Per Grufman

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.

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.

T cell competition for the antigen-presenting cell as a model for immunodominance in the cytotoxic T lymphocyte response against minor histocompatibility antigens

We recently demonstrated that spleen cells primed against dominant BALB.B antigens can inhibit the cytotoxic T lymphocyte (CTL) response against subdominant antigens in vitro. In this study, we show that this interference is dependent on CD8+, but not CD4+, T cells directed against dominant antigens. Similar to immunodominance in vivo, T cell interference in vitro required presentation of dominant and sobdominant antigens by the same antigen‐presenting cell. In vivo priming with cells expressing dominant and subdominant antigens did not induce long‐lasting unresponsiveness against the latter. These results support a model in which immunodominance is mediated by T cell competition. In line with this, we found that the immunodominance effects in the CTL response against these minor histocompatibility antigens could be broken by immunization with live bone marrow‐derived dendritic cells.

A Structural Basis for LCMV Immune Evasion: Subversion of H-2Db and H-2Kb 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 peptides 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.

Innate and adaptive immunity to tumors: IL-12 is required for optimal responses

We have investigated the importance of endogenously produced IL‐12 in innate and adaptive immunity to tumor transplants. The immunogenic lymphoma RMA and its TAP‐deficient variant RMA‐S were tested for rejection responses by normal and IL‐12‐deficient mice. IL‐12 was crucial for the immunity induced by one immunization with irradiated RMA cells, as well as for in vivo priming of a CTL response in mixed lymphocyte tumor cultures against this MHC class I‐expressing tumor. The defective in vivo response could be overcome by multiple immunizations. In further studies of in vitro CTL responses, we found that IL‐12 production from either the antigen‐pulsed dendritic cells or from host cells was necessary to obtain strong CTL responses. In the complete absence of IL‐12, no or only very weak responses could be detected. NK cell‐mediated innate resistance, as assessed in non‐immunized mice inoculated with a threshold dose of RMA‐S cells, also required IL‐12. However, NK cells with reduced activity were present in IL‐12‐deficient mice and contributed to innate resistance, as demonstrated with lower cell dose challenges. In conclusion, IL‐12 is required for optimal adaptive and innate responses against tumors.

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.

IMMUNIZATION WITH DENDRITIC CELLS BREAKS IMMUNODOMINANCE IN CTL RESPONSES AGAINST MINOR HISTOCOMPATIBILITY AND SYNTHETIC PEPTIDE ANTIGENS

We have examined the mechanisms involved in immunodominance in two different experimental models: the cytotoxic T lymphocyte (CTL) response in B6 mice against minor histocompatibility antigens of BALB.B mice, and the response of B6 mice against a mixture of five synthetic peptides corresponding to well‐defined immunogenic epitopes. The CTL responses in these models focus on a few dominant epitopes, whereas no or only weak responses can be detected against other subdominant epitopes. Neither of these immunodominance phenomena can be explained by insufficient presentation of subdominant epitopes in the presence of the dominant ones. Immunodominance could also be demonstrated in an in vitro system, in which B6 splenocytes primed with BALB.B could interfere with the CTL response against subdominant antigens. This interference was dependent on CD8+ T cells and on the simultaneous presentation of dominant and subdominant antigens on the same antigen‐presenting cell, suggesting T cell competition around the antigen‐presenting cell as a potential explanation. The immunodominance in both systems could be broken by immunization with dendritic cells (from BALB.B or from B6 loaded with peptides). This procedure allowed detection of CTL responses against both dominant and previously subdominant antigens. J. Leukoc. Biol. 66: 268–271; 1999.

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.

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.