Forced expression of HLA-DM at the surface of dendritic cells increases loading of synthetic peptides on MHC class II molecules and modulates T cell responses.

Adoptive transfer of autologous dendritic cells (DCs) loaded with tumor-associated CD4 and CD8 T cell epitopes represents a promising avenue for the immunotherapy of cancer. In an effort to increase the loading of therapeutic synthetic peptides on MHC II molecules, we used a mutant of HLA-DM (DMY) devoid of its lysosomal sorting motif and that accumulates at the cell surface. Transfection of DMY into HLA-DR(+) cells resulted in increased loading of the exogenously supplied HA(307-318) peptide, as well as increased stimulation of HA-specific T cells. Also, on transduction in mouse and human DCs, DMY increased loading of HEL(48-61) and of the tumor Ag-derived gp100(174-190) peptides, respectively. Interestingly, expression of DMY at the surface of APCs favored Th1 differentiation over Th2. Finally, we found that DMY(-) and DMY(+) mouse APCs differentially stimulated T cell hybridomas sensitive to the fine conformation of peptide-MHC II complexes. Taken together, our results suggest that the overexpression of HLA-DMY at the plasma membrane of DCs may improve quantitatively, but also qualitatively, the presentation of CD4 T cell epitopes in cellular vaccine therapies for cancer.

Evidence for a human leucocyte antigen-DM-induced structural change in human leucocyte antigen-DObeta.

Human leucocyte antigen (HLA)-DO is a non-classical major histocompatibility complex class II molecule which modulates the function of HLA-DM and the loading of antigenic peptides on molecules such as HLA-DR. The bulk of HLA-DO associates with HLA-DM and this interaction is critical for HLA-DO egress from the endoplasmic reticulum. HLA-DM assists the early steps of HLA-DO maturation presumably through the stabilization of the interactions between the N-terminal regions of the alpha and beta chains. To evaluate a possible role for HLA-DM in influencing the conformation of HLA-DO, we made use of a monoclonal antibody, Mags.DO5, that was raised against HLA-DO/DM complexes. Using transfected cells expressing mismatched heterodimers between HLA-DR and -DO chains, we found that the epitope for Mags.DO5 is located on the DObeta chain and that Mags.DO5 reactivity was increased upon cotransfection with HLA-DM. Our results suggest that HLA-DM influences the folding of HLA-DO in the endoplasmic reticulum. A mutant HLA-DO showing reduced capacity for endoplasmic reticulum egress was better recognized by Mags.DO5 in the presence of HLA-DM. On the other hand, an HLA-DO mutant capable of endoplasmic reticulum egress on its own was efficiently recognized by Mags.DO5, irrespective of the presence of HLA-DM. Taken together, our results suggest that HLA-DM acts as a private chaperone, directly assisting the folding of HLA-DO to promote egress from the endoplasmic reticulum.

Frequency of cystathionine beta-synthase 844INS68 polymorphism in Southern Iran.

Iranian population with an Indo-European origin is one of the oldest populations in the world. Historical evidence suggests the close similarity in the origin of Iranian, European and north Indian population. However, there are few anthropological and genetic evidences on this subject. This study, which is the first report from Iran, was performed to investigate the genetic origin of Iranian population using a polymorphism in Cystathionine beta synthase (CBS) gene known as 844INS68bp in this respect, genomic DNA was extracted from the whole blood of 480 healthy normal blood donors referred to Fars Blood Transfusion Center, using a salting out method. The fragment containing 844INS68bp was amplified, the normal fragment was 174 bp and the fragment containing the insertion was 242 bp in length. Results indicated that 418 (87.08%) out of 480 individuals had a normal (N/N) genotype, 59 (12.29%) individuals were heterozygote (N/I) and 3 (0.63%) had homozygote a mutated genotype (I/I). The total frequency of 844INS68bp allele was found 6.8% which is similar to with the reported in White Caucasians. Comparison of the genotype of this study with the polymorphism in other populations revealed that Southern Iranian population has a great similarity with other Caucasians populations' especially South Italy and North America while differed from East Asian and African populations. These results are in agreement with the result of other studied polymorphisms. Therefore, despite the great admixture of Iranian population with the neighboring non-Caucasian populations during the time, Iranian population still share a genetic background with other Caucasian populations.

The frequency of CCR5Delta32 and CCR2-64I in southern Iranian normal population.

Host genetic control of HIV infection involves certain polymorphisms of some chemokine receptor genes that are associated with susceptibility and progression of HIV-1 infection. Recent data suggest that two important polymorphisms in CCR2 and CCR5 chemokine receptors, CCR5Delta32 and CCR2-64I, prevent HIV transmission and delay disease progression. In this study allele and haplotype frequencies of the CCR5Delta32 and CCR2-64I mutations were determined in southern Iranian normal population using PCR and PCR restriction fragment length polymorphism (PCR-RFLP) assays. Allele frequencies and the fit to the Hardy-Weinberg equilibrium (HWE) were evaluated by Arlequin population genetic software. Frequencies of CCR5Delta32 and CCR2-64I alleles were 0.0146 and 0.1221, respectively. Moreover, higher and lower haplotype frequencies in 341 normal individuals were CCR2/CCR5 (0.8636) and CCR5/CCR2-64I (0.1217), respectively. Only one case with CCR5Delta32/CCR2-64I haplotype was found among the studied normal population. This data is the first finding on the frequencies of CCR5Delta32 and CCR2-64I alleles in Iranian population. Results of the present study suggest that low frequency of CCR5Delta32 allele may be related to higher genetic susceptibility to the HIV-1 infection in Iranians. Results also suggest that the CCR2-64I mutation is sufficiently common in Iranians and may be associated with slower HIV infection progression in Iran.

The expression of p53, c-erbB-1 and c-erbB-2 molecules and their correlation with prognostic markers in patients with head and neck tumors.

Molecular prognostic and predictive factors have extensively been studied in different cancers during the past decades, some of which were found to be useful in diagnosis, follow up or even treatment of some malignant tumors. To assess the significance of c-erbB-1, c-erbB-2 and p53 expression in head and neck tumors among Iranian patients and their correlation with known prognostic factors, samples from 53 patients with squamous cell carcinomas of larynx and tongue were studied immunohistochemically. Strong immunoreactivity of c-erbB-1, c-erbB-2 and p53 was observed in 37 (70%), 40 (76%) and 37 (70%) of cases, respectively. The coexpression of these molecules was detected in 27 (50.9%) samples. Neither histological grading nor nodal involvement revealed correlation with c-erbB-1 and/or c-erbB-2 expression. No correlation was found between p53 expression and histological grade. However, a significant positive association was observed between p53 expression and nodal involvement. This data, which is the first report on head and neck squamous cell carcinomas (HNSCC) in Iran, indicates the significance of p53 protein expression which may result from p53 tumor suppressor gene inactivation in lymph node metastasis of HNSCC among Iranian patients.

The promoter region (-800, -509) polymorphisms of transforming growth factor-beta1 (TGF-beta1) gene and recurrent spontaneous abortion.

Recurrent spontaneous abortion (RSA) is regarded as a common pregnancy complication in southern Iran. The exact causes of RSA are not yet known. Transforming growth factor-beta1 (TGF-beta1) is produced by T regulatory lymphocytes (Treg), which play an important role in the physiology of pregnancy. Several polymorphisms of the TGF-beta1 gene have been reported, some with important correlation with disease severity. In this investigation, the polymorphism of the TGF-beta1 gene at promoter region positions -800 (G/A) and -509 (C/T) was studied in 111 RSA and 110 normal female subjects from southern Iran by PCR-RFLP. Results indicated that at position -800 (G/A) polymorphism, 75.7% of RSA cases and 77.3% of normals were homozygote GG. In addition, 23.4% of cases and 22.7% of normal individuals were heterozygote AG. Only one of the patients appeared to be homozygote AA. None of the normal individuals were found to be homozygote AA at this position. In the case of the -509 (C/T) polymorphism, 38.7% of patients and 28.2% of controls were homozygote CC. While 40.6% of cases and 50.9% of normal individuals were heterozygote CT, 20.7% of RSA cases and 20.9% of controls were homozygote TT. The results indicate that there are no statistically significant differences of genotype distribution and allele frequency between RSA cases and controls at both polymorphic sites. In conclusion, the promoter region polymorphisms of TGF-beta1 at positions -800 (G/A) and -509 (C/T) may not be associated with RSA.

Establishment and characterization of a human large cell lung cancer cell line with neuroendocrine differentiation.

Characterization of a human lung cancer cell line is reported. This cell line was established from a patient referred to Nemazi Hospital of Shiraz University of Medical Sciences with a diagnosis of poorly differentiated carcinoma. Sterile sample from peritoneal effusion was taken and immediately cultured in RPMI-1640 medium containing 20% FBS, at 37 degrees C with 5% CO2. This cell line has been in continuous culture for more than one year and has been named as Mehr-80. Several features of the cell line were investigated, including growth characteristics, electron microscopic features, cloning efficiency in soft agar, expression of various antigenic markers, chromosomal and DNA analysis. On the basis of morphological and immunohistochemical analysis of Mehr-80, it is possible to conclude that this cell line is characterized by features similar to those reported for large cell carcinoma with neuroendocrine differentiation (LCCND). This cell line will be a valuable in vitro tool for further studies on lung cancers.

The Endoplasmic Reticulum Chaperone GRP170: From Immunobiology to Cancer Therapeutics.

Glucose-regulated protein 170 (GRP170) is the largest member of glucose-regulated protein family that resides in the endoplasmic reticulum (ER). As a component of the ER chaperone network, GRP170 assists in protein folding, assembly, and transportation of secretory or transmembrane proteins. The well documented cytoprotective activity of intracellular GRP170 due to its intrinsic chaperoning property has been shown to provide a survival benefit in cancer cells during tumor progression or metastasis. Accumulating evidence shows that extracellular GRP170 displays a superior capacity in delivering tumor antigens to specialized antigen-presenting cells for cross-presentation, resulting in generation of an anti-tumor immune response dependent on cytotoxic CD8(+) T cells. This unique feature of GRP170 provides a molecular basis for using GRP170 as an immunostimulatory adjuvant to develop a recombinant vaccine for therapeutic immunization against cancers. This review summarizes the latest findings in understanding the biological effects of GRP170 on cell functions and tumor progression. The immunomodulating activities of GRP170 during interactions with the innate and adaptive arms of the immune system as well as its therapeutic applications in cancer immunotherapy will be discussed.

HLA-DO increases bacterial superantigen binding to human MHC molecules by inhibiting dissociation of class II-associated invariant chain peptides.

HLA-DO (H2-O in mice) is an intracellular non-classical MHC class II molecule (MHCII). It forms a stable complex with HLA-DM (H2-M in mice) and shapes the MHC class II-associated peptide repertoire. Here, we tested the impact of HLA-DO and H2-O on the binding of superantigens (SAgs), which has been shown previously to be sensitive to the structural nature of the class II-bound peptides. We found that the binding of staphylococcal enterotoxin (SE) A and B, as well as toxic shock syndrome toxin 1 (TSST-1), was similar on the HLA-DO(+) human B cell lines 721.45 and its HLA-DO(-) counterpart. However, overexpressing HLA-DO in MHC class II(+) HeLa cells (HeLa-CIITA-DO) improved binding of SEA and TSST-1. Accordingly, knocking down HLA-DO expression using specific siRNAs decreased SEA and TSST-1 binding. We tested directly the impact of the class II-associated invariant chain peptide (CLIP), which dissociation from MHC class II molecules is inhibited by overexpressed HLA-DO. Loading of synthetic CLIP on HLA-DR(+) cells increased SEA and TSST-1 binding. Accordingly, knocking down HLA-DM had a similar effect. In mice, H2-O deficiency had no impact on SAgs binding to isolated splenocytes. Altogether, our results demonstrate that the sensitivity of SAgs to the MHCII-associated peptide has physiological basis and that the effect of HLA-DO on SEA and TSST-1 is mediated through the inhibition of CLIP release.

Tollip-induced down-regulation of MARCH1.

In addition to their classical antigen presenting functions, MHC class II molecules potentiate the TLR-triggered production of pro-inflammatory cytokines. Here, we have addressed the effect of Tollip and MARCH1 on the regulation of MHC II trafficking and TLR signaling. Our results show that MARCH1-deficient mice splenocytes are impaired in their capacity to produce pro-inflammatory cytokines in response to poly(I:C) and that TLR3 and MHC II molecules interact in the endocytic pathway. Knocking down Tollip expression in human CIITA(+) HeLa cells increased expression of HLA-DR but reduced the proportion of MHC II molecules associated with the CLIP peptide. Truncation of the HLA-DR cytoplasmic tails abrogated the effect of Tollip on MHC class II expression. While overexpression of Tollip did not affect HLA-DR levels, it antagonized the function of co-transfected MARCH1. We found that Tollip strongly reduced MARCH1 protein levels and that the two molecules appear to compete for binding to MHC II molecules. Altogether, our results demonstrate that Tollip regulates MHC class II trafficking and that MARCH1 may represent a new Tollip target.

CD1a and CD1d genes polymorphisms in breast, colorectal and lung cancers.

CD1 molecules might contribute to anti-tumor immune response by presentation of tumor-derived lipid and glycolipid antigens to T cells and NKT cells. Polymorphisms in CD1 genes have been suggested to modify ligand binding of CD1 molecules and thereby change the antigen presenting ability of these molecules. The aim of this study was to investigate the exon 2 polymorphisms of CD1a and CD1d in several high incident cancers in Iran. For this purpose, 201 female breast cancer patients and 207 healthy women, 64 lung cancer patients and 95 healthy individuals and 109 patients with colorectal cancer and 109 healthy controls were recruited to this study. Using PCR-SSP method, no significant correlation was found in genotype and allele frequencies of CD1a between all three studied groups and their control counterparts. Moreover, a dominant frequency of CD1d 01 (A) allele was observed in the majority of studied individuals. No significant association between the CD1 polymorphisms and prognostic factors in breast, lung and colorectal cancers was detected. Our results highlight the conserved nature of CD1 genes and may point to the immuoregulatory functions of CD1 molecules in cancer that can be exerted through fine tuning of NK, T and NKT cells.

Cytotoxic T lymphocyte antigen-4 gene in breast cancer.

The exon 1 polymorphism (49A/G) of ctla-4 gene corresponds to an amino acid exchange (threonine to alanine) in the leader peptide of the expressed protein. There are reports concerning the higher level of G allele in subjects with various autoimmune diseases, which has resulted in the hypothesis that CTLA-4 may play a role in regulating self-tolerance by the immune system and in the pathogenesis of autoimmune disorders. This study was undertaken to investigate the correlation of exon 1 (49A/G) polymorphism in the ctla-4 gene and breast cancer. The ctla-4 49A/G polymorphism was studied in 197 women with primary breast cancer and 151 age/sex matched normal individuals. The results indicated a significant difference between frequency of ctla-4 genotypes in patients and controls. The frequency of GG genotype was significantly decreased in breast cancer patients compared to controls (4.6% v.s. 12.6%, P = 0.012). There was also a significant positive correlation between tumor size and the existence of AA genotype in patients (P = 0.016). In addition, a positive correlation between AA genotype and lymph node involvement was observed (P = 0.042). The observed decrease in the frequency of GG genotype in the breast cancer patients is contrary to the frequently reported increase of GG genotype in autoimmune diseases. In addition, the data implies that polymorphism of ctla-4 exon 1 contributes in tumor progression.