Itaru Hirai

Tumor-derived heat shock protein 70-pulsed dendritic cells elicit tumor-specific cytotoxic T lymphocytes (CTLs) and tumor immunity

Vaccination with autologous tumor‐derived heat shock proteins (Hsp), such as Hsp70, Hsp90 and gp96, has been demonstrated to elicit specific immune responses against the tumor from which the Hsps were isolated. The effect of Hsp immunization is wholly dependent on the presence of functional antigen‐presenting cells (APCs) in the immunized host, and Hsp receptors on APCs have recently been identified. Here we show that bone marrow‐derived dendritic cells (DCs) are able to internalize HSP‐peptide complex and that peptides are re‐presented by DCs via the major histocompatibility complex (MHC) class I presentation pathway. In addition, immunization with tumor‐derived HSP‐pulsed DCs induces strong cytotoxic T cell (CTL) responses against multiple antigenic peptides in a transporter‐associated antigen processing (TAP)‐dependent manner. The results of the present study provide strong evidence of an efficient cross‐priming activity of Hsp70, which could be exploited in the development of new and more effective immunotherapeutic strategies for cancer patients. (Cancer Sci 2004; 95: 248–253)

Human CD8 and CD4 T cell epitopes of epithelial cancer antigens

Abstract Recent human tumor immunology research has identified several genes coding immunogenic peptides recognized by CD8 cytotoxic T lymphocytes (CTLs) in melanoma tumors. Very recently, CD4u2009T cell antigenic epitopes were also determined in certain melanoma tumors. The use of these peptides in conjunction with human immunotherapy could prove to be of great benefit. However, such peptides in clinically common tumors of epithelial cell origin, such as of the stomach, colon, lung, etc., have not yet been determined extensively. We describe for the first time an HLA-A31 (A*31012)-restricted natural antigenic peptide recognized by the CD8 CTL TcHST-2 of gastric signet ring cell carcinoma cell line HST-2. We also identified the HLA-DRB1*08032-restricted peptide recognized by the CD4u2009T cell line TcOSC-20 of squamous cell carcinoma OSC-20 derived from the oral cavity. The antigenic peptide of HST-2, designated F4.2, is composed of 10 amino acid residues with two anchor motif residues necessary for binding to HLA-A31 molecules. The synthetic F4.2 peptide enhanced the reactivity of TcHST-2 against HST-2 cells. Furthermore, introduction of an expression minigene coding F4.2 peptide to HLA-A31(+) cells conferred cytotoxic susceptibility to TcHST-2 on the cells. Some stomach cancer lines into which the HLA-A31 gene had been introduced, such as MKN28-A31-2, were lysed by TcHST-2, suggesting the presence of F4.2 peptide in at least some HLA-A31(+) stomach cancers. Furthermore, F4.2 peptide induced an F4.2 peptide-specific CTL response in at least 30–40% of HLA-A31(+) peripheral blood lymphocytes from gastric cancer patients, suggesting that F4.2 peptide could be used as a cancer vaccine for gastric tumors. The natural antigenic peptide of OSC-20 was also determined using acid extraction and biochemical separation and by mass spectrometry. Consequently, OSC-20 peptide was designated as the 6-1-5 peptide, an HLA-DRB1*08032-restricted 16-mer peptide with two possible anchor motifs. It has an amino acid sequence identical to that of human α-enolase, suggesting that it was derived from the processed parental α-enolase protein. We are presently attempting to determine the genes that code tumor rejection antigens recognized by HLA-A24- and A26-restricted T cells, including those of pulmonary and pancreatic carcinomas. The search for these antigenic peptides may lead to the identification of immunogenic peptide antigens that would be suitable for clinical use in commonly occurring epithelial cancers.

The cell surface-expressed HSC70-like molecule preferentially reacts with the rat T-cell receptor Vδ6 family

We previously showed that the cell surface-expressed Mrxa070,000 heat shock cognate (hsc70, a constitutively expressed member of the hsp70 family) protein-like molecule (#067 molecule) interacts with rat CD3+, CD4–, CD8–, T-cell receptor (TCR)αβ–, natural killer recetor-P1– T cells. This 70hsc-like molecule was also suggested to present cellular peptide antigens to these T cells. In the present study, we identified the genetic structure of the TCR by establishing T-cell hybridomas between these T cells and mouse BW5147 cells. Our data indicated that these T cells preferentially used TCRs with the Vδ6 family. Analysis of the nucleotide sequence of the CDR3 junctional portion showed that there are substantial diversities, with insertion of seven to nine amino acid residues. These data provide indirect evidences for our hypothesis that an hsc70-like molecule could be presented together with cellular peptide antigens to particular T cells with TCR γδ chains. Since the expression of this hsc70-like #067 antigen on the cell surface is usually induced along with cell transformation by activated oncogenes, T cells with the TCR Vδ6 family are likely to contribute to host resistance to tumor cells.

Molecular Cloning of Rat NK Target Structure —The Possibility of CD44 Involvement in NK Cell‐Mediated Lysis

The nature of target molecules of natural killer (NK) cell‐mediated lysis remains to be elucidated. As we previously reported, mAb 109 recognizes one of the tumor‐associated antigens, designated as 109 antigen (Ag), expressed on the cell surface of rat fibrosarcomas W31 and W14, which are transformants of WFB (rat fetal fibroblast cell line) with H‐ras oncogene. 109Ag was thought to be a target structure of NK cells since mAb 109 inhibited NK cell‐mediated lysis against W31 and W14. Here, we demonstrate by molecular cloning that 109Ag is identical to rat CD44. Immunoprecipitation and immunoblotting studies also showed that mAb 109 and anti‐rat CD44 mAb OX‐50 recognize the same protein of W31 cell lysates with an 86 kDa molecular size. CD44 was suggested to be a target structure of NK cell‐mediated lysis; however, rat CD44 cDNA transfection alone into CD44 null cell lines did not result in up‐regulation of target cell susceptibility to NK cell‐mediated lysis. Our results therefore indicated that CD44 may play a crucial role as one of the target structures in our rat fibrosarcoma system though the cell surface expression of CD44 alone does not affect NK susceptibility of the target cells.

Inhibition of Natural Killer Cell Cytotoxicity by Cell Growth-related Molecules

Certain MHC class I molecules on target cells are known to inhibit the cytotoxic action of NK cells. By using monoclonal antibody (mAb) Cho‐1, we have found inhibitory non‐MHC class I cell surface molecules that are noncovalently‐associated with 200 kDa and 40 kDa antigens. Poly I‐C‐induced rat NK cells were not cytotoxic to rat fetus‐derived fibroblast WFB cell line. In contrast, NK cells were cytotoxic to H‐ras oncogene‐induced transformants of WFB, W14 and W31. FACS analysis indicated that mAb Cho‐1 reacts with WFB, but not with W14 and W31 cells. Thus, this antigen may disappear concomitantly with cell growth and transformation. Cho‐1 antigens were also expressed on other NK‐resistant lines, such as mouse BALB3T3 fibroblast, EL‐4 lymphoma and human fibroblast HEPM. However, they were not expressed on NK‐sensitive mouse YAC‐1 and H‐ras transformant (Brash) of BALB3T3 cells. Furthermore, treatment of target cells with IFN‐γ clearly induced the cell surface expression of Cho‐1 antigens, and conferred a resistance to NK cytolysis on target cells. These data strongly suggest that Cho‐I antigen expression may correlate with target cell susceptibility to NK cells. Indeed, treatment of NK‐resistant WFB as well as HEPM cells with F(ab)2 fragments of mAb Cho‐1 resulted in the acquisition of susceptibility to NK cytolysis. Cho‐1 antigens may be novel molecules that regulate the NK resistance of cells.

Immunosuppressive effect on T cell activation by interleukin-16- and interleukin-10-cDNA-double-transfected human squamous cell line.

It is well known that induction of immunotolerance with allogeneic skin transplantation is generally difficult. This study attempted to find an immunosuppressive protocol for skin allograft rejection involving interleukin-16 (IL-16) and interleukin-10 (IL-10), because both are known to inhibit mixed lymphocyte reaction (MLR). The data indicated that IL-16 enhanced the immunosuppressive effect of IL-10. IL-16-cDNA- and IL-10-cDNA-double-transfected squamous cell carcinoma cell line were used as an in vitro model and they produced more than 20 ng/ml of IL-16 and 100 pg/ml of IL-10 in the supernatant, which significantly inhibited MLR and also the activation of allogeneic lymphocytes, which were stimulated directly by allogeneic double-cDNA-transfectant cells. Thus allogeneic skin graft producing IL-16 and IL-10 might have a local immunosuppressive action that could prolong graft survival.

Identification of germline mutation of PTEN gene and analysis of apoptosis resistance of the lymphocytes in a patient with Cowden disease.

Mutations of the tumor suppressor gene PTEN have been reported in patients with Cowden disease (CD) and in several malignant tumors. We analyzed a germline mutation of the PTEN gene in a patient with CD and identified a 4-bp deletion in exon 8 of the PTEN gene. The same germline mutation was detected in 3 members of her family. The mutated gene was predicted to encode a C-terminal truncated PTEN protein. Immunoblotting analysis revealed that the expression level of the wild-type PTEN protein in the patient’s lymphocytes was reduced to almost half the level of the control lymphocytes, and the predicted truncated mutant PTEN could not be detected. Since PTEN is known to function as a negative regulator of the phosphatidylinositol-3-kinase signal pathway that promotes cell survival, the patient’s lymphocytes were tested for the resistance against the apoptotic stimulus. It was shown that the patient’s lymphocytes were more resistant to apoptosis induced by calcium ionophore than the healthy control lymphocytes. These results indicate that the germline mutation of the PTEN gene and the consequent loss of heterozygous expression may lead to an increase in the survival potential of cells, thereby elucidating a role of PTEN in the pathogenesis of tumor generation and hyperplasia of lymphoid tissue in CD.

Disruption of the association of 73 kDa heat shock cognate protein with transporters associated with antigen processing (TAP) decreases TAP-dependent translocation of antigenic peptides into the endoplasmic reticulum.

Major histocompatibility complex class I‐bound antigenic peptides generated in the cytosol are translocated into the ER by TAP. In the present study, the physical association of HSC73 with TAP in human lymphoblastoid T1 cells was demonstrated. The dissociation was induced in the presence of 10u2003mM ATP, indicating that the ADP‐binding form of HSC73 might be associated with TAP. We found that HSC73‐binding immunosuppressant, MeDSG disrupted the HSC73‐TAP association, whereas it did not affect the binding of HSC73 to a substrate protein. MHC class I expression on the cell surface was also downregulated. Then, the effect of MeDSG on the TAP‐mediated ER translocation was examined using two homologous model peptides, NGT‐Bw4 and NGT‐Bw6, which had distinct binding affinity to HSC73. Although high‐affinity peptide NGT‐Bw4 was translocated by TAP, low‐affinity peptide NGT‐Bw6 was not. The TAP‐dependent translocation of NGT‐Bw4 was abolished in the presence of MeDSG. Decreased presentation on the cell surface was shown for the human leukocyte antigen (HLA)‐A31‐restricted natural antigenic peptide F4.2, which had high affinity to HSC73, in the presence of MeDSG. It was indicated that disruption of the HSC73‐TAP association resulted in inhibition of TAP‐dependent translocation of HSC73‐bound peptides. Our findings highlighted an important role of HSC73 for feeding antigenic peptides to TAP, and suggested a possibility that a synthetic polyamine might inhibit the function of HSC73, thereby suppressing MHC class I‐restricted presentation of HSC73‐bound antigenic peptides.

Establishment of shared antigen reactive cytotoxic T lymphocyte using co-stimulatory molecule introduced autologous cancer cells

Cytotoxic T lymphocytes (CTLs) play an essential role in immunological responses for tumor rejection. In the past decade, many tumor-associated antigens (TAAs) have been identified predominantly in melanomas. Several clinical trials based on such antigenic peptides with or without adjuvants brought about partially favorable results, suggesting that identification of more immunogenic TAAs is needed. We show here the successful establishment of human leukocyte antigen (HLA)-A24-restricted CTL (TcLHK2 line1) from a pleural effusion of lung cancer patient, using B7.1 (CD80) transduced autologous lung cancer cells as an antigen-presenting cell (APC). TcLHK2 line1 recognized autologous lung adenocarcinoma cell line LHK2 in an HLA-A24-restricted fashion. Moreover, this CTL line also recognized allogeneic HLA-A24-positive lung adenocarcinoma cell line, gastric carcinoma cell line and melanoma cell line. These data raise the possibility that co-stimulatory molecule B7.1 (CD80) plays important role to overcome the immunological tolerance. Furthermore, TcLHK2 line1 is a useful tool for the identification of widely expressed shared antigens restricted by HLA-A24. Further analysis of this CTL and autologous cancer cell line will bring about novel TAAs.

T-Cell Receptor Variable γ Chain Gene Expression in the Interaction between Rat γδ-Type T Cells and Heat-Shock Protein 70-Like Molecule

We previously reported that rat T‐cell receptor (TCR) Vδ6 of T‐cell hybridomas was preferentially involved in recognition of the cell surface‐expressed 70 kDa rat heat‐shock cognate (hsc70, a constitutively expressed member of the hsp 70 family) protein‐like molecule (#067 molecule). In the present study, we analyzed usage of the TCR Vγ family of #067‐restricted T‐cell hybridomas. Our data indicated that most of these hybridomas expressed transcripts of TCR Vγ1 and/or Vγ2. However, some of the Vγ2 transcripts were out‐of‐frame, suggesting that the TCR Vγ1 family may be important for the recognition of #067‐defined molecules. TCR Vγ1 transcripts were detected in not only #067‐restricted T‐cell hybridomas, but #067‐non restricted ones as well. However, V‐J nucleotide sequences of #067‐restricted and #067‐non restricted T‐cell hybridomas suggested that #067‐restricted T‐cell hybridomas showed limited insertion of nucleotide stretch as compared with #067‐non restricted ones. In terms of amino acids, only one amino acid was added in #067‐restricted T‐cell hybridomas, whereas two or three amino acids were added in #067‐non restricted ones. These data suggest that the heterodimer of the TCR relatively short stretch form of Vγ1 molecule and TCR Vδ6 may participate in recognition of the #067 molecule.