Association of CD8+ T cell infiltration in oesophageal carcinoma lesions with human leucocyte antigen (HLA) class I antigen expression and survival

Oesophageal cancer is one of the most aggressive tumours with a poor prognosis. However, little is known about the immune response in the tumour microenvironment. To investigate the role of immunosurveillance in the clinical course of oesophageal squamous cell carcinoma, 98 formalin-fixed, paraffin-embedded primary tumours were analysed using immunohistochemical methods for human leucocyte antigen (HLA) class I heavy chain and β2-microglobulin expression and for CD4-, CD8- and CD57-positive cell infiltration. HLA class I expression of tumour cells was correlated positively with infiltration of CD8(+) T cells into the cancer nest, but not with the clinical course of disease. However, CD8(+) and CD4(+) T cell infiltration was correlated with prognosis. These results suggest that tumour antigen-specific cellular immune response plays a role in the clinical course of the disease and that HLA class I antigen expressed on tumour cells contribute to this association most probably by mediating the interactions between tumour cells and CD8(+) T cells.     

A gp120 HIV peptide with high similarity to HLA class II beta chains enhances PPD-specific and autoreactive T cell activation.

The recent report that anti-gp120 antibodies can be induced by allogeneic stimuli in experimental animals in the absence of HIV, has focused attention on the structural similarities between gp120 and MHC. Here we report that some HIV+ individuals develop antibodies which similarly react with the gp120 HIV sequence (aa 254-263) and with the HLA-DR beta chains (aa 142-151). As these two peptides share a high level of similarity, we have investigated the role of this gp120 region on HLA class II mediated T cell recognition. The synthetic peptide corresponding to the gp120 HIV sequence aa 254-263 has been tested on T cell line (TCL) activation. Both the PPD-specific and the self-HLA reactive TCL proliferation increased in the presence of this peptide. Prepulsing experiments indicate that this enhancing effect carried out by HIV peptide is exerted at the level of antigen presentation. Moreover, the specificity of this interaction is supported by the fact that a MoAb specific for this HIV peptide blocked the autoreactive TCL proliferation, similarly to the inhibition carried out by anticlass II antibody. These data support the hypothesis that the functional homology between the HIV peptide and the HLA beta chain described may be involved in the pathogenesis of AIDS.     

Thymic selection and peptide-induced activation of T cell receptor-transgenic CD8 T cells in interleukin-2-deficient mice

The requirement for interleukin-2 (IL-2) in repertoire selection and peripheral activation of CD8 T cells was tested in mice rendered IL-2 deficient by gene targeting and expressing a transgenic T cell receptor (TcR) (F5) specific for influenza nucleoprotein (NP) 366-374 + H-2D^b. Positive selection of the transgenic F5 TcR into the CD8 compartment proceeded normally. Both in vivo and in vitro, the antigenic peptide induced depletion of immature thymocytes and proliferation of mature CD8 T cells regardless of the presence of an intact IL-2 gene. In contrast, cytotoxic T lymphocyte (CTL) activity was only generated by T cells from IL-2⁺ F5 transgenic mice. Exogenous IL-2 was able to fully restore the CTL response of IL-2^?/? responder cells in vitro. Thus, both in vivo and in vitro, clonal expansion of CD8 T cells can proceed in the absence of IL-2, whereas in peptide-immunized F5 transgenic mice, induction of cytotoxic effector function is IL-2 dependent.     

Normal T cell receptor Vβ usage in a primary immunodeficiency associated with HLA class II deficiency

The human Tcell receptor was studied using an anchored-polymerase chain reaction (A-PCR) and hybridization with Vβ-specific oligonucleotide probes, together with the few anti-Vβ monoclonal antibodies (mAb) available. After confirming the semiquantitative and reproducible nature of the A-PCR technique, we assessed the complete Vβ repertoire in sorted CD4⁺ and CD8⁺ lymphocyte populations from three normal donors. These experiments confirmed the absence of Vβ-restricted deletions in human peripheral cells, in contrast to several mouse strains. This feature makes it difficult to study negative selection in man, given the apparent absence of an endogenous superantigen corresponding to the Mis system in the mouse. To investigate human Vβ repertoire shaping, we studied VP usage in CD4+ and CD8⁺ T cells from children with an inherited immunodeficiency characterized by defective expression of human leukocyte antigen class II molecules. An initial study using anti-Vβ monoclonal antibodies failed to show significant abnormalities in Vβ usage. Four patients analyzed using the A-PCR method all had a polyclonal Vβ repertoire, suggesting normal positive selection and raising questions as to the importance of VP major histocompatibility complex (MHC) interactions and the role of thymic MHC density in shaping the Vβ repertoire.     

Interleukin-12 increases interleukin-4 production by established human ThO and Th2-like T cell clones

Interleukin (IL)-12 is a potent inducer of cell-mediated immunity: it favors the generation of interferon (IFN)-γ-producing T cells, increases IFN-γ production by T cells and natural killer cells and prevents the generation of a Th2 response in murine in vivo models. Nevertheless, the effects of IL-12 on an established Th2 response remain poorly documented. In the present paper, we analyzed the effect of IL-12 on the profile of lymphokines produced by established IL-4-producing Th0 and Th2-like human T cell clones (TCC) and by polyclonal T cells. We found that IL-12 (i) enhances, as previously reported, IFN-γ production by Th0 TCC and, to a smaller extent, by Th2-like TCC, (ii) increases the proliferation of Th0 and Th2-like TCC and (iii) unexpectedly, synergizes with T cell receptor-associated or nonspecific stimuli in increasing IL-4 production by these TCC. Thus, IL-12 potentiates the production of IFN-γ and also of IL-4 by established IL-4-producing TCC. Although IL-12 has been widely reported to induce a Th1 response and to prevent the development of a Th2 response in vivo, IL-12 may on the contrary potentiate an established Th2 response in humans.     

Soluble HLA class I antigen secretion by normal lymphocytes: relationship with cell activation and effect of interferon-gamma

HLA class I antigens are thought to be integral membrane proteins. However, soluble forms of these molecules have been detected. Our laboratory has recently shown that the predominant form of these soluble proteins present in human serum, spleen tissue and culture supernatant of activated lymphocytes exhibits molecular weight and structure similar to classical HLA class I antigens, but lacks HLA A or B polymorphic determinants. In the present study, the secretion of such soluble proteins by lymphocytes has been further explored. Phytohaemagglutinin-stimulated normal lymphocytes secrete considerable quantities of soluble HLA (sHLA) class I proteins. This secretion seems to be a general property of lymphocytes, since activation of T as well as B cells by appropriate mitogens equally induce sHLA I secretion. Lymphocytes require RNA and protein synthesis, but not DNA synthesis, for the secretion to occur. Kinetic studies reveal that maximal sHLA I secretion precedes the peak of DNA synthesis by 24 h. In vitro stimulation with antigens or alloantigens also provokes sHLA I secretion. Moreover, this phenomenon has also been detected for in vivo-activated lymphocytes, as enhanced spontaneous sHLA I secretion was observed in cultures of low-density blastic B and T cells, and of blood lymphocytes obtained from normal subjects who had received a booster immunization 5 days earlier. Interferon-gamma (IFN-gamma) increases the expression of membrane-bound class I antigens but does not induce any sHLA I secretion, suggesting that both molecules are under different regulatory mechanisms. Our results indicate that human lymphocytes, upon stimulation, actively secrete considerable amounts of a soluble form of these biologically relevant proteins.     

Ligation of HLA class II molecules promotes sensitivity to CD95 (Fas antigen,APO-1)-mediated apoptosis

CD95 (Fas antigen/APO-1) is up-regulated in activated lymphocytes, and monoclonal antibody (mAb) to CD95 induces apoptosis. HLA class II molecules play a key role in antigen presentation, ligation of which induces signal transduction. We examined 18 lymphoid cell lines (15 B cell and 3 T cell lines) to investigate the effects of ligation of HLA class II molecules on CD95-mediated apoptosis. All of the five immature B cell lines were sensitive to anti-CD95 mAb, and ligation of HLA class II molecules promoted CD95-mediated apoptosis. In seven B-blastoid cell lines, two Burkitt lines were resistant to anti-CD95 mAb in spite of high expression of CD95. In three of five non-Burkitt B-blastoid lines, CD95-mediated apoptosis was augmented by treatment with anti-HLA class II mAb, while the other two lines lacking CD95 were resistant to anti-CD95 mAb. Three plasmacytic cell lines showed CD95-mediated apoptosis, but enhancement by anti-HLA class II mAb was slight in one cell line and was not observed in the other two lines. Out of three HLA class II antigen-positive T cell lines, CD95-mediated apoptosis was observed to some degree in one cell line but was not promoted by the treatment with anti-HLA class II mAb, and the other two cell lines were resistant to anti-CD95 mAb. Ligation of HLA class II molecules did not alter CD95 expression in the five cell lines examined, except Su-DHL-4 originated from a follicular lymphoma, which showed slight up-regulation. Taken together, ligation of HLA class II molecules apparently promotes CD95-mediated apoptosis in immature B cells and non-Burkitt B blasts. These findings highlight the role of HLA class II molecules in CD95-mediated apoptosis, which may facilitate rapid clearance of functionally useless cells from the immune system and might be involved in negative selection of B cells.     

Differential effects of interleukin-10 on the expression of HLA class II and CD1 molecules induced by granulocyte/macrophage colony-stimulating factor/interleukin-4

Interleukin (IL)-10 down-regulates HLA class II molecules, whether constitutively expressed or up-regulated by interferon-γ or IL-4 on monocytes but not on B lymphocytes. In this study we show that IL-10 does not inhibit HLA class II expression induced by the combination granulocyte/macrophage colony-stimulating factor and IL-4 on monocytes, although it simultaneously abrogates the expression of CD1 molecules induced by the same combination of cytokines. CD1 molecules can act as element of genetic restriction for CD4^? CD8^? T lymphocytes, and the suppression of CD1 expression by IL-10 abolished antigen presentation to CD1-restricted CD4^? CD8^? T cell receptor-positive T cells. Although HLA class II expression was not down-regulated by IL-10, the antigen specific proliferative response of CD4⁺ T cells was nevertheless decreased. This was not caused by down-regulation of known co-stimulatory molecules such as B7.1, B7.2 and ICAM-1. IL-10 decreased the antigen specific proliferative response further by directly influencing the T lymphocytes. Our results indicate that IL-10 exerts some of its immunoregulatory functions by differential modulation of antigen presenting molecules, induced by the same combination of cytokines.     

The susceptibility to natural killer cell-mediated lysis of HLA class I-positive melanomas reflects the expression of insufficient amounts of different HLA class I alleles

NK cells selectively lyse tumor cells which do not express one or more MHC class I alleles. The ability to discriminate between self normal or tumor cells is due to the expression of MHC class I-specific killer inhibitory receptors (KIR). In the present study we analyzed melanoma cell lines which were highly susceptible to NK cell-mediated lysis in spite of the expression of a complete set of HLA class I alleles. Quantitative analysis of the HLA class I expression using allele-specific monoclonal antibodies (mAb) revealed a down-regulation of all HLA class I molecules. Treatment of melanoma cells with IFN-γ resulted in up-regulation of all HLA class I alleles that was paralleled by the acquisition of resistance to lysis. That resistance to lysis reflected the up-regulation of HLA class I molecules was revealed by the finding that mAb-mediated masking of either KIR or their HLA class I ligands completely restored the melanoma cell lysis. These results were obtained by the use of selected NK cell clones derived either from allogeneic or autologous donors. In addition, similar results were obtained using in vitro expanded autologous NK cell populations. Our data indicate that NK cells can lyse not only melanoma cells which have lost the expression of one or more HLA class I alleles but also cells expressing a decreased amount of class I molecules.     

A sensitive fluorometric assay for quantitatively measuring specific peptide binding to HLA class I and class II molecules

A sensitive, highly reproducible assay was developed for measuring binding of peptides to various HLA class I and II alleles. The assay is based on competition for binding to HLA between a peptide of interest and a fluorescent labelled standard peptide. This mixture is incubated with HLA to obtain equilibrium binding, and subsequently separated on an HPLC size-exclusion column in (i) a protein fraction containing HLA and bound peptide and (ii) a free peptide fraction. Each assay uses only 100 fmol labelled peptide and approximately 10 pmol of HLA. The analytical system contains an autosampler that samples from 96-well microtiter plates. Injections and data recording/evaluation is fully automated. Typical analysis time is 10–12 min per sample. The fluorescence in the HLA-bound peptide and free peptide containing fractions is measured on-line. The ratios of fluorescence signal in protein and peptide fractions at various concentrations of the peptide of interest are determined. IC₅₀ values are calculated from the binding curve as obtained by curve fitting of the data. Here we show results for peptide binding to HLA-DR1 and -DR17 molecules purified from detergent solubilized cell lysates, and for recombinant HLA-A^* 0201 and HLA-A^* 0301 expressed in E. coli.

The assay reported is sensitive and reproducible. It is non-radioactive and is non-labor intensive due to the high degree of automation.     

Identification of nine new HLA class I alleles in volunteers from the Singapore stem cell donor registries

Based on unusual probe hybridization patterns, two human leukocyte antigen (HLA)-A, five HLA-B, and two HLA-C alleles (A*240208, A*3211Q; B*1822, B*3938Q, B*4606, B*4607N, B*5139; Cw*0321, Cw*0734) were identified in individuals from the Singapore Bone Marrow Donor Program and Singapore Cord Blood Bank. Eight of the nine alleles encode amino acid substitutions altering the antigen-binding region including three alleles with changes altering a cysteine at codon 164 (A*3211Q, B*3938Q, B*4607N). This substitution either eliminates a key disulfide bond or results in a stop codon, both likely affecting the expression of the HLA molecules. Only one allele (A*240208) carries a synonymous substitution.     

Expression of HLA class I/II antigens and T cell immune response in human neuroendocrine tumors of the pancreas

Peptide presentation by HLA class I and II antigens regulates specific antigen recognition by T cells. The present study aimed to investigate T cell infiltration and its relation to HLA antigen expression in pancreatic neuroendocrine tumors. Fresh tissue samples were collected from five insulinomas and six other neuroendocrine tumors (one gastrinoma, one glucagonoma, two carcinoid, and two neuroendocrine carcinomas). Normal pancreatic and splenic tissue samples were used as controls. Investigation of infiltrating lymphocyte populations, as well as staining of HLA class I and II antigens, were performed by standard immunohistochemistry. The majority of investigated tumors demonstrated an intratumoral infiltration by CD3+, CD4+ and CD8+ T cells that was significantly higher than in normal pancreatic islets. Only a minority of tumor-infiltrating T cells showed the CD45RO+ phenotype. The expression of HLA class I antigen was altered in 10 of 11 tumors. A loss of beta-2microglobulin represented the most frequent type of alteration to HLA class I expression, although the total loss of HLA class I was found in only one case of neuroendocrine carcinoma. HLA class II molecules were expressed by endothelial and lymphoid cells and not by tumor cells. In conclusion most neuroendocrine pancreatic tumors induce a T cell mediated immune response resulting in an intratumoral infiltration with CD3+, CD4+ and CD8+ T cells. Loss of beta-2microglobulin is a frequent alteration in these tumors, which may influence the normal function of the HLA class I antigen complex. In contrast to malignant tumors of the exocrine pancreas, expression of HLA class II was absent in neuendocrine pancreatic tumor cells.     

Functional CD40 ligand expression on T lymphocytes in the absence of T cell receptor engagement: involvement in interleukin-2-induced interleukin-12 and interferon-γ production

Despite the fact that the great majority of T cells at the site of an inflammatory response are not antigen specific, the mechanisms leading to activation and recruitment of these bystander T cells are poorly understood. We previously reported that soluble (s)CD23 potentiated the interleukin (IL)-2-induced interferon (IFN)-γ production by T cells co-cultured with autologous monocytes in the absence of T cell receptor (TCR) engagement. Our present data demonstrate that the IL-2-induced IFN-γ secretion, in the presence but also in the absence of sCD23, is strictly IL-12 dependent, inasmuch as anti-IL-12 antibody abrogated both responses. Most interestingly, anti-CD40 ligand (CD40L) monoclonal antibody significantly inhibited IL-2-induced IL-12 as well as IFN-γ production. These results suggest that CD40L was expressed on T cells in the absence of TCR engagement. Indeed, purified unstimulated T cells readily expressed CD40L. IL-2 and monocytes did not up-regulate CD40L on resting T cells. It is proposed that low levels of CD40L expression on non-antigen stimulated T cells are sufficient to signal through CD40 molecules on accessory cells and to induce IL-12 secretion, which in turn can synergize with IL-2 for the induction of IFN-γ production, thus contributing to the inflammatory process.     

HLA class I and II polymorphisms in Azores show different settlements in Oriental and Central islands

Human leucocyte antigen-A, -B, -Cw, -DRB1, -DQA1 and -DQB1 polymorphisms were examined in the Azorean population. The data were obtained at high-resolution level, using polymerase chain reaction (PCR) with sequence-specific primer, PCR-sequence-specific oligonucleotides and sequence-based typing. The most frequent allele in each locus was: A*0201 (24.5%), B*510101 (9.8%), Cw*0401 (14.8%), DRB1*070101 (18.3%), DQA1*0201 (17.4%) and DQB1*0301 (19.4%). The predominant extended haplotype was A*0202-B*1503-Cw*0202-DRB1*090102-DQA1*0303- DQB1*0202 (1.9%), which was found to be absent in the Portuguese mainland. The present study corroborates historical sources that say the Azores were populated not only by Portuguese but also by other Europeans, mostly Flemish people. Despite dendrogram analysis showing some remote Asian genetic affinities, the lack of specific alleles and haplotypes from those populations does not allow us to conclude for direct influence. Haplotype and allele frequencies in Azores show no homogeneous distribution between Oriental and Central islands of this archipelago. The Oriental islands harbour several haplotypes already found in mainland Portugal and identified as Mediterranean and European. The Central group of islands on the contrary clearly shows an influence of north Europeans (most probably derived from a well-documented Flemish settlement), with much less affinity to mainland Portugal.     

Increased beta2-microglobulin-free HLA class I heavy chain serum levels in the course of immune responses to viral antigens and to mismatched HLA antigens

Besides being present in serum in association with beta2-mu, HLA class I heavy chains are also present in serum as beta2-micro-free moieties. The increase in serum levels of beta2-micro-associated HLA class I heavy chains in conditions associated with an activation of the immune system have prompted us to measure the serum levels of beta2-mu-free HLA class I heavy chains in the course of immune responses to viral antigens and to mismatched histocompatibility antigens. The serum level of beta2-mu-free HLA class I heavy chains, like that of beta2-mu-associated HLA class I heavy chains was significantly increased in patients affected by advanced HIV-1 infection or by chronic hepatitis C (CHC). In the latter group of patients an association was found between a reduction in the beta2-mu-free HLA class I heavy chain serum level and response to therapy with interferon alpha and ribavirin. Moreover, the beta2-mu-free HLA class I heavy chain serum level was increased more than that of beta2-mu-associated HLA class I heavy chains during episodes of liver ischemia following liver transplantation and in the course of acute graft rejection and of acute graft-versus-host-disease (GVHD) after allogeneic bone marrow transplantation (BMT). These results suggest that the serum levels of beta2-mu-free and beta2-mu-associated HLA class I heavy chains are independently regulated. Furthermore, beta2-mu-free HLA class I heavy chain serum level may be a useful marker to monitor response to therapy in CHC patients and the clinical course of liver and bone marrow grafts.     

C2_4_4 microheterogeneity and HLA Class I

The microheterogeneity of the tetranucleotide repeat locus C2_4_4 situated in the HLA class I region (6p21.3) was investigated by sequencing 50 alleles in an Austrian population sample of 240 unrelated Caucasoid individuals. Several different sequences were found in alleles of the same length. Analysis of the associations between the sequenced C2_4_4 alleles and HLA class I showed a strong linkage disequilibrium between the C2_4_4*9 sequence variants and two different HLA class I haplotypes, as well as between the most common *17 sequence and one HLA-ABC haplotype. No clear cut association could be observed in C2_4_4*16 and *18. The results of this study demonstrate that the exclusive use of microsatellite polymorphisms for the definition of HLA haplotypes is generally not possible.     

Efficient cloning and expression of HLA class I cDNA in human B-lymphoblastoid cell lines

Analysis of HLA restriction specificity is one of the important steps in characterizing T cell clones. This usually requires either a panel of HLA-typed cells or HLA cDNA transfectants. Although preparation of HLA cDNA transfectants is laborious, utilization of transfectants is advantageous when a suitable panel is not available due to linkage disequilibrium or rarity of the HLA allele of interest. In this report, we describe an efficient and rapid HLA cloning and expression system. Three sets of PCR primers specific for HLA-A, B and C loci were designed by extensively sequencing 5'- and 3'- untranslated regions of HLA class I genes. The PCR-amplified products were introduced into modified Phoenix retrovirus vectors containing a puromycin resistant gene under the control of a LTR promotor. Gibbon ape leukemia virus (GALV)-pseudotyped retrovirus was produced and infected into B-lymphoid cell lines. Following expansion in selection media, more than 80% of cells expressed transduced HLA at a comparable level to that normally expressed. These results indicate that locus-specific PCR cloning and utilization of GALV-pseudotyped retroviral vector can be an effective and relatively efficient tool for constructing a panel of different HLA transfectants.     

Serological and molecular analysis of HLA class I and II alleles in Thai patients with psoriasis vulgaris

Abstract: The HLA class I and class II alleles in 67 patients with type I psoriasis vulgaris, 23 patients with type II psoriasis vulgaris and 140 healthy individuals were analyzed. The frequencies of HLA-A2, -B46, -B57 and DQB1*0303 were significantly increased in type I psoriasis compared to the controls (Pc<0.05). Molecular analysis of HLA-A2 alleles showed an increase in HLA-A*0207 and a decrease in HLA-A*0203 in type I psoriasis. HLA-DQBl*0301 was significantly decreased in type I psoriasis compared to the normal controls (Pc<0.05). No association of any alleles with type II psoriasis was observed. This data demonstrated two susceptible haplo-types: HLA-A1-B57-DRB1*0701-DQA1*0201-DQB1*0303 (AH57.1) and HLA-A2-B46-DRB1*0901-DQA1*0301-DQB1*0303 (AH46.1) for type I psoriasis in the Thai population. Besides, the haplotype AH46.1 was also associated with type II psoriasis.     

T cell subset distribution and B cell hyperreactivity in mice expressing interleukin-4 under the control of major histocompatibility complex class I regulatory sequences

Transgenic mice in which interleukin-4 (IL-4) is expressed under the control of the major histocompatibility complex (MHC) class I regulatory sequences show low level expression of IL-4 in all organs investigated. Several weeks after birth the animals develop thymus hypoplasia with a loss of CD4⁺CD8⁺ double-positive cells and a relative increase in the mature population, especially, and in contrast to previously published lines, the CD4⁺ single-positive cells. In the periphery, T lymphocytes eventually decline, CD8⁺ cells being more strongly affected. Many of the residual T cells exhibit the CD44^highMel-14^low phenotype of antigenically experienced T cells. B cells also show an activated phenotype with respect to size, MHC class II and CD23 expression, are more readily stimulated by anti-μ F(ab′)₂ antibodies than are B cells from control littermates, and show a higher spontaneous and antigen-induced production of IgG1 and IgE.