Behavior of bone marrow-derived cells following in vivo transplantation: differentiation into stromal cells with roles in organ maintenance.

After injection of green fluorescent protein-positive (GFP(+)) bone marrow (BM) cells into lethally irradiated wild-type mice, the organs of the recipient mice [BM transplantation (BMT) mice] were regenerated; however, irradiation of the cecum or spleen (only) blocked their regeneration with loss of injected BM cells. These results suggest that the donor cells first enter the BM and then migrate to the peripheral organs. The maintenance of epithelial structure and function is controlled by interactions between stromal cells and the epithelia; the organ is stable only if the stroma is functioning normally. In BMT mice, intestinal GFP(+) stromal cells were regenerated fairly rapidly although GFP(+) cells were observed only rarely in the intestinal epithelium even if it passes several weeks or months post BMT, indicating that BM-derived stromal cells play a pivotal role in epithelial renewal and are crucial for maintaining organ structure and function. BM-derived cells in the periphery possess a special key to return to the BM and then to migrate to various organs to become resident cells.

Mismatched human leukocyte antigen class II-restricted CD8⁺ cytotoxic T cells may mediate selective graft-versus-leukemia effects following allogeneic hematopoietic cell transplantation.

Partial human leukocyte antigen (HLA)-mismatched hematopoietic stem cell transplantation (HSCT) is often performed when an HLA-matched donor is not available. In these cases, CD8(+) or CD4(+) T cell responses are induced depending on the mismatched HLA class I or II allele(s). Herein, we report on an HLA-DRB1*08:03-restricted CD8(+) CTL clone, named CTL-1H8, isolated from a patient following an HLA-DR-mismatched HSCT from his brother. Lysis of a patient Epstein-Barr virus-transformed B cell line (B-LCL) by CTL-1H8 was inhibited after the addition of blocking antibodies against HLA-DR and CD8, whereas antibodies against pan-HLA class I or CD4 had no effect. The 1H8-CTL clone did not lyse the recipient dermal fibroblasts whose HLA-DRB1*08:03 expression was upregulated after 1 week cytokine treatment. Engraftment of HLA-DRB1*08:03-positive primary leukemic stem cells in non-obese diabetic/severe combined immunodeficient/γc-null (NOG) mice was completely inhibited by the in vitro preincubation of cells with CTL-1H8, suggesting that HLA-DRB1*08:03 is expressed on leukemic stem cells. Finally, analysis of the precursor frequency of CD8(+) CTL specific for recipient antigens in post-HSCT peripheral blood T cells revealed a significant fraction of the total donor CTL responses towards the individual mismatched HLA-DR antigen in two patients. These findings underscore unexpectedly significant CD8 T cell responses in the context of HLA class II.

HapMap scanning of novel human minor histocompatibility antigens.

Minor histocompatibility antigens (mHags) are molecular targets of allo-immunity associated with hematopoietic stem cell transplantation (HSCT) and involved in graft-versus-host disease, but they also have beneficial antitumor activity. mHags are typically defined by host SNPs that are not shared by the donor and are immunologically recognized by cytotoxic T cells isolated from post-HSCT patients. However, the number of molecularly identified mHags is still too small to allow prospective studies of their clinical importance in transplantation medicine, mostly due to the lack of an efficient method for isolation. Here we show that when combined with conventional immunologic assays, the large data set from the International HapMap Project can be directly used for genetic mapping of novel mHags. Based on the immunologically determined mHag status in HapMap panels, a target mHag locus can be uniquely mapped through whole genome association scanning taking advantage of the unprecedented resolution and power obtained with more than 3 000 000 markers. The feasibility of our approach could be supported by extensive simulations and further confirmed by actually isolating 2 novel mHags as well as 1 previously identified example. The HapMap data set represents an invaluable resource for investigating human variation, with obvious applications in genetic mapping of clinically relevant human traits.

Epstein-Barr virus nuclear antigen 1-specific CD4+ T cells directly kill Epstein-Barr virus-carrying natural killer and T cells.

Epstein-Barr virus (EBV) nuclear antigen (EBNA)1 is expressed in every EBV-infected cell, regardless of the state of EBV infection. Although EBNA1 is thought to be a promising antigen for immunotherapy of all EBV-associated malignancies, it is less clear whether EBNA1-specific CD4(+) T cells can act as direct effectors. Herein, we investigated the ability of CD4(+) T-cell clones induced with overlapping peptides covering the C-terminal region of EBNA1, and identified minimal epitopes and their restricted major histocompatibility complex class II molecules. Of these, a novel epitope, EYHQEGGPD, was found to be presented by DRB1*0401, 0403 and 0406. Five CD4(+) T-cell clones recognized endogenously processed and presented antigens on EBV-transformed lymphoblastoid cell lines (LCL) and one example proved capable of killing EBV-carrying natural killer (NK) and T-cell lines derived from patients with chronic active EBV infection (CAEBV). Identification of minimal epitopes facilitates design of peptide-based vaccines and our data suggest that EBNA1-specific CD4(+) T cells may play roles as direct effectors for immunotherapy targeting EBV-carrying NK and T-cell malignancies.

CD137-guided isolation and expansion of antigen-specific CD8 cells for potential use in adoptive immunotherapy.

The efficient isolation and ex vivo expansion of antigen-specific T cells are crucial for successful adoptive immunotherapy against uncontrollable infections and cancers. Several methods have been reported for this purpose, for example, employing MHC-multimeric complexes, interferon-gamma secretion, and antibodies specific for molecules expressed on T-cell surfaces, including CD25, CD69, CD107a, CD137, and CD154. Of the latter, CD137 has been shown to be one of the most promising targets since it is only expressed on CD8(+) T cells early after encountering antigen, while being almost undetectable on resting cells. However, detailed comparisons between CD137-based and other methods have not yet been conducted. In this study, we therefore compared three approaches (with CD137, CD107a, and tetramers) using HLA-A24-restricted CMV pp65 and EBV BRLF1 epitopes as model antigens. We found that the CD137-based isolation of antigen-stimulated CD8(+) T cells was comparable to tetramer-based sorting in terms of purity and superior to the other two methods in terms of subsequent cell expansion. The method was less applicable to CD4(+) T cells since their CD137 upregulation is not sufficiently high. Collectively, this approach is most likely to be optimal among the methods tested for the isolation and expansion of antigen-specific CD8(+) cells.

Aberrant expression of BCL2A1-restricted minor histocompatibility antigens in melanoma cells: application for allogeneic transplantation.

It has been shown that allogeneic hematopoietic stem cell transplantation (HSCT) can be one of the therapeutic options for patients with metastatic solid tumors, such as renal cancer. However, the development of relatively severe GVHD seems to be necessary to achieve tumor regression in the current setting. Thus, it is crucial to identify minor histocompatibility antigens (mHags) only expressed in tumor cells but not GVHD target organs. In this study, we examined whether three mHags: ACC-1 and ACC-2 encoded by BCL2A1, and HA-1 encoded by HMHA1, could serve as such targets for melanoma. Real-time PCR and immunohistochemical analysis revealed that the expression of both BCL2A1and HMHA1 in melanoma cell lines and primary melanoma cells was comparable to that of hematopoietic cells. Indeed, melanoma cell lines were efficiently lysed by cytotoxic T lymphocytes specific for ACC-1, ACC-2, and HA-1. Our data suggest that targeting mHags encoded not only by HMHA1, whose aberrant expression in solid tumors has been reported, but also BCL2A1 may bring about beneficial selective graft-versus-tumor effects in a population of melanoma patients for whom these mHags are applicable.

Identification of human minor histocompatibility antigens based on genetic association with highly parallel genotyping of pooled DNA.

Minor histocompatibility (H) antigens are the molecular targets of allo-immunity responsible both for the development of antitumor effects and for graft-versus-host disease (GVHD) in allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, despite their potential clinical use, our knowledge of human minor H antigens is largely limited by the lack of efficient methods of their characterization. Here we report a robust and efficient method of minor H gene discovery that combines whole genome association scans (WGASs) with cytotoxic T-lymphocyte (CTL) assays, in which the genetic loci of minor H genes recognized by the CTL clones are precisely identified using pooled-DNA analysis of immortalized lymphoblastoid cell lines with/without susceptibility to those CTLs. Using this method, we have successfully mapped 2 loci: one previously characterized (HMSD encoding ACC-6), and one novel. The novel minor H antigen encoded by BCL2A1 was identified within a 26 kb linkage disequilibrium block on chromosome 15q25, which had been directly mapped by WGAS. The pool size required to identify these regions was no more than 100 individuals. Thus, once CTL clones are generated, this method should substantially facilitate discovery of minor H antigens applicable to targeted allo-immune therapies and also contribute to our understanding of human allo-immunity.

Effective induction of anti-tumor immune responses with oligomannose-coated liposome targeting to intraperitoneal phagocytic cells.

We recently established a novel drug delivery system (DDS) using oligomannose-coated liposomes (OMLs) which are probably taken up by macrophages (Mvarphi) to carry anti-cancer drugs to milky spots known as preferential metastatic sites of gastric cancers [Y. Ikehara, T. Niwa, L. Biao, S.K. Ikehara, N. Ohashi, T. Kobayashi, Y. Shimizu, N. Kojima, H. Nakanishi, A carbohydrate recognition-based drug delivery and controlled release system using intraperitoneal macrophages as a cellular vehicle, Cancer Res. 66 (2006) 8740-8748]. In the present study, we applied this intraperitoneal DDS for systemic cancer immunotherapy employing ovalbumin (OVA) as a model antigen. The cells taking up the OMLs containing FITC-OVA injected into the peritoneal cavity were predominantly Mvarphi, as they showed adhesive characteristics and expressed F4/80 and CD11b almost exclusively. The phagocytic cells also took up bare OVA directly to the same extent as OML-enclosed OVA (OML-OVA), as it is a highly mannosilated protein. The phagocytic cells taking up OML-OVA, however, could activate OVA-specific CD8+ (from OT-I: H-2Kb/OVA257-264-specific) and CD4+ (from OT-II: H-2Ab/OVA323-339-specific) T cells much more effectively in vitro than those taking up bare OVA. Furthermore, only the mice pre-immunized with OML-OVA rejected E.G7-OVA (OVA-transfected EL4) but not EL4. These results indicate that the OMLs can also be used as an effective antigen delivery system for cancer immunotherapy activating both CTL and Th subsets.

Minor histocompatibility antigens as targets for immunotherapy using allogeneic immune reactions.

Minor histocompatibility antigens (mHag) were originally identified as antigens causing graft rejection or graft-versus-host disease in human leukocyte antigen (HLA)-matched allogeneic transplantation. Molecular identification has revealed most to be major histocompatibility complex (MHC)-bound short peptide fragments encoded by genes which are polymorphic due to single nucleotide polymorphisms (SNP). Genotypic disparity of SNP between transplantation donors and recipients gives rise to mHag as non-self antigens for both the donor and the recipient. Subsequently, mHag have been explored as immunotherapeutic antigens for use against recurring hematological malignancies after allogeneic hematopoietic cell transplantation (HCT), because mHag expressed only on hematopoietic cells are considered to augment graft-versus-leukemia/lymphoma (GVL) effects without increasing the risk of life-threatening graft-versus-host disease (GVHD). Accumulating evidence suggests that T-cell responses to mHag aberrantly expressed on solid tumor cells are also involved in the eradication of sensitive tumors such as renal cell carcinomas following HCT. Over the past decade, the number of putative GVL-directed mHag has increased to a level that covers more than 30% of the Japanese patient population, so that clinical trials may now be executed in the setting of either vaccination or adoptive immunotherapy. As it is expected that immune responses to alloantigens are more powerful than to tumor antigens mostly derived from overexpressed self-proteins, mHag-based immunotherapy may lead to a new treatment modality for high-risk malignancies following allogeneic HCT.

Alternative splicing due to an intronic SNP in HMSD generates a novel minor histocompatibility antigen.

Here we report the identification of a novel human leukocyte antigen (HLA)-B44-restricted minor histocompatibility antigen (mHA) with expression limited to hematopoietic cells. cDNA expression cloning studies demonstrated that the cytotoxic T lymphocyte (CTL) epitope of interest was encoded by a novel allelic splice variant of HMSD, hereafter designated as HMSD-v. The immunogenicity of the epitope was generated by differential protein expression due to alternative splicing, which was completely controlled by 1 intronic single-nucleotide polymorphism located in the consensus 5' splice site adjacent to an exon. Both HMSD-v and HMSD transcripts were selectively expressed at higher levels in mature dendritic cells and primary leukemia cells, especially those of myeloid lineage. Engraftment of mHA(+) myeloid leukemia stem cells in nonobese diabetic/severe combined immunodeficient (NOD/SCID)/gammac(null) mice was completely inhibited by in vitro preincubation with the mHA-specific CTL clone, suggesting that this mHA is expressed on leukemic stem cells. The patient from whom the CTL clone was isolated demonstrated a significant increase of the mHA-specific T cells in posttransplantation peripheral blood, whereas mHA-specific T cells were undetectable in pretransplantation peripheral blood and in peripheral blood from his donor. These findings suggest that the HMSD-v-encoded mHA (designated ACC-6) could serve as a target antigen for immunotherapy against hematologic malignancies.

Full-length EBNA1 mRNA-transduced dendritic cells stimulate cytotoxic T lymphocytes recognizing a novel HLA-Cw*0303- and -Cw*0304-restricted epitope on EBNA1-expressing cells.

Epstein-Barr virus (EBV)-encoded nuclear antigen 1 (EBNA1) is an attractive target for immunotherapy against EBV-associated malignancies because it is expressed in all EBV-positive cells. Although CD8+ cytotoxic T-lymphocyte (CTL) epitope presentation is largely prevented by its glycine-alanine-repeat domain (GAr), the use of mRNA-transduced dendritic cells (DCs) would offer the advantage of priming EBNA1-specific CTLs. After stimulation with GAr-containing EBNA1-transduced monocyte-derived DCs, two EBNA1-specific CTL clones, B5 and C6, were isolated successfully from a healthy donor. These CTLs recognize peptides in the context of HLA-B*3501 and HLA-Cw*0303, respectively. A novel epitope, FVYGGSKTSL, was then identified, presented by both HLA-Cw*0303 and -Cw*0304, which are expressed by >35% of Japanese, >20% of Northern Han Chinese and >25% of Caucasians. The mixed lymphocyte-peptide culture method revealed that FVYGGSKTSL-specific CTL-precursor frequencies in HLA-Cw*0303- or -Cw*0304-positive donors were between 1x10(-5) and 1x10(-4) CD8+ T cells. Moreover, both CTL clones inhibited growth of HLA-matched EBV-transformed B lymphocytes in vitro, and B5 CTLs produced a gamma interferon response to EBNA1-expressing gastric carcinoma cells in the context of HLA-Cw*0303. These data demonstrate that EBNA1 mRNA-transduced DCs may be useful tools for inducing EBNA1-specific CTLs that might be of clinical interest for CTL therapy of EBV-associated malignancies.

Identification of an HLA-A24-restricted cytotoxic T lymphocyte epitope from human papillomavirus type-16 E6: the combined effects of bortezomib and interferon-gamma on the presentation of a cryptic epitope.

About 50% of cervical cancers are associated with human papillomavirus type 16 (HPV-16), and since the HPV-16 E6 and E7 oncoproteins are constitutively expressed in the tumor cells, they are attractive targets for cytotoxic T lymphocyte (CTL)-mediated immunotherapy. Nevertheless, only a limited number of HPV-16 E6 epitopes have been identified to date. Using reverse immunological methods, we have generated a CTL clone against the HPV-16 E6(49-57) epitope restricted by HLA-A*2402, which is the most common allele in Japan and relatively frequent worldwide, capable of lysing 293T cells transduced with HLA-A*2402 and HPV-16 E6. Although it was unable to recognize the SiHa cervical cancer cell line positive for HPV-16 and HLA-A*2402, the cells became susceptible to lysis when transduced with E6-E7 genes, which was unexpectedly offset by pretreatment with interferon (IFN)-gamma alone. Interestingly, however, combined pretreatment with a proteasome inhibitor, bortezomib and IFN-gamma fully restored CTL-mediated lysis of the original SiHa cells. Furthermore, such intervention of 2 of 4 other cervical cancer cell lines expressing HPV-16 E6 and HLA-A*2402 was found to induce IFN-gamma production by specific CTLs. Tetramer analysis further revealed that induction of E6(49-57)-specific T cells was possible in 5 of 7 patients with HPV-16-positive high grade cervical intraepithelial neoplasia or cervical cancer by in vitro stimulation with E6(49-57) peptide. Thus, these findings together indicate that E6(49-57) is a candidate epitope for immunotherapy and immunological monitoring of such patients.

Antitumor NK activation induced by the Toll-like receptor 3-TICAM-1 (TRIF) pathway in myeloid dendritic cells.

Myeloid dendritic cells (mDCs) recognize and respond to polyI:C, an analog of dsRNA, by endosomal Toll-like receptor (TLR) 3 and cytoplasmic receptors. Natural killer (NK) cells are activated in vivo by the administration of polyI:C to mice and in vivo are reciprocally activated by mDCs, although the molecular mechanisms are as yet undetermined. Here, we show that the TLR adaptor TICAM-1 (TRIF) participates in mDC-derived antitumor NK activation. In a syngeneic mouse tumor implant model (C57BL/6 vs. B16 melanoma with low H-2 expresser), i.p. administration of polyI:C led to the retardation of tumor growth, an effect relied on by NK activation. This NK-dependent tumor regression did not occur in TICAM-1(-/-) or IFNAR(-/-) mice, whereas a normal NK antitumor response was induced in PKR(-/-), MyD88(-/-), IFN-beta(-/-), and wild-type mice. IFNAR was a prerequisite for the induction of IFN-alpha/beta and TLR3. The lack of TICAM-1 did not affect IFN production but resulted in unresponsiveness to IL-12 production, mDC maturation, and polyI:C-mediated NK-antitumor activity. This NK activation required NK-mDC contact but not IL-12 function in in vivo transwell analysis. Implanted tumor growth in IFNAR(-/-) mice was retarded by adoptively transferring polyI:C-treated TICACM-1-positive mDCs but not TICAM-1(-/-) mDCs. Thus, TICAM-1 in mDCs critically facilitated mDC-NK contact and activation of antitumor NK, resulting in the regression of low MHC-expressing tumors.

A phase I radioimmunolocalization trial of humanized monoclonal antibody huA33 in patients with gastric carcinoma.

In order to determine the in vivo characteristics of huA33, an open label dose escalation biopsy-based phase I clinical trial and radioimmunolocalization study were conducted with a complement determinant region-grafted humanized monoclonal antibody against the A33 antigen in patients with gastric carcinoma. Thirteen patients were entered onto one of four dose levels (1.0, 2.0, 5.0 or 10.0 mg/m(2)). Patients with locally advanced (UICC-TNM [International Union Against Cancer-tumor, node, metastasis] stage over 2 but resectable at clinical diagnosis) gastric carcinoma received a single infusion of (131)I-huA33 1 week prior to surgery. Adverse events were monitored, and imaging studies with gamma camera plus ex vivo scintigraphy of the resected specimen, biodistribution study by dosimetry analysis of the biopsied and resected tissues, and immunohistochemical analysis were carried out and evaluated. No dose-limiting toxicity was observed during the trial. Therefore, the maximum tolerated dose was not reached. Although cancer tissues with + intensity and <25% extent by immunostaining in biopsied frozen sections did not show positive imaging or postoperative dosimetry findings, cancers with ++ or +++ intensity or wide (>25%) extent by frozen and paraffin sections in the biopsied specimen showed positive ex vivo tumor images and positive antigen expression in resected gastric cancer specimens, and the biodistribution analysis showed tumor uptake of (131)I-huA33. In conclusion, humanized monoclonal antibody huA33 demonstrated selective localization to gastric cancer that expressed A33 antigen strongly. These excellent targeting characteristics of huA33 indicate potential for targeted therapy of advanced gastric cancer that is refractory to cytotoxic therapy, and could also be exploitable for curatively resected early gastric cancer in an adjuvant setting.

Generation of peptide-specific CD8+ T cells by phytohemagglutinin-stimulated antigen-mRNA-transduced CD4+ T cells.

Functional analysis of antigen-specific CD8(+) T cells is important for understanding the immune response in various immunological disorders. To analyze CD8(+) T cell responses to a variety of antigens with no readily defined peptides available, we developed a system using CD4(+) phytohemagglutinin (PHA) blasts transduced with mRNA for antigen molecules. CD4(+) PHA blasts express MHC class I and II, and also CD80 and CD86 and are thus expected to serve as potent antigen presenting cells. EGFP mRNA could be transduced into and the protein expressed by more than 90% of either LCL or CD4(+) PHA blasts. Its expression stably persisted for more than 2 weeks after transduction. In experiments with HLA-A*2402 restricted CD8(+) CTL clones for either EBNA3A or a cancer-testis antigen, SAGE, mRNA-transduced lymphoid cells were appropriate target cells in ELISPOT assays or (51)Cr releasing assays. Finally, using CD4(+) PHA blasts transduced with mRNA of a cancer-testis antigen MAGE-A4, we successfully generated specific CTL clones that recognized a novel HLA-B*4002 restricted epitope, MAGE-A4(223-231). Messenger RNA-transduced CD4(+) PHA blasts are thus useful antigen presenting cells for analysis of CD8(+) T cell responses and induction of specific T cells for potential immunotherapy.

Characterization of murine CD160+ CD8+ T lymphocytes.

CD160 is an Ig-like glycoprotein expressed on NK, NKT and TCRgammadelta T cells, as well as intestinal intraepithelial T lymphocytes. In addition, a minor subset of CD8(+) but not CD4(+) T cells in the periphery is also known to express CD160, but the subset has not been fully characterized. In this study, we prepared anti-murine CD160 mAbs and investigated the expression profile of CD160 on various subsets of CD8(+) T cells. The amount of CD160 on almost all CD8(+) T cells was increased upon CD3-mediated stimulation in vitro, and soluble CD160 was found to be released. Flow cytometric analysis revealed most CD8(+) T cells expressing CD160 to show a CD44(high) phenotype in vivo. On further analysis, both CD44(high)CD62L(low) effector memory T cells (T(EM)) and CD44(high)CD62L(high) central memory T cells (T(CM)) expressed CD160 at an intermediate level. High levels were evident with recently activated CD8(+) T(EM). Naïve CD8(+) T cells presumably immediately after stimulation (CD44(low)CD62L(low)CD69(+)) also expressed CD160, but only at a low level. Purified CD160(+) CD8(+) T cells from OT-1 transgenic mice expressing TCR against OVA residues 257-264 presented by H-2K(b) produced IFN-gamma more rapidly than CD160(-) CD8(+) T cells upon antigen stimulation. These results together show that CD160 is expressed on the majority of CD8(+) memory T cells as well as recently activated CD8(+) T cells.

Epstein-Barr virus (EBV) latent membrane protein-1-specific cytotoxic T lymphocytes targeting EBV-carrying natural killer cell malignancies.

Epstein-Barr virus (EBV)-encoded latent membrane protein (LMP) 1 is a potential target for immunotherapy of some proportion of Hodgkin's disease cases, nasopharyngeal carcinomas, EBV-associated natural killer (NK)/T lymphomas, and chronic active EBV infection (CAEBV). Since it is unknown whether EBV-infected NK/T cells are susceptible to lysis by LMP1-specific cytotoxic T lymphohcytes (CTL), we here tested the ability of mRNA-transduced antigen-presenting cells (APC) to stimulate rare LMP1-specific CTL. A 43-amino acid N-terminal deletion mutant LMP1 (DeltaLMP1) could be efficiently expressed in dendritic cells and CD40-activated B cells upon mRNA electroporation. DeltaLMP1-expressing APC were found to stimulate LMP1-specific CTL from a healthy donor and a CTL clone recognized a peptide, IIIILIIFI, presented by HLA-A*0206 molecules. Processing and presentation of the antigenic peptide proved dependent on expression of an immunoproteasome subunit, low-molecular-weight protein-7, as confirmed by RNA interference gene silencing. Furthermore, an EBV-infected NK cell line derived from a patient with CAEBV, and another from an NK lymphoma with enforced HLA-A*0206 expression, were specifically lysed by the CTL. Overall, these data suggest that immunotherapy targeting LMP1 in EBV-associated NK lymphomas and CAEBV might serve as an alternative treatment modality.