Induction of CTL responses by simultaneous administration of liposomal peptide vaccine with anti-CD40 and anti-CTLA-4 mAb

Activation of APC via CD40-CD40 ligand pathway induces up-regulation of costimulatory molecules such as B7 and production of IL-12. Interaction between B7 on APC and CD28 on naive T cells is necessary for priming the T cells. On the other hand, interaction between B7 on APC and CTLA-4 on activated T cells transduces a negative regulatory signal to the activated T cells. In the present study, we attempted to generate tumor-specific CTL by s.c. administration of antigenic peptides encapsulated in multilamellar liposomes (liposomal peptide vaccine) with anti-CD40 mAb and/or anti-CTLA-4 mAb. Liposomal OVA257-264 and anti-CD40 mAb or anti-CTLA-4 mAb were administrated to C57BL/6 mice and the splenocytes were cocultured with OVA257-264 for 4 days. The splenic CD8+ T cells showed a significant cytotoxicity against EL4 cells transfected with cDNA of OVA. In addition, administration of both anti-CD40 and anti-CTLA-4 mAb enhanced the CTL responses. Considerable CTL responses were induced in MHC class II deficient mice by the same procedure. This finding indicated that CTL responses could be generated even in the absence of Th cells. When BALB/c mice were immunized with pRL1a peptide that are tumor-associated Ag of RLmale symbol1 leukemia cells using the same procedure, significant CTL responses were induced and prolonged survival of the BALB/c mice was observed following RLmale symbol1 inoculation. These results demonstrate that anti-CD40 mAb and anti-CTLA-4 mAb function as immunomodulators and may be applicable to specific cancer immunotherapy with antitumor peptide vaccine.     

Identification of a novel HLA-A2-restricted cytotoxic T lymphocyte epitope from cancer-testis antigen PLAC1 in breast cancer

Identification of cytotoxic T lymphocyte (CTL) epitopes from tumor antigens is essential for the development of peptide vaccines against tumor immunotherapy. Among all the tumor antigens, the caner-testis (CT) antigens are the most widely studied and promising targets. PLAC1 (placenta-specific 1, CT92) was considered as a novel member of caner-testis antigen, which expressed in a wide range of human malignancies, most frequently in breast cancer. In this study, three native peptides and their analogues derived from PLAC1 were predicted by T cell epitope prediction programs including SYFPEITHI, BIMAS and NetCTL 1.2. Binding affinity and stability assays in T2 cells showed that two native peptides, p28 and p31, and their analogues (p28-1Y9?V, p31-1Y2L) had more potent binding activity towards HLA-A*0201 molecule. In ELISPOT assay, the CTLs induced by these four peptides could release IFN-γ. The CTLs induced by these four peptides from the peripheral blood mononuclear cells (PBMCs) of HLA-A*02⁺ healthy donor could lyse MCF-7 breast cancer cells (HLA-A*0201⁺, PLAC1⁺) in vitro. When immunized in HLA-A2.1/K^b transgenic mice, the peptide p28 could induce the most potent peptide-specific CTLs among these peptides. Therefore, our results indicated that the peptide p28 (VLCSIDWFM) could serve as a novel candidate epitope for the development of peptide vaccines against PLAC1-positive breast cancer.     

Cancer immunotherapy targeting Wilms' tumor gene WT1 product

The Wilms' tumor gene WT1 is expressed at high levels not only in acute myelocytic and lymphocytic leukemia and in chronic myelocytic leukemia but also in various types of solid tumors including lung cancers. To determine whether the WT1 protein can serve as a target Ag for tumor-specific immunity, three 9-mer WT1 peptides (Db126, Db221, and Db235), which contain H-2Db-binding anchor motifs and have a comparatively higher binding affinity for H-2Db molecules, were tested in mice (C57BL/6, H-2Db) for in vivo induction of CTLs directed against these WT1 peptides. Only one peptide, Db126, with the highest binding affinity for H-2Db molecules induced vigorous CTL responses. The CTLs specifically lysed not only Db126-pulsed target cells dependently upon Db126 concentrations but also WT1-expressing tumor cells in an H-2Db-restricted manner. The sensitizing activity to the Db126-specific CTLs was recovered from the cell extract of WT1-expressing tumor cells targeted by the CTLs in the same retention time as that needed for the synthetic Db126 peptide in RP-HPLC, indicating that the Db126-specific CTLs recognize the Db126 peptide to kill WT1-expressing target cells. Furthermore, mice immunized with the Db126 peptide rejected challenges by WT1-expressing tumor cells and survived for a long time with no signs of autoaggression by the CTLs. Thus, the WT1 protein was identified as a novel tumor Ag. Immunotherapy targeting the WT1 protein should find clinical application for various types of human cancers.     

In vivo anti-melanoma efficacy of allo-restricted CTLs specific for melanoma expanded by artificial antigen-presenting cells

Cytotoxic CD8⁺ T cells are key effectors in the immunotherapy of malignant and viral diseases. However, autologous T cell responses to tumor antigens presented by self-MHC are usually weak and ineffective. Allo-restricted T cells represent a potent source of tumor-specific T cells for adoptive immunotherapy. This study reports in vivo anti-melanoma efficacy of the pTRP2-specific allo-restricted CTLs expanded from the BALB/c splenocytes by multiple stimulations with aAPCs made by coating H-2K^b-Ig/pTRP2 dimeric complexes, anti-CD28 antibody, 4-1BBL molecules and CD83 molecules to cell-sized latex beads. The induced allo-restricted CTLs exhibited specific lysis against RMA-S cells pulsed with the peptide pTRP2 and H-2K^b+ melanoma cells expressing TRP2, while a murine Lewis lung carcinoma cell line 3LL could not be recognized by the CTLs. The peptide-specific activity was inhibited by anti-H-2K^b monoclonal antibody Y3. Adoptive transfer of the allo-restricted CTLs specific for malignant melanoma expanded by the aAPCs can mediate effective anti-melanoma response in vivo. These results suggested that the specific allo-restricted CTLs expanded by aAPCs coated with an MHC-Ig/peptide complex, anti-CD28 antibody, 4-1BBL and CD83 could be a potential option of specific immunotherapy for patients with malignant melanoma. X.-l. Lu and X.-b. Jiang have contributed equally to this work. An erratum to this article can be found at     

CD4+ T cells from (New Zealand Black x New Zealand White)F1 lupus mice and normal mice immunized against apoptotic nucleosomes recognize similar Th cell epitopes in the C terminus of histone H3

We have previously reported that peptide 88-99 of histone H4 represents a minimal T cell epitope recognized by Th cells from nonautoimmune BALB/c (H-2(d/d)) mice immunized with nucleosomes. In this study, we tested a panel of overlapping peptides spanning the whole sequences of H4 and H3 for recognition by CD4(+) T cells from unprimed (New Zealand Black (NZB) x New Zealand White (NZW))F(1) lupus mice (H-2(d/z)). None of the 11 H4 peptides was recognized by CD4(+) T cells from (NZB x NZW)F(1) mice. In contrast, these cells proliferated and secreted IL-2, IL-10, and IFN-gamma upon ex vivo stimulation with H3 peptides representing sequences 53-70, 64-78, and 68-85. Peptides 56-73 and 61-78 induced the production of IFN-gamma and IL-10, respectively, without detectable proliferation, suggesting that they may act as partial agonist of the TCR. Th cells from unprimed BALB/c mice and other lupus-prone mice such as SNF(1) (H-2(d/q)) and MRL/lpr (H-2(k/k)) mice did not recognize any peptides present within the H3 region 53-85. We further demonstrated that immunization of normal BALB/c mice with syngeneic liver nucleosomes and spleen apoptotic cells, but not with nonapoptotic syngeneic cells, induced Th cell responses against several peptides of the H3 region 53-85. Moreover, we found that this conserved region of H3, which is accessible at the surface of nucleosomes, is targeted by Abs from (NZB x NZW)F(1) mice and lupus patients, and contains motifs recognized by several distinct HLA-DR molecules. It might thus be important in the self-tolerance breakdown in lupus.     

Identification of a minimal T cell epitope recognized by antinucleosome Th cells in the C-terminal region of histone H4

Autoreactive T cells responding to systemic autoantigens have been characterized in patients and mice with autoimmune diseases and in healthy individuals. Using peptides covering the whole sequence of histone H4, we characterized several epitopes recognized by lymph node Th cells from nonsystemic lupus erythematosus-prone mice immunized with the same peptides, the H4 protein, or nucleosomes. Multiple T epitopes were identified after immunizing H-2d BALB/c mice with H4 peptides. They spanned residues 28-42, 30-47, 66-83, 72-89, and 85-102. Within the region 85-102, a minimal CD4+ T epitope containing residues 88-99 was characterized. Although Abs to peptide 88-99 recognized H4, this peptide does not contain a dominant B cell epitope recognized by anti-H4 Abs raised in BALB/c mice or Abs from NZB/NZW H-2d/z lupus mice. Th cells primed in vivo with H4 responded to H4, but not to peptide 88-99. However, this peptide was able to stimulate the proliferation and IL-2 secretion of Th cells generated after immunization with nucleosomes. H488-99 thus represents a cryptic epitope with regard to H4 and a supradominant epitope presented by nucleosome, a supramolecular complex that plays a key role in lupus. This study shows that in the normal repertoire of naive BALB/c mice, autoreactive Th cells specific for histones are not deleted. The reactivity of these Th cells seems to be relatively restricted and resembles that of Th clones generated from SNF1 ((SWR x NZB)F1; I-Ad/q) lupus mice described earlier.     

Acute rejection of allografted CTL-susceptible leukemia cells from perforin/Fas ligand double-deficient mice

The generation of knockout mice demonstrated that CD4(+), but not CD8(+), T cells were essential for the rejection of allografted skin or heart, presumably because these targets were CTL resistant. In the case of CTL-susceptible targets (e.g., P815 mastocytoma cells and EL-4 or RLmale1 T lymphoma cells), however, it is assumed that the CTL is the effector cell responsible for allograft rejection and that perforin and Fas ligand (FasL) pathways are the killing mechanisms. In the present study, we examined the role of these cytotoxic molecules in the rejection of i.p. allografted CTL-susceptible leukemia cells. Unexpectedly, the allografted leukemia cells were acutely rejected from gld (a mutation of FasL), perforin(-/-), or double-deficient mice. The peritoneal exudate cells from gld or normal mice showed T cell-, TCRalphabeta-, and perforin-dependent cytotoxic activity against the allograft, whereas the exudate cells from perforin(-/-) mice exhibited almost full cytotoxic activity in the presence of Fas-Fc. Furthermore, the infiltrates from double-deficient mice showed a high cytotoxic activity against the allografted cells even in the presence of anti-TCRalphabeta Ab or in the absence of T cells. The cytotoxic cells appeared to be macrophages, because they were Mac-1(+) mononuclear cells with a kidney- or horseshoe-shaped nucleus and because the cytotoxic activity was completely suppressed by the addition of N(G)-monomethyl-l-arginine, an inhibitor of inducible NO synthase. These results indicate that macrophages are ready and available to kill CTL-susceptible allografts when CTLs lack both perforin and FasL molecules.     

A cyclophilin B gene encodes antigenic epitopes recognized by HLA-A24-restricted and tumor-specific CTLs.

We have studied Ags recognized by HLA class I-restricted CTLs established from tumor site to better understand the molecular basis of tumor immunology. HLA-A24-restricted and tumor-specific CTLs established from T cells infiltrating into lung adenocarcinoma recognized the two antigenic peptides encoded by a cyclophilin B gene, a family of genes for cyclophilins involved in T cell activation. These two cyclophilin B peptides at positions 84-92 and 91-99 induced HLA-A24-restricted CTL activity against tumor cells in PBMCs of leukemia patients, but not in epithelial cancer patients or in healthy donors. In contrast, the modified peptides at position 2 from phenylalanine to tyrosine, which had more than 10 times higher binding affinities to HLA-A24 molecules, could induce HLA-A24-restricted CTL activity against tumor cells in PBMCs from leukemia patients, epithelial cancer patients, or healthy donors. PHA-activated normal T cells were resistant to lysis by the CTL line or by these peptide-induced CTLs. These results indicate that a cyclophilin B gene encodes antigenic epitopes recognized by CTLs at the tumor site, although T cells in peripheral blood (except for those from leukemia patients) are immunologically tolerant to the cyclophilin B. These peptides might be applicable for use in specific immunotherapy of leukemia patients or that of epithelial cancer patients.     

Identification of cross-reactive peptides using combinatorial libraries circumvents tolerance against Her-2/neu-immunodominant epitope

The majority of the currently defined tumor-associated Ags are often overexpressed products of normal cellular genes. Therefore, tolerance deletes high-affinity T cells directed against the TAAs, leaving only a low-affinity repertoire. We have demonstrated previously that the T cell repertoire against the immunodominant p773-782 A2.1-Her-2/neu-restricted peptide has low affinity in A2xneu mice (Her-2/neu mice crossed with A2.1/Kb mice), compared with A2xFVB mice (A2.1/Kb crossed with FVB-wild-type mice). Immunizations with this peptide have a minor impact in preventing tumor growth in A2xneu mice. Therefore, attempts to expand these responses may be of little clinical value. We hypothesized that if not all possible cross-reactive peptides (CPs) are naturally processed and presented, the possibility exists that T cells against these CPs persist in the repertoire and can be used to induce antitumor responses with higher avidity against native epitopes present on the tumor cells. We have used the positional scanning synthetic peptide combinatorial library methodology to screen the p773-782 T cell clone. The screening data identified potential amino acids that can be substituted in the primary sequences of the p773-782 peptide. The designed CPs induce CTL responses of higher affinity in A2xneu mice compared with the native p773-783 peptide. These CTLs recognize A2+-Her-2/neu(+) tumors with high efficiency. Moreover, multiple immunizations with CPs significantly prolonged the survival of tumor-bearing A2xneu mice. These results have demonstrated that it was possible to circumvent tolerance with the identification of CPs and that these peptides could be of significant clinical value.     

Immunization of mice with vaccinia virus-M2 recombinant induces epitope-specific and cross-reactive Kd-restricted CD8+ cytotoxic T cells.

The M2 protein of respiratory syncytial virus (RSV) is a protective antigen in H-2d, but not H-2b or H-2k mice. None of the other RSV proteins, excluding the surface glycoproteins that induce neutralizing antibodies, is protective in mice bearing these haplotypes. Thus, the M2 protein stands alone as a nonglycoprotein-protective antigen of RSV. The M2 protein is a target for murine Kd-restricted cytotoxic T lymphocytes (CTLs), and the resistance induced by infection with a vaccinia virus-RSV M2 (vac-M2) recombinant is mediated by CD8+ CTLs. Since the nonameric consensus sequence for H-2 Kd-restricted T-cell epitopes and the amino acid sequence of the M2 protein of subgroup A and B strains of RSV are known, the present study sought to identify the specific epitope(s) on the M2 protein recognized by CD8+ CTLs. This was done by examining the ability of four predicted Kd-specific motif peptides present in the M2 amino acid sequence of an RSV subgroup A strain to sensitize target cells for lysis by pulmonary or splenic CTLs obtained from mice infected with RSV or vac-M2. The following observations were made. First, two of the four peptides sensitized target cells for lysis by pulmonary or splenic CTLs induced by infection with either vac-M2 or RSV. Second, one of the two peptides, namely the 82-90 (M2) peptide, sensitized targets at a very low peptide concentration (10(-10) to 10(-12) M). Third, cold-target competition experiments revealed that the predominant CTL population induced by infection with vac-M2 or RSV recognized the 82-90 (M2) peptide, and this CTL population appeared to recognize the 71-79 (M2) peptide in a cross-reactive manner. Fourth, CTL recognition of targets sensitized with either the 71-79 (M2) or the 82-90 (M2) peptide was Kd restricted. Fifth, CTLs induced by infection with RSV subgroup A or B strains recognized the two M2 peptides. The findings suggest that the M2 protein of RSV contains an immunodominant Kd-restricted CTL epitope consisting of amino acid residues 82 to 90 (SYIGSINNI), which are shared by subgroup A and B RSVs.     

A stable single-chain variable fragment expressing transfectoma demonstrates induction of idiotype-specific cytotoxic T-cells during early growth stages of a murine B-lymphoma

The idiotypic determinants associated with the variable regions of antibody molecules are known to function as tumor-associated antigens (TAAs). However, there is no clear-cut evidence documenting their efficacy in inducing TAA-specific cytotoxic T-lymphocytes (CTLs). In most previous studies, idiopeptides were implicated in elicitation of TAA-specific CD4+ T-cells. Using a murine B-cell lymphoma, 2C3, we earlier demonstrated induction of splenic CD4+ and CD8+ T-lymphocytes directed to idiotypic Ig of the tumor. In the present study, we provide more direct evidence of the existence of Id-specific CTLs in the spleens of 2C3 bearing BALB/c mice using an scFv-transfectoma, P815A4, as a target. While both P815A4 and 2C3 cells were equally susceptible to cytolysis by the effector cells, lysis was evident only during early tumor progression. Moribund animals at the late stage of tumor growth failed to demonstrate any significant cytotoxic immune response against either tumor. Antibodies to MHC class I alleles Kd, Dd, Ld, beta2m and CD8 molecules all inhibited cytotoxicity. The CTL population from early tumor-bearers recognized 2C3 tumor in the context of all major H-2d alleles; however, in case of P815A4 cells, it was restricted to Kd and Dd alleles only. Based on these antibody inhibition studies, it appears that the idiopeptides generated in both tumors are in some way different, yet they were recognized equally by CTLs not only from the tumor-bearers but also by CTLs from 2C3-hyperimmune mice. It appears that scFv-containing transfectomas expressing antibody variable region epitopes would be useful for both elucidating CTL-defined idiopeptides and monitoring TAA-specific CTL response in tumor-bearing animals.     

Spontaneous rejection of intradermally transplanted non-engineered tumor cells by neutrophils and macrophages from syngeneic strains of mice

It is not surprising that tumors arising spontaneously are rarely rejected by T cells, because in general they lack molecules to elicit a primary T-cell response. In fact, cytokine-engineered tumors can induce granulocyte infiltration leading to tumor rejection. In the present study, we i.d. injected seven kinds of non-engineered tumor cells into syngeneic strains of mice. Three of them (i.e. B16, KLN205, and 3LL cells) continued to grow, whereas four of them (i.e. Meth A, I-10, CL-S1, and FM3A cells) were spontaneously rejected after transient growth or without growth. In contrast to the i.d. injection of B16 cells into C57BL/6 mice, which induces infiltration of TAMs into the tumors, the i.d. injection of Meth A cells into BALB/c mice induced the invasion of cytotoxic inflammatory cells, but not of TAMs, into or around the tumors leading to an IFN-γ-dependent rejection. On day 5, the cytotoxic activity against the tumor cells reached a peak; and the effector cells were found to be neutrophils and macrophages. The i.d. Meth A or I-10 cell-immunized, but not non-immunized, mice rejected i.p.- or i.m.-transplanted Meth A or I-10 cells without growth, respectively. The main effector cells were CTLs; and there was no cross-sensitization between these two kinds of tumor cells, suggesting specific rejection of tumor cells by CTLs from i.d. immunized mice. These results indicate that infiltration of cytotoxic myeloid cells (i.e. neutrophils and macrophages, but not TAMs) into or around tumors is essential for their IFN-γ-dependent spontaneous rejection.     

Two Distinct Populations of Primary Cytotoxic Cells Infiltrating into Allografted Tumor Rejection Sites: Infiltration of Macrophages Cytotoxic against Allografted Tumor Precedes That of Multiple Sets of Cytotoxic T Lymphocytes with Distinct Specificity to Alloantigens

It has been reported that the rejection of tumor allografts is mainly mediated by cytotoxic T lymphocytes (CTLs). Here, we characterized the cytotoxic effector cells of C57BL/6 (B6; H-2^b) mice infiltrating into the rejection site of the i.p. allografted Meth A fibrosarcoma (or P815 mastocytoma) cells of H-2^d origin. Two types of cytotoxic cells (i.e., CD8⁺ CTLs and macrophages (Mφs)) were identified by flow cytometric fractionation of the infiltrates or by specific in vitro elimination of cells either with antibody (Ab)-coated beads or with an Ab-plus complement. Of particular interest, these effector cells showed distinct and unique target specificities. First, the CTLs were inactive against transplanted tumor (e.g., Meth A) cells, whereas they were cytotoxic against donor-related concanavalin A (Con A) blasts as well as CTLL-2 (H-2^b) cells transfected with a class I gene of H-2^d origin. A cold target competition assay suggested that the CTLs were composed of multiple sets of T cells, each of which specifically recognized different allo-antigens. Second, the Mφs lysed the allografted tumor cells but were inert toward the Con A blasts and the CTLL-2 transfectants. Unexpectedly, the infiltration of Mφs preceded the infiltration of CTLs by several days during the course of rejection. These results indicate that two distinct populations of unique cytotoxic cells (i.e., CTLs and Mφs) are induced in the allografted tumor rejection site, and that the infiltration of cytotoxic Mφs responsible for rejection precedes that of the CTLs cytotoxic against cells expressing donor-related allo-antigens.     

Identification of an immunodominant cytotoxic T-lymphocyte recognition site in glycoprotein B of herpes simplex virus by using recombinant adenovirus vectors and synthetic peptides.

Cytotoxic T-lymphocyte (CTL) responses to herpes simplex virus (HSV) polypeptides play an important role in recovery from infection and in preventing latency. We have previously shown that glycoprotein B (gB) is a major target recognized by HSV-specific CTLs in C57BL/6 (H-2b) and BALB/c (H-2d) mice but not in CBA/J (H-2k) mice (L. A. Witmer, K. L. Rosenthal, F. L. Graham, H. M. Friedman, A. Yee, and D. C. Johnson, J. Gen. Virol. 71:387-396, 1990). In this report, we utilize adenovirus vectors expressing gB with various deletions to localize an immunodominant site in gB, recognized by H-2b-restricted anti-HSV CTLs, to a region between residues 462 and 594. Overlapping peptides spanning this region were synthesized and used to further localize the immunodominant site to residues 489 to 515, a region highly conserved in HSV type 1 (HSV-1) and HSV-2 strains. The 11-amino-acid peptide was apparently associated exclusively with the Kb major histocompatibility complex gene product and not the Db gene product. In contrast, H-2d-restricted CTLs recognized an immunodominant site between residues 233 and 379.     

T cell tolerance based on avidity thresholds rather than complete deletion allows maintenance of maximal repertoire diversity

Given the flexible nature of TCR specificity, deletion or permanent disabling of all T cells with the capacity to recognize self peptides would severely limit the diversity of the repertoire and the capacity to recognize foreign Ags. To address this, we have investigated the patterns of CD8+ CTL reactivity to a naturally H-2Kb-presented self peptide derived from the elongation factor 1alpha (EF1alpha). EF1alpha occurs as two differentially expressed isoforms differing at one position of the relevant peptide. Low avidity CTLs could be raised against both variants of the EF1alpha peptide. These CTLs required 100-fold more peptide-H-2Kb complexes on the target cell compared with CTLs against a viral peptide, and did not recognize the naturally expressed levels of EF1alpha peptides. Thus, low avidity T cells specific for these self peptides escape tolerance by deletion, despite expression of both EF1alpha isoforms in dendritic cells known to mediate negative selection in the thymus. The low avidity in CTL recognition of these peptides correlated with low TCR affinity. However, self peptide-specific CTLs expressed elevated levels of CD8. Furthermore, CTLs generated against altered self peptide variants displayed intermediate avidity, indicating cross-reactivity in induction of tolerance. We interpret these data, together with results previously published by others, in an avidity pit model based on avidity thresholds for maintenance of both maximal diversity and optimal self tolerance in the CD8+ T cell repertoire.     

Determination of T cell epitopes on the S1 subunit of pertussis toxin.

To understand the immunologic characteristics of pertussis toxin molecule and to explore the possibility of developing a synthetic vaccine, T cell epitopes on the enzymatic S1 subunit of pertussis toxin were studied by measuring the proliferative response of immune murine lymph node cells and T cell lines to Ag and to synthetic peptides. The maximum in vitro T cell proliferative response was obtained by stimulating immune lymphoid cells with 20 nM of the enzymatic S1 subunit. When the T cell proliferative response of murine lymphoid cells with different MHC backgrounds was tested, only mice bearing the H-2d haplotype were high responder to the S1 subunit. To determine T cell epitopes on the S1 subunit, the proliferative response of BALB/c immune lymphoid cells to several synthetic S1 peptides was measured. Only the peptide containing amino acid residues, 65-79, was recognized by BALB/c lymphoid cells and was confirmed to contain a T cell epitope by generating S1 specific BALB/c T cell line. By using this T cell line, the response of BALB/c mice to the S1 subunit as well as to peptide 65-79 was shown to be restricted to the I-Ad sublocus of class II Ag. Finally, we showed that lymph node cells of mice immunized with peptide 65-79 respond to the native S1 subunit.     

Identification of an HLA-A2-Restricted Epitope Peptide Derived from Hypoxia-Inducible Protein 2 (HIG2)

We herein report the identification of an HLA-A2 supertype-restricted epitope peptide derived from hypoxia-inducible protein 2 (HIG2), which is known to be a diagnostic marker and a potential therapeutic target for renal cell carcinoma. Among several candidate peptides predicted by the HLA-binding prediction algorithm, HIG2-9-4 peptide (VLNLYLLGV) was able to effectively induce peptide-specific cytotoxic T lymphocytes (CTLs). The established HIG2-9-4 peptide-specific CTL clone produced interferon-γ (IFN-γ) in response to HIG2-9-4 peptide-pulsed HLA-A*02:01-positive cells, as well as to cells in which HLA-A*02:01 and HIG2 were exogenously introduced. Moreover, the HIG2-9-4 peptide-specific CTL clone exerted cytotoxic activity against HIG2-expressing HLA-A*02:01-positive renal cancer cells, thus suggesting that the HIG2-9-4 peptide is naturally presented on HLA-A*02:01 of HIG-2-expressing cancer cells and is recognized by CTLs. Furthermore, we found that the HIG2-9-4 peptide could also induce CTLs under HLA-A*02:06 restriction. Taken together, these findings indicate that the HIG2-9-4 peptide is a novel HLA-A2 supertype-restricted epitope peptide that could be useful for peptide-based immunotherapy against cancer cells with HIG2 expression.     

Cytotoxic T cell immunity against the non-immunogenic, murine, hepatocellular carcinoma Hepa1-6 is directed towards the novel alternative form of macrophage colony stimulating factor

Mouse Hepa1-6 hepatocellular carcinoma (HCC) cells were transduced with the membrane form of macrophage colony stimulating factor (mM-CSF). When mM-CSF transduced Hepa1-6 cells were injected subcutaneously into mice, these cells did not form tumors. The spleens of these immunized mice contained cytotoxic CD8⁺ T lymphocytes (CTL) that killed the unmodified Hepa1-6 cells. We show that the alternative form of macrophage colony stimulating factor (altM-CSF) induced CTL-mediated immunity against Hepa1-6 cells. AltM-CSF is restricted to the H-2D^b allele. CTLs killed RMA-S cells loaded with exogenous altM-CSF peptide. Vaccination of mice with dendritic cells pulsed with the altM-CSF peptide stimulated anti-Hepa1-6 CTLs. Hyper-immunization of mice with mM-CSF Hepa1-6 cells showed inflammation of the liver and kidneys. Although altM-CSF was expressed within liver and kidney cells, its intensity was lower than Hepa1-6 cells. AltM-CSF was detected within the human HepG2 cell line. These studies suggest that altM-CSF may be a tumor antigen for HCC.     

Leukocyte integrin-dependent and antibody-independent cytotoxicity of macrophage against allografts

Macrophages (Mphis), but not T cells, infiltrating into the rejection site of either i.p. allografted Meth A (H-2d) fibrosarcoma cells in C57BL/6 (B6) (H-2b) mice or BALB/c (H-2d) skin onto B6 mice are cytotoxic against allografts with H-2d specificity. To determine the mechanisms of specific killing of allografts by allograft-induced Mphi (AIM), we raised approximately 5,000 rat monoclonal antibodies (mAbs) against AIM and selected three of them (R1-73, R2-40 and R1-34), each of which inhibited cytotoxic activity against allografts in a dose-dependent manner. The antigens recognized by R1-73, R2-40 and R1-34 mAbs were defined by immunoprecipitation and Western blot analyses as CD11a, CD18 and CD11b, respectively; and the allografts expressed CD54, a ligand of CD11a or CD11b, suggesting leukocyte integrin-dependent killing. Although Ab-dependent cellular cytotoxicity has been recognized as a mechanism of specific killing by Mphis, the infiltration of AIM into the rejection site of allografts far (approximately 6 days) preceded the appearance of serum IgG Ab specific for the allograft. AIM exhibiting full cytotoxic activity against allografts was also induced in the transplantation site of Fcgamma receptor knockout [(B6x129) F1] mice as well as B10.D2 (H-2 compatible with allograft) and B6-xid (X-linked immunodeficiency with B cell-specific defect) strains of mice. In the latter two strains of mice, the levels of serum IgG Ab to the allograft were negligible. Moreover, the cytotoxic activity of AIM against allografts was not affected by pretreatment of the cells with anti-mouse IgG serum, suggesting Ab-independent cytotoxicity.