No abstract

To identify HLA markers that may contribute to the genetic susceptibility of Koreans to psoriasis, we studied 84 psoriasis patients, with serologic HLA types of A, B, and genotypes of HLA-Cw, DRB1, DQA1, DQB1, DPB1 alleles. The distribution of HLA markers and the associated haplotypes were analyzed according to age and sex. HLA-Cw*0602 showed the strongest association with psoriasis (relative risk = 36.0, p < 10-8, Pc < 8 x 10-7). The frequencies of A1 (relative risk = 17.0, p < 9 x 10-7, Pc < 7 x 10-5), A30 (relative risk = 5.5, p < 2 x 10-5, Pc < 0.001), B13 (relative risk = 5.6, p < 4 x 10-6, Pc < 3 x 10-4), B37 (relative risk = 30.3, p < 7 x 10-7, Pc < 6 x 10-5), DRB1*07 (relative risk = 5.9, p < 2 x 10-6, Pc < 8 x 10-5), DRB1*10 (relative risk = 26.4, p < 4 x 10-6, Pc < 3 x 10-4), DQA1*02 (relative risk = 6.2, p < 5 x 10-7, Pc < 4 x 10-4), DQB1*02 (relative risk = 2.5, p < 0.005, Pc = ns) and DPB1*1701 (relative risk = 24.6, p < 9 x 10-6, Pc < 7 x 10-4) were also significantly increased in Korean psoriasis patients. Type I and type II psoriasis were subdivided into groups of below and above 30 y of age, because of the significant difference found in HLA-Cw*0602 phenotype frequency between the two groups (83.9% vs. 54.5%, p < 0. 009). In addition to HLA-Cw*0602, the frequencies of B37 and DPB1*1701 were significantly higher in type I as opposed to type II psoriasis. HLA-A30-B13-Cw*0602-DRB1*07-DQA1* 02-DQB1*02 was identified as a high risk haplotype. This was particularly true at an early age in the female. HLA-A33-B44-Cw*1401-DRB1*13-DQA1* 01-DQB1*06-DPB1*0401 was defined as a protective haplotype for psoriasis. The extended haplotype HLA-A1-B37-Cw*0602-DRB1*10-DQA1*01-DQB1*05 was discovered to be a high-risk factor in Koreans. To summarize, this study demonstrates the differential association of HLA according to sex, and identifies a newly found high-risk haplotype and a protective haplotype in Korean psoriasis patients.

To define whether individual human leukocyte antigen (HLA) class I allotypes are used preferentially in human cytomegalovirus (CMV)-specific cytotoxic T lymphocyte responses, CD8+ T cell responses restricted by up to six HLA class I allotypes in an individual were measured in parallel using K562-based artificial antigen-presenting cells expressing both CMV pp65 antigen and one of 32 HLA class I allotypes (7 HLA-A, 14 HLA-B, and 11 HLA-C) present in 50 healthy Korean donors. The CD8+ T cell responses to pp65 in the HLA-C allotypes were lower than responses to those in HLA-A and -B allotypes and there was no difference between the HLA-A and HLA-B loci. HLA-A*02:01, -B*07:02, and -C*08:01 showed the highest magnitude and frequency of immune responses to pp65 at each HLA class I locus. However, HLA-A*02:07, -B*59:01, -B*58:01, -B*15:11, -C*03:02, and -C*02:02 did not show any immune responses. Although each individual has up to six different HLA allotypes, 46% of the donors showed one allotype, 24% showed two allotypes, and 2% showed three allotypes that responded to pp65. Interestingly, the frequencies of HLA-A alleles were significantly correlated with the positivity of specific allotypes. Our results demonstrate that specific HLA class I allotypes are preferentially used in the CD8+ T cell immune response to pp65 and that a hierarchy among HLA class I allotypes is present in an individual.

Most tumors express an array of antigens that act as targets for their immune-mediated destruction, and a number of potential therapies have emerged to exploit this (22). The immunotherapeutic strategy used to induce an immune response against tumors is quite attractive because it offers the potential for a high level of tumor-specific cytotoxicity, minimal side effects, and a durable effect.Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) in the induction of primary immune responses (29, 33). Because of their central role in controlling cell-mediated immunity, DCs hold much promise as cellular adjuvants in therapeutic cancer vaccines. DC-based immunotherapy has been reported to induce strong antitumor immune responses in animal experiments and in selected clinical trials involving malignant gliomas (2, 11, 36). However, its clinical effects on patients with malignancies have not been up to the expectations because of immune tolerance, the sheer physical burden of tumor antigens, and the mechanisms of tumor escape from the immune surveillance system, among others (10,20).Calreticulin (CRT) acts as a danger signal for DCs, allowing them to phagocytose tumor cells and to prime tumor antigenspecific cytotoxic T cells (CTLs) (12). It was recently reported that CRT exposure on the surfaces of dying tumor cells may determine whether chemotherapy is immunogenic (26). The capacity of chemotherapies to induce immunogenic tumor cell death is associated with the expression of CRT on the tumor cell surface. Furthermore, it was shown with an animal tumor model that the provision of CRT from an exogenous CRT exposure source as enforcement for endogenous CRT exposure could improve the efficacy of chemotherapy by stimulating antitumor immunity (27). Thus, whether chemotherapy triggers such an immunogenic effect depends on the exposure of CRT on the cell surface. The use of multimodality treatments that combine conventional antitumor therapies with immunotherapy, such as vaccination with DC-based vaccines, has emerged as a potentially plausible approach to the treatment of tumors (3, 5). We previously reported that the use of a multimodality treatment regimen with a DC-based vaccine in combination with the chemotherapeutic agent temozolomide (TMZ) leads to enhanced tumor-specific CTL responses and enhanced antitumor effects, resulting in a cure rate higher than that achieved with either a DC-based vaccine or TMZ alone (17,28). However, the immunological factors relating to the antitumor effect of TMZ chemoimmunotherapy in a murine glioma model are still unclear.

Interleukin (IL)-12 has been reported to induce cellular immune responses for protection against tumor formation. Here we investigate the utility of adenoviral delivery of IL-12 as an adjuvant for a human papillomavirus E7 subunit vaccine in a mouse tumor challenge model. Direct intratumoral injection of AdIL-12 resulted in a significant suppression of tumor growth compared to the control group. Injection of E7 protein into either a tumor site or the distance site along with AdIL-12 further enhanced antitumor effects significantly higher than either AdIL-12 or E7 injection alone. This combined injection resulted in complete regression of 9-mm-sized tumor in 40% of animals as well as lasting antitumor immunity against tumor recurrence. We also evaluated immune responses induced by these injections. AdIL-12 plus E7 enhanced E7-specific antibody responses significantly higher than AdIL-12 or E7 injection. In particular, the production level of interferon (IFN)-gamma from E7-specific CD4(+) T cells was similar between AdIL-12 group and AdIL-12 + E7 group. However, IFN-gamma production from E7-specific CD8(+) T cells was the most significant when injected with AdIL-12 + E7. This was consistent with intracellular IFN-gamma staining levels of CD8(+) T cells, suggesting that AdIL-12 + E7 injection enhances antitumor immunity in the human papillomavirus (HPV) 16 tumor model through increased expansion of the cytotoxic T-lymphocyte (CTL) subset. This enhanced protection appeared to be mediated by CD8(+) T cells, as determined by in vivo T-cell subset deletion. Thus, these studies demonstrate that E7 vaccines can induce CTL responses responsible for antitumor effects in the presence of IL-12 delivered via adenovirus vectors. This likely provides one additional approach for immune therapy against cervical cancers.

Extranodal NK/T-cell lymphoma (ENKTCL) is associated with latent Epstein-Barr virus (EBV) infection and frequent relapse even after complete response (CR) to intensive chemotherapy and radiotherapy. The expression of EBV proteins in the tumor provides targets for adoptive immunotherapy with antigen-specific cytotoxic T cells (CTL). To evaluate the efficacy and safety of EBV latent membrane protein (LMP)-1 and LMP-2a-specific CTLs (LMP1/2a CTLs) stimulated with LMP1/2a RNA-transferred dendritic cells, we treated 10 ENKTCL patients who showed complete response to induction therapy. Patients who completed and responded to chemotherapy, radiotherapy, and/or high-dose therapy followed by stem cell transplantation (HDT/SCT) were eligible to receive eight doses of 2 × 107 LMP1/2a CTLs/m2. Following infusion, there were no immediate or delayed toxicities. The 4-year overall survival (OS) and progression-free survival (PFS) were 100%, and 90% (95% CI: 71.4 to 100%) respectively with a median follow-up of 55·5 months. Circulating IFN-γ secreting LMP1 and LMP2a-specific T cells within the peripheral blood corresponded with decline in plasma EBV DNA levels in patients. Adoptive transfer of LMP1/2a CTLs in ENKTCL patients is a safe and effective postremission therapeutic approach. Further randomized studies will be needed to define the role of EBV-CTLs in preventing relapse of ENKTCL.

HLA genes play a pivotal role for successful hematopoietic stem cell transplantation (HSCT). There is an increasing need for sophisticated screening of donor HLA genotypes for unrelated HSCT. Next generation sequencing (NGS) has emerged as an alternative for classical Sanger sequence for HLA typing. In this study, HLA‐A, ‐B, and ‐DRB1 alleles were genotyped at the allelic (6‐digit) level using MiSeqDx in 26,202 volunteers from the Korean Network for Organ Sharing. Exon 2 and 3 of HLA‐A and ‐B and exon 2 of HLA‐DRB1 were amplified by polymerase chain reaction (PCR) and each allele was determined by matching the targeted exons and the reference sequence consisting of the IPD‐IMGT/HLA Database. Seventy alleles of HLA‐A, 102 alleles of HLA‐B, and 69 alleles of HLA‐DRB1 were identified. According to common and well‐documented catalogs, 34 alleles in HLA‐A, 61 in HLA‐B, and 45 in HLA‐DRB1 locus were common alleles, and 12, 14, and 11 kinds, were well‐documented alleles, respectively. Thirteen novel alleles including 3 alleles in HLA‐A, 8 alleles in HLA‐B, and 2 alleles in HLA‐DRB1 loci were found. Ten haplotypes with a frequency of more than 1.0% accounted for 22.4% of the total haplotype frequencies. Cis/trans ambiguities of HLA‐A and ‐B loci by combination of exons 2 and 3 were analyzed to be 0.17% of 3 and 3.95% of 22 genotypes, respectively. This information on rare and novel alleles found by accurate HLA typing with NGS may be helpful for unrelated HSCT among Koreans.

Myeloid-derived suppressor cells (MDSCs) are increased in tumor patients. Studies have shown generation of MDSCs from human peripheral blood mononuclear cells (PBMCs) by various cytokine combinations. However, large scale expansion of human MDSCs has not been demonstrated or applied in clinic settings. We investigated which cytokine combinations among GM-CSF/SCF, G-CSF/SCF, or M-CSF/SCF efficiently expand and differentiate human MDSCs following culture CD34 + cells of umbilical cord blood (CB). GM-CSF/SCF showed the greatest expansion of MDSCs. Up to 10 8 MDSCs (HLA-DR low CD11b + CD33 + ) could be produced from 1 unit of CB following 6 weeks of continuous culture. MDSCs produced from culture of CD34 + cells with GM-CSF/SCF for 6 weeks had the greatest suppressive function of T cell proliferation and had the highest expression of immunosuppressive molecules including iNOS, arginase 1 and IDO compared to those differentiated with G-CSF/SCF or M-CSF/SCF. MDSCs secreted IL-10, TGB-β, and VEGF. The infusion of expanded MDSCs significantly prolonged the survival and decreased the GVHD score in a NSG xenogeneic model of GVHD. Injected MDSCs increased IL-10 and TGF-β but decreased the level of TNF-α and IL-6 in the serum of treated mice. Notably, FoxP3 expressing regulatory T (Treg) cells were increased while IFN-γ (Th1) and IL-17 (Th17) producing T cells were decreased in the spleen of MDSC treated mice compared to untreated GVHD mice. Our results demonstrate that human MDSCs are generated from CB CD34 + cells using GM-CSF/SCF. These MDSCs exhibited potent immunosuppressive function, suggesting that they are useable as a treatment for inflammatory diseases such as GVHD.