Selective cytotoxic T-lymphocyte targeting of tumor immune escape variants.

Defects in major histocompatibility complex (MHC) class I-restricted antigen presentation are frequently observed in human cancers and result in escape of tumors from cytotoxic T lymphocyte (CTL) immune surveillance in mice. Here, we show the existence of a unique category of CTLs that can prevent this escape. The CTLs target an alternative repertoire of peptide epitopes that emerge in MHC class I at the surface of cells with impaired function of transporter associated with antigen processing (TAP), tapasin or the proteasome. These peptides, although derived from self antigens such as the commonly expressed Lass5 protein (also known as Trh4), are not presented by normal cells. This explains why they act as immunogenic neoantigens. The newly discovered epitopes can be exploited for immune intervention against processing-deficient tumors through adoptive T-cell transfer or peptide vaccination.

Is there a clinical need for a diagnostic test allowing detection of chain type-specific anti-A and anti-B?

BACKGROUND: Hemagglutination for detection and semiquantification of ABO antibodies is associated with large center-to-center variations and poor reproducibility. Because acceptance for transplantation and diagnosis of rejection in ABO-incompatible transplantation rely on the levels and specificity of ABO antibodies, reproducible tests that allow their detection and specificity determination are required. STUDY DESIGN AND METHODS: The level of chain type-specific anti-A and anti-B were analyzed in the sera of 44 healthy individuals of known ABO blood group using an enzyme-linked immunosorbent assay (ELISA) with polyacrylamide (PAA) conjugates of blood group A and B trisaccharides or Type 2 chain A and B tetrasaccharides. Selected sera were further analyzed by hemagglutination and in an ELISA with Types 1 to 4 chain A or B neoglycolipids (NGL) as antigens. RESULTS: Immunoglobulin (Ig)G anti-A and anti-B levels were higher (p ≤ 0.05) in blood group O than in B and A individuals. More IgM anti-A and anti-B cross-reactivity was detected in AB serum on PAA-conjugated A and B trisaccharides than on the tetrasaccharides. One of 11 blood group B and two of 12 A individuals had IgG antibodies binding the tetrasaccharide despite lack of, or very low reactivity with, the trisaccharides. IgG antibodies preferring the A and B Type 2 tetrasaccharides were of the IgG2 subclass. The NGL ELISA further supported the presence of chain type-specific anti-A and -B antibodies among nonsensitized, healthy individuals. CONCLUSION: An ELISA with structurally defined ABH antigens will allow the antibody class and fine specificity of ABO antibodies to be determined, which may improve risk assessment in ABO-incompatible transplantation.

Induction of protective CTL immunity against peptide transporter TAP-deficient tumors through dendritic cell vaccination.

A large proportion of human cancers show deficiencies in the MHC class I antigen-processing machinery. Such defects render tumors resistant to immune eradication by tumoricidal CTLs. We recently identified a unique population of CTL that selectively targets tumor immune-escape variants through recognition of MHC-presented peptides, termed TEIPP (T cell epitopes associated with impaired peptide processing), expressed on cells lacking functional TAP-peptide transporters. Previously, we showed that vaccination with TEIPP peptides mediates protection against TAP-deficient tumors. Here, we further explored the concept of TEIPP-targeted therapy using a dendritic cell (DC)-based cellular vaccine. Impairment of TAP function in DC induced the presentation of endogenous TEIPP antigens by MHC class I molecules, and immunization with these DCs protected mice against the outgrowth of TAP-deficient lymphomas and fibrosarcomas. Immune analysis of vaccinated mice revealed strong TEIPP-specific CTL responses, and a crucial role for CD8(+) cells in tumor resistance. Finally, we show that TEIPP antigens could be successfully induced in wild-type DC by introducing the viral TAP inhibitor UL49.5. Our results imply that immune intervention strategies with TAP-inhibited DC could be developed for the treatment of antigen processing-deficient cancers in humans.

Quantification of blood group A and B antibodies by flow cytometry using beads carrying A or B trisaccharides.

In the clinical management of patients receiving blood group ABO-incompatible organ allografts, it is of importance to determine the levels of blood group A and B antibodies before and after transplant. Currently used methods, which are mostly based on hemagglutination, are inexact and are associated with large intercenter variations. Here, we describe preliminary data from our efforts to establish a flow cytometry-based assay for the semiquantification of blood group A and B antibodies using beads carrying synthetic A or B trisaccharides. In agreement with previous investigations, blood group O individuals had greater levels of anti-A immunoglobulin G (IgG) than B individuals, whereas the levels of anti-A immunoglobulin M (IgM) were similar in sera from blood group O and B individuals.

Analytical Performance Characteristics of the Cepheid GeneXpert Ebola Assay for the Detection of Ebola Virus.

BACKGROUND: The recently developed Xpert® Ebola Assay is a novel nucleic acid amplification test for simplified detection of Ebola virus (EBOV) in whole blood and buccal swab samples. The assay targets sequences in two EBOV genes, lowering the risk for new variants to escape detection in the test. The objective of this report is to present analytical characteristics of the Xpert® Ebola Assay on whole blood samples. METHODS AND FINDINGS: This study evaluated the assay's analytical sensitivity, analytical specificity, inclusivity and exclusivity performance in whole blood specimens. EBOV RNA, inactivated EBOV, and infectious EBOV were used as targets. The dynamic range of the assay, the inactivation of virus, and specimen stability were also evaluated. The lower limit of detection (LoD) for the assay using inactivated virus was estimated to be 73 copies/mL (95% CI: 51-97 copies/mL). The LoD for infectious virus was estimated to be 1 plaque-forming unit/mL, and for RNA to be 232 copies/mL (95% CI 163-302 copies/mL). The assay correctly identified five different Ebola viruses, Yambuku-Mayinga, Makona-C07, Yambuku-Ecran, Gabon-Ilembe, and Kikwit-956210, and correctly excluded all non-EBOV isolates tested. The conditions used by Xpert® Ebola for inactivation of infectious virus reduced EBOV titer by ≥6 logs. CONCLUSION: In summary, we found the Xpert® Ebola Assay to have high analytical sensitivity and specificity for the detection of EBOV in whole blood. It offers ease of use, fast turnaround time, and remote monitoring. The test has an efficient viral inactivation protocol, fulfills inclusivity and exclusivity criteria, and has specimen stability characteristics consistent with the need for decentralized testing. The simplicity of the assay should enable testing in a wide variety of laboratory settings, including remote laboratories that are not capable of performing highly complex nucleic acid amplification tests, and during outbreaks where time to detection is critical.

Multicenter evaluation of a novel endothelial cell crossmatch test in kidney transplantation.

BACKGROUND: Despite their clinical importance, clinical routine tests to detect anti-endothelial cell antibodies (AECA) in organ transplantation have not been readily available. This multicenter prospective kidney transplantation trial evaluates the efficacy of a novel endothelial cell crossmatch (ECXM) test to detect donor-reactive AECA associated with kidney allograft rejection. METHODS: Pretransplant serum samples from 147 patients were tested for AECA by a novel flow cytometric crossmatch technique (XM-ONE) using peripheral blood endothelial progenitor cells as targets. Patient enrolment was based on acceptance for transplantation determined by donor lymphocyte crossmatch results. RESULTS: Donor-reactive AECA were found in 35 of 147 (24%) patients. A significantly higher proportion of patients with a positive ECXM had rejections (16 of 35, 46%) during the follow-up of at least 3 months compared with those without AECA (13 of 112, 12%; P<0.00005). Both IgG and IgM AECAs were associated with graft rejections. Mean serum creatinine levels were significantly higher in patients with a positive ECXM test at 3 and 6 months posttransplant. CONCLUSIONS: XM-ONE is quick, easy to perform on whole blood samples and identifies patients at risk for rejection and reduced graft function not identified by conventional lymphocyte crossmatches.

A case of acute vascular rejection caused by endothelial-reactive non-HLA antibodies.

We describe a female patient who, despite negative conventional cross-matches, lost her first kidney graft in an acute humoral rejection. Prior to the second, AB0-incompatible (A1B to A1) living-donor kidney transplant, the patient had negative T- and B-cell cross-matches but had a positive donor-reactive endothelial cell cross-match. Following pre-transplant protein A and GlycoSorb-ABO immunoadsorptions to remove blood group B and anti-endothelial cell antibodies, Mabthera, and IVIG administrations, she was successfully transplanted. By the second post-operative day, creatinine levels were down to 96 micromol/L from 611 micromol/L pre-operatively. On day 9 creatinine rose again, and on the same day the endothelial cell crossmatch became positive for IgG, whereas the T-cell cross-match remained negative and the anti-A1B titers remained low. A kidney biopsy taken on day 10 post-transplant showed a picture of an acute vascular, antibody-mediated rejection. Following rejection treatment and repeated protein A and Glyco-Sorb-ABO immunoadsorptions, the patient's kidney function was again normalized. The use of a recently developed kit (XM-ONE) for the detection of anti-endothelial cell antibodies allowed us to identify a patient at risk for developing acute antibody-mediated rejection as well as to monitor treatment efficacy and post-transplant complications.

A structural basis for LCMV immune evasion: subversion of H-2D(b) and H-2K(b) presentation of gp33 revealed by comparative crystal structure.Analyses.

LCMV infection of H-2(b) mice generates a CD8(+) CTL response mainly directed toward three immunodominant epitopes. One of these, gp33, is presented by both H-2D(b) and H-2K(b) MHC class I molecules. The virus can escape immune recognition in the context of both these MHC class I molecules through single mutations of the peptide. In order to understand the underlying structural mechanism, we determined the crystal structures of both complexes. The structures reveal that the peptide is presented in two diametrically opposed manners by H-2D(b) and H-2K(b), with residues used as anchor positions in one MHC class I molecule interacting with the TCR in the other. Importantly, the peptide's N-terminal residue p1K protrudes from the binding cleft in H-2K(b). We present structural evidence that explains the functional consequences of single mutations found in escape variants.