Expression Of HLA Antigens Research Articles

Formation of Genetically Determined Resistance against Human Norovirus Infection through Polymorphism of the FUT2 gene: a Review of the Literature

Relevance. Human Noroviruses (HuNoV) are highly contagious pathogens responsible of acute human norovirus infection. HuNoV is the cause of every fifth case of acute non-bacterial gastroenteritis, annually causing about 699 million cases of the disease and more than 200 thousand deaths worldwide. Controlled expression of the HBGA antigens by the FUT2 gene causes resistance to human norovirus. Polymorphisms of the FUT family genes contribute to partial or complete immunity to certain genogroups/ genotypes of norovirus.Aims. To characterize the effect of FUT2 gene polymorphisms on susceptibility to HuNoV.Results. Nonsensemutations of G428A in two homologous alleles contribute to the formation of a secretory-negative phenotype (se), which is a factor determining immunity to noroviruses. Some missense-mutations in the nucleotide positions se385,571 form partial resistance against certain genotypes. People with a secretory-negative phenotype are immune to infection by the GII.4 genotype and its genovariants.Conclusions. The expression of HLA antigens by the functionally inactive FUT2 gene plays a key role in the resistance of the human population to HuNoV. Susceptibility to HuNoV largely depends on the prevalence of HBGA phenotypic diversity among ethnic populations around the world. Targeted screening aimed at identifying polymorphisms of the FUT family will allow identifying risk groups more susceptible to HuNoV.

Expression of Class II Human Leukocyte Antigens on Human Endothelial Cells Shows High Interindividual and Intersubclass Heterogeneity.

Donor-specific antibodies against class II HLA are a major cause of chronic kidney graft rejection. Nonetheless, some patients presenting with these antibodies remain in stable histological and clinical condition. This study describes the use of endothelial colony-forming cell lines to test the hypothesis of the heterogeneous expression of HLA molecules on endothelial cells in humans. Flow cytometry and immunofluorescence staining revealed substantial interindividual and interlocus variability, with HLA-DQ the most variable. Our data suggest that the expression of HLA class II is predicted by locus. The measurement of endothelial expression of HLA class II in the graft could present a novel paradigm in the evaluation of the alloimmune risk in transplantation and certain diseases. HLA antigens are important targets of alloantibodies and allospecific T cells involved in graft rejection. Compared with research into understanding alloantibody development, little is known about the variability in expression of their ligands on endothelial cells. We hypothesized individual variability in the expression of HLA molecules. We generated endothelial colony forming cell lines from human peripheral blood mononuclear cells ( n =39). Flow cytometry and immunofluorescence staining were used to analyze the cells, and we assessed the relationship between HLA-DQ expression and genotype. Two cohorts of kidney transplant recipients were analyzed to correlate HLA-DQ mismatches with the extent of intragraft microvascular injury. Large variability was observed in the expression of HLA class II antigens, not only between individuals but also between subclasses. In particular, HLA-DQ antigens had a low and heterogeneous expression, ranging from 0% to 85% positive cells. On a within-patient basis, this expression was consistent between endothelial cell colonies and antigen-presenting cells. HLA-DQ5 and -DQ6 were associated with higher levels of expression, whereas HLA-DQ7, -DQ8, and -DQ9 with lower. HLA-DQ5 mismatches among kidney transplant recipients were associated with significant increase in graft microvascular. These data challenge the current paradigm that HLA antigens, in particular HLA class II, are a single genetic and post-translational entity. Understanding and assessing the variability in the expression of HLA antigens could have clinical monitoring and treatment applications in transplantation, autoimmune diseases, and oncology.

HLA AND CANCER

This review provides updated information on HLA class I and II antigens in cancer. The expression of HLA antigens in normal and tumor tissues, the physiological organization of the components of HLA antigen-processing machinery, the expression patterns of HLA antigens associated with the molecular and regulatory defects identified to date, as well as their functional and clinical significance, are described. This review summarizes clinical and experimental data on the complexity of immune escape mechanisms used by tumour cells to avoid T and natural killer cell responses. The variety of class I HLA phenotypes that can be produced by tumor cells during this process is presented. We also discuss here the potential capacity of metastatic lesions to recover MHC/HLA class I expression after immunotherapy, which depends on the reversible/ soft or irreversible/hard nature of the molecular mechanism responsible for the altered HLA class I phenotypes, and which determines the progression or regression of metastatic lesions in response to treatment. HLA сlass II genes play key roles in connecting innate and adaptive immunity in tumor rejection and when the escape route via HLA-I is already established. Antigens сlass II HLA expression in tumor cells and gives tumor cells the ability to present antigens, becoming less aggressive, and improves prognosis. Malignant tumors, as a genetic disease, are caused by structural alterations of the genome which can give rise to the expression of tumor-associated antigens in the form of either structurally altered molecules or of overexpressed normal molecules. Tumor associated antigens recognized by the immune system and induce a T-cell-mediated immune response. Outgrowing cancers use different strategies to evade destruction by the immune system. Immune evasion mechanisms affecting the expression and/or function of HLA-antigens are of special interest to tumor immunologists, since these molecules play a crucial role in the interaction of malignant cells with immune cells. This review describes the potential role of immunity control points in immunosuppression and therapeutic strategies for restoring the cytotoxicity of immune cells.

The expression of human leukocyte antigen by human ejaculated spermatozoa.

BackgroundAfter coitus and insemination, an inflammatory response is evident in the female reproductive tract (FRT). Semen contains a variety of immune‐activating components that have a major role in the induction of an immune response in the FRT. One of the most important is (human leukocyte antigen) HLA molecules which are present in soluble form in seminal plasma and in membrane form on the surface of cells (such as epithelial and leukocytes) existing in semen. Nevertheless, there is considerable debate over the expression of HLA antigens by human spermatozoa. Considering the critical role of HLA molecules in reproduction and the induction of an immune response, it is very important to clearly define HLA expression by spermatozoa and the role of these molecules in sperm morphology, motility, and strength to fertilize an egg. Therefore, the objective of this study was to determine HLA expression by ejaculated spermatozoa. The results of this study will facilitate the design of future studies.MethodSemen samples were collected from 50 healthy men with normal semen status by masturbation after 2–3 days of sexual abstinence. After purification of normal spermatozoa, HLA class I & II expression was evaluated by quantitative real‐time PCR and flow cytometry methods.ResultsThe results showed the expression of both HLA class I & class II by spermatozoa. The results also showed that the expression of HLA class Ⅱ was significantly more than HLA class Ⅰ.ConclusionSpermatozoa express both HLA class I & class II molecules.

A phase I study of PolyPEPI1018 vaccine plus maintenance therapy in patients with metastatic colorectal cancer with a predictive biomarker (OBERTO).

3557 Background: The goal of this study was to evaluate the safety, tolerability and immunogenicity of a single dose of PolyPEPI1018 as an add-on to maintenance therapy in subjects with metastatic colorectal cancer (mCRC). PolyPEPI1018 is a peptide vaccine containing 12 unique epitopes derived from 7 conserved cancer testis antigens (CTAs) frequently expressed in mCRC. These epitopes were designed to be Personal EPItopes (PEPIs), i.e. predicted by our novel PEPI test to bind to at least three autologous HLA alleles and more likely to induce T-cell responses than epitopes presented by a single HLA. Methods: mCRC patients in the first line setting received the vaccine (dose: 0.2 mg/peptide) just after the transition to maintenance therapy with a fluoropyrimidine and bevacizumab. Vaccine-specific T-cell responses were first predicted by the PEPI test (using the patient’s complete HLA genotype and antigen expression rate) and then measured by ELISpot after one cycle of vaccination. Results: Eleven patients were vaccinated with PolyPEPI1018. The most common adverse events were transient skin reactions (local inflammation at the site of the injections, e.g. erythema, redness and itchiness) and flu-like syndrome. No grade 3 or higher adverse events related to the vaccine occurred. Initial analysis on 4 patients demonstrated that T-cell responses were elicited by 96% of vaccine peptides. The overall percentage agreement between PEPI test-predicted and Elispot-measured CD8+ T cell responses was 71%, consistent with our retrospective analysis on 64 vaccine clinical trials involving 1,790 patients. Two of these 4 patients had unexpected tumor size reduction. Based on these encouraging results, the trial was amended to administer 3 doses of PolyPEPI1018 given 12 weeks apart. Conclusions: PolyPEPI1018 combined with maintenance therapy was safe and well-tolerated in mCRC patients. Unprecedented immune responses were induced after single dose, with broad CRC-specific T cell responses and high accuracy prediction of CD8+ T cell responses. This promising activity in mCRC patients led to a trial amendment to administer 3 doses of PolyPEPI1018 in combination with systemic therapy. Clinical trial information: NCT03391232.

P164 Repetitive in-vitro exposure to DFP affects MHC class I and class II antigen expression

O,O-Diisopropyl phosphofluoridate also known as Diisopropylfluorophosphate (DFP) a surrogate compound of nerve agents (Cyclosarin, VX, Sarin), is an organophosphate, used in this study to understand the mechanisms of toxicity on T cells, B cells and monocytes. Our aim was to study MHC class I & II antigen expression on T cells, B cells and monocytes after repetitive exposure to sub-lethal doses of DFP in-vitro. This study was conducted as a proof of concept since this compound was used in rodent models to study Gulf War Illness (GWI), which is a chronic multisymptom disorder affecting military veterans and civilian worker that served during 1990–91 Gulf War. We isolated T cells, B cells, and macrophages from 10 healthy donors. Freshly isolated cells were maintained in culture and were exposed to 0.1–100 μM DFP for 3 days at a 24 h interval. HLA typing was performed both by using the PCR-sequence specific primer (SSP) and by serological methods before exposure to DFP. After exposure, the cells were screened for the presence of the specific HLA-B and HLA-DR antigen. We isolated the culture supernatant to study the soluble HLA antigens. HLA typing was done for both A and B allele for T cells and DR for B cells using serology and by PCR-SSP. Once the antigens were delineated, after exposure they were tested for the specific antigen. Doses above 50 μM of DFP showed weak antigen expression. The cell surface antigen appeared weak by serology. The cell supernatant tested positive for HLA antigens, although they were combined with other proteins, it will be interesting to see how these HLA cell surface markers detach from the cell. We have isolated the supernatant for LC–MS analysis. Control cells that were not treated with DFP gave a consistently positive result and the HLA A, B, and DR were same as pre-exposure. There was no detectable sHLA in the supernatant. Exposure to organophosphate-based pesticides can cause immunotoxicity. Although we suspected and were trying to understand the GWI, these results offer a valid clue that there is relevance to HLA antigen expression and to look for soluble HLA antigen in the serum/plasma, as this sHLA, may very well initiate an autoimmune response by triggering inflammatory signals.

OR10 Data-driven modeling of flow cytometric crossmatch: Enhanced virtual crossmatching

Aim Increasingly HLA laboratories are using virtual crossmatching to predict recipient and donor compatibility using HLA antibody data and donor HLA type. However, virtual crossmatch interpretation is based on HLA experience and expertise of individual transplant centers. The purpose of this study is to develop data-driven algorithms (DDA) that predict flow cytometric crossmatch (FXM) outcomes using HLA antibody mean fluorescent intensity (MFI) data and donor HLA typing. These methods are independent of human intuition or experience and may provide insights that are otherwise imperceptible. Methods Two data sets consisting of 222 and 109 FXM with single antigen bead data for both HLA class I and II antibodies were used. Single antigen bead data was compiled against donor HLA antigens using the OneLambda assay and Luminex. The first DDA found the optimal MFI threshold using summation of mean fluorescent intensity data for class I and/or II that predicted either T cell or B cell mean channel shifts (MCS) above FXM cutoff. The second DDA applied a least-squares regression model to the HLA locus-specific data (second data set) to predict the actual T or B cell MCS. Results The threshold method yielded between 84.9% and 91.1% accuracy when using class I donor specific antibody (DSA) data to predict T cell outcome, and class I and II DSA data to predict B cell outcome. Optimal MFI thresholds of 3450 and 7560 were found for T and B cell prediction. For quality assurance, prediction of T cell MCS was attempted using class II data, resulting in 61.9% accuracy. The least-squares model increased accuracy to 93.6% and 97.2% for T and B cell MCS, respectively. Class I DSA influenced T and B cell MCS more than class II. The relative importance of an individual HLA locus on T cell prediction was found to be HLA-B > -A > -C. In the least-squares fitting, B64 had a large positive effect while A34 had a large negative effect on T cell MCS. Conclusions Utilizing DDA can expand accuracy of biologic systems beyond human experience and expertise. We showed how DDA can improve HLA crossmatch by generating high-level accuracy T and B cell FXM outcomes. Further improvements to the algorithm will incorporate HLA antigen expression and HLA antibody avidity.

Enhancement of anti-tumor immunity and survival prolongation by neoadjuvant 5-FU, oxaliplatin and irinotecan (folfirinox) in pancreatic ductal adenocarcinoma patients

Background: Due to the encouraging results in the metastatic setting, patients with locally advanced PDAC (LA PDAC) have been treated with FOLFIRINOX, often followed by radiation, with the hope of getting them to resection. Our aim was to characterize the immunological profile of the tumor microenvironment, by analyzing: i) the expression of HLA antigens by PDAC cells, since these molecules play a crucial role in the interactions of tumor cells with host’s immune system and ii) the infiltration of tumors by immune cells which has been shown to reflect patients’ antitumor immune response in the tumor microenvironment. Methods: Clinicopathologic data were collected from 63 FOLFIRINOX treated patients between 2012 and 2014, 18 photon patients (50.4Gy) between 1998 and 2010 and 154 no neoadjuvant patients between 1998 and 2012. Immunohistochemical staing was performed for HLA-A, HLA-B, HLA-C, CD8, CD4, and FoxP3. Results: Of the 235 patients the median age was 66 yo and 50% were women. Immunohistochemical staining demonstrated defects in both HLA-A and HLA-B and C expression in more than 60% of the PDACs analyzed the frequency of HLA-A defects in PDACs from FOLFIRINOX patients was significantly lower than in tumors from the other two cohorts (p = 0.001). Both CD8 + and CD4 + T cell density in tumors from FOLFIRINOX patients was significantly higher than that in tumors from photon and no neoadjuvant patients. FoxP3 + cell density in PDACs from FOLFIRINOX patients was significantly lower than that in tumors from photon and no neoadjuvant patients (p = 0.001). In the FOLFIRINOX cohort CD8 + T cell density, but not CD4 + , correlated with HLA-A expression (p < 0.001). Conclusion: FOLFIRINOX increases the expression of HLA class I and the immune infiltrate. The altered immune microenvironment may make FOLFIRINOX an attractive therapy to combine with an immune modulator in PDAC.TableResults of immunohistochemical staining of 218 surgically removed PDACs treated with neoadjuvant FOLFIRINOX alone or followed by radiotherapy (photon or proton) (n = 63), photon radiotherapy (n = 18) and no neoadjuvant therapy (n = 137)FOLFIRINOX +/− proton or photon radiationPhoton radiationNo neoadjuvantOverallp-valueCells/HPF medianIQRCells/HPF medianIQRCells/HPF medianIQRCells/HPF medianIQRFoxP3+10–231–431–521–4<0.001CD8+4024–8071–15179–302111–37<0.001N%N%N%N%HLA-A0.001 Negative11.6527.82317.63313.7 Heterogeneous3758.71161.17658.014660.3 Positive2539.7211.13224.46326.0HLA-B/C0.855 Negative57.915.664.6145.8 Heterogeneous3555.61161.17255.413857.3 Positive2336.5633.35240.08936.9HPF: high Power Field; IQR: inter-Quartile Range. p-values derived from Fisher's exact test or Kruskal Wallis test, as appropriate. Staining was not obtained for all slides for all markers (that is why Ns do not add up to group N). Open table in a new tab HPF: high Power Field; IQR: inter-Quartile Range. p-values derived from Fisher's exact test or Kruskal Wallis test, as appropriate. Staining was not obtained for all slides for all markers (that is why Ns do not add up to group N).

Preliminary findings from part 1 of COMBI-i: A phase III study of anti–PD-1 antibody PDR001 combined with dabrafenib (D) and trametinib (T) in previously untreated patients (pts) with advanced BRAF V600-mutant melanoma.

189 Background: Checkpoint inhibitors and targeted therapies have improved outcomes in pts with advanced BRAF V600–mutant melanoma; however, many still progress and die from this disease. Thus, new treatment strategies are needed. BRAF and MEK inhibitor combinations (eg, D+T) may reverse immunosuppressive phenotypes induced by oncogenic BRAF and improve sensitivity to checkpoint inhibitors by enhancing HLA and melanocytic antigen expression and tumor antigen–specific T-lymphocyte recognition. Methods: The phase 3 COMBI-i study (NCT02967692) is evaluating the anti–PD-1 antibody PDR001 in combination with D+T in treatment-naive pts with BRAF V600–mutant unresectable or metastatic melanoma in 3 parts: 1, safety run-in; 2, biomarker cohort; and 3, randomized, double-blind, placebo-controlled part. Here we report preliminary findings for 9 pts in part 1 dosed with PDR001 400 mg Q4W + D 150 mg BID + T 2 mg QD. Response was assessed at wk 12 and Q8W thereafter. Results: At data cutoff (16 Jul 2017; median follow-up, 2.7 mo), all 9 pts completed the 8-wk dose-limiting toxicity (DLT) period, during which 1 DLT (transaminitis [AST and ALT &gt; 8 × ULN]; n = 1) occurred. Adverse events (AEs) of any grade occurring in &gt; 3 pts included pyrexia (n = 9), headache (n = 6), chills (n = 4), and vomiting (n = 4). Grade 3/4 AEs reported in &gt; 1 pt included hepatitis (n = 3), increased lipase (n = 2), and increased transaminases (n = 2). AEs leading to discontinuation occurred in 2 pts (22%; transaminitis, n = 1; grade 3 hepatitis, n = 1) who permanently discontinued PDR001 but were still receiving D+T at the data cutoff. All 9 pts responded: 3 (33%) achieved a complete response (confirmed, n = 1), and 6 (67%) had partial responses (confirmed, n = 1). Additional safety and efficacy results for these 9 pts, all ongoing at the data cutoff, will be presented. Conclusions: These preliminary results indicate that PDR001 can be combined with D+T with a manageable safety profile and demonstrate promising activity in pts with BRAF V600–mutant melanoma. Clinical trial information: NCT02967692.

HLA and Histo-Blood Group Antigen Expression in Human Pluripotent Stem Cells and their Derivatives

One prerequisite for a successful clinical outcome of human pluripotent stem cell (hPSC) based therapies is immune compatibility between grafted cells/tissue and recipient. This study explores immune determinants of human embryonic stem cell lines (hESC) and induced human pluripotent stem cell (hiPSC) lines and hepatocyte- and cardiomyocyte-like cells derived from these cells. HLA class I was expressed on all pluripotent hPSC lines which upon differentiation into hepatocyte-like cells was considerably reduced in contrast to cardiomyocyte-like cells which retained class I antigens. No HLA class II antigens were found in the pluripotent or differentiated cells. Histo-blood group carbohydrate antigens SSEA-3/SSEA-4/SSEA-5, Globo H, A, Lex/Ley and sialyl-lactotetra were expressed on all hPSC lines. Blood group AB(O)H antigen expression was in accordance with ABO genotype. Interestingly, only a subpopulation of A1O1 cells expressed A. During differentiation of hPSC, some histo-blood group antigens showed congruent alteration patterns while expression of other antigens differed between the cell lines. No systematic difference in the hPSC cell surface tissue antigen expression was detected. In conclusion, hPSC and their derivatives express cell surface antigens that may cause an immune rejection. Furthermore, tissue antigen expression must be established for each individual stem cell line prior to clinical application.

P064 Serum treatment in a flow crossmatch

Aim Autoantibodies present in patient sera may cause a positive flow cytometric crossmatch that is not clinically relevant. Previous studies have shown that pronase treatment of lymphocytes reduces autoreactivity. However, in addition to removing Fc receptors, treatment with pronase can affect HLA antigen expression. More recent studies show that pronase treatment can also create cryptic epitopes, and many laboratories that treat unseparated T and B cells have reported an increased incidence of positive T cell and negative B cell crossmatches. For these reasons, our laboratory has focused on treatment of serum to enhance assay performance. Hypotonic dialysis (HD) is one of many serum treatments that are used to reduce non-specific reactivity. In this study we assess the ability of HD treatment to remove autoantibodies and improve the specificity of the flow crossmatch. Method We identified T cell ( n = 68) and B cell crossmatches ( n = 56) performed between January 2015 and May 2016 in which untreated and hypotonically dialyzed (HD) sera were used to perform an autologous crossmatch. The crossmatch was considered positive if the test serum to negative control ratio was ⩾2.5. Results There was a significant decrease in the ratio of HD compared to untreated sera in B cell autocrossmatches (mean ratio 1.16 versus 1.76; p = 0.002) and no significant difference between with the T cell autocrossmatches (mean ratio 1.05 versus 1.1; p = 0.06). Eleven (19%) B cell autocrossmatches were positive with the untreated serum (mean ratio 3.5 versus 1.8 for the HD; p = 0.005). Four autocrossmatches (7%) remained positive with HD serum. Conclusion Hypotonic dialysis reduced auto-reactivity in B cell autocrossmatches. Treatment of sera to remove interference offers several advantages; the same serum can be used in solid-phase immunoassays, an untreated serum can be used as a control and most importantly it maintains integrity of lymphocytes used in the crossmatch.

Identification and characterization of novel immunosensitizing therapeutics for melanoma (VAC3P.1060)

Abstract Recent breakthroughs in the field of immunotherapy have revolutionized the treatment of melanoma patients. Specifically, immune checkpoint blockade of CTLA-4 and PD-1 have prolonged overall survival in a subset of melanoma patients. Despite dramatic and durable results in some cases, low overall response rates to these therapies leave the majority of melanoma patients without effective immunotherapeutic treatment options. In order to identify immunotherapies which might offer improved response rates with the potential to be rapidly translated to the clinic, we developed a high-throughput screen and tested over 2000 compounds, many of which are FDA-approved for various malignancies or other diseases. The screen revealed distinct families of multikinase inhibitors and DNA-damage repair inhibitors as novel melanoma immunosensitizing agents. We show that these two classes of drugs can reduce the expression of various immunosuppressive molecules (e.g. PD-L1, CD155) on the cell surface of a wide variety of genetically distinct human melanoma cell lines. Notably, the drugs also increased the expression of HLA and melanoma antigens in most cases. Importantly, preliminary data suggests that drug treatment sensitizes melanoma cells to T cell mediated cytotoxicity. Further preclinical studies are underway to verify drug targets, identify mechanisms of action, and establish in vivo anti-tumor activity.

P028 : IMPROVING VIRTUAL CROSSMATCHES: USING TWO DIFFERENT CUTOFFS FOR RESOLUTION OF ANTI-DQ AND CLASS I/DR UNACCEPTABLE ANTIBODIES INCREASE ACCURACY IN PREDICTION OF FLOW CROSSMATCHES RESULTS

Aim Antibodies (Abs) against the HLA system is associated with an increased risk of acute and chronic rejection of the allograft plus an increase of time on the waiting transplant list. Solid phase immunoassays based single-antigen beads (SAB) are now commonly used to identify Abs and characterise their specificity. However, despite several advantages, there are still some concerns about their clinical reliability, allowing the detection of clinically irrelevant Abs. Importantly, a robust cutoff is needed to determine unacceptable Abs in patients’ sera. Before performing virtual crossmatches with our patients, we decided to review the cutoff value of 1000 mean fluorescence intensity (MFI) used in our lab. Our goal was to identify a cutoff value with a sensitivity of 95% or more associated with the highest specificity. Methods In order to find the lowest MFI value related with a positive flow crossmatch, 18 sera from our patients list showing one or few antibody specificities (>2000 MFI) were selected and serially diluted. Each serum condition was analysed by SAB and by flow crossmatch. Fresh donor cells were chosen for their expression of HLA antigens that would presumably lead to a positive flow crossmatch by the presence of a single donor specific antibody. Data were analysed by receiver operating characteristic. Results Preliminary results revealed that using a universal cutoff for the HLA system does not appear to be as predictive as the use of cutoffs specific for a group of loci or for an individual locus. In particular, the anti-DQ Abs seem to require a significantly higher cutoff to achieve an acceptable specificity. When cutoffs are calculated independently, both DQB1 and class I/DRB1 cutoffs revealed a superior predictive ability. Conclusions With the dual-purpose of safety and accessibility to organs for patients on the waiting transplant list, our data suggest that the 1000 MFI cutoff used in our lab showed good predictability in flow crossmatch results for anti-class I and DR Abs. The use of higher cutoff in the interpretation of unacceptable anti-DQ Abs shows decent sensitivity but still weak specificity. The difficulty of establishing a robust cutoff for anti-DQ Abs lead us to explore whether we can still consider anti-DQ Abs as a homogeneous group.

Expression of HLA Class I Antigen, Aspirin Use, and Survival After a Diagnosis of Colon Cancer

Use of aspirin (which inhibits platelet function) after a colon cancer diagnosis is associated with improved overall survival. Identifying predictive biomarkers of this effect could individualize therapy and decrease toxic effects. To demonstrate that survival benefit associated with low-dose aspirin use after a diagnosis of colorectal cancer might depend on HLA class I antigen expression. A cohort study with tumor blocks from 999 patients with colon cancer (surgically resected between 2002 and 2008), analyzed for HLA class I antigen and prostaglandin endoperoxide synthase 2 (PTGS2) expression using a tissue microarray. Mutation analysis of PIK3CA was also performed. Data on aspirin use after diagnosis were obtained from a prescription database. Parametric survival models with exponential (Poisson) distribution were used to model the survival. Overall survival. The overall survival benefit associated with aspirin use after a diagnosis of colon cancer had an adjusted rate ratio (RR) of 0.53 (95% CI, 0.38-0.74; P < .001) when tumors expressed HLA class I antigen compared with an RR of 1.03 (0.66-1.61; P = .91) when HLA antigen expression was lost. The benefit of aspirin was similar for tumors with strong PTGS2 expression (0.68; 0.48-0.97; P = .03), weak PTGS2 expression (0.59; 0.38-0.97; P = .02), and wild-type PIK3CA tumors (0.55; 0.40-0.75; P < .001). No association was observed with mutated PIK3CA tumors (0.73; 0.33-1.63; P = .44). Contrary to the original hypothesis, aspirin use after colon cancer diagnosis was associated with improved survival if tumors expressed HLA class I antigen. Increased PTGS2 expression or the presence of mutated PIK3CA did not predict benefit from aspirin. HLA class I antigen might serve as a predictive biomarker for adjuvant aspirin therapy in colon cancer.

Adult and cord blood endothelial progenitor cells have different gene expression profiles and immunogenic potential.

Endothelial colony-forming cells (ECFC) are endowed with vascular regenerative ability in vivo and in vitro. In this study we compared the genotypic profile and the immunogenic potential of adult and cord blood ECFC, in order to explore the feasibility of using them as a cell therapy product. ECFC were obtained from cord blood samples not suitable for haematopoietic stem cell transplantation and from adult healthy blood donors after informed consent. Genotypes were analysed by commercially available microarray assays and results were confirmed by real-time polymerase chain reaction analysis. HLA antigen expression was evaluated by flow-cytometry. Immunogenic capacity was investigated by evaluating the activation of allogeneic lymphocytes and monocytes in co-cultures with ECFC. Microarray assays revealed that the genetic profile of cord blood and adult ECFC differed in about 20% of examined genes. We found that cord blood ECFC were characterised by lower pro-inflammatory and pro-thrombotic gene expression as compared to adult ECFC. Furthermore, whereas cord blood and adult ECFCs expressed similar amount of HLA molecules both at baseline and after incubation with γ-interferon, cord blood ECFC elicited a weaker expression of pro-inflammatory cytokine genes. Finally, we observed no differences in the amount of HLA antigens expressed among cord blood ECFC, adult ECFC and mesenchymal cells. Our observations suggest that cord blood ECFC have a lower pro-inflammatory and pro-thrombotic profile than adult ECFC. These preliminary data offer level-headed evidence to use cord blood ECFC as a cell therapy product in vascular diseases.

Immune Escape Mechanisms in Diffuse Large B-Cell Lymphoma

Diffuse large B-cell lymphoma (DLBCL) is the most frequent subtype of non-Hodgkin lymphomas in Western countries. Implementation of immunotherapy using monoclonal antibodies to therapeutic protocols has led to dramatic improvements in overall survival. DLBCL became a model of a successful immunochemotherapy concept. Despite this fact, there is still a proportion of patients who do not respond to or relapse early after treatment. Growing evidence suggests that host antitumor immunity is suppressed by lymphoma cells in many ways. First, host cytotoxic T cells are directly suppressed by interaction with programmed cell death (PD) ligand on lymphoma cell surface and a similar mechanism enhances the activity of suppressive regulatory T cells (Tregs). Second, tumor cells escape host cytotoxic cells due to lower immunogenicity caused by reduced expression of HLA antigens. Both mechanisms have an origin in primary genetic events in lymphomagenesis. Rearrangement of MHC class II transcriptional activator (CIITA) gene and amplification of Janus kinase (JAK2) gene lead to enhanced expression of PD ligands 1 and 2, higher proliferation activity, and lower expression of HLA. This paper summarizes current knowledge about clinically relevant immune escape mechanisms in DLBCL.

HLA antigen expression in melanocytic lesions: Is acquisition of HLA antigen expression a biomarker of atypical (dysplastic) melanocytes?

Although criteria are established for the histologic diagnosis of atypical nevi (AN), consensus about the criteria in the diagnosis of and in the definition of AN is limited. Moreover, intraobserver and interobserver differences in the application of these criteria for the diagnosis of AN have been observed. We sought to determine the usefulness of HLA antigen expression as a biomarker of AN. The immunoperoxidase reaction was used to mark common nevi and AN with HLA class I heavy chain-, β2microglobulin (β2m)-, and HLA class II β chain-specific monoclonal antibodies. HLA class I heavy chain, β2m, and HLA class II β chain were expressed in 5 (8.6%) of the 58 common nevi and in 46 (∼72%) of the 64 atypical melanocytic lesions. Among common lesions, only halo nevi expressed HLA class I heavy chain, β2m, and HLA class II β chain. The level of HLA class I heavy chain β2m and of HLA class II β chain expression correlated with the degree of cytologic atypia and architectural disorder. The number of lesions tested and the subjective nature of the analysis of immunohistochemical staining of tissue sections are both limitations. The data presented suggest that HLA antigen expression is an objective biomarker that correlates well with the degree of cytologic atypia in AN and may: (1) be useful to distinguish common nevi from AN, and (2) represent a more objective measure to determine which AN should be excised.

HLA antigen and NK cell activating ligand expression in malignant cells: a story of loss or acquisition

Malignant transformation of cells is often associated with changes in classical and non-classical HLA class I antigen, HLA class II antigen as well as NK cell activating ligand (NKCAL) expression. These changes are believed to play a role in the clinical course of the disease since these molecules are critical to the interactions between tumor cells and components of both innate and adaptive immune system. For some time, it has been assumed that alterations in the expression profile of HLA antigens and NKCAL on malignant cells represented loss of classical HLA class I antigen and induction of HLA class II antigen, non-classical HLA class I antigen and/or NKCAL expression. In contrast to these assumptions, experimental evidence suggests that in some cases dysplastic and malignant cells can acquire classical HLA class I antigen expression and/or lose the ability to express HLA class II antigens. In light of the latter findings as well as of the revival of the cancer immune surveillance theory, a reevaluation of the interpretation of changes in HLA antigen and NKCAL expression in malignant lesions is warranted. In this article, we first briefly describe the conventional types of changes in HLA antigen and NKCAL expression that have been identified in malignant cells to date. Second, we discuss the evidence indicating that, in at least some cell types, classical HLA class I antigen expression can be acquired and/or the ability to express HLA class II antigens is lost. Third, we review the available evidence for the role of immune selective pressure in the generation of malignant lesions with changes in HLA antigen expression. This information contributes to our understanding of the role of the immune system in the control of tumor development and to the optimization of the design of immunotherapeutic strategies for the treatment of cancer.

99 Phosphorylated Signaling Proteins as Biomarkers of Antibody-Mediated Heart Allograft Rejection

Purpose Ligation of class I molecules on endothelial cells (EC) by anti-HLA Ab stimulates phosphorylation of p70 S6K, S6RP, ERK1/2 and Akt that in turn mediate cell survival and proliferation. We postulate that the capacity of Ab to mediate these signals is dependent upon the HLA Ab titer and level of HLA antigen expression on the endothelium. Methods and Materials Purified IgG fractions of heart recipient sera containing HLA Ab were tested for their ability to induce phosphorylation of S6RP, Akt, S6K and ERK in primary EC bearing relevant HLA antigens. Phosphorylation was measured by Western blot and normalized to EC treated with IgG containing no HLA Ab. The Ab binding capacity (ABC) of IgG fractions to EC was measured by flow-cytometry. Phosphorylation of paraffin embedded endomyocardial biopsies was determined by immunohistochemical staining. Results DSA with ABC values >=14000 triggered higher levels of phosphorylation of S6K (T421/S424), S6RP (S235/6), and ERK1/2 (T202/Y204). In contrast, DSA with low ABC values = Conclusions These results provide mechanistic insights into how DSA contribute to the process of AMR. High levels of DSA activate cell proliferation pathways which may play a role in the development of transplant vasculopathy. Low levels of DSA may play a role in accommodation by activating cell survival pathways. The study also emphasizes the value of immunostaining of phosphorylated kinases, such as S6K, as biomarkers of AMR.

A Retrospective Study on Standard Regimen for Vaccination in Celiac Children

Background: HLA system plays an essential role in the human immune system activity and the expression of some spe-cific HLA antigens could modify the immune response to vaccinations. Celiac Disease is included among the diseases associated to specific HLA profiles, principally characterized by the expression of the HLA DQ2 antigen. Object: Our study was a retrospective study, leaded on a group of celiac children, with the object to evaluate their immunological response to both obligatory and recommended vaccinations in childhood. Materials and methods: It was a retrospec-tive study, including 66 patients affected by celiac disease, between 3 and 15 years of age, and a control group of 50 children of the same age. All patients performed both obligatory and recommended vaccinations as indicated in the Italian standard regimen for vaccinations. The immunologic response to each vaccine was analysed and compared in the two groups. Moreover, authors also studied the immunologic response to vaccines in celiac children comparing patients whose diagnosis was made before 18 months of age with those whose disease was diagnosed after 18 months of age. Results: Our results showed that in celiac patients the immunological response to vaccine is similar to that one found in general population, except for HBV vaccine. Conclusions: the valuation of immunological response to HBV vaccine should be regularly effectuated in celiac children and revaccination should be recommended.