anti-TCR Antibody Research Articles

Pulmonary TCR γδ T cells induce the early inflammation of granuloma formation by a glycolipid trehalose 6,6′-dimycolate (TDM) isolated from Mycobacterium tuberculosis

We previously showed that formation of pulmonary granulomas in mice in response to a mycobacterial glycolipid, trehalose 6,6′-dimycolate (TDM) is due to the action of TNF-α and not of IFN-γ. However, the mechanisms of formation and maintenance of pulmonary granulomas are not yet clear. The purpose of the present study is to evaluate the mechanisms of granuloma formation by TDM at the early phase. Histological analysis showed that inflammatory cells infiltrated the murine pulmonary interstitium on day 2 after an intravenous injection with TDM as a w/o/w emulsion. Clear granuloma formation was observed on day 7 after the injection. The mRNA expression of IL-17, IFN-γ and macrophage inflammatory protein 2 was found in lung mononuclear cells at the day after TDM injection. The major IL-17-producing cells were T-cell receptor (TCR) γδ T cells expressing Vγ6. In mice depleted of γδ T cells by treatment with anti-TCR γδ monoclonal antibody, the number of TDM-induced granuloma was decreased, but the size of granuloma was not affected. Our results suggest that the mycobacterial glycolipid TDM causes activation of IL-17-producing TCR γδ T cells and stimulates chemotaxis of inflammatory cells including neutrophils in to lung.

Abstract 3527: Potent anti-tumor activity of AbGn-100, an anti-CD326 x anti-TCR bispecific antibody to CD326-expressing solid tumors

Abstract CD326 is one of the first and prominent immunotherapeutic targets in cancer therapy due to its frequent and high-level expression on most carcinomas of various origins. The dominant role of cytotoxic T cells for tumor suppression and surveillance in vivo is well documented in literature. A novel bi-specific antibody AbGn-100 was constructed, in which the antigen-binding domain of an anti- human CD326 antibody was linked to the antigen-binding domain of an anti-human T cell receptor (TCR) ≤ chain antibody. Human serum albumin was incorporated into this construct to prolong the half life in vivo. This bi-specific antibody is able to direct T cells toward CD326-expressing cancer cells to execute T cell cytotoxic function after TCR cross-linking mediated by CD326 expressed on cancer cells while it remains harmless when either T cells or CD326-expressing cancer cells is absent. In vitro cytotoxicity assays with PBMCs from different donors have demonstrated potent (up to 60% above the background) cytotoxicity eliciting activity of AbGn-100 to lung (NCI-H358), ovarian (OMC3) and colorectal (COLO205) cancer cells lines. In the absence of cancer cells, the addition of AbGn-100 didn't trigger the up-regulation of T cell activation markers such as CD25 or CD69, nor did it increase the secretion of cytokines such as IL-2 and Interferon β, demonstrating the safety profile of AbGn-100. In vivo activity of AbGn-100 was tested with established lung (NCI-H358) and ovarian (OMC-3) cancer xenografts in NOD-SCID mice. AbGn-100, in the presence of human PBMC, significantly reduced the size of the established lung and ovarian tumors. Most notably, in some of the mice, tumor was not detectable after 50 days of treatment, suggesting the possibility of tumor eradication. PK studies in mice showed the half-life of AbGn-100 to be around 20 hours, which is substantially longer than conventional single-chain Fv bi-specific antibodies. These data support the potential of further development of AbGn-100 as therapeutics for the treatment of CD326-expressing solid tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3527. doi:1538-7445.AM2012-3527

Widespread expressions of immunoglobulin superfamily proteins in cancer cells.

RP215 monoclonal antibody (Mab) was shown to recognize a carbohydrate-associated epitope of cancer cell-expressed glycoproteins, known as CA215. Extensive MALDI-TOF MS analysis was performed to search for the molecular identity of CA215. Besides immunoglobulin (Ig) heavy chains, homology to human T-cell receptors (TCR) and Ig-like cell adhesion molecules was also detected. By using RT-PCR and cDNA sequencing, it was observed that as many as 80% of cancer cell lines showed significant levels of gene expressions of TCR-α and TCR-β. Selected Ig-like cell adhesion molecules such as CD47, CD54, CD58 and CD 147 were also highly expressed among all the cell lines tested. In contrast, co-receptors and co-stimulators of TCR such as CD3, CD4 and CD8 were rarely expressed demonstrating the non-functional nature of TCR in cancer cells. Results of immunohistochemical staining and Western blot assays of cancer cell lines as well as cancerous tissue sections were consistent with these observations. Anti-TCR and anti-human IgG antibodies were shown to induce complement-dependent cytotoxicity and apoptosis of cultured cancer cells indicating the surface nature of Ig-like proteins. Based on these experimental observations, it was hypothesized that the expressions of these immunoglobulin superfamily (IgSF) proteins may be relevant to the immune protection and proliferations of cancer cells during carcinogenesis or cancer progression. Surface-bound TCR-like proteins as well as immunoglobulins may be the potential targets for RP215-based anti-cancer drugs.

A natural anti-T-cell receptor monoclonal antibody protects against experimental autoimmune encephalomyelitis

The therapeutic potential of natural anti-T-cell receptor (TCR) antibodies is largely unknown. We investigated whether passive administration of C1-19, a novel natural anti-TCRVβ8 monoclonal antibody, could interfere with the development of EAE. Treatment with C1-19 prevented myelin basic protein (MBP)-induced EAE in Vβ8-sufficient B10.PL but not in Vβ8-deficient SJL mice. Furthermore, C1-19 reduced disease severity when administrated shortly after disease onset. These protective effects of C1-19 correlated with a Th2 bias of the cytokine response, in the absence of T-cell deletion or anergy. Together, these findings indicate that natural anti-TCR antibodies could function as therapeutic tools in autoimmune inflammatory diseases.

B-Cell-Delivered Gene Therapy Induces Functional T Regulatory Cells and Leads to a Loss of Antigen-Specific Effector Cells

Previous reports have shown that B-cell-mediated gene therapy can induce tolerance in several animal models for autoimmune diseases and inhibitory antibody formation in hemophilia A mice. We know from our previous work that the induction of tolerance following B-cell therapy is dependent upon CD25(+) regulatory T cells (Tregs). To extend these studies and identify the effects of this gene therapy protocol on the target CD4 T cells, we have adapted in vitro suppression assays using Tregs isolated from treated and control mice. Using carboxyfluorescein succinimidyl ester (CFSE) dilution as a measure of T-cell responsiveness to FVIII, we show that CD25(+) Tregs from treated mice are more suppressive than those from control animals. To monitor the induction of antigen-specific Tregs, we repeated these studies in ovalbumin (OVA) peptide-specific DO11.10 T-cell receptor (TCR) transgenic mice. Tregs from DO11.10 mice treated with a tolerogenic OVA-Ig construct are better than polyclonal Tregs at suppressing the proliferation of responder cells stimulated with OVA peptide 323-339 (pOVA). Furthermore, we show that following B-cell therapy, there is an increase in antigen-specific FoxP3(+) Tregs, and there is also a distinct decrease in antigen-specific CD4(+) effector T cells. These changes in the lymphocyte population shift the balance away from effector function toward a tolerogenic phenotype.

Analysis of the inflammatory response in HY-TCR transgenic mice highlights the pathogenic potential of CD4− CD8− T cells

Transgenic mice expressing a rearranged T cell receptor (TCR)-αβ prematurely at the double-negative stage develop an abnormal population of peripheral T cells that lack CD4 and CD8 expression and are hyper-reactive to anti-TCR antibody stimulation. One such example is the HY-TCR transgenic mice. These mice express a TCR transgenic specific for the HY antigen that is expressed in male but not in female mice. As a result, male mice have an abnormal population of HY+/CD4− 8− or HY+/CD4− CD8low T cells that are much lower in female mice. In this study, we investigated the potential patho/physiological function of these cells in vivo using a model of delayed-type hypersensitivity (DTH) reaction: the λ-carrageenan-induced paw edema. Interestingly, while both male and female HY-TCR mice develop a classical biphasic inflammatory response to λ-carrageenan, the degree of inflammation in the former was much higher than that in the latter. This was accompanied by a selective expansion of HY+/CD4− 8− and HY+/CD4− CD8low T cells in male mice and by a markedly increased production of typical DTH cytokines compared with cells from female mice. These results were specific since analysis of the inflammatory response of HY-TCR transgenic mice subjected to zymosan-induced peritonitis showed no differences between male and female mice. Together, these findings provide novel evidence for the pathological role of self-reactive CD4− CD8− T cells, previously described in several autoimmune strains and recently identified in patients suffering from autoimmune diseases such as systemic lupus erythematosus.

Thy-1 signaling in murine T cells promotes cytokine polarization (90.20)

Abstract Glycosylphosphatidylinositol-anchored proteins (GPI-AP) that provide activating signals to T cells have been implicated in T cell-mediated immune responses. The effect of GPI-AP signaling on cytokine production and the development of effector T lymphocyte phenotypes remains unclear. In this study, highly pure resting T cells were stimulated with either anti-Thy-1 or anti-TcR monoclonal antibody (mAb) along with costimulatory signals provided by LPS-matured dendritic cells. At concentrations of mAb that induced optimal proliferation, Thy-1-activated T cells proliferated ~2-fold less than TcR-activated T cells. Analysis of cytokine production using cytokine arrays and ELISA showed that Thy-1 signaling induced significantly less IL-2 and IFN-γ synthesis than TcR signaling. Interestingly, Thy-1-stimulated T cells produced significantly more IL-17 and IL-4 than TcR-stimulated T cells. Cytokine polarization was unique to Thy-1, as T cells stimulated with mAb against the GPI-AP Ly6A/E did not produce appreciable levels of IL-17 or IL-4. These results support previous reports that Thy-1 signaling differs from TcR signaling and suggest that Thy-1 may provide polarizing signals that promote the development of Th17 and Th2 cells. Thy-1 and other GPI-AP proteins require further study to elucidate their possible roles in influencing the nature of T cell-mediated immune responses. Research was funded by NSERC

A Biophysical Mechanosensor Model for T-Cell Receptor Signaling

T-cells (cytolytic, helper and others) are vital components in adaptive mammalian immune defense systems. T-cell receptor (TCR) signaling which follows recognition of a peptide antigen bound to an MHC molecule (pMHC) on an antigen presenting cell (APC) is critical for T-cell activation, differentiation, and proliferation. For decades, the mechanism for TCR signaling across the T-cell membrane by this multi-subunit receptor has remained a mystery. Based on our recently obtained structural data on stimulatory and non-stimulatory anti-TCR antibodies and their binding footprint to TCR components obtained by NMR, we present here a simple but all-inclusive “dynamic torque transducer” model for TCR signaling. Extracellular mechanical torque can result in quaternary structure changes between the pMHC-binding TCR alpha/beta chains and the non-convalently linked CD3 chains within the TCR complex, triggering downstream signaling via ITAMs in the CD3 cytoplasmic tails. In this process, the pMHC behaves as a detachable effort arm of this complex-lever system; its binding is determined by the specificity of an individual TCR but is not sufficient to mediate signal transduction per se. An external torque is additionally required to provide the energy for this signaling. Such a torque can be generated either via a shear force or vertical pressure between the opposing APC and T cell membranes. The former “bind-and-tug” mode may be responsible for initial signaling during T-cell scanning of APCs, while the latter “bind-and-bend” mode is responsible for sustained signaling inside immune-synapses during T-cell activation. Our model can also explain T-cell signaling mediated by antibody or multimeric pMHC cross-linking. This mechanism of converting mechanical energy to a biochemical signal, mediated and controlled by a detachable interface, may be generally applicable in other cell-cell signaling systems both within and outside of the immune system.

Bacterial Infection of Smad3/Rag2 Double-Null Mice with Transforming Growth Factor-β Dysregulation as a Model for Studying Inflammation-Associated Colon Cancer

Alterations in genes encoding transforming growth factor-beta-signaling components contribute to colon cancer in humans. Similarly, mice deficient in the transforming growth factor-beta signaling molecule, Smad3, develop colon cancer, but only after a bacterial trigger occurs, resulting in chronic inflammation. To determine whether Smad3-null lymphocytes contribute to increased cancer susceptibility, we crossed Smad3-null mice with mice deficient in both B and T lymphocytes (Rag2(-/-) mice). Helicobacter-infected Smad3/Rag2-double knockout (DKO) mice had more diffuse inflammation and increased incidence of adenocarcinoma compared with Helicobacter-infected Smad3(-/-) or Rag2(-/-) mice alone. Adoptive transfer of WT CD4(+)CD25(+) T-regulatory cells provided significant protection of Smad3/Rag2-DKO from bacterial-induced typhlocolitis, dysplasia, and tumor development, whereas Smad3(-/-) T-regulatory cells provided no protection. Immunohistochemistry, real-time reverse transcriptase-polymerase chain reaction, and Western blot analyses of colonic tissues from Smad3/Rag2-DKO mice 1 week after Helicobacter infection revealed an influx of macrophages, enhanced nuclear factor-kappaB activation, increased Bcl(XL)/Bcl-2 expression, increased c-Myc expression, accentuated epithelial cell proliferation, and up-regulated IFN-gamma, IL-1alpha, TNF-alpha, IL-1beta, and IL-6 transcription levels. These results suggest that the loss of Smad3 increases susceptibility to colon cancer by at least two mechanisms: deficient T-regulatory cell function, which leads to excessive inflammation after a bacterial trigger; and increased expression of proinflammatory cytokines, enhanced nuclear factor-kappaB activation, and increased expression of both pro-oncogenic and anti-apoptotic proteins that result in increased cell proliferation/survival of epithelial cells in colonic tissues.

Localization of NK1.1 + invariant Vα19 TCR + cells in the liver with potential to promptly respond to TCR stimulation

Previously, we found that more than a half of the NK1.1 + T cell lines prepared from CD1 −/− livers expressed invariant Vα19-Jα33 TCR α chains. Over-expression of the invariant Vα19-Jα33 TCR α transgene (Tg) with a natural TCR α promoter and an enhancer in mice induced the development of NK1.1 + T cells (Vα19 NKT cells) in the lymphoid organs, especially in the liver. Preferential usage of the Vα19 Tg by NKT cells in the transgenic mouse livers was indirectly indicated by the observation that few NK1.1 + TCRαβ + cells of the Vα19 Tg livers were stained with a cocktail of anti-TCR Vα antibodies in the FACS analysis. Upon invariant TCR engagement in vivo following injection of mice with anti-CD3 antibody, NKT cells of the Tg mouse livers as well as spleens promptly produced immunoregulatory cytokines such as IL-4 and IFN-γ and altered surface receptor expression. Collectively, localization of Vα19 NKT cells in the liver is suggested that are ready to immediately response against antigen stimulation.

STAT6 Inhibits TGF-β1-mediated Foxp3 Induction through Direct Binding to the Foxp3 Promoter, Which Is Reverted by Retinoic Acid Receptor

It has been shown that transforming growth factor beta1 (TGF-beta1) is critical in the generation of CD4(+)CD25(+)Foxp3(+)-inducible regulatory T cells (iTregs) from naïve CD4(+)T cells. However, in contrast to natural Tregs, TGF-beta1-induced iTregs rapidly lose both Foxp3 expression and suppression activity. We found that TGF-beta1-induced Foxp3 levels were maintained by the addition of the anti-interleukin 4 (IL-4) antibody or by STAT6 gene deletion. Thus, IL-4 is an important suppressor of Foxp3 induction, and T helper 2 development is a major cause for the disappearance of iTreg during long culture. Using promoter analysis in EL4 cells and primary T cells, we identified a silencer region containing a STAT6 binding site. STAT6 binding to this site reduced TGF-beta1-mediated Foxp3 promoter activation and chromatin modification. Retinoic acid has also been shown to suppress loss of Foxp3 induced by TGF-beta1. Retinoic acid in the presence of TGF-beta1 reduced STAT6 binding to the Foxp3 promoter and enhanced histone acetylation, thereby reverting the effect of IL-4. We propose that antagonistic agents for neutralizing IL-4 could be a novel strategy to facilitate inducible Treg cell generation and the promotion of tolerance in Th2-dominated diseases such as allergy.

T cell receptor biochemistry, repertoire selection and general features of TCR and Ig structure.

T cell recognition is a central event in the development of most immune responses, whether appropriate or inappropriate (i.e. autoimmune). We are interested in reducing T cell recognition to its most elemental components and relating this to biological outcome. In a model system involving a cytochrome c-specific I-Ek restricted T cell receptor (TCR) derived from the 2B4 hybridoma, we have studied the interaction of soluble TCR and soluble peptide-MHC complexes using surface plasmon resonance. We find a striking continuum in which biological activity correlates best with the dissociation rate of the TCR from the peptide-MHC complex. In particular, we have found that weak agonists have significantly faster off-rates than strong agonists and that antagonists have even faster off-rates. This suggests that the stability of TCR binding to a given ligand is critically important with respect to whether the T cell is stimulated, inhibited or remains indifferent. It also suggests that the phenomenon of peptide antagonists might be explained purely by kinetic models and that conformation, either inter- or intramolecular, may not be a factor. We have also studied TCR repertoire selection during the establishment of a cytochrome c response, initially using an anti-TCR antibody strategy, but more recently using peptide-MHC tetramers as antigen-specific staining reagents. These tetramers work well with either class I or class II MHC-specific TCRs and have many possible applications. Lastly, we have also tried to correlate the structural and genetic features of TCRs with their function. Recent data on TCR structure as well as previous findings with antibodies suggest that both molecules are highly dependent on CDR3 length and sequence variation to form specific contacts with antigens. This suggests a general "logic' behind TCR and Ig genetics as it relates to structure and function that helps to explain certain anomalous findings and makes a number of clear predictions.

Defective integration of activating signals derived from the T cell receptor (TCR) and costimulatory molecules in both CD4+ and CD8+ T lymphocytes of common variable immunodeficiency (CVID) patients.

CVID is characterized by hypogammaglobulinaemia and impaired antibody production. Previous studies demonstrated defects at the T cell level. In the present study the response of purified CD4+ and CD8+ T lymphocytes to stimulation with anti-TCR monoclonal antibody (the first signal) in combination with anti-CD4 or anti-CD8, anti-CD2 and anti-CD28 MoAbs (the costimulatory signals) was investigated. Both CD4+ and CD8+ T cells from the patients showed significantly reduced IL-2 release following stimulation via TCR and costimulation via CD4 or CD8 and CD2, respectively. However, normal IL-2 production following TCR plus phorbol myristate acetate (PMA) costimulation and normal expression of an early activation marker, CD69, after TCR+CD28 stimulation indicated that TCR was able to transduce a signal. Furthermore, both IL-2 and IL-4 release were impaired in CD4+ lymphocytes following TCR+CD28 stimulation. In addition, stimulation via TCR+CD28 resulted in significantly decreased expression of CD40 ligand in the patients. These results suggest that the integration of activating signals derived from the TCR and costimulatory molecules is defective in CVID patients; the defect is not confined to costimulation via a single molecule, or restricted to cells producing Th1-type cytokines such as IL-2, and is expressed in both CD4+ and CD8+ T cell subsets.

Lysis of a Broad Range of Epithelial Tumour Cells by Human γδ T Cells: Involvement of NKG2D ligands and T‐cell Receptor‐ versus NKG2D‐dependent Recognition

Human gammadelta T cells expressing a V gamma 9V delta 2 T-cell receptor (TCR) kill various tumour cells including autologous tumours. In addition to TCR-dependent recognition, activation of NKG2D-positive gammadelta T cells by tumour cell-expressed NKG2D ligands can also trigger cytotoxic effector function. In this study, we investigated the involvement of TCR versus NKG2D in tumour cell recognition as a prerequisite to identify tumour types suitable for gammadelta T-cell-based immunotherapy. We have characterized epithelial tumour cells of different origin with respect to cell surface expression of the known NKG2D ligands MHC class I-chain-related antigens (MIC) A/B and UL16-binding proteins (ULBP), and susceptibility to gammadelta T-cell killing. Most tumour cells expressed comparable levels of MICA and MICB as well as ULBP with the exception of ULBP-1 which was absent or only weakly expressed. Most epithelial tumours were susceptible to allogeneic gammadelta T-cell lysis and in the case of an established ovarian carcinoma to autologous gammadelta T-cell killing. Lysis of resistant cells was enhanced by pre-treatment of tumour cells with aminobisphosphonates or pre-activation of gammadelta T cells with phosphoantigens. A potential involvement of TCR and/or NKG2D was investigated by antibody blockade. These experiments revealed three patterns of inhibition, i.e. preferential inhibition by anti-TCR antibody, preferential inhibition by anti-NKG2D antibody, or additive blockade by anti-TCR plus anti-NKG2D antibodies. Our results indicate for the first time that the NKG2D pathway is involved in the lysis of different melanomas, pancreatic adenocarcinomas, squamous cell carcinomas of the head and neck, and lung carcinoma.

Depletion of γδ T Cells Exacerbates Murine Adriamycin Nephropathy

It has been reported that the presence of gammadelta T cells in kidney is associated with kidney damage in human IgA nephropathy and in rat models of chronic renal injury, including Adriamycin nephropathy (AN), but the functional role of gammadelta T cells in this setting is unknown. This study examined the functional role of gammadelta T cells in tissue injury in a murine model of AN. Murine AN was induced in BALB/c mice by a single injection of Adriamycin. gammadelta T cells as a proportion of CD3(+) T cells were significantly increased in AN kidneys (16.8 +/- 3.9%) but not in lymph nodes (1.3 +/- 0.8%; P < 0.001). The proportion of gammadelta T cells in AN kidney correlated positively with serum creatinine and glomerular sclerosis. The Vgammadelta T cell receptor (TCR) repertoire in kidney showed expansion of a subset of cells that expressed Vgamma6/Vdelta1 genes and that used canonical TCR Vgamma6/Vdelta1 sequences in the CDR3 region of the TCR. gammadelta T cells that were sorted from the kidneys expressed TGF-beta but not IL-4, IL-10, or IFN-gamma. gammadelta T cells also expressed the activating receptor NKG2D and the NKG2D adaptor molecule DAP12. RAE-1, a ligand of NKG2D, was upregulated in AN kidney. Depletion of gammadelta T cells using anti-TCR gammadelta antibody resulted in worsening of serum creatinine, glomerulosclerosis, and interstitial inflammation. These studies indicate the involvement of the gammadelta T cell in innate recognition and regulation of inflammation in AN.

Distinctive T Cell-suppressive Signals from Nuclearized Type 1 Sphingosine 1-Phosphate G Protein-coupled Receptors

Sphingosine 1-phosphate (S1P) generated by cells of innate immunity and the type 1 S1P G protein-coupled receptor (S1P(1)) on mobile T cells constitute a major system for control of lymphoid organ traffic and tissue migration of T cells. Now we show that T cell activation mediated by the T cell antigen receptor translocates plasma membrane S1P(1) to nuclear envelope membranes for association there with G(i/o), Erk (1/2), and other proteins that plasma membrane S1P(1) uses to signal T cell proliferation. However, nuclear S1P(1) and plasma membrane S1P(1) transduce opposite effects of S1P on T cell proliferation and relevant signaling as exemplified by respective decreases and increases in T cell nuclear concentrations of both phospho-Erk and active (phosphorylated) c-Jun. T cell antigen receptor-mediated activation of T cells therefore both eliminates migration responses to S1P by down-regulation of plasma membrane S1P(1) and translocates the S1P-S1P(1) axis into the nuclear domain where signals are directed to transcriptional control of immune functions other than migration.

Protection Against SEB-Induced Toxic Shock by Anti-TCR Vb8 Antibody

Protection Against SEB-Induced Toxic Shock by Anti-TCR Vb8 Antibody

Discovery and SAR of 2-amino-5-(thioaryl)thiazoles as potent and selective Itk inhibitors

A series of structurally novel aminothiazole based small molecule inhibitors of Itk were prepared to elucidate their structure–activity relationships (SARs), selectivity, and cell activity in inhibiting IL-2 secretion in a Jurkat T-cell assay. Compound 3 is identified as a potent and selective Itk inhibitor which inhibits anti-TCR antibody induced IL-2 production in mice in vivo and was previously reported to reduce lung inflammation in a mouse model of ovalbumin induced allergy/asthma.

Generation of tumor-reactive effector lymphocytes using tumor RNA-introduced dendritic cells in gastric cancer patients

Anti-tumor effector cells were generated by stimulating peripheral blood lymphocytes with cultured dendritic cells (DCs) and mRNA extracted from the gastric cancer cell line MKN45 or ascites tumor cells of gastric cancer patients. DCs were generated from an adherent fraction of peripheral blood mononuclear cells (PBMCs) in the presence of GM-CSF and IL-4. mRNA was extracted from tumor cells and subjected to T7-amplification. The DCs were electroporated (150 V/150 microF) with amplified mRNA and used after maturation with TNF-alpha to stimulate PBMCs to generate tumor RNA-introduced DC-activated killer (TRiDAK) cells. It was found that tumor RNA could efficiently be introduced into cultured DCs by electroporation (55% efficiency, 78% viability), and tumor RNA-introduced DCs could reproducibly stimulate lymphocytes to be tumor-reactive TRiDAK cells. The TRiDAK cells expressed an IFN-gamma response specific for tumor cells, but not for normal cells. Mock DCs or normal cell RNA-introduced DCs did not induce any killer cells. RNA-specific recognition of the effector cells generated was demonstrated using an amplified EGFP-mRNA system. The tumor killing activity of TRiDAK cells was inhibited not only with the anti-HLA class I antibody but also with the anti-HLA class II antibody as well as the anti-TCR antibody. TRiDAK cells reactive with autologous tumor cells could be generated in a CEA-positive gastric cancer patient with malignant ascites, in whom effector cell generation using DCs and CEA peptides had failed. These results suggest that TRiDAK cell generation is safe, feasible, and active in gastric cancer patients with malignant ascites, and is superior to other effector cell generation systems using DCs and epitope peptides. The adoptive immunotherapy of cancer using TRiDAK cells may be warranted in a clinical setting. This is the first study investigating anti-tumor effector cell generation using cultured DCs and tumor mRNA from gastric cancer cells.

Alterations in the T-Cell Receptor Variable β Gene–Restricted Profile of CD8+ T Lymphocytes in the Peripheral Circulation of Patients with Squamous Cell Carcinoma of the Head and Neck

Apoptosis of activated CD8(+) T cells is often seen in tumor-infiltrating lymphocytes and circulating peripheral blood mononuclear cells (PBMC) in patients with squamous cell carcinoma of the head and neck (SCCHN). We investigated whether T-cell receptor (TCR) variable beta chain (Vbeta)-restricted T cells were more sensitive to apoptosis than non-TCR Vbeta-restricted T cells. Flow cytometry analysis with anti-TCR Vbeta antibodies was used to define expansions and contractions of Vbeta-restricted T cells in patients with SCCHN relative to normal donors. This staining was combined with Annexin V binding to indicate early T-cell apoptosis. The TCR Vbeta profiles of CD3(+) T cells in tumor-infiltrating lymphocytes and PBMCs of patients with SCCHN were altered relative to controls, with one to five expansions and numerous contractions of TCR Vbeta-restricted T cells detected. These types of alterations were significantly greater in CD8(+) than CD4(+) T cells. Enhanced Annexin V binding to CD8(+) T cells was evident in PBMCs obtained from all patients, with 3 of 13 showing preferential targeting for apoptosis of TCR Vbeta-restricted T cells. TCR Vbeta profiles of CD8(+) T cells were altered in patients with SCCHN relative to normal controls. This may reflect increased apoptosis of expanded or contracted CD8(+) T cells, which define the TCR Vbeta profile of antigen-responsive T-cell populations in patients with cancer.