Antigen-specific Killing Research Articles

Detection and Characterization of a Novel Subset of CD8+CD57+ T Cells in Metastatic Melanoma with an Incompletely Differentiated Phenotype

Tumor-specific T cells are frequently induced naturally in melanoma patients and infiltrate tumors. It is enigmatic why these patients fail to experience tumor regression. Given that CD8(+) T cells mediate antigen-specific killing of tumor cells, the focus of this study was to identify alterations in the differentiation of CD8(+) residing at the tumor site, with emphasis on a population expressing CD57, a marker for terminal differentiation. We conducted flow cytometric analysis of CD8(+) tumor-infiltrating lymphocytes (TIL) isolated from 44 resected melanoma metastases with known T-cell differentiation markers. For comparison, peripheral blood mononuclear cells were isolated from matched melanoma patients. We sorted different CD8(+) subsets found in TIL and determined their effector functions. In addition, we carried out Vβ clonotype expression analysis of T-cell receptors to determine lineage relationship between the CD8(+) TIL subsets. The majority of CD8(+) TIL was in the early-effector memory stage of differentiation. A significant population consisted of an oligoclonal subset of cells coexpressing CD27, CD28, CD57, and Granzyme B, with little or no perforin. These cells could be induced to proliferate, produce a high level of IFN-γ, and differentiate into CD27(-)CD57(+), perforin(high) mature CTL in vitro. Addition of TGF-β1 prevented further differentiation. Our studies identified a novel subset of incompletely differentiated CD8(+) CTL coexpressing early effector memory and late CTL markers. This population resembles that found in patients with uncontrolled chronic viral infections. TGF-β1, frequently produced by melanoma tumors, may be a key cytokine inhibiting further maturation of this subset.

Delineation of antigen-specific and antigen-nonspecific CD8+ memory T-cell responses after cytokine-based cancer immunotherapy

AbstractMemory T cells exhibit tremendous antigen specificity within the immune system and accumulate with age. Our studies reveal an antigen-independent expansion of memory, but not naive, CD8+ T cells after several immunotherapeutic regimens for cancer resulting in a distinctive phenotype. Signaling through T-cell receptors (TCRs) or CD3 in both mouse and human memory CD8+ T cells markedly up-regulated programmed death-1 (PD-1) and CD25 (IL-2 receptor α chain), and led to antigen-specific tumor cell killing. In contrast, exposure to cytokine alone in vitro or with immunotherapy in vivo did not up-regulate these markers but resulted in expanded memory CD8+ T cells expressing NKG2D, granzyme B, and possessing broadly lytic capabilities. Blockade of NKG2D in mice also resulted in significantly diminished antitumor effects after immunotherapy. Treatment of TCR-transgenic mice bearing nonantigen expressing tumors with immunotherapy still resulted in significant antitumor effects. Human melanoma tissue biopsies obtained from patients after topically applied immunodulatory treatment resulted in increased numbers of these CD8+ CD25− cells within the tumor site. These findings demonstrate that memory CD8+ T cells can express differential phenotypes indicative of adaptive or innate effectors based on the nature of the stimuli in a process conserved across species.

Increased PRAME antigen-specific killing of malignant cell lines by low avidity CTL clones, following treatment with 5-Aza-2'-Deoxycytidine.

The cancer testis antigen Preferentially Expressed Antigen of Melanoma (PRAME) is overexpressed in many solid tumours and haematological malignancies whilst showing minimal expression in normal tissues and is therefore a promising target for immunotherapy. HLA-A0201-restricted peptide epitopes from PRAME have previously been identified as potential immunogens to drive antigen-specific autologous CTL responses, capable of lysing PRAME expressing tumour cells. CTL lines, from 13 normal donors and 10 melanoma patients, all of whom were HLA-A0201 positive, were generated against the PRAME peptide epitope PRA(100-108). Specific killing activity against PRA(100-108) peptide-pulsed targets was weak compared with CTL lines directed against known immunodominant peptides. Moreover, limiting dilution cloning from selected PRAME-specific CTL lines resulted in the generation of a clone of only low to intermediate avidity. Addition of the demethylating agent 5-aza-2'-Deoxycytidine (DAC) increased PRAME expression in 7 out of 11 malignant cell lines including several B lineage leukaemia lines and also increased class I expression. Pre-treatment of target cells was associated with increased sensitivity to antigen-specific killing by the low avidity CTL. When CTL, as well as of the target cells, were treated, the antigen-specific killing was further augmented. Interestingly, one HLA-A0201-negative DAC-treated line (RAJI) showed increased sensitivity to killing by clones despite a failure of expression of PRAME or HLA-A0201. Together these data point to a general increased augmentation of cancer immunogenocity by DAC involving both antigen-specific and non-specific mechanisms.

Cytotoxic T cells – Stroma interactions

The tumor microenvironment is a complex system playing an important role in tumor development and progression. Besides tumor cells, the tumor microenvironment harbours a variety of host-derived cells, such as endothelial cells, fibroblasts, innate and adaptive immune cells, as well as extracellular matrix (ECM) fibers, cytokines, and other mediators. This review discusses the potential role of hypoxia and endothelial cells within tumor microenvironment and emphasizes their interaction with antigen specific killer cells.

Pathogenesis of oral lichen planus - a review

Oral lichen planus (OLP) is a T-cell-mediated chronic inflammatory oral mucosal disease of unknown etiology. OLP presents as white striations, white papules, white plaques, erythema, erosions, or blisters affecting predominantly the buccal mucosa, tongue and gingiva. Both antigen-specific and non-specific mechanisms are hypothesized to be involved in the pathogenesis of oral lichen planus (OLP). Antigen-specific mechanisms in OLP include antigen presentation by basal keratinocytes and antigen-specific keratinocyte killing by CD8(+) cytotoxic T cells. Non-specific mechanisms include mast cell degranulation and matrix metalloproteinase activation in OLP lesions. These mechanisms may combine to cause T cell accumulation in the superficial lamina propria, basement membrane disruption, intra-epithelial T cell migration and keratinocyte apoptosis in OLP. The various hypotheses proposed for pathogenesis of oral lichen planus are discussed in this review.

Abstract 5616: Soluble, high affinity TCRs fused to anti-CD3 redirect T cells to kill cancer cells presenting MAGE-A3 and NY-ESO antigens

Abstract In the last decade, major efforts in the fight against cancer have focussed on galvanising the adaptive immune system to kill tumours. Many of these endeavours are based on the development and clinical use of monoclonal antibodies (mAb) which are the most successful class of immune modulating agent identified to date. However, while mAbs show promise against certain cancers, their specificity is limited to integral membrane proteins; this hinders their extensive development for the purposes of targeting cancer cells. In contrast to mAbs, T cell receptors (TCRs) recognise peptides bound to major histocompatibility complex class I (MHC I) molecules. These peptides are derived from endogenously processed proteins, and therefore represent a different repertoire of targets to those recognised by mAbs. This alternate spectrum of antigens provides the potential to target cancers using an untapped source of well-validated epitopes. Naturally occurring TCRs, however, have relatively low affinities for their antigen compared to antibody binding. Advances in engineering techniques have allowed the generation of high affinity monoclonal TCRs (mTCRs) with picomolar affinities for their antigen. Using targeted mutagenesis and phage display, we have generated a number of soluble, high affinity mTCRs specific for several reported tumour-associated antigens. Through mTCR fusion to an anti-CD3 single chain variable fragment (scfv), we produced bifunctional proteins that redirect T cell immune specificity. These novel proteins are termed ImmTACs (Immune-mobilising mTCRs Against Cancer). We present data demonstrating the potential of two such ImmTAC molecules, NY-ESO-ImmTAC and MAGE-A3-ImmTAC, to treat certain cancers. NY-ESO1 and MAGE-A3 are both cancer testes antigens and therefore represent potentially very clean molecular targets. We demonstrate that both NY-ESO-ImmTAC and MAGE-A3-ImmTAC are capable of potently redirecting unstimulated peripheral blood mononuclear cells (PBMC) or CD8+ T cells against multiple myeloma, colorectal carcinoma and non-small cell lung cancer cell lines despite the presentation of extremely low antigen numbers (<100 epitopes/cell) on the surface of these cells. ImmTAC-redirected T cells respond with multiple effector functions including the production of granzyme B, IFNγ and IL-2, resulting in a high level of antigen-specific cancer cell killing over the course of 24 hours. The mechanism of killing is at least partly contributed by the caspase-3/7 apoptotic pathway. We also show that NY-ESO-ImmTAC and MAGE-A3-ImmTAC were inert by all our measures in the presence of normal primary cells including melanocytes, hepatocytes and astrocytes. Thus both MAGE-A3 and NY-ESO-ImmTACs possess the potential to be highly specific, potent cancer immunotherapies offering a targeting and therapeutic approach distinct from any other biologic in development. 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 5616.

Ad5-vectored FMDV vaccine elicits antigen-specific CTL responses in swine. (43.10)

Abstract Foot-and-mouth disease virus (FMDV) is a highly infectious picornavirus that causes disease in cloven-hoofed animals. Neutralizing antibodies are thought to play a primary role in FMDV immunity, and their induction forms the basis for current vaccine approaches. However, the role of cytotoxic T-lymphocytes (CTLs) in recovery and protection has remained largely unexplored. Immune evasion strategies of FMDV are predicted to impair the development of functional CTL assays. Specifically, virus encoded proteases inhibit expression of Class I MHC molecules by infected cells. In order to overcome these difficulties, we have analyzed the response to a recombinant, human Adenovirus 5 (huAd5) vectored, FMDV vaccine. Swine homozygous for MHC were vaccinated with a replication-defective huAd5 expressing the unprocessed capsid polyprotein of FMDV, strain A24. An MHC-matched cell line was used for antigen stimulation and as a source of target cells in cellular cytotoxicity assays. PBMCs from vaccinated animals were incubated with Ad5-infected target cells for 3-5 days. CD6+ T cells were isolated and mixed with Ad5-FMDV infected target cells. Antigen-specific killing was detected relative to control target cells. This suggests that vaccination with adenovirus vectored vaccine constructs can induce a functional CTL response against epitopes of FMDV in a MHC restricted manner.

Paclitaxel inhibits killing by murine cytotoxic T lymphocytes in vivo but not in vitro

To kill antigen-specific target cells (TCs), cytotoxic T lymphocytes (CTLs) reorganise their microtubule cytoskeleton to deliver lytic granules to the TCs. We used two drugs that stabilise microtubules, paclitaxel and peloruside, to determine how the stabilising microtubule network affects CTL function in vitro and in vivo. In vitro, neither paclitaxel nor peloruside inhibited antigen-specific killing, lytic granule delivery to the cell surface, nor interferon-gamma release by murine CTLs. In contrast, in an in vivo murine model of antigen-induced killing, a single dose of paclitaxel had a significant inhibitory effect on killing by CTLs. Furthermore, the inhibitory effect of paclitaxel was not caused by specific deletion of the effector CTL population in drug-treated mice. The findings reveal that microtubule-stabilising drug treatment can lead to immediate impairment of CTL function without affecting lytic granule release. The results also suggest that patients undergoing taxane anti-cancer therapy may be impaired in their ability to fight infection before the anti-mitotic effects of paclitaxel are apparent.

Development of a Wilms' tumor antigen-specific T-cell receptor for clinical trials: engineered patient's T cells can eliminate autologous leukemia blasts in NOD/SCID mice

The Wilms' tumor antigen (WT1) is an attractive target for immunotherapy of leukemia. In the past, we isolated and characterized the specificity and function of a WT1-specific T-cell receptor. The goal of this translational study was to develop a safe and efficient WT1-T-cell receptor retroviral vector for an adoptive immunotherapy trial with engineered T cells. We generated a panel of retroviral constructs containing unmodified or codon-optimized WT1-T-cell receptor alpha and beta genes, linked via internal ribosome entry sites or 2A sequences, with or without an additional inter-chain disulfide bond in the T-cell receptor constant domains. These constructs were functionally analyzed in vitro, and the best one was tested in an autologous primary leukemia model in vivo. We identified a WT1-T-cell receptor construct that showed optimal tetramer staining, antigen-specific cytokine production and killing activity when introduced into primary human T cells. Fresh CD34(+) cells purified from a patient with leukemia were engrafted into NOD/SCID mice, followed by adoptive immunotherapy with patient's autologous T cells transduced with the WT1-T-cell receptor. This therapeutic treatment evidently decreased leukemia engraftment in mice and resulted in a substantial improvement of leukemia-free survival. This is the first report that patient's T cells, engineered to express the WT1-T-cell receptor, can eliminate autologous leukemia progenitor cells in an in vivo model. This study provides a firm basis for the planned WT1-T-cell receptor gene therapy trial in leukemia patients.

Topical resiquimod promotes priming of CTL to parenteral antigens

We explored the topical use of resiquimod (R-848), a Toll-like receptor (TLR) 7/8 agonist, in gel formulation, to enhance cross-priming to subcutaneously administered protein antigen in a murine model. Resiquimod application at the time of subcutaneous administration of ovalbumin generated robust antigen-specific CTL as detected by tetramers, IFN-γ ELISPOT assays and standard cytotoxicity assays. Induced CTL were capable of mediating antigen-specific killing in vivo as measured by in vivo cytotoxicity assays and an ability to protect against B16-OVA tumor challenge. Multiple serial applications of topical resiquimod increased the frequency of antigen-specific CTL when compared to single application. This enhanced frequency was noted despite a marked inhibition of adjuvant mediated pro-inflammatory cytokine release following repeated administration. Topical resiquimod is a potent adjuvant for locally administered subcutaneous vaccines, inducing clinically relevant CTL responses following single application at the time of subcutaneous vaccination.

Combining the Antigen Processing Components TAP and Tapasin Elicits Enhanced Tumor-Free Survival

Tpn is a member of the MHC class I loading complex and functions to bridge the TAP peptide transporter to MHC class I molecules. Metastatic human carcinomas often express low levels of the antigen-processing components Tapasin and TAP and display few functional surface MHC class I molecules. As a result, carcinomas are unrecognizable by effector CTLs. The aim of this study is to examine if Tapasin (Tpn) plays a critical role in the escape of tumors from immunologic recognition. To test our hypothesis, a nonreplicating adenovirus vector encoding human Tpn (AdhTpn) was constructed to restore Tpn expression in vitro and in vivo in a murine lung carcinoma cell line (CMT.64) that is characterized by down-regulation of surface MHC class I due to deficiency in antigen-processing components. Ex vivo, Tpn expression increased surface MHC class I and restored susceptibility of tumor cells to antigen-specific CTL killing, and AdhTpn infection of dendritic cells also significantly increased cross-presentation and cross-priming. Furthermore, tumor-bearing animals inoculated with AdhTpn demonstrated a significant increase in CD8(+) and CD4(+) T cells and CD11c(+) dendritic cells infiltrating the tumors. Provocatively, whereas syngeneic mice bearing tumors that were inoculated with AdhTpn a significant reduction in tumor growth and increased survival compared with vector controls, combining AdhTpn inoculation with AdhTAP1 resulted in a significant augmentation of protection from tumor-induced death than either component alone. This is the first demonstration that Tpn alone can enhance survival and immunity against tumors but additionally suggests that Tpn and TAP should be used together as components of immunotherapeutic vaccine protocols to eradicate tumors.

Matrilysin (MMP-7) Is a Novel Broadly Expressed Tumor Antigen Recognized by Antigen-Specific T Cells.

Identification and targeting of antigens involved in malignant transformation, survival advantage of tumor cells or their metastatic spread might represent a promising approach in the development of immunotherapeutic strategies to treat malignant diseases. Using oligonucleotide microarray analysis we found that matrix metalloproteinase- (MMP-)7 is extensively upregulated in a broad variety of malignant tissues. MMPs are a family of endopeptidases that degrade extracellular matrix (ECM). MMP-7 can promote cancer invasion by proteolytic cleavage of ECM and basement membrane proteins. In addition, MMP-7 has been shown to accelerate the proliferation of umbilical vein endothelial cells in vitro and promote angiogenesis. Thus, targeting MMP-7 may affect both tumor cells and tumor vessels. In order to analyze the feasibility to induce MMP-7-specific CTLs, monocyte derived dendritic cells (DCs) were generated from a healthy donor, electroporated with full length MMP-7-in-vitro-transcript (IVT) that coded for the entire MMP-7-protein and used as antigen presenting cells (APCs) for the in vitro induction of antigen-specific CTLs. These in vitro generated CTLs demonstrated an antigen-specific and HLA class I-restricted cytolytic activity against tumor cells endogenously expressing MMP-7. The specificity and HLA-restriction was confirmed in cold target inhibition assays and by using blocking antibodies. In the next set of experiments, we wanted identify HLA class I-presented MMP-7-peptides that are recognized by our CTL lines. To accomplish this, peptides bound to HLA class I molecules were isolated from surgically removed RCC specimens that overexpressed MMP-7. By applying mass spectrometry-based peptide sequencing of the HLA ligands, a HLA-A3-binding peptide derived from the novel antigen MMP-7 was identified. To analyze the immunogenicity of the new MMP-7 derived peptide with respect to its ability to elicit primary T cell responses, we induced MMP-7-specific CTLs in vitro. DCs derived from a HLA-A3+ healthy donor were pulsed with the HLA-A3-binding antigenic peptide and utilized as APCs. The induced CTL lines demonstrated antigen-specific killing: T cells only recognized autologous DCs loaded with the cognate MMP-7-A3 peptide or HLA matched tumor cells expressing MMP-7, whereas they did not lyse target cells pulsed with an irrelevant HLA-A3-binding peptide. The objective of our study is the development of a DC-based adoptive immunotherapy for patients with malignant diseases. Therefore, we sought to analyze, whether our previous findings could be applied in patients with malignant diseases in an autologous setting. To achieve this, we generated DCs from an HLA-A3+ ALL patient in complete remission after chemotherapy and used them as APCs for CTL induction after electroporation with MMP-7-IVT. The autologous ALL blasts, frozen at the time of diagnosis, were used as targets in Cr-release assays. These cells were efficiently lysed by the CTLs specific for MMP-7, confirming the expression analysis by RT-PCR. Importantly, the normal autologous purified DCs, CD3+ T and CD19+ B cells were not recognized. In conclusion, our study describes the identification of a novel broadly expressed T-cell epitope derived from the MMP-7-protein that represents an interesting candidate to be applied in immunotherapies of human malignancies capable of targeting neo-vascularization and malignant cells in developing tumors.

CD8α/α homodimers fail to function as co‐receptor for a CD8‐dependent TCR

In this study, we have started to dissect the molecular basis of CD8 dependence of a high and low avidity CTL clone specific for the same peptide epitope. Using anti-CD8alpha and anti-CD8beta antibodies, we found that cytotoxicity and IFN-gamma production by high but not by low avidity CTL was strongly CD8 dependent. We isolated the TCR genes of both types of CTL clones and used retroviral gene transfer to analyse the function of these TCR in primary T cells of wild-type and CD8beta-deficient mice. Both TCR triggered antigen-specific killing in wild-type T cells, and blocking experiments showed that CD8 dependence/independence co-transferred with the TCR into primary T cells, indicating that it was dictated by the TCR itself. Gene transfer experiments into CD8beta-deficient T cells revealed that only the TCR derived from the CD8-independent CTL clone elicited antigen-specific cytotoxicity, while the CD8-dependent TCR was non-functional in the absence of the CD8beta-chain. These data indicate a striking difference between CD8alpha/beta heterodimers and CD8alpha/alpha homodimers as only the former were able to provide co-receptor function for the CD8-dependent TCR.

PankoMab: a potent new generation anti-tumour MUC1 antibody.

Recently, we described a new carbohydrate-induced conformational tumour-epitope on mucin-1 (MUC1) with the potential for improvement of immunotherapies [29, 30]. PankoMab is a novel antibody, which binds specifically to this epitope and was designed to show the highest glycosylation dependency and the strongest additive binding effect when compared to other MUC1 antibodies. This enables PankoMab to differentiate between tumour MUC1 and non-tumour MUC1 epitopes. It has a high-affinity towards tumour cells (e.g. KD [M] of 0.9 and 3x10(-9 )towards NM-D4 and ZR75-1, respectively) and detects a very large number of binding sites (e.g. 1.0 and 2.4x10(6 )for NM-D4 and ZR75-1, respectively). PankoMab is rapidly internalised, and after toxin coupling is able to induce very effectively toxin-mediated antigen-specific tumour cell killing. PankoMab reveals a potent tumour-specific antibody-dependent cell cytotoxicity (ADCC). PankoMab is, therefore, distinguished by a combination of advantages compared to other MUC1 antibodies in clinical development, including higher tumour specificity, higher affinity, a higher number of binding sites, largely reduced binding to shed MUC1 from colon and pancreatic carcinoma patients, no binding to mononucleated cells from peripheral blood (except approximately 7% of activated T cells), stronger ADCC activity and rapid internalisation as required for toxin-mediated cell killing. This renders it a superior antibody for in vivo diagnostics and various immunotherapeutic approaches.

HLA type-independent generation of antigen-specific T cells for adoptive immunotherapy

Adoptive immunotherapy with antigen-specific T cells has been successfully used to treat certain infectious diseases and cancers. Although more patients may profit from T cell therapy, its more frequent use is restricted by limitations in current T cell generation strategies. The most commonly applied peptide-based approaches rely on the knowledge of relevant epitopes. Therefore, T cells cannot be generated for diseases with unknown epitopes or for patients with unfavorable HLA types. We developed a peptide-based approach for HLA type-independent generation of specific T cells against various proteins. It is based on short-time stimulation with peptide libraries that cover most CD4(+) and CD8(+) T cell epitopes of given proteins. The procedure requires no prior knowledge of epitopes because libraries are synthesized solely on the basis of the protein's amino acid sequence. Stimulation is followed by immunomagnetic selection of activated IFN-gamma-secreting cells and nonspecific expansion. To evaluate the protocol, we generated autologous T cells specific for a well-characterized antigen, the human cytomegalovirus phosphoprotein 65 (pp65). Generated T cell lines consisted of pp65-specific CD4(+) and CD8(+) lymphocytes that displayed antigen-specific killing and proliferation. The protocol combines the biosafety of peptide-based approaches with HLA type independence and may help to advance adoptive immunotherapy in the future.

Exploiting T cell receptor genes for cancer immunotherapy

Adoptive antigen-specific immunotherapy is an attractive concept for the treatment of cancer because it does not require immunocompetence of patients, and the specificity of transferred lymphocytes can be targeted against tumour-associated antigens that are poorly immunogenic and thus fail to effectively trigger autologous T cell responses. As the isolation and in vitro expansion of antigen-specific lymphocytes is difficult, 'conventional' adoptive T cell therapy can only be carried out in specialized centres in small numbers of patients. However, T cell receptor (TCR) genes isolated from antigen-specific T cells can be exploited as generic therapeutic molecules for 'unconventional' antigen-specific immunotherapy. Retroviral TCR gene transfer into patient T cells can readily produce populations of antigen-specific lymphocytes after a single round of polyclonal T cell stimulation. TCR gene modified lymphocytes are functionally competent in vitro, and can have therapeutic efficacy in murine models in vivo. TCR gene expression is stable and modified lymphocytes can develop into memory T cells. Introduction of TCR genes into CD8(+) and CD4(+) lymphocytes provides an opportunity to use the same TCR specificity to produce antigen-specific killer and helper T lymphocytes. Thus, TCR gene therapy provides an attractive strategy to develop antigen-specific immunotherapy with autologous lymphocytes as a generic treatment option.

Off the Shelf, GMP Grade Artificial APC Efficiently Generates Large Numbers of Antigen Specific CTLs Sufficient for the Treatment of Cancer and Infectious Disease.

Generating antigen specific cytotoxic T lymphocytes (CTLs) for adoptive immunotherapy requires the use of antigen presenting cells (APCs) such as dendritic cells, activated B cells, and/or artificial APC engineered to express immunoaccessory molecules. Although clearly useful, each approach presents certain drawbacks preventing their widespread application to the treatment of minimal residual disease in cancer and the treatment and prophylaxis of infectious disease. For instance, generating autologous APC for each patient is costly and is dependent on a limited supply of patient material. Most artificial APC approaches, while bypassing the individualized preparation of APC, are limited by the requirement to produce clinical grade HLA/peptide tetramers, monoclonal antibodies, polystyrene beads, and/or non-human cell lines. To circumvent all these issues, we have developed an immortalized APC line that can uniquely support the priming and prolonged expansion of antigen specific T cells through the engagement of TCR, CD28, and CD83 ligand. Co-engagement of CD28 and CD83 ligand preferentially enriches and significantly amplifies the number of antigen specific T cells, obviating the necessity to perform any tetramer sorting procedures. Furthermore, we are consistently successful in generating immunity to a wide array of peptide epitopes derived from antigens such as MART1, NY-ESO-1, Her-2/neu, influenza virus, telomerase, and HIV. Since this strategy can provide an unlimited APC source that can be used to prime, expand and select for antigen specific CTLs, we have generated a GMP grade APC for the treatment of patients. Our CASE (clinical grade antigen specific expansion) line was produced by simultaneously transfecting K562 with linearized plasmids encoding the HLA-A*0201 (A2), CD80, CD83, and puromycin resistance genes using a lipofection method. Following drug selection and cloning by limiting dilution, 5 CASE clones were then studied for their ability to induce expansion of antigenic peptide specific CTL, using MART1 as a model antigen. HLA-A2+ CD8 T cells were stimulated with irradiated, peptide pulsed CASE clones, and the percentage of tetramer staining T cells was determined by flow cytometry. The stable CASE clone, #33, was found to consistently generate the highest number of antigen specific T cells. Using the CASE33 clone, we successfully generated several CTL lines, succeeding in 8/8 cases. The generated CTL lines achieved several logs of antigen specific expansion, with tetramer staining up to 57%, and function has been demonstrated by high antigen specific killing and gamma-interferon secretion. Both master and working cell banks for CASE33 have been established and have successfully passed extensive testing including in vitro cultures, testing for bovine adventitious agents, in vivo testing and PCR for viral pathogens, karyotype analysis, and analysis of cellular morphology and growth. The highly effective APC, CASE33, was generated in a relatively short timeframe, and the production of additional clinical grade APCs expressing HLA molecules such as A11, A24, and A30, would be technically straightforward. With such an APC bank, most patients encountered in the clinic could be treated using one or more of these APCs and the widespread testing of adoptive immunotherapy in cancer and infectious diseases will commence.

Input Virion Proteins: Cryptic Targets of Antivector Immune Responses in Preimmunized Subjects

Input Virion Proteins: Cryptic Targets of Antivector Immune Responses in Preimmunized Subjects

Predominant cell-mediated immunity in the oral mucosa: gene gun-based vaccination against infectious diseases

Background: Direct immunization via epithelial surfaces has been considered for many vaccine approaches, including DNA vaccines. It remains to be determined, however, which body site is suitable for genetic vaccination. Objective: To characterize the effects of the oral mucosa-mediated genetic vaccination, we compared antigen-specific immune responses of the oral mucosal DNA vaccine to the flank skin vaccination against influenza virus and malaria parasite. Methods: DNA vaccines against the influenza A/WSN/33 (H1N1) hemagglutinin and the malaria Plasmodium berghei circumsporozoite protein were administered respectively three times at 3-week intervals into the oral mucosa, skin, or liver of hamsters. The effects of their vaccine were evaluated by antigen-specific antibody production and cell-mediated killing activity. Furthermore, the in vivo malaria challenge test was also performed after the vaccination. Results: Significant specific antibody production was not observed in each case, but interferon-gamma production and cell-mediated killing activity were strongly induced in splenic lymphocytes from hamsters with the oral vaccination. The in vivo malaria challenge after the oral mucosal vaccination significantly delayed the blood-appearance day of the parasites in comparison with other immunization sites ( P<0.05). Conclusion: These results suggest that gene immunization via the oral mucosa may induce cell-mediated immunity more efficiently than via the skin or liver, and that the oral mucosa may be one of the most suitable tissues for gene gun-based DNA vaccination against infectious diseases.

Cell-mediated fas-based lysis of dendritic cells which are apparently resistant to anti-Fas antibody.

In this report, the controversy concerning the sensitivity of dendritic cells (DCs) to Fas-dependent induction of apoptosis was examined using murine DCs. Although DCs could not be lysed when exposed to an anti-Fas antibody, Jo2, the observed resistance turned out to reflect their lack of the expression of Fc(gamma)R necessary for crosslinking the antibody, rather than their intrinsic resistance. Thus, at least a fraction of DCs was sensitive to Jo2 in the presence of Fc(gamma)R-expressing by-standers. Consistently, a significant fraction of DCs was sensitive to Fas-dependent lysis mediated by T cells including the antigen-specific killing by CD4+ T cells. Both immature (class II MHClow) and mature (class II MHChigh) DCs were sensitive to the Fas-based induction of apoptosis.