Endoplasmic reticulum (ER) chaperones of the antigen processing machinery play a crucial role in HLA class I antigen complex assembly and antigen presentation. The characterization of the expression of these chaperones in normal tissues and malignant lesions has been hampered by the lack or limited availability of ER chaperone-specific monoclonal antibodies (mAb) that are suitable for immunohistochemical staining. To overcome this limitation, we have generated human calnexin, ERp57, calreticulin and tapasin-specific mAb-secreting hybridomas from BALB/c mice immunized with peptides and recombinant proteins. The mAb TO-5, TO-2, TO-11 and TO-3 were shown to be specific for calnexin, ERp57, calreticulin and tapasin, respectively, as they react specifically with the corresponding immunizing peptides in ELISA and with the corresponding proteins when tested with human lymphoid cell lysates in Western blotting. Furthermore, the reactivity of the four mAb with the corresponding intracellular antigens yielded intracellular staining when the mAb were tested with formalin-fixed, microwave-treated and saponin-permeabilized cells in indirect immunofluorescence and with formalin-fixed, paraffin-embedded tissue sections in the immunoperoxidase reaction. These results suggest that the ER chaperone-specific mAb we have developed are useful probes for characterizing the expression of ER chaperones of the antigen processing machinery in normal and pathological cells. This information will contribute to defining the effects of abnormalities in their expression on HLA class I antigen expression and function and on the interactions of target cells with the host's immune system.

The human high molecular weight-melanoma associated antigen (HMW-MAA) is a membrane-bound chondroitin sulfate proteoglycan that is variably expressed in a high percentage of melanoma cell lines and tumors. Since the mechanism(s) regulating HMW-MAA expression has(ve) not been defined, in this study, we have examined whether promoter DNA methylation regulates the level of HMW-MAA expression. In melanoma cell lines, the level of HMW-MAA mRNA and protein expression is coordinately regulated, implicating a transcriptional control mechanism. Consistent with a role for regulation by DNA methylation, we have found that a dense CpG island flanks the human HMW-MAA gene transcriptional start site. Methylation-specific PCR and sodium bisulfite DNA sequencing analyses indicate that the HMW-MAA promoter is heavily methylated in melanoma cell lines, melanoma lesions and normal lymphocytes that do not express HMW-MAA; in contrast, the HMW-MAA promoter is not methylated in melanoma cell lines and tumors that express this antigen. In addition, HMW-MAA expression is markedly induced in HMW-MAAnegative melanoma cell lines by incubation with the DNA methyltransferase inhibitor 5-aza-2 0 -deoxycytidine. In summary, our results establish DNA methylation as a key regulator of HMW-MAA expression by human melanoma cells. This information represents a useful background to optimize immunotherapeutic strategies targeting HMW-MAA.

Only three anti-HLA-A monoclonal antibodies (mAbs) have been described in the literature. Two of them recognize determinants shared by only a few HLA-A allospecificities. The third one, mAb 3G11, recognizes a determinant shared by most HLA-A allospecificities. Being an IgM, the latter mAb is not likely to be a useful probe in immunohistochemical reactions and in functional assays. Therefore, in the present study we have characterized the specificity of the mAbs LGIII-147.4.1 and LGIII-220.6.2. The two mAbs that do not share idiotypic determinants recognize distinct but spatially close antigenic determinants expressed on most of the gene products of the HLA-A locus. Specifically, the determinant recognized by mAb LGIII-220.6.2 is expressed on HLA-A1, -A2, -A3, -A26, -A28, -A29, -A30, -A33, -A36, -A74 and -A80 allospecificities. The determinant recognized by mAb LGIII-147.4.1, which appears to be located on the amino-acid residues 79-83 of the heavy chain, is expressed on all HLA-A allospecificities but HLA-A23, -A24, -A25 and -A32. Because of its broad reactivity, the mAb LGIII-147.4.1 was characterized in a number of assays. It was found to be a useful probe to measure the HLA-A antigen level in serum, to assess the HLA-A restriction of cytotoxic T lymphocytes (CTL) and to monitor HLA-A antigen expression in normal and malignant lesions.

Major histocompatibility complex (MHC) class I chain-related gene A (MICA), a ligand for the activating immunoreceptor natural killer group 2D (NKG2D), is expressed on stressed cells such as tumor cells. Study of expression of this molecule on tumor cells and patients’ sera is useful to define patients’ stages leading to proper selection of therapy. In this study, mouse anti-MICA monoclonal antibodies (mAbs) were produced by DNA immunization using a gene gun. Screening of anti-MICA-producing mouse and hybridomas were performed by immunoblot and cell enzyme-linked immunosorbent assay (ELISA) against MICA-positive HeLa and -negative Me1386 cell lines. MAbs were characterized against MICA-positive and -negative cell lines by immunoblot, cell ELISA and flow cytometry. The mAbs were also characterized for locus and allele specificities of MICA and MHC class I chain-related gene B (MICB) as well as for their ability to stain formalin-fixed paraffin-embedded tissues by immunohistochemistry. Although all mouse immune sera were positive with MICA-positive cells by both immunoblot and cell ELISA methods, some hybridomas were positive only with one method. The mAbs had diverse specificities to detect MICA and MICB and different abilities to stain formalin-fixed paraffin-embedded tissues. Thus, DNA immunization by gene gun is an effective method to generate immune mice for the production of mAbs with a variety of specificities against native and denatured forms of MIC proteins.

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