Functional analysis of HLA-A*0201/Melan-A peptide multimer+ CD8+ T cells isolated from an HLA-A*0201- donor: exploring tumor antigen allorestricted recognition.

Recent studies in mouse models have suggested that genetic transfer of tumor antigen-specific high affinity T cell receptors (TCR) into host lymphocytes could be a viable strategy for the rapid induction of tumor-specific immunity. A previously proposed approach for the isolation of such TCRs consists in circumventing tolerance to self-restricting HLA/peptide complexes by deriving them from PMBCs of allogenic donors. Towards this aim, we used fluorescent HLA-A2 class-I/peptide soluble multimers to isolate A2-restricted CD8+ T cells specific for a previously described Melan-A peptide enhanced analog (Melan-A 26-35 A27L) from an HLA-A*0201 (A2) negative donor. We isolated two distinct groups of Melan-A 26-35 A27L-specific clones. Clones from the first group recognized the analog peptide with high avidity but showed very low recognition of Melan-A parental peptides. In contrast, clones from the second group efficiently recognized Melan-A parental peptides. Surprisingly however, most clones recognized not only A2+ Melan-A+ targets, but also A2+ Melan-A- targets suggesting that they can also recognize endogenous peptides other than Melan-A. In addition, one clone showed full cross-recognition of an antigenically unrelated peptide. Together, our data show that HLA-A2/peptide multimers can be successfully used for the isolation of allorestricted CD8+ T cells reactive with tumor antigen-derived peptides. However, as the cross-reactivity of these apparently peptide-specific allorestricted TCRs is presently unpredictable, a careful in vitro analysis of their reactivity to the host's normal cells is recommended.

Détermination de l'antigène HLA-G soluble dans le liquide folliculaire et dans le milieu de culture d'embryons comme indicateur du succès d'un traitement par FIV-ICSI [Soluble human leukocyte antigen-G (sHLA-G) in follicular fluid and embryo culture medium and its impact on pregnancy prediction in IVF-ICSI treatment]

In IVF around 70% of embryos fail to implant. Often more than one embryo is transferred in order to enhance the chances of pregnancy, but this is at the price of an increased multiple pregnancy risk. In the aim to increase the success rate with a single embryo, research projects on prognostic factors of embryo viability have been initiated, but no marker has found a routine clinical application to date. Effects of soluble human leukocyte antigen-G (sHLA-G) on both NK cell activity and on Th1/Th2 cytokine balance suggest a role in the embryo implantation process, but the relevance of sHLA-G measurements in embryo culture medium and in follicular fluid (FF) are inconsistent to date. In this study, we have investigated the potential of sHLA-G in predicting the achievement of a pregnancy after IVF-ICSI in a large number of patients (n = 221). sHLA-G was determined in media and in FF by ELISA. In both FF and embryo medium, no significant differences in sHLA-G concentrations were observed between the groups "pregnancy" and "implantation failure", or between the groups "ongoing" versus "miscarried pregnancies". Our results do not favour routine sHLA-G determinations in the FF nor in embryo conditioned media, with the current assay technology available.

Harmonisation of short-term in vitro culture for the expansion of antigen-specific CD8(+) T cells with detection by ELISPOT and HLA-multimer staining.

Ex vivo ELISPOT and multimer staining are well-established tests for the assessment of antigen-specific T cells. Many laboratories are now using a period of in vitro stimulation (IVS) to enhance detection. Here, we report the findings of a multi-centre panel organised by the Association for Cancer Immunotherapy Immunoguiding Program to investigate the impact of IVS protocols on the detection of antigen-specific T cells of varying ex vivo frequency. Five centres performed ELISPOT and multimer staining on centrally prepared PBMCs from 3 donors, both ex vivo and following IVS. A harmonised IVS protocol was designed based on the best-performing protocol(s), which was then evaluated in a second phase on 2 donors by 6 centres. All centres were able to reliably detect antigen-specific T cells of high/intermediate frequency both ex vivo (Phase I) and post-IVS (Phase I and II). The highest frequencies of antigen-specific T cells ex vivo were mirrored in the frequencies following IVS and in the detection rates. However, antigen-specific T cells of a low/undetectable frequency ex vivo were not reproducibly detected post-IVS. Harmonisation of the IVS protocol reduced the inter-laboratory variation observed for ELISPOT and multimer analyses by approximately 20 %. We further demonstrate that results from ELISPOT and multimer staining correlated after (P < 0.0001 and R (2) = 0.5113), but not before IVS. In summary, IVS was shown to be a reproducible method that benefitted from method harmonisation.

Mapping Bias Overestimates Reference Allele Frequencies at the HLA Genes in the 1000 Genomes Project Phase I Data.

Next-generation sequencing (NGS) technologies have become the standard for data generation in studies of population genomics, as the 1000 Genomes Project (1000G). However, these techniques are known to be problematic when applied to highly polymorphic genomic regions, such as the human leukocyte antigen (HLA) genes. Because accurate genotype calls and allele frequency estimations are crucial to population genomics analyses, it is important to assess the reliability of NGS data. Here, we evaluate the reliability of genotype calls and allele frequency estimates of the single-nucleotide polymorphisms (SNPs) reported by 1000G (phase I) at five HLA genes (HLA-A, -B, -C, -DRB1, and -DQB1). We take advantage of the availability of HLA Sanger sequencing of 930 of the 1092 1000G samples and use this as a gold standard to benchmark the 1000G data. We document that 18.6% of SNP genotype calls in HLA genes are incorrect and that allele frequencies are estimated with an error greater than ±0.1 at approximately 25% of the SNPs in HLA genes. We found a bias toward overestimation of reference allele frequency for the 1000G data, indicating mapping bias is an important cause of error in frequency estimation in this dataset. We provide a list of sites that have poor allele frequency estimates and discuss the outcomes of including those sites in different kinds of analyses. Because the HLA region is the most polymorphic in the human genome, our results provide insights into the challenges of using of NGS data at other genomic regions of high diversity.

Shared HLA Class II in Six Autoimmune Diseases in Latin America: A Meta-Analysis

The prevalence and genetic susceptibility of autoimmune diseases (ADs) may vary depending on latitudinal gradient and ethnicity. The aims of this study were to identify common human leukocyte antigen (HLA) class II alleles that contribute to susceptibility to six ADs in Latin Americans through a meta-analysis and to review additional clinical, immunological, and genetic characteristics of those ADs sharing HLA alleles. DRB1∗03:01 (OR: 4.04; 95%CI: 1.41–11.53) was found to be a risk factor for systemic lupus erythematosus (SLE), Sjogren’s syndrome (SS), and type 1 diabetes mellitus (T1D). DRB1 ¨ ∗04:05 (OR: 4.64; 95%CI: 2.14–10.05) influences autoimmune hepatitis (AIH), rheumatoid arthritis (RA), and T1D; DRB1∗04:01 (OR: 3.86; 95%CI: 2.32–6.42) is a susceptibility factor for RA and T1D. Opposite associations were found between multiple sclerosis (MS) and T1D. DQB1∗06:02 and DRB1∗15 alleles were risk factors for MS but protective factors for T1D. Likewise, DQB1∗06:03 allele was a risk factor for AIH but a protective one for T1D. Several common autoantibodies and clinical associations as well as additional shared genes have been reported in these ADs, which are reviewed herein. These results indicate that in Latin Americans ADs share major loci and immune characteristics.

Estudo dos genes do complexo do antígeno leucocitário humano (hla) associados à susceptibilidade ao diabetes mellitus tipo 1

Of all of the genes associated with the development of Diabetes mellitus type 1 (T1D), the largest contribution comes from the genes in the Human Leukocyte Antigen (HLA) region, mostly the class II DR e DQ genes. Specific combinations of alleles DRB1, DQA1 and DQB1 constituting haplotypes, and further, a combination of more than one haplotype, providing multilocus genotypes are associated with susceptibility, protection and neutrality to DM1. Thus, the aim of present study was to verified the association of polymorphisms of HLA genes class II with susceptibility to type 1 diabetes mellitus (T1D). Ninety-two patients with T1D and 100 individuals normoglycemics (NG) aged between 6 and 20 years were studied. Genomic DNA was obtained from peripheral whole blood, collected in EDTA tube, using the extraction kit Illustra Triple Prep®, GE Healthcare. For HLA typing was used DNA LABType system by One Lambda kit applying Luminex® technology to the method of PCRSSO typing reverse. The alleles DRB1*03:01, *04:05, *04:01, *04:02, DQA1*03:01g, *05:01g, DQB1*02:01g, *03:02, the haplotypes DRB1*03:01-DQA1*05:01-DQB1*02:01, DRB1*04:05-DQA1*03:01g-DQB1*03:02, DRB1*04:02-DQA1*03:01g-DQB1*03:02, DRB1*04:01-DQA1*03:01g-DQB1*03:02 and DR3-DQ2/DR4-DQ8 genotype were significantly associated with the chance of developing T1D. The alleles DRB1*11:01, *15:03, *15:01, *13:01, DQA1*01:02, *04:01g, *01:03, DQB1*06:02, *03:01g, *06:03, *04:02, the haplotypes DRB1*11:01-DQA1*05:01-DQB1*03:01, DRB1*13:01-DQA1*01:03-DQB1*06:03 and DRX-DQX/DRX-DQX genotype, formed by other than the DR3-DQ2 or DR4-DQ8 haplotypes, were significantly associated with T1D protection Despite the major racial Brazilian, even at the regional level, these results are similar to the majority of alleles, genotypes and haplotypes of HLA class II-related susceptibility or resistance to T1D, extensively described in the literature for Caucasian population. Children with age at diagnosis less than 5 years of age had significantly higher frequency of the heterozygous genotype DR3-DQ2/DR4-DQ8 compared to children with age at diagnosis than 5 years old. These results also demonstrate strong association of the genetic profile of the class II HLA for this age group, possibly associated with the severity and rapid progression to the onset of T1D. The knowledge of HLA class II genes may be useful in genetic screens that allow the prediction of T1D

Frequency of human leukocyte antigen (hla) in patients with malaria and in the general population of Humaitá county, Amazonas state, Brazil

Em agosto de 1983 foram observados 85 habitantes do Município de Humaitá, Estado do Amazonas, Brasil, com a finalidade de estudar a prevalência dos antígenos de HLA -A, -B, -C e DR, dentre os quais 38 eram doentes com malária causada pelo Plasmodium falciparum Todos eles foram examinados para avaliação de esplenomegalia, exame parasitológico de sangue e pesquisa de anticorpos de malária. Foram constituídos três grupos: (I) 25 indivíduos nascidos na região Amazônica que nunca tiveram malária; (II) 38 indivíduos naturais da Amazônia que tinham sido tratados de malária no passado, ou que estavam tendo malária atual, e (III) 22 doentes com malária que contraíram na Amazônia e eram procedentes de outras regiões do Brasil. Foram colhidas amostras de sangue de cada um deles, separados os linfôcitos e os antígenos de HLA foram tipados pelo teste de microlinfocitotoxidade. Houve elevada freqüência de antígenos não identificados, nos grupos estudados, o que sugere ou a existência de homozigoze, oufenôtipo não identificado nessa população. Houve alta freqüência fenotípica de antígeno deAg(W24) (44,7%) no Grupo II, quando comparado ao Grupo 1(32%) ou Grupo III (9%). Os indivíduos do Grupo II mostraram também elevada freqüência do antígeno DR4 (80%) quando comparado ao Grupo 1(36,3%) ou Grupo III(16,6%). Essas observações sugerem a possibilidade de suscetibilidadegenética ã malária entre os nativos da Amazônia e indicam a necessidade da realização de inquéritos mais extensos sobre a freqüência de antígenos de HLA em habitantes de zona endêmica de malária.

Efeito quimiopreventivo e modulador de HLA-G (Human Leukocyte Antigen - G) de Morelloflavona

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Greater expression of the human leukocyte antigen-G (HLA-G) and interleukin-17 (IL-17) in cervical intraepithelial neoplasia: analytical cross-sectional study

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Low expression of human histocompatibility soluble leukocyte antigen-G (HLA-G5) in invasive cervical cancer with and without metastasis, associated with Papilloma Virus (HPV)

Human leukocyte antigen-G (HLA-G) is a non-classical major histocompatibility complex class Ib molecule that acts as a specific immunosuppressor. Some studies have demonstrated that human papillomavirus (HPV) seems to be involved in lower or absent HLA-G expression, particularly in cervical cancer. In this study, we performed a cross-sectional study, systematically comparing the qualitative expression of the HLA-G5 isoform in invasive cervical carcinoma (ICC), stratifying patients according to the presence [ICC with metastasis (ICCW)] and absence [ICC without metastasis (ICCWT)] of metastasis, correlating these findings with interference of HPV and demographic and clinical variables. Seventy-nine patients with a diagnosis of ICC were stratified into two groups: ICCWT (n=52 patients) and ICCW (n=27). Two biopsies were collected from each patient (one from the tumor lesion and one from a lymph node). Immunohistochemistry analyses were performed for the HLA-G5 isoform, for HPV detection, and virus typing. HLA-G5 isoform molecules were detected in 25 cases (31.6%), 17 (32.7%) without metastasis and 8 (29.6%) with metastasis. HPV was detected in the cervical lesions of 74 patients (93.7%), but low expression of the HLA-G5 isoform was observed in all HPV-related cases. These findings are important; however, additional studies are necessary to identify the influence of HPV with HLA-G5 isoform expression on invasive cervical malignancies.

Charakterisierung von T-Zell-Antworten gegen humane Nierenzellkarzinome sowie Identifizierung von HLA-G als tumorassoziiertes Antigen und Immunevasionsmechanismus des Nierenzellkarzinoms

Humane Nierenzellkarzinom-(NZK)-Zelllinien wurden etabliert, um sie zur Generierung von autologen zytotoxischen T-Zelllinien einzusetzen. Erst nach Modifikation mit dem kostimulierenden B7-1-Molekül wurden mit der NZK-Zelllinie MZ1257RC autologe, tumorspezifische T-Zelllinien generiert und charakterisiert. Die Aufklärung eines T-Zell-definierten TAA eines autologen, zytotoxischen T Zellklons wurde mittels Expressionsklonierung einer hergestellten cDNS-Expressionsbank begonnen. Nach in vitro-Sensibilisierung von peripheren Blutmonozyten mit der autologen NZK-Zelllinie MZ2733RC wurde die HLA-Klasse I-restringierte T Zelllinie XIE6 generiert, die die autologe und verschiedene allogene NZK- sowie Zervixkarzinom-Zelllinien, jedoch nicht autologe Nierenzellen lysiert. Die T Zellen exprimieren TZR Vβ13.6-Ketten und sezernieren GM-CSF und IL-10 nach Antigenstimulation. Jedoch ist die NZK-Zelllinie MZ2733RC wenig sensitiv gegenüber autologen und allogenen Effektorzellen. Erst die Blockade ihrer HLA Klasse I-Moleküle auf der Zelloberfläche erhöht ihre Sensitivität gegenüber allogenen lymphokin-aktivierten Killer-Zellen. Verantwortlich dafür können nicht-klassische HLA Klasse Ib-Moleküle, insbesondere HLA-G sein, dessen Transkripte in der RNS der NZK-Zellen, jedoch nicht in Nierenzellen detektiert wurden. In einer detaillierten Studie wurden HLA-G-Transkripte in NZK-Zelllinien (58%), in NZK-Biopsien (80%), und nur in wenigen Nierenepithelbiopsien (10%) nachgewiesen. In der NZK-Zelllinie MZ2733RC wurde eine konstitutive HLA-G1-Proteinexpression beobachtet, die durch eine IFN-γ-Behandlung induzierbar ist.

HLA class II mismatch alleles as targets of the alloreactive CD4 T-cell response

In allogeneic hematopoietic stem cell transplantation (allo-HSCT), alloreactive T lymphocytes of donor origin mediate the beneficial graft-versus-leukemia effect but also induce graft-versus-host disease (GvHD). Since human leukocyte antigens (HLA) mismatch alleles represent major targets of alloreactive T lymphocytes, patient and donor are usually matched for the class I molecules A, B, C, and for the class II molecules DRB1 and DQB1, in order do reduce the risk of GvHD. The HLA-DPB1 locus, however, is still ignored in donor selection. Interestingly, clinical studies have demonstrated that disparities at HLA-DQB1 alleles as well as distinct HLA DPB1 mismatch constellations do not adversely affect the outcome of allo-HSCT. It has also been shown that HLA class II is predominantly expressed on hematopoietic cells under non-inflammatory conditions. Therefore, this PhD thesis focused on the application of CD4 T cells in adoptive immunotherapy of leukemias.rnIn the first part of this thesis we developed a rapid screening approach to detect T-cell reactivity of donors to single HLA class II mismatch alleles. Allo-HLA reactivity was measured in naive, memory, and entire CD4 T cells isolated from PBMC of healthy donors by flow cytometric cell sorting according to expression of the differentiation markers CD45RA, CD45RO, CD62L, and CCR7. T-cell populations were defined by a single marker to facilitate translation into a clinical-grade allo-depletion procedure. Alloreactivity to single HLA-DR/-DQ mismatch alleles was analyzed in short-term mixed lymphocyte reactions (MLR) in vitro. As standard antigen-presenting cells, we used the HLA-deficient cell line K562 upon electroporation with single HLA-DR/-DQ allele mRNA. We observed in IFN-γ ELISpot assays that allo-HLA-reactivity preferentially derived from subsets enriched for naive compared to memory T cells in healthy donors, irrespective of the HLA mismatch allele. This separation was most efficient if CD62L (P=0.008) or CD45RA (P=0.011) were used as marker. Median numbers of allo-HLA-reactive effector cells were 3.5-fold and 16.6-fold lower in CD62Lneg and CD45RAneg memory CD4 T cells than in entire CD4 T cells, respectively. In allele-specific analysis, alloreactivity to single HLA-DR alleles clearly exceeded that to HLA-DQ alleles. In terms of alloproliferation no significant difference could be observed between individual CD4 T-cell subsets. rnThe second part of this thesis dealed with the generation of allo-HLA-DQ/-DP specific CD4 T cells. Naive CD45RApos CD4 T cells isolated from healthy donor PBMC by flow cytometric cell sorting were stimulated in MLR against single allo-HLA-DQ/-DP alleles transfected into autologous mature monocyte-derived dendritic cells by mRNA electroporation. Rapidly expanding HLA-DQ/-DP mismatch reactive T cells significantly recognized and cytolysed primary acute myeloid leukemia (AML) blasts, fibroblasts (FB) and keratinocytes (KC) in IFN-γ ELISpot and 51chromium release assays if the targets carried the HLA DQ/ DP allele used for T cell priming. While AML blasts were recognized independent of pre-incubating them with IFN-γ, recognition of FB and KC required IFN-γ pre treatment. We further investigated HLA class II expression on hematopoietic and non-hematopoietic cells by flow cytometry. HLA class II was not detected on primary FB, KC, and non-malignant kidney cells, but was expressed at significant levels on primary AML blasts and B-LCL. Up-regulation of HLA class II expression was observed on all cell types after pre-incubation with IFN-γ.rnIn summary, the novel K562-HLA based MLR approach revealed that naive-depleted CD4 T-cell subsets of healthy individuals contain decreased allo-HLA reactivity in vitro. We propose the application of CD45RAneg naive-depleted CD4 T cells as memory T cell therapy, which might be beneficial for HLA-mismatched patients at high-risk of GvHD and low-risk of leukemia relapse. Memory T cells might also provide important post-transplant immune functions against infectious agents. Additionally, the screening approach could be employed as test system to detect donors which have low risks for the emergence of GvHD after allo-HSCT. In the second part of this thesis we developed a protocol for the generation of allo-HLA-DQ/-DP specific CD4 T cell lines, which could be applied in situations in which patient and donor are matched in all HLA alleles but one HLA-DQ/-DP allele with low GvHD potential. These T cells showed lytic activity to leukemia cells while presumably sparing non-hematopoietic tissues under non-inflammatory conditions. Therefore, they might be advantageous for allo-HSCT patients with advanced stage AML after reduced-intensity conditioning and T-cell depletion for the replenishment of anti-leukemic reactivity if the risk for disease relapse is high. rn

HLA class II antigen expression in colorectal carcinoma tumors as a favorable prognostic marker.

The goal of this study was to determine the frequency of HLA class II antigen expression in colorectal carcinoma (CRC) tumors, its association with the clinical course of the disease, and the underlying mechanism(s). Two tissue microarrays constructed with 220 and 778 CRC tumors were stained with HLA-DR, DQ, and DP antigen-specific monoclonal antibody LGII-612.14, using the immunoperoxidase staining technique. The immunohistochemical staining results were correlated with the clinical course of the disease. The functional role of HLA class II antigens expressed on CRC cells was analyzed by investigating their in vitro interactions with immune cells. HLA class II antigens were expressed in about 25% of the 220 and 21% of the 778 tumors analyzed with an overall frequency of 23%. HLA class II antigens were detected in 19% of colorectal adenomas. Importantly, the percentage of stained cells and the staining intensity were significantly lower than those detected in CRC tumors. However, HLA class II antigen staining was weakly detected only in 5.4% of 37 normal mucosa tissues. HLA class II antigen expression was associated with a favorable clinical course of the disease. In vitro stimulation with interferon gamma (IFNγ) induced HLA class II antigen expression on two of the four CRC cell lines tested. HLA class II antigen expression on CRC cells triggered interleukin-1β (IL-1β) production by resting monocytes. HLA class II antigen expression in CRC tumors is a favorable prognostic marker. This association may reflect stimulation of IL-1β production by monocytes.

Interaction of Class I Human Leukocyte Antigen (HLA-I) Molecules with Insulin Receptors and Its Effect on the Insulin-Signaling Cascade

Insulin receptor (IR) and class I major histocompatibility complex molecules associate with one another in cell membranes, but the functional consequences of this association are not defined. We found that IR and human class I molecules (HLA-I) associate in liposome membranes and that the affinity of IR for insulin and its tyrosine kinase activity increase as the HLA:IR ratio increases over the range 1:1 to 20:1. The same relationship between HLA:IR and IR function was found in a series of B-LCL cell lines. The association of HLA-I and IR depends upon the presence of free HLA heavy chains. All of the effects noted were reduced or abrogated if liposomes or cells were incubated with excess HLA-I light chain, β2-microglobulin. Increasing HLA:IR also enhanced phosphorylation of insulin receptor substrate-1 and the activation of phosphoinositide 3-kinase. HLA-I molecules themselves were phosphorylated on tyrosine and associated with phosphoinositide 3-kinase when B-LCL were stimulated with insulin.