 Polymerase during the North American Epidemic

Human leukocyte antigen (HLA) class I-associated polymorphisms in HIV-1 that persist upon transmission to HLA-mismatched hosts may spread in the population as the epidemic progresses. Transmission of HIV-1 sequences containing such adaptations may undermine cellular immune responses to the incoming virus in future hosts. Building upon previous work, we investigated the extent of HLA-associated polymorphism accumulation in HIV-1 polymerase (Pol) through comparative analysis of linked HIV-1/HLA class I genotypes sampled during historic (1979 to 1989; n = 338) and modern (2001 to 2011; n = 278) eras from across North America (Vancouver, BC, Canada; Boston, MA; New York, NY; and San Francisco, CA). Phylogenies inferred from historic and modern HIV-1 Pol sequences were star-like in shape, with an inferred most recent common ancestor (epidemic founder virus) sequence nearly identical to the modern North American subtype B consensus sequence. Nevertheless, modern HIV-1 Pol sequences exhibited roughly 2-fold-higher patristic (tip-to-tip) genetic distances than historic sequences, with HLA pressures likely driving ongoing diversification. Moreover, the frequencies of published HLA-associated polymorphisms in individuals lacking the selecting HLA class I allele was on average ∼2.5-fold higher in the modern than in the historic era, supporting their spread in circulation, though some remained stable in frequency during this time. Notably, polymorphisms restricted by protective HLA alleles appear to be spreading to a greater relative extent than others, though these increases are generally of modest absolute magnitude. However, despite evidence of polymorphism spread, North American hosts generally remain at relatively low risk of acquiring an HIV-1 polymerase sequence substantially preadapted to their HLA profiles, even in the present era. HLA class I-restricted cytotoxic T-lymphocyte (CTL) escape mutations in HIV-1 that persist upon transmission may accumulate in circulation over time, potentially undermining host antiviral immunity to the transmitted viral strain. We studied >600 experimentally collected HIV-1 polymerase sequences linked to host HLA information dating back to 1979, along with phylogenetically reconstructed HIV-1 sequences dating back to the virus' introduction into North America. Overall, our results support the gradual spread of many-though not all-HIV-1 polymerase immune escape mutations in circulation over time. This is consistent with recent observations from other global regions, though the extent of polymorphism accumulation in North America appears to be lower than in populations with high seroprevalence, older epidemics, and/or limited HLA diversity. Importantly, the risk of acquiring an HIV-1 polymerase sequence at transmission that is substantially preadapted to one's HLA profile remains relatively low in North America, even in the present era.

The modification of Tapasin enhances cytotoxic T lymphocyte activity of intracellularly delivered CTL epitopes <italic>via</italic> cytoplasmic transduction peptide

Previous studies have demonstrated that the therapeutic vaccine based on the enhancement of hepatitis B virus (HBV)-specific cytotoxic T lymphocyte (CTL) activity may lead to viral clearance in HBV-infected individuals. The endoplasmic reticulum (ER) chaperone Tapasin plays an important role in major histocompatibility complex (MHC) class I assembly and enhances specific MHC class I-restricted CTL activity by allowing more peptides to be translocated into the ER. Combining the specificity of hepatitis B core antigen (HBcAg) CTL epitope, the cell-penetrating property of cytoplasmic transduction peptide (CTP), and chaperone Tapasin may elicit robust specific HBV immune responses. In the present study, we confirmed the cytoplasmic localization preference of CTP-HBcAg(18-27)-Tapasin fusion protein in vitro and evaluated the effects on promoting bone marrow-derived dendritic cells (BMDCs) maturation and enhancing T cells response to generate specific CTLs. Our results showed that CTP-HBcAg(18-27)-Tapasin fusion protein could not only penetrate into the cytoplasm exactly and effectively to elevate Tapasin expression, but also increase the expression of surface molecules (CD80, CD83, CD86, and MHC-I) and secretion of cytokine (IL-12p70) of DCs. Moreover, DCs treated with the above fusion proteins increased significantly the cytokine secretion of proliferated T cells in vitro, the percentages of IFN-γ(+)CD8(+) T cells and specific CTL responses compared with control groups. In conclusion, the modification of Tapasin can enhance the presentation of targeting antigens via intracellular delivery to DCs and elicit specific CTL immune responses efficiently.

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.

Multiple, Non-conserved, Internal Viral Ligands Naturally Presented by HLA-B27 in Human Respiratory Syncytial Virus-infected Cells

Cytotoxic T lymphocyte (CTL)-mediated death of virus-infected cells requires prior recognition of short viral peptide antigens that are presented by human leukocyte antigen (HLA) class I molecules on the surface of infected cells. The CTL response is critical for the clearance of human respiratory syncytial virus (HRSV) infection. Using mass spectrometry analysis of complex HLA-bound peptide pools isolated from large amounts of HRSV-infected cells, we identified nine naturally processed HLA-B27 ligands. The isolated peptides are derived from six internal, not envelope, proteins of the infective virus. The sequences of most of these ligands are not conserved between different HRSV strains, suggesting a mechanism to explain recurrent infection with virus of different HRSV antigenic subgroups. In addition, these nine ligands represent a significant fraction of the proteome of this virus, which is monitored by the same HLA class I allele. These data have implications for vaccine development as well as for analysis of the CTL response.

Maturation Pathways of Dendritic Cells Determine TAP1 and TAP2 Levels and Cross-presenting Function

Ability to cross-present exogenous antigens in the human leukocyte antigen class I pathway is key to the antigen presenting function of mature tumor cell-loaded dendritic cells (DC). Conditions of DC maturation have been shown to be important for DCs ability to produce proinflammatory cytokines and induce T cell effector functions. However, it remains unknown if the different pathways of maturation are associated with modulation of the ability of mature DCs to cross-present tumor antigens (TA). Here, we compare DC matured with 3 clinically relevant cytokine combinations including interleukin (IL)-1 beta, tumor necrosis factor-alpha, IL-6 (termed DC-0), DC-0 cells incubated with prostaglandin-2 (termed DC-0+prostaglandin-2), or DC treated with interferon-gamma, interferon-alpha, tumor necrosis factor-alpha, Poly I:C, and IL1-beta (termed DC-1). We found that these DC vary in their ability to cross-present TA to cytotoxic T lymphocytes (CTL), with the DC-1 cytokine combination being significantly more effective than the other 2. TA cross presentation and CTL priming were strongly correlated with level of expression of the antigen processing machinery components, TAP1 and TAP2, indicating that these components could be used as biomarkers to standardize DC preparations for optimal function. However, the up-regulation of TAP1/TAP2 was not sufficient to explain the enhanced cross-presentation ability of DC-1 cells, as the use of IFN-gamma alone to up-regulate TAP1/TAP2 did not generate DC as effective at cross-presentation as the full DC-1 maturation cytokine combination. These data indicate for the first time that the pathways of DC maturation modulate antigen processing machinery component expression to different extents and that differently matured DC vary in the ability to cross-present TA to human leukocyte antigen class I-restricted CTL.

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.

Whole-tumor-antigen-pulsed dendritic cells elicit cytotoxic T-cell response against pediatric nasopharyngeal carcinoma in vitro

Nasopharyngeal carcinoma (NPC) is endemic in Southeast Asia. Although dendritic cell (DC)-based vaccine has emerged as a promising immunotherapy for various malignancies, its use in pediatric nasopharyngeal carcinoma (PNPC) has not been addressed. In this study, DCs isolated from peripheral blood monocytes of three pediatric patients with advanced (stage IV) NPC were incubated with whole-tumor-antigen preparations and differentiated into immature DCs in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4, and then underwent maturation when exposed to tumor necrosis factor-alpha. Upon maturation, DCs acquired the ability to stimulate T-cell proliferation as examined by [(3)H]-thymidine incorporation and the ability of these T-cells to secrete interferon-gamma as determined by enzyme-linked immunosorbent spot assay. Cytotoxic assay revealed that mature tumor-antigen-pulsed DCs induced cytotoxic activity of the T-cells against both autologous and allogeneic NPC tumor cells (including NPC tumor cells from a different individual or from CNE-2Z, a poorly differentiated human NPC cell line). Blocking HLA class I molecules by W6/32 inhibited T-cell-mediated cytotoxic activity in both autologous and allogeneic settings. Our results indicate that DCs pulsed with whole-tumor-antigen can effectively activate HLA class I-restricted cytotoxic T-cells in vitro, and thus provide experimental basis for their future clinical use in PNPC.

Cytotoxic T-lymphocyte–mediated killing of human pancreatic islet cells in vitro

Cytotoxic T lymphocytes (CTL) are believed to play an essential role in beta-cell destruction leading to development of type 1 diabetes and allogeneic islet graft failure. We aimed to identify the mechanisms used by CTL to kill human beta cells. CTL clones that recognize epitopes from influenza virus and Epstein-Barr virus restricted by human leukocyte antigen (HLA)-A0201 and -B0801, respectively, were used to investigate the susceptibility of human beta cells to CTL. In a short-term (5-hour) assay, CTL killed human islet cells of the appropriate major histocompatibility complex (MHC) class I type that had been pulsed with viral peptides. Killing was increased by pretreating islets with interferon gamma that increases MHC class I on target cells. Killing was abolished by incubation of CTL with the perforin inhibitor concanamycin A. The Fas pathway did not contribute to killing because blocking with neutralizing anti-Fas ligand antibody did not significantly reduce beta-cell killing. In conclusion, we report a novel way of investigating the interaction between CTL and human islets. Human islets were rapidly killed in vitro by MHC class I-restricted CTL predominantly by the granule exocytosis pathway.

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.

Cancer immunotherapy targeting Wilms’ tumor gene WT1 product

The Wilms’ tumor gene WT1 is expressed at high levels in leukemic blast cells in most acute myeloid and lymphoblastic leukemias. In myelodysplastic syndrome, WT1 mRNA expression levels increase along with disease progression; thus, WT1 mRNA is a tumor marker for leukemic blast cells. WT mRNA is also expressed at high levels in various types of solid cancers, including cancers of the lung, breast, colon and pancreas. Patients with WT1-expressing tumors produce antibodies and cytotoxic T-lymphocytes against WT1 protein, indicating that WT1 protein is highly immunogenic and a promising tumor antigen. Major histocompatibility complex class I-restricted cytotoxic T-lymphocyte and class II-restricted helper epitopes of WT1 protein were identified, and clinical studies of cancer immunotherapy using these cytotoxic T-lymphocyte epitope peptides were performed without significant adverse effect and with clinical results promising enough to encourage further clinical trials. The clinical efficacy of cancer immunotherapy targeting the WT1 protein should be clarified by a large-scale clinical study.

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.

Induction of tumor-specific cytotoxicity in tumor infiltrating lymphocytes by HPV16 and HPV18 E7-pulsed autologous dendritic cells in patients with cancer of the uterine cervix

Objective To evaluate the potential of autologous dendritic cells (DC) pulsed with HPV16 and HPV18 E7 oncoprotein in restoring tumor-specific cytotoxicity in populations of tumor infiltrating lymphocytes (TIL) for adoptive immunotherapy of cervical cancer patients. Methods Full-length E7-pulsed DC-stimulated CD8 + T cells derived from peripheral blood (PBL) and from tumor tissues (TIL) were tested and compared for their ability to induce a HLA class-I-restricted cytotoxic T lymphocyte (CTL) response against autologous tumor cells. In addition, in order to correlate cytotoxic activity by CTL with a particular lymphoid subset, analysis of surface antigens and intracellular CD3 ζ chain and two-color flow cytometric analysis of intracellular cytokine expression (IFN-γ vs IL-4) at the single cell level were performed. Results DC stimulation induced powerful cytotoxicity against autologous tumor target cells by TIL-derived CD8 + T cells from all three cervical cancer patients, while autologous Epstein–Barr virus-transformed lymphoblastoid cell lines were not lysed. Killing of autologous tumor cells was higher by CD8 + T cells from TIL compared to PBL ( P > 0.01) and was more strongly inhibited by anti-HLA class I MAb ( P > 0.05). Phenotypically, all CTL populations were CD3 +/CD8 +, with higher levels of CD56 expression by TIL-derived CTL. Finally, although a marked Type 1 cytokine bias (i.e., IFN-γ high/IL-4 low) was observable in both PBL- and TIL-derived DC-stimulated CD8 + T cell populations, TIL-derived CD8 + T cells showed a higher percentage of IFN-γ-positive cells compared to PBL. Conclusions Full-length E7-pulsed DC can consistently restore strong CD8 + CTL responses against autologous HPV16- and HPV18-infected cervical cancer cells. DC-stimulated TIL may represent a superior source of tumor-specific CTL compared to PBL for adoptive T cell immunotherapy of patients harboring metastatic or recurrent cervical cancer refractory to standard treatment modalities.

The early expression of glycoprotein B from herpes simplex virus can be detected by antigen-specific CD8+ T cells.

The immune response to cutaneous herpes simplex virus type 1 (HSV-1) infection begins with remarkable rapidity. Activation of specific cytotoxic T lymphocytes (CTL) begins within hours of infection, even though the response within the draining lymph nodes peaks nearly 5 days later. HSV gene products are classified into three main groups, alpha, beta, and gamma, based on their kinetics and requirements for expression. In C57BL/6 mice, the immunodominant epitope from HSV is derived from glycoprotein B (gB(498-505)). While gB is considered a gamma or "late" gene product, previous reports have indicated that some level of gene expression may occur soon after infection. Using brefeldin A as a specific inhibitor of viral antigen presentation to major histocompatibility complex class I-restricted CTL, we have formally addressed the timing of gB peptide expression in an immunologically relevant manner following infection. Presentation of gB peptide detected by T-cell activation was first observed within 2 h of infection. Comparison with another viral epitope expressed early during infection, HSV-1 ribonucleotide reductase, demonstrated that gB is presented with the same kinetics as this classical early-gene product. Moreover, this rapidity of gB expression was further illustrated via rapid priming of naïve transgenic CD8(+) T cells in vivo after HSV-1 infection of mice. These results establish that gB is expressed rapidly following HSV-1 infection, at levels capable of effectively stimulating CD8(+) T cells.

Successful generation of sperm protein 17 (Sp17)-specific cytotoxic T lymphocytes from normal donors: implication for tumour-specific adoptive immunotherapy following allogeneic stem cell transplantation for Sp17-positive multiple myeloma.

Sperm protein 17 (Sp17) is a highly immunogenic cancer-testis antigen expressed by tumour cells from up to 30% of patients with multiple myeloma (MM). We recently successfully generated Sp17-specific human leucocyte antigen (HLA)-A1 and B27-restricted cytotoxic T lymphocytes (CTLs) from the peripheral blood of a healthy donor. Because CTLs were able to kill HLA-matched fresh myeloma cells, it may be possible to generate and administer myeloma-specific donor T cells to MM patients following allogeneic stem cell transplantation to enhance graft-versus-myeloma (GVM) without inducing graft-versus-host disease (GVHD). To determine how widely applicable this approach is, we have determined the ability to generate Sp17-specific CTLs from four consecutive healthy donors with other HLA class I phenotypes. We found that Sp17-specific HLA class I-restricted CTLs could be easily generated from all four donors. Sp17-specific CTLs were primarily CD8 in phenotype and produced interferon-gamma and very little interleukin-4. These T cells killed target cells primarily via the perforin-mediated route. These results therefore suggest that myeloma-specific donor T-cell infusion that targets Sp17 to selectively enhance GVM could be applicable to patients with Sp17+ MM.

Salmonella enterica serovar Typhi live vector vaccines delivered intranasally elicit regional and systemic specific CD8+ major histocompatibility class I-restricted cytotoxic T lymphocytes.

We investigated the ability of live attenuated Salmonella enterica serovar Typhi strains delivered to mice intranasally to induce specific cytotoxic T-lymphocyte (CTL) responses at regional and systemic levels. Mice immunized with two doses (28 days apart) of Salmonella serovar Typhi strain Ty21a, the licensed oral typhoid vaccine, and genetically attenuated mutants CVD 908 (DeltaaroC DeltaaroD), CVD 915 (DeltaguaBA), and CVD 908-htrA (DeltaaroC DeltaaroD DeltahtrA) induced CTL specific for Salmonella serovar Typhi-infected cells in spleens and cervical lymph nodes. CTL were detected in effector T cells that had been expanded in vitro for 7 days in the presence of Salmonella-infected syngeneic splenocytes. A second round of stimulation further enhanced the levels of specific cytotoxicity. CTL activity was observed in sorted alphabeta+ CD8+ T cells, which were remarkably increased after expansion, but not in CD4+ T cells. CTL from both cervical lymph nodes and spleens failed to recognize Salmonella-infected major histocompatibility complex (MHC)-mismatched cells, indicating that the responses were MHC restricted. Studies in which MHC blocking antibodies were used showed that H-2L(d) was the restriction element. This is the first demonstration that Salmonella serovar Typhi vaccines delivered intranasally elicit CD8+ MHC class I-restricted CTL. The results further support the usefulness of the murine intranasal model for evaluating the immunogenicity of typhoid vaccine candidates at the preclinical level.

Stimulation of autologous antitumor T-cell responses against medullary thyroid carcinoma using tumor lysate-pulsed dendritic cells.

Dendritic cells (DCs) have attracted wide interest because of their unique capacity to elicit primary and secondary antitumor responses. We have generated autologous tumor lysate-pulsed DCs from three patients with medullary thyroid carcinoma (MTC) and tested them for their ability to stimulate cytotoxic T-cell responses against autologous MTC tumor cells in vitro. The aim of our investigations was to evaluate the potential efficacy of DC-based immunotherapy in patients with MTC. DCs were generated from peripheral blood monocytes using GM-CSF and IL-4 (immature DCs) or GM-CSF, IL-4, and TNFalpha (mature DCs). Our results indicate that mature tumor lysate-pulsed DCs are able to elicit a human leukocyte antigen class I-restricted cytotoxic T-cell response against autologous MTC tumor cells, whereas immature tumor lysate-pulsed DCs do not stimulate significant antitumor activity. We feel that our data may be relevant for future clinical trials of active immunotherapy using tumor lysate-pulsed DCs in patients with MTC who have residual or distant disease after surgical treatment. The fact that mature DCs displayed a substantially higher capacity to stimulate autologous antitumor T-cell responses than immature DCs underlines the importance of a maturation step in immunotherapy protocols based on DCs.

Serine proteinase inhibitor 9 can be recognized by cytotoxic T lymphocytes of epithelial cancer patients.

Serine proteinase inhibitor 9 (PI–9) inhibits granzyme B‐mediated apoptosis and interleukin–lβ‐converting enzyme activity. In this study, we report that the PI–9 gene encodes antigenic epitopes recognized by the HLA‐A24–restricted and tumor‐reactive cytotoxic T lymphocytes (CTLs) of epithelial cancer patients. Screening of an autologous cDNA library using a CTL line recognizing HLA‐A24+ tumor cells resulted in the isolation of a cDNA, which had an identical coding region to the previously described PI–9 genes. PI–9 gene was expressed in approximately three‐fourths of epithelial cancer cell lines and all leukemic cell lines tested. It was also expressed in normal peripheral blood mononuclear cells (PBMCs), but not in a normal fibroblast cell line. CTL sublines contained T cells capable of recognizing the PI–9292–300 and PI–9348–356 peptides among 13 different peptides having the HLA‐A24 binding motifs. These two peptides were recognized by the CTL line in a dose‐dependent and HLA class‐I‐restricted manner, and also possessed the ability to induce HLA class I‐restricted and tumor‐reactive CTLs in PBMCs from HLA‐A24+ cancer patients. These results demonstrate that PI–9 is recognized by HLA class I‐restricted and tumor‐reactive CTLs of epithelial cancer patients.