Association of CTLA-4 polymorphisms with improved overall survival in melanoma patients treated with CTLA-4 blockade: a pilot study.

CTLA-4 blockade with monoclonal antibodies can lead to cancer regression in patients with metastatic melanoma (MM). CTLA-4 gene polymorphisms may influence the response to anti-CTLA-4 antibodies although few data are available regarding this issue. We analyzed six CTLA-4 single nucleotide polymorphisms (-1661A > G, -1577G > A, -658C > T, -319C > T, +49A > G, and CT60G > A) in 14 Italian MM patients and 45 healthy subjects. We found a significant association between the -1577G/A and CT60G/A genotypes and improved overall survival (Pc < 0.006, Bonferroni corrected), further confirmed by the diplotype analysis (-1577 & CT60 GG-AA diplotype, p < 0.001). A positive trend toward an association between these genotypes and response to therapy was also observed.

The predictive and prognostic role of single nucleotide gene variants of PD-1 and PD-L1 in patients with advanced melanoma treated with PD-1 inhibitors.

Background: Despite having revolutionized the treatment paradigm for advanced melanoma, not all patients benefit from immune checkpoint inhibitor therapy. To date, there are no predictive biomarkers for response or the occurrence of immune-related adverse events (irAEs) to programmed cell death protein 1 (PD-1) inhibitors. Our aim was to investigate the predictive and prognostic role of single nucleotide variants (SNVs) of genes involved in the PD-1 axis. Methods: We analysed, in metastatic melanoma patients treated with nivolumab or pembrolizumab, five PD-1 SNVs, namely PD1.3 G>A (rs11568821), PD1.5 C>T (rs2227981), PD1.6 G>A (rs10204525), PD1.7 T>C(rs7421861), PD1.10 C>G (rs5582977) and three programmed death-ligand 1 (PD-L1) SNVs: +8293 C>A (rs2890658), PD-L1 C>T (rs2297136) and PD-L1 G>C (rs4143815). Association of SNV genotypic frequencies with best overall response to PD-1 inhibitors and development of irAEs were estimated through a modified Poisson regression. A Cox regression modelling approach was applied to evaluate the SNV association with OS. Results: A total of 125 patients with advanced melanoma were included in the analysis. A reduction in irAEs risk was observed in patients carrying the PD-L1 +8293 C/A genotype compared with those carrying the C/C genotype (risk ratio = 0.45; 95% CL 0.22-0.93; P = 0.031). A trend for a reduction in irAEs was also observed with the PD1.5 T allele (risk ratio = 0.70, 95% confidence limits 0.48-1.01 versus C allele). None of the SNVs was associated with response to therapy. Finally, a survival benefit was observed in patients harbouring the PD1.7 C/C genotype (hazard ratio = 0.37; 95% confidence limits 0.14-0.96; P = 0.028) in the homozygous model. Conclusions: Our study showed that PD-1.5 and PD-L1 +8293 SNVs may play a role as a predictive biomarker of development of irAEs to PD-1 inhibitors. PD1.7 SNV may also be associated with a reduction of the risk of death, although further translational research is needed to confirm these results.

The human antibody repertoire: heavy and light chain variable region gene usage in six alloantibodies specific for human HLA class I and class II alloantigens.

Peripheral blood B lymphocytes have been isolated from healthy individuals who were immunized with lymphocytes from HLA-incompatible donors and transformed with Epstein-Barr virus to produce human monoclonal cell lines specific for human HLA molecules. The cell lines have been previously characterized and are known to bind to various class I and class II alloantigens. In this report we describe the molecular characterization of the heavy and light chain variable region gene segments that are utilized by these monoclonal antibodies. Using the polymerase chain reaction and primer pairs specific for the respective constant region and VH or VL family, rearranged variable region gene segments were amplified from cDNA from individual cell lines. Products were then subcloned, sequenced and analysed for gene usage and apparent somatic mutation. The results show that the VH3 gene family predominates in a group of six heavy chains (four out of six) with one VH1 and one VH4 gene segment. The light chain variable region gene family usage is more diverse with 2 V kappa 3, 1 V kappa 1, 2 V lambda 2 and 1 V lambda 3. The extent of apparent somatic mutation is minimal, relative to our previous observations in a group of high affinity human monoclonal antibodies specific for pathogenic organisms.

Proliferation and immunoglobulin secretion of lymphoblastoid cell lines are differently affected by soluble cytokines.

The aim of our study was to investigate whether supernatant from lipopolysaccharide-activated monocytes (monocyte-factor) and/or cytokines could enhance secretion of human monoclonal antibodies specific to HLA antigens produced by Epstein-Barr virus lymphoblastoid cell lines (EBV-LCLs). In a low cell density culture system, the monocyte-factor significantly stimulated cell growth of three monoclonal and two polyclonal EBV-LCLs while no enhancement of immunoglobulin production was observed. The enhancement of proliferation was completely neutralized by an antiserum to human IL-6 suggesting that IL-6 was required for the stimulation of growth of LCLs. The effect of cytokines on proliferation showed large variations among the cell lines, with IL-1beta generally inducing the highest response. Of the cytokines tested, only IL-2 was able to enhance total immunoglobulin secretion due to the induction of a higher production of light chains. The specific anti-HLA activity was slightly increased by IL-10 although this cytokine had no effect on total immunoglobulin concentration or proliferation.

High yields of anti-HLA human monoclonal antibodies can be provided by SCID mice.

In an attempt to develop a suitable model for increasing the yield of human anti-HLA mAbs, we have used mice with SCID for i.p. injection of two human-mouse heterohybridomas. HMP1 hybridoma secretes a DQB1*0201 allele-specific human mAb whereas HMP12 secretes a human mAb recognizing the DRB1*1101, 1102, 1103, and 1104 alleles. Both hybridomas could be grown in SCID mice as localized tumors with no apparent alteration in the morphology of the cells or in the immunoglobulin secretion. Ascitic fluid was produced that showed a 600- to 1000-fold increase in monoclonal antibody cytotoxic titer as compared with that obtained in tissue culture. HLA-DQB1* and DRB1* alleles recognized by ascites and supernatants from SCID-derived cultures were analyzed by microlymphocytotoxicity assay on a small panel of B-lymphoblastoid cell lines. The results show that HLA specificity was retained after in vivo passage.

A supertypic HLA class II determinant shared by DR1 and DRw9, and crossreactive with DR2, defined by human monoclonal antibody.

A human monoclonal antibody Pez.2F5, produced by a lymphoblastoid cell line, has been established in vitro by Epstein-Barr virus (EBV) transformation of B lymphocytes isolated from the blood of a volunteer immunized with allogeneic peripheral blood leukocytes (PBLs). The antibody reacted with a new supertypic determinant expressed on all lymphoblastoid cell lines homozygous for HLA-DR1, -2, and -w9. The genetic linkage of the Pez.2F5 determinant to the HLA region was demonstrated by family segregation studies. Quantitative absorption studies indicated that DR2-positive cells required more Pez.2F5 antibody for lysis, and since their absorption capacity was significantly lower than that of DR1- or DRw9-positive cells, it is likely that the Pez.2F5 determinant of the DR2 haplotype is crossreactive but not identical with the determinant found on the latter haplotypes. In addition, on a test panel of HLA-typed B lymphocytes, Pez.2F5 showed perfect correlation with DR1 and DRw9, but reacted with only a fraction of DR2-positive cells. The Pez.2F5 determinant was found to be absent from resting T lymphocytes, but its expression could be identified on IL-2-dependent T-cell lines by cytotoxicity and flow cytofluorometric analysis. By sequential immunoprecipitation and SDS gel analysis of antigens of DR1 cells it was determined that the Pez.2F5 determinant is carried by HLA class II DR molecules. Thus, the Pez.2F5 is the first described human monoclonal antibody able to immunoprecipitate HLA class II-related molecules.

In vitro production of a human HLA alloantibody of restricted specificity (DQw2) via Epstein-Barr virus transformation.

This paper describes the in vitro production of the human alloantibody 454A.C5 with HLA-DQw2 specificity produced by a stable EBV-transformed cell line. Immune B cells for transformation were generated by planned immunization of a blood donor volunteer with repeated HLA-immunizations over the course of 9 yr. Reactivity of 454A.C5 was in exact concordance with the presence of the DQw2 antigen as defined by microcytotoxicity assay on a panel of 21 B lymphoblastoid cell lines with well-characterized HLA antigens. Family studies showed the segregation according to the HLA-DQw2 haplotype; thus we demonstrate by serological and formal genetics criteria, that the alloantibody is specifically directed against the DQw2. 454A.C5 antibody may be employed as a monospecific HLA typing reagent.

Human anti-HLA-DQw2 monoclonal antibody secreted by an Epstein-Barr-virus--transformed lymphoblastoid cell line: assessment of the monoclonality, allospecificity, and target.

IgM molecules were purified by the use of anti-IgM antibody-coupled Sepharose from the culture supernatant of an Epstein-Barr-virus-transformed lymphoblastoid cell line, MP1, that secretes alloantibodies possessing HLA-DQw2 specificity as defined by the cytotoxicity assay. The obtained IgM preparation was labeled with radioactive iodine-125I and fractionated by gel filtration. It contained pentameric IgM and smaller oligomeric IgMs. When tested by the direct cellular binding assay against a panel of HLA-typed cell lines, they all showed the DR3 and DR7 association pattern characteristic of DQw2. A weak but significant binding was detected for DR1, DR6, and DR9. On isoelectrofocusing, MP1 pentameric IgM gave a restricted banding pattern comparable to monoclonal IgM obtained from a patient with Waldenström's syndrome. Moreover, the pattern was identical to that of IgM purified from the culture supernatant of a defined hybrid clone, 162, that was generated by fusing MP1 cells with heteromyeloma D33 cells. The target class II molecules showed the dimeric structure that conforms to DQw2 molecules.

Biochemical analysis of HLA class I subunits expression in breast cancer tissues.

The expression of HLA class I alpha-chain and beta(2)-m subunits was studied at the protein level by a semiquantitative Western blot (WB) approach, in 25 primary breast tumors. The results indicated three pathways of alterations defined comparing the tumor WB gel band with the corresponding PBL gel band: (i) high downregulation pattern (the tumor WB gel band was < or =50% relative to the PBL band), which was found in 44% and 36% of tumors for alpha-chain and beta(2)-m, respectively; (ii) low downregulation pattern (the tumor gel band was between 51% and 75%), which was found in 24% and 20% of tumors for alpha-chain and beta(2)75%), which was found in 32% and 44% of tumors for alpha-chain and beta(2)-m, respectively. The concordance rate with immunohistochemistry (IHC) performed on the same tissue samples was 72% for alpha-chain and 64% for beta(2)-m. This study shows that the use of a semiquantitative WB technique can well define the levels of HLA class I antigens in an autologous setting allowing the biochemical analysis of HLA class I downregulation directly in solid tumor tissues. In addition, the WB technique can be a valuable tool to objectively support the IHC method.

Production of two human hybridomas secreting antibodies to HLA-DRw11 and--DRw8+w12 specificities.

In this study we describe the production of two human monoclonal antibodies (mAbs) HMP12 and HMP14, that recognize polymorphic HLA-DR specificities. These mAbs have been produced by hybridization of antibody-secreting Epstein-Barr virus-transformed cells with SHM-D33 human-mouse heteromyeloma. By microcytotoxicity assay HMP12 mAb was found to react with all DRw11-positive cells and HMP14 mAb with all cells bearing the DRw8 or the DRw12 specificity. Cytotoxic activity of HMP14 was completely removed after absorption with DRw8- or DRw12-positive cells and unaffected by absorption with cells carrying different DR specificities. The HLA specificity was further analyzed by cytofluorometry on mouse transfectant cells. The reactivity of the two mAbs was correlated with the presence of a particular polymorphic amino acid residue in the DR beta chain and by this approach the epitopes possibly involved in the antibody binding sites were predicted.

A novel approach to human anti-HLA mABs production: use of phage display libraries.

The production of human monoclonal antibodies was previously limited to very laborious and time-consuming processes involving EBV-transformation and/or hybridoma generation. Due to the development of molecular cloning techniques, it is now possible to produce human monoclonal antibody fragments quickly by panning phage display libraries against predefined antigenic specificities. Therefore, we tested this technology for producing human single chain Fv fragments (scFvs) against HLA-DR1 purified molecules immobilized on solid phase. Enrichment of DR1-specific phages was measured through five selection rounds of a synthetic library and revealed a 100-fold amplification. Soluble antibody fragments were then expressed and 7 out of 48 clones were found to secrete scFvs which specifically bind to DR1 molecules in ELISA. Further analysis revealed binding of the scFvs also to DR3 but not to DR5 or DR7 molecules correlating with the presence of particular polymorphic aminoacid residues in the DR beta chain. Western blot analysis indicated that the 7 scFvs react with the DR1 alpha/beta-dimer but not with free alpha- or beta- chains. This study shows that the innovative approach of phage display libraries can efficiently provide scFv fragments as useful reagents for the identification and dissection of HLA polymorphic epitopes.

Investigation of HLA class I downregulation in breast cancer by RT-PCR.

Downregulation of HLA class I antigen expression has been reported in a significant proportion of primary breast carcinomas suggesting an escape mechanism from CTL mediated lysis leading to tumor dissemination and metastasis. We have previously reported the biochemical and immunohistochemical analysis of HLA total class I (W6/32 mAb), alpha-chain (Q1/28,TP25.99 mAbs) and beta(2)-microglobulin (Namb-1 mAb) subunits expression in 25 primary breast carcinomas. This study at protein level resulted in the observation of three different HLA class I expression patterns by both techniques: high, low, and absent downregulation patterns. To better characterize the HLA class I antigens downregulation we extended such analysis also at RNA level by RT-PCR using HLA-A, HLA-B, HLA-C, and beta(2)-microglobulin specific primers either in breast cancer or normal tissues derived from the same patient. None (100%) of the alpha-chain genes analyzed in patient tumor tissues showed significant reduction of expression. In 10 patients out of 25 (40%) the beta(2)-microglobulin gene showed complete loss of expression compared with the corresponding normal tissue counterpart, which showed a constitutive expression, whereas in 2 patients (12.5%) its expression was comparable with the normal counterpart. Sequence analysis at genomic level revealed no defects affecting beta(2)-microglobulin gene in those patients showing lack of expression. Also TAP1 and TAP2 genes expression were investigated in order to confirm or exclude involvement of the MHC class I molecules assembling machinery. The RT-PCR approach mainly confirmed our beta(2)-microglobulin biochemical analysis indicating that in breast cancer specimens it is possible to address the HLA class I gene downregulation as a phenomenon occurring at post-transcriptional level mainly affecting the beta(2)-microglobulin gene expression.

Production and characterization of murine monoclonal antibodies recognizing HLA-DQ polymorphisms obtained by immunizing mice with transfected L cells.

Two polymorphic anti-HLA-DQB1 mAbs, TM 902 and TM 903, have been produced by immunizing F1 mice (Balb/C x C3H) with HLA-DQ-transfected mouse L cells. Cytotoxic analysis on a panel of HLA-typed cell lines has shown that TM 902 reacts with all the DQB1* alleles except DQB1*0501, *0502, and *0503, and DQB1*0601, *0602, *0603, and *0604, whereas TM 903 reacts with the DQB1*0501, *0502, and *0503, DQB1*0601, *0602, *0603, and *0604, and DQB1*0401 and *0402 alleles. The same reactivity pattern has been confirmed by cytofluorimetric analysis. Indirect immunofluorescence with various class-II-transfected cell lines showed no binding of both mAbs to the DR or DP products, suggesting their reactivity to the DQ products. The use of transfectants expressing HLA-DR/DQ heterodimers demonstrates that TM902 and TM903 mAbs are both specific for the DQ-beta chain. Comparison of the amino acid sequences of the DQ-beta chain suggests the involvement of residues 84-90 (QLELRTT) in the formation of TM902 epitope and of residues 54-55 (GR) in the formation of TM903 epitope.

Critical role of HLA-DR beta 1 residue 58 in multiple polymorphic epitopes recognized by xenogeneic and allogeneic antibodies.

In a previous study, we identified glutamic acid at position 58 in DR (beta 1*1101) as critical for the epitopes recognized by the DRw11-specific mAb GS88.2, as well as the I-LR1 mAb that recognizes a polymorphic epitope on DR(alpha,beta 1*1101) and some DP molecules. The purpose of this study was to determine whether other polymorphic residues contribute to these epitopes and whether DR beta glutamic acid or alanine 58 and DP beta glutamic acid 56, the analogous position in DP beta, contribute to epitopes recognized by other anti-class-II mAb and allosera. Site-directed mutagenesis and transfection were used to produce cells bearing wild-type or mutant class II molecules that were analyzed with mAbs by flow cytometry and with human allosera by absorption and subsequent microcytotoxicity assays. These studies demonstrate that the residue at DR beta position 58 plays a central role in at least three different mAb epitopes and an epitope recognized by anti-DRw11 allosera. Substitution of glutamic acid for alanine at position 58 of eight DR beta chains caused gain of binding of four mAbs to all of the mutant molecules, except DR(alpha,beta 4*0101). These data suggest that the side chains of DR beta 58 and DP beta 56 point outward from the alpha-helix and directly contact antibody.

Diverse locations of amino acids in HLA-DR beta chains involved in polymorphic antibody binding epitopes on DR(alpha, beta 1*0101), DR(alpha, beta 1*1101), and DR(alpha,beta 3*0202) molecules.

In a previous study, we used transfectants expressing hybrid HLA-DR(beta 1*0403)/DR(beta 1*0701) chains to map sequences involved in polymorphic antibody binding epitopes on DR(alpha, beta 1*0403) or DR(alpha, beta 1*0701) molecules. Amino acids 1-40 of the beta 1 domain were found to make the major contributions to most of the antibody binding epitopes studied. To begin to localize sequences that contribute to polymorphic antibody epitopes on DR(alpha,beta 1*0101), DR(alpha,beta 1*1101) and DR(alpha,beta 3*0202) molecules, we used indirect immunofluorescence and flow cytometry to assess the binding of mAb to transfectants expressing hybrid DR(beta 1*0101)/DR(beta 1*1101) or DR(beta 1*1101)/DR(beta 3*0202) chains that divide the DR beta chain into three segments: amino acids 1-40, 41-97, and the beta 2 domain. The results indicate that amino acids 41-97 of the beta 1 domain on DR(beta 1*0101), DR(beta 1*1101), or DR(beta 3*0202) are critical in most of the epitopes, including those recognized by human antibodies MP4 and MP12, and mouse mAb GS88.2, I-LR1, 21r5, and 7.3.19.1, whereas amino acids 1-40 of DR(beta 1*1101) are critical in the epitope recognized by the MCS-7 mAb, and both segments 1-40 and 41-97 of DR(beta 1*1101) are important in the epitopes recognized by the I-LR2 and UL-52 mAbs. Based on these data and comparison of DR beta allelic protein sequences, the residues that may play critical roles in these antibody binding epitopes are predicted.

The "in situ" expression of human leukocyte antigen class I antigens is not altered by cryopreservation in human arterial allografts.

This study was aimed to establish whether the cryopreservation procedure we currently use in clinics can modify arterial homograft antigenicity. To this purpose, we performed an immunohistochemical study on fresh and cryopreserved human arterial homografts to visualize the expression of HLA class I heavy and light chains "in situ" by using the HC-10 and Namb-1 monoclonal antibodies. Human femoral arteries and thoracic aortas were harvested from 18 heart-beating donors and sampled before and after cryopreservation. Arterial segments were frozen in liquid nitrogen vapors in a controlled rate freezing system. After thawing, samples were processed for routine immunohistochemistry. To standardize immunostaining, flow-cytometry indirect immunofluorescence analysis was performed on HUVEC; immunohistochemistry of human ovarian cortical vessels was performed as an additional positive control. Negative controls were performed by omitting tissue incubation with primary antibodies. HLA-class I antigens were markedly expressed by endothelial cells lining surface intima and adventitial vasa vasorum; a moderate expression was found in medial smooth muscle cells. Except for the surface unreactivity caused by loss of endothelium, results from cryopreserved arterial allografts were strictly comparable to those observed in fresh, unfrozen tissues. These results support the view that cryopreserved arterial allografts are immunogenic as their fresh counterparts; apart from smooth muscle cells which retained a moderate expression of HLA class I antigens following cryopreservation, our study suggests that the highly HC-10 positive endothelial cells we found to line the rich adventitial network of vasa vasorum are expected to be one of the major targets of the serological response in the recipient.

Association of -318 C/T and +49 A/G cytotoxic T lymphocyte antigen-4 (CTLA-4) gene polymorphisms with a clinical subset of Italian patients with systemic sclerosis.

Systemic sclerosis (SSc) is a complex and heterogeneous autoimmune disorder with a multi-factorial pathogenesis. Like other autoimmune disorders, the possible role of specific cytotoxic T lymphocyte antigen-4 (CTLA-4) gene polymorphisms in predisposing to SSc has been hypothesized, but it remains controversial. CTLA-4 promoter (-318C/T) and exon 1 (+49 A/G) polymorphisms have been analysed in 43 Italian females with SSc and in 93 unrelated matched healthy controls by a newly designed tetra-primer amplification refractory mutation system-polymerase chain reaction (T-ARMS-PCR) method. No significant association has been found with either polymorphisms.Nevertheless, SSc patients without concomitant Hashimoto's thyroiditis (HT) were carrying both the -318T allele (P = 0.031) and the +49 G allele (P = 0.076) more frequently than SSc patients with HT [defined by positivity for anti-thyroperoxidase (TPO) and anti-thyroglobulin (TGA) autoantibodies] than controls. Haplotype analysis confirms this association (P = 0.028), and suggests the predominant role of the -318T, whereas that of the +49 G, if any, seems weak. Thus, in Italian SSc patients the CTLA-4 -318C/T promoter polymorphism appears to be associated with the susceptibility to develop SSc without thyroid involvement. Larger studies are needed to confirm these findings and to clarify whether the -318C/T polymorphism is the functional responsible or whether it reflects the presence of another linked genetic element in the same chromosomal region.