François A. Lemonnier

Quantification by flow cytofluorimetry of HLA class I molecules at the surface of murine cells transformed by cloned HLA genes

A method allowing quantitative analysis of the expression of foreign antigens at the surface of transformed cells is described. Aminoethyl-Sephadex G-25 beads labelled with different amounts of fluorescein isothiocyanate (FITC) were used to calibrate an Epics V cell sorter. The quantity of FITC molecules bound per bead was found to be a linear function of relative fluorescence intensity expressed by channel number for intermediate and high levels of fluorescence and a non-linear function for low levels. The lowest limit of detectable fluorescence was 3400 FITC molecules bound per bead. Using FITC-conjugated monoclonal antibodies (m.Ab.) the number of HLA class I molecules expressed at the surface of murine LMTK- (H-2Kk) cells transfected by cloned HLA class I genes was estimated and compared with the amount expressed on human B (Raji) and T (MOLT 4) lymphoblastoid reference cells. Murine transformed cells expressed approximately 3 times less HLA class I determinants per surface unit (micrometer 2) than Raji cells and 1.4 times less than MOLT 4 cells. Similar results were obtained by Scatchard analysis of the same populations using radiolabelled m.Ab. A detailed analysis of fluorescent cells showed that 5-20% of transformed cells expressed less than 33,000 HLA class I molecules/cell whereas most MOLT 4 cells exhibited at least 280,000 molecules/cell and the majority of Raji cells more than 700,000 molecules/cell. The expression of foreign antigen did not reduce the amount of H-2Kk molecules at the surface of transformed cells (mean of 350,000 molecules/cell).

Relationships between private and public H-2 specificities on the cell surface

Differential redistribution was used to investigate relationships between private specificity H-2.4 and public specificity H-2.28, in the product of aD region allele of theH-2 complex. Monospecific anti-H-2 antisera and fluorochrome conjugated antimouse Ig antibodies were used to induce redistribution of H-2 antigens on the surface of peripheral T lymphocytes fromH-2a andH-2d mice. Results showed that redistribution of specificity H-2.4 into patches and caps did not induce concomittant redistribution of specificity H-2.28, which remain diffusely scattered on the cell surface outside the caps of H-2.4. Redistribution of H-2.28 induced redistribution of H-2.4, which was no longer detectable outside the caps of H-2.28. These data indicate that (a) at least some of the H-2.28 sites are expressed on polypeptide chains independent from those carrying H-2.4 and (b) other H-2.28 sites may be linked to molecules carrying H-2.4. Since, onH-2a cells, both specificities are products of the D region of theH-2 gene complex, our results suggest that there are at least two genes in theD region.

Uncovering the Peptide-Binding Specificities of HLA-C: A General Strategy To Determine the Specificity of Any MHC Class I Molecule

MHC class I molecules (HLA-I in humans) present peptides derived from endogenous proteins to CTLs. Whereas the peptide-binding specificities of HLA-A and -B molecules have been studied extensively, little is known about HLA-C specificities. Combining a positional scanning combinatorial peptide library approach with a peptide–HLA-I dissociation assay, in this study we present a general strategy to determine the peptide-binding specificity of any MHC class I molecule. We applied this novel strategy to 17 of the most common HLA-C molecules, and for 16 of these we successfully generated matrices representing their peptide-binding motifs. The motifs prominently shared a conserved C-terminal primary anchor with hydrophobic amino acid residues, as well as one or more diverse primary and auxiliary anchors at P1, P2, P3, and/or P7. Matrices were used to generate a large panel of HLA-C–specific peptide-binding data and update our pan-specific NetMHCpan predictor, whose predictive performance was considerably improved with respect to peptide binding to HLA-C. The updated predictor was used to assess the specificities of HLA-C molecules, which were found to cover a more limited sequence space than HLA-A and -B molecules. Assessing the functional significance of these new tools, HLA-C*07:01 transgenic mice were immunized with stable HLA-C*07:01 binders; six of six tested stable peptide binders were immunogenic. Finally, we generated HLA-C tetramers and labeled human CD8+ T cells and NK cells. These new resources should support future research on the biology of HLA-C molecules. The data are deposited at the Immune Epitope Database, and the updated NetMHCpan predictor is available at the Center for Biological Sequence Analysis and the Immune Epitope Database.

High-affinity HLA-A(*)02.01 peptides from parathyroid hormone-related protein generate in vitro and in vivo antitumor CTL response without autoimmune side effects.

Parathyroid hormone-related protein (PTH-rP), a protein produced by prostate carcinoma and other epithelial cancers, is a key agent in the development of bone metastases. We investigated whether the protein follows the self-tolerance paradigm or can be used as a target Ag for anticancer immunotherapy by investigating the immunogenicity of two HLA-A(*)02.01-binding PTH-rP-derived peptides (PTR-2 and -4) with different affinity qualities. PTH-rP peptide-specific CTL lines were generated from the PBMC of two HLA-A(*)02.01+ healthy individuals, stimulated in vitro with PTH-rP peptide-loaded autologous dendritic cells and IL-2. The peptide-specific CTLs were able to kill PTH-rP+HLA-A(*)02.01+ breast and prostate carcinoma cell lines. The two peptides were also able to elicit a strong antitumor PTH-rP-specific CTL response in HLA-A(*)02.01 (HHD) transgenic mice. The vaccinated mice did not show any sign of side effects due to cell-mediated autoimmunity or toxicity. In this study we describe two immunogenic and toxic-free PTH-rP peptides as valid candidates for the design of peptide-based vaccination strategies against prostate cancer and bone metastases from the most common epithelial malignancies.

Altered structure of HLA class I heavy chains associated with mouse beta-2 microglobulin

The serological reactivities of HLA-A3, -B7, and -CW3 heavy chains associated with either mouse, bovine, or human beta-2 microglobulin (β2m) and expressed on the surface of transfected mouse fibroblasts were analyzed. All reactivities associated with one cluster (defined by monoclonal antibody W6/32) of antigenic determinants expressed by these HLA class I molecules were lost, or profoundly reduced, after each heavy chain associated with mouse β2-m. Expression by the transfected fibroblasts of the HLA-A3, -B7, and -CW3 heavy chains in association with human β2m restores these reactivities. Since most of the amino acid differences between mouse and human β2m probably correspond to externally oriented hydrophilic residues, these results suggest that critical interactions in the three-dimensional structure of HLA class I molecules occur between the light chain and the first two external domains of the class I heavy chains, to which some of the altered reactivities have been mapped.

Comparative effects of glucose and fructose on growth and morphological aspects of cultured skin fibroblasts

The growth rate of human skin fibroblasts was evaluated when glucose was replaced by fructose in the culture medium. Four mediums containing respectively 5.5 mmol/l glucose (G1), 27.5 mmol/l glucose (G5), 5.5 mmol/l fructose (F1), and 27.5 mmol/fructose (F5) were used. Skin fibroblasts from fourteen subjects were continuously cultured for 20 days and the number of cells was counted at days 1, 3, 7, 10, 15 and 20 after plating. The morphological patterns were observed and compared, the pH values of the medium were calculated, as were hexose consumption and lactate production. The results established clear differences in cell growth, pH and morphology: up to day 7, the growth rate was lower in fructose than in glucose medium, and the pH values were higher. In addition, marked steatosis appeared, with increased pyruvate dehydrogenase (PDH) activity. After day 10, the mean values gave a significant increase in the number of cells grown in fructose mediums, even if variations occurred between different cell strains. This increase was accompanied by loss of density-dependent growth inhibition and a reduction in the quantity and size of the vacuoles caused by steatosis. These findings were also established for other cell types, like aponeurosis fibroblasts. In addition, the longevity of the strains increased. These observations indicate that intermediary metabolism is considerably influenced by the carbohydrate present in the cell culture medium and that there are also repercussions on the growth rate. Under our experimental conditions, metabolism pathways seemed to differ on day 7 and on day 20. The various metabolic events suggested by the differences in the pH values are now being studied in our laboratory.

HLA Class I Genes: From Structure to Expression, Serology and Function

HLA class I genes have been isolated from phage and cosmid libraries and assayed by transfection into murine L cells. The transfection step proved to be very important because of the large number of genes (and pseudogenes) in this family. All functional genes characterized so far in this way are classical class I genes, i.e. members of the HLA-A, -B or -C families. Three of these have been sequenced (HLA-A3, -Aw24; HLA-Cw3) in addition to the pHLA 12.4 pseudogene. Sequence comparisons indicate, in particular, extreme conservation of the 3 non-coding region between allelic HLA-A locus genes; the general organization of all these genes (8 exons) is very similar. Restriction mapping around the functional genes has been performed to investigate the degree of conservation (e.g. between HLA-A3 regions from 2 different individuals) and examine allelism at the DNA level (e.g. between HLA-A3 and HLA-Aw24 regions). Exon shuffling experiments followed by serological analysis of the expressed product indicate that, as expected, specificities are determined by the first two domains of the molecule. However, further constructs show that as soon as a single exon is exchanged most specific reactivities disappear. CTL analysis of murine cells expressing HLA molecules has run into many difficulties but still holds promise for the study of structure-function relationships in this system.

Associations of HLA-A, HLA-B and HLA-C alleles frequency with prevalence of herpes simplex virus infections and diseases across global populations: implication for the development of an universal CD8+ T-cell epitope-based vaccine.

A significant portion of the worlds population is infected with herpes simplex virus type 1 and/or type 2 (HSV-1 and/or HSV-2), that cause a wide range of diseases including genital herpes, oro-facial herpes, and the potentially blinding ocular herpes. While the global prevalence and distribution of HSV-1 and HSV-2 infections cannot be exactly established, the general trends indicate that: (i) HSV-1 infections are much more prevalent globally than HSV-2; (ii) over a half billion people worldwide are infected with HSV-2; (iii) the sub-Saharan African populations account for a disproportionate burden of genital herpes infections and diseases; (iv) the dramatic differences in the prevalence of herpes infections between regions of the world appear to be associated with differences in the frequencies of human leukocyte antigen (HLA) alleles. The present report: (i) analyzes the prevalence of HSV-1 and HSV-2 infections across various regions of the world; (ii) analyzes potential associations of common HLA-A, HLA-B and HLA-C alleles with the prevalence of HSV-1 and HSV-2 infections in the Caucasoid, Oriental, Hispanic and Black major populations; and (iii) discusses how our recently developed HLA-A, HLA-B, and HLA-C transgenic/H-2 class I null mice will help validate HLA/herpes prevalence associations. Overall, high prevalence of herpes infection and disease appears to be associated with high frequency of HLA-A(∗)24, HLA-B(∗)27, HLA-B(∗)53 and HLA-B(∗)58 alleles. In contrast, low prevalence of herpes infection and disease appears to be associated with high frequency of HLA-B(∗)44 allele. The finding will aid in developing a T-cell epitope-based universal herpes vaccine and immunotherapy.

HLA-A*01:03, HLA-A*24:02, HLA-B*08:01, HLA-B*27:05, HLA-B*35:01, HLA-B*44:02, and HLA-C*07:01 Monochain Transgenic/H-2 Class I Null Mice: Novel Versatile Preclinical Models of Human T Cell Responses

We have generated a panel of transgenic mice expressing HLA-A*01:03, -A*24:02, -B*08:01, -B*27:05, -B*35:01, -B*44:02, or -C*07:01 as chimeric monochain molecules (i.e., appropriate HLA α1α2 H chain domains fused with a mouse α3 domain and covalently linked to human β2-microglobulin). Whereas surface expression of several transgenes was markedly reduced in recipient mice that coexpressed endogenous H-2 class I molecules, substantial surface expression of all human transgenes was observed in mice lacking H-2 class I molecules. In these HLA monochain transgenic/H-2 class I null mice, we observed a quantitative and qualitative restoration of the peripheral CD8+ T cell repertoire, which exhibited a TCR diversity comparable with C57BL/6 WT mice. Potent epitope-specific, HLA-restricted, IFN-γ–producing CD8+ T cell responses were generated against known reference T cell epitopes after either peptide or DNA immunization. HLA-wise, these new transgenic strains encompass a large proportion of individuals from all major human races and ethnicities. In combination with the previously created HLA-A*02:01 and -B*07:02 transgenic mice, the novel HLA transgenic mice described in this report should be a versatile preclinical animal model that will speed up the identification and optimization of HLA-restricted CD8+ T cell epitopes of potential interest in various autoimmune human diseases and in preclinical evaluation of T cell–based vaccines.

Transformation of murine LMTK− cells with purified HLA class I genes

Viabilite de lapproche gene hybride pour localiser les reactivites serologiques sur les molecules de classe I humaines. Ces reactivites sont determinees par les deux premiers domaines de la molecule (trois premiers exons du gene). Ces deux domaines sont ceux qui montrent la plus grande diversite de sequence ou lon sattend a trouver des reactivites allospecifiques