A general hindrance to progress in adoptive cellular therapy is the lack of detailed knowledge of the fate of transferred cells in the body of the recipient. In this study, we present a novel technique for tracking of 124I-labeled cells in situ, which combines the high spatial resolution of magnetic resonance imaging with the high sensitivity and spatial accuracy of positron emission tomography. We have used this technique, together with determination of tissue radioactivity, flow cytometry, and microscopy, to characterize and quantitate the specific accumulation of transferred CD8+ T cells in tumor tissue in a mouse model. Transgenic CD8+ T cells, specific for the ovalbumin peptide SIINFEKL, were adoptively transferred to recipients carrying a subcutaneous tumor of the ovalbumin-expressing malignant melanoma cell line B16-OVA. The number of SIINFEKL-specific CD8+ cells in the tumor tissue was determined by flow cytometry each day for 8 consecutive days after adoptive transfer. From low levels 1 day after injection, their number gradually increased until day 5 when an average of 3.3×106 SIINFEKL-specific cells per gram tumor tissue was found. By applying the combined positron emission tomography/magnetic resonance imaging technique we were able to determine the position of the transferred, 124I-labeled SIINFEKL-specific T cells in 3 dimensions in recipient mice, and could demonstrate a highly significant accumulation of the 124I label in and around the subcutaneous B16-OVA tumors compared with normal tissue. Accumulation of 124I was significantly higher in B16-OVA than in B16 tumors not expressing the OVA antigen.

s for the 19th Annual Scientific Meeting of the International Society for Biological Therapy of Cancer, San Francisco, California, November 4-7, 2004: Imaging

A method for precise and sensitive organ specific quantification of 124I (124IdUR) activities in small animals has been developed. The method is based upon high precision co-registration between PET and MR imagery, utilizing an innovative mouse fixation system with external point sources visible in both modalities. The methodology is generic, and thus can easily be extended to other types of small animal studies. The methodology comprises PET/MR rigid co-registration utilising external fiducial markers, measurement of activities within small 3D volumes manually drawn in MR data and extracted from the co-aligned PET data, and subsequently corrected for Partial Volume Effect.

PURPOSE:We wanted to study whether an allogeneic melanoma lysate would be a feasible stimulatory antigen source for detection of a peripheral CD4+ T-cell immune response in patients with medically untreated malignant melanoma. The lysate was produced from a melanoma cell line (FM3.29) which expresses high amounts of melanoma antigens.METHODS:Fresh peripheral blood was incubated with and without lysate for 6 h in the presence of anti-CD28/anti-CD49d MoAb (for costimulation). After flow cytometric estimation of the frequency of CD69+/IFN-gamma+ cells in the CD4+ population, the response to lysate was calculated as the difference between the number of activated IFN-gamma-producing CD4+ cells in the lysate-stimulated and the nonstimulated sample.RESULTS:An immune response to lysate was observed in blood samples from 11 of 15 patients (73%) with metastatic melanoma. A weak response was found in 1 of 4 patients radically operated for localized disease, whereas no responders were seen among 7 healthy donors. The fraction of circulating lysate-activated T cells ranged from 0.0037% to 0.080% of the CD4+ population. A negative result of the assay was found occasionally, especially in donors with high background levels of spontaneous IFN-gamma production, indicating an inhibitory effect of the lysate.CONCLUSIONS:This method for detection of a peripheral T-cell immune response in melanoma patients has several advantages for clinical use. The tumor lysate preparations may contain large numbers of stimulating antigens (known, as well as unknown) and are easily prepared and handled. Potentially, the assay might be useful as a diagnostic tool, a marker of residual or recurrent disease, a prognostic factor, or a predictor or monitor of the effect of antineoplastic therapy including immune-modulating therapy.

In the attempt to identify genes associated with metastasis, we have compared gene expressions of two metastatic cell lines, 4T1 and 66cl4, and one noninvasive, nonmetastatic cell line, 67NR, which originate from the same mouse mammary adenocarcinoma. Using the technique of differential display, we identified a novel member of the semaphorin/collapsin family in the two metastatic cell lines. We have named it M-semaH. Northern hybridization to a panel of tumor cell lines revealed transcripts in 12 of 12 metastatic cell lines but in only 2 of 6 nonmetastatic cells and none in immortalized mouse fibroblasts. To our knowledge, this is the first time that the expression of a semaphorin gene has been shown to correlate positively with tumor progression. We have characterized two transcripts present in the tumor cells. One transcript, M-semaH-v, is a putative splice variant, which is less abundant in normal tissue and lacks 478 bp in the 3' untranslated region. Both transcripts encode the same 775 amino acids with the features of a secreted glycoprotein. Northern analysis suggests that the M-semaH gene is involved in embryonic development and in situ hybridization locates the M-semaH expression to the developing lungs, to developing skeletal elements, and to the ventral horns of the developing neural tube.

We report the cloning of the mouse surface GPI-anchored Ly-6E.1 protein from a highly metastatic mouse adenocarcinoma cell line CSML-100 by differential display. The expression is specific for the metastatic cell line as the closely related, non-metastatic mouse adenocarcinoma cell line CSML-0 does not express Ly-6E.1. Northern blot analysis reveals expression in a number of mouse tumour cell lines, exclusively metastatic ones. To date, active Ly-6A/E has only been described in lymphoid cells. The correlation between Ly-6E.1 expression, and the ability to metastasize, is discussed.

The mtsl gene is one of the genes specifically expressed in mouse metastatic tumors and tumor cell lines. In this paper, we present data on cloning and sequencing of two variants of human mtsl cDNAs (hu-mtsl and hu-mtsl (rar)), as well as of the corresponding region in the human genome. Comparison of the genomic sequence with the sequence of the mtsl cDNAs demonstrates presence of two alternatively spliced variants of the mtsl in the human osteosarcoma cell line (OHS). The alternative splicing occurs within the 5′-untranslated region (UTR) of human mtsl pre-mRNA. Both splice variants, hu-mtsl and hu-mtsl (rar), retain similar stability in the cells, contain one open reading frame coding for the MTS1 protein and differ only slightly in their translational capacity. The splice variants demonstrate dramatic variations in the level of expression in different human tissues and in human tumor cell lines. Although we have not revealed substantial differences in the mode of action of the two splice variants in the cells, the observed tissue specificity of expression supports the notion that it plays an important role in determining the activity of mtsl in different tissues.

A novel competition ELISA for detection of antibodies against HIV-1 was developed. The assay is based on competition at the single epitope level and utilises a human monoclonal antibody and an E. coli-produced fragment of the transmembrane glycoprotein gp41. The sensitivity of the assay was 100% in tests on 247 serum samples obtained from 219 individuals previously shown to be HIV-1 antibody positive by both conventional indirect ELISA and the immunoblotting test. The patients represented various clinical and immunological stages of HIV-1 infection. Likewise, the specificity of the assay was 100% in tests on 105 serum samples from normal individuals previously tested negative by indirect ELISA. Further, among 105 serum samples selected due to consistent false positive reactions in the indirect ELISA only 2 samples (1.9%) demonstrated false positive reactions in the competition ELISA, i.e. 98.1% specificity. Finally, only 2 of 57 (3.5%) serum samples from HIV-2 infected individuals showed positive reactions in the assay, while 54 (94.7%) had absorbance values similar to the negative controls. These results demonstrate that human monoclonal antibodies may form the basis for highly sensitive and specific assays for detection of antibodies to HIV-1.

A human monoclonal antibody, 41-7 [immunoglobulin G1(kappa)], directed against the transmembrane glycoprotein gp41 of the human immunodeficiency virus type 1 (HIV-1) has been produced by direct fusion of lymph node cells from an HIV-1-infected individual with a human B-lymphoblastoid cell line. The minimal essential epitope for 41-7 was mapped to a conserved seven-amino acid sequence, N-CSGKLIC-C, located within the N-terminal part of gp41. Antibodies blocking the binding of 41-7 could be detected in the serum of all HIV-1-infected individuals tested, irrespective of the stage of the infection. The epitope is located externally to the plasma membrane, and it is accessible to antibody in the native conformation of the glycoprotein. Despite this, no neutralizing activity of 41-7 could be demonstrated in vitro. These data indicate, directly and indirectly, that this immunodominant epitope on gp41, although exposed on the viral surface, elicits antibodies lacking antiviral activity and, hence, should be avoided in future vaccine candidates.