Should HLA mismatch acceptability for sensitized transplant candidates be determined at the high-resolution rather than the antigen level?

Defining HLA mismatch acceptability of organ transplant donors for sensitized recipients has traditionally been based on serologically defined HLA antigens. Now, however, it is well accepted that HLA antibodies specifically recognize a wide range of epitopes present on HLA antigens and that molecularly defined high resolution alleles corresponding to the same low resolution antigen can possess different epitope repertoires. Hence, determination of HLA compatibility at the allele level represents a more accurate approach to identify suitable donors for sensitized patients. This approach would offer opportunities for increased transplant rates and improved long term graft survivals.

Solid phase testing in the HLA laboratory: implications for organ allocation.

This piece was originally requested as a white paper from the Scientific and Clinical Affairs Committee of the American Society for Histocompatibility and Immunogenetics (ASHI), of which the author was then Chairman. Upon review by the ASHI Board of Directors and the Editors of their journal, it was considered too controversial for publication. It is intended to be provocative and controversial; it is not intended as a review of the literature. Though written with a decided 'American point of view', it is of importance that the issues facing US transplantation and laboratory testing efforts are shared to varying degrees by the international community, and who, unlike some of their American cousins, may be able to tolerate a spirited discussion. Sadly, we sometimes forget that dissent from dogma can be fun!

MHC: function and implication on vaccine development.

The purpose of this State of the Art review is to present an overview of structural and functional aspects of human MHC molecules and how polymorphisms in HLA molecules determine the nature of the antigenic peptides bound and how such may impact upon rational vaccine development. The following areas will be discussed: elements of immune recognition, class I and class II structure and function, the class I antigen processing pathway, class II antigen processing and presentation, functional divergence among T-cells recognizing class I or class II MHC molecules, and rational vaccine development. While the first section will review our current understanding of MHC structure and function, the latter part will draw on my own work with Hepatitis C Virus (HCV) antigens.

Restoration of alloreactivity of melanoma by transduction with B7.1.

Melanoma cells are unusual because, unlike most epithelial tumors, constitutive expression of human leukocyte antigen (HLA) class II molecules is common. To elucidate the role of HLA class II expression in the immunopathogenesis of melanoma, the authors compared HLA class II+ melanoma cells to autologous B cells with respect to their ability to stimulate primary (naïve) histoincompatible lymphocytes and T-cell clones (antigen experienced). Using primary lymphocytes (peripheral blood lymphocytes [PBLs]), melanoma cells were nonstimulatory when compared to autologous B cells. To determine whether this was caused by defective antigen processing, the authors used alloreactive T-cell clones, which require alloantigen presentation by a histocompatible stimulator cell but not costimulation. Melanoma cells stimulated the alloreactive T-cell clones in two of three clones tested, indicating that they processed and presented alloantigen. To determine whether the failure of melanoma cells to stimulate primary lymphocytes was caused by their inability to costimulate the T cells, the authors transduced the melanoma cells with B7.1 and achieved stable expression in more than 95% of the cells. The transduced cells were highly stimulatory, eliciting a 17- to 25-fold increase in proliferation by the peripheral blood lymphocytes compared with controls. Indeed, B7-expressing melanoma cells were more stimulatory than autologous B cells, which elicited an 11- to 15-fold increase compared with controls. These data indicate that melanoma cells fail to stimulate primary lymphocytes because they do not deliver costimulatory signals. Engineering HLA class II+ melanoma cells to express high levels of B7.1 may provide a way to elicit primary T-cell responses to melanoma-associated antigens.

Sequence-based typing of HLA class II DQB1.

Due to the expanding number of known HLA class II DQB1 alleles, high-resolution oligotyping is becoming ineffective, therefore a sequence-based typing (SBT) strategy was developed to provide rapid and definitive typing of HLA-DQB1. HLA-DQB1*02, *03, *04, *05, and *06 alleles were individually amplified by polymerase chain reaction (PCR) using exon 2 group-specific primers. Forward and reverse PCR primers were tailed with M13 universal and M13 reverse sequences, respectively. Subsequent bi-directional cycle-sequencing was carried out using Cy5.5-labeled M13 universal primer and Cy5.0-labeled M13 reverse primer. Automated sequencing was performed in 30 min using a Visible Genetics, Inc. (VGI) MicroGene Clipper Sequencer. Full concordance was observed between this SBT method and oligotyping among 151 individuals.

Immunobiology of hepatitis C virus (HCV) infection: the role of CD4 T cells in HCV infection.

Hepatitis C virus (HCV) is yet another example of a pathogen that persists in the presence of a readily apparent immune response. As evidence for both humoral and cellular immune responsiveness is quite strong, our studies have begun to examine whether qualitative defects in CD4 T-cell responses to viral antigens may help to explain why HCV is not eliminated in the vast majority of infections. Direct evidence that CD4 T cells play a role in HCV persistence is lacking, but several observations are consistent with this possibility. Importantly, it does not exclude the role of antibody or killer T cells in the immunopathogenesis of HCV infection. In addition, we discuss the consequences of viral mutation and how naturally occurring variants in immunodominant viral epitopes can effectively suppress helper T-cell responses to wild type virus.

CD4(+) T cells kill HLA-class-II-antigen-positive melanoma cells presenting peptide in vitro.

PURPOSE: Most melanoma cell lines express HLA class II antigens constitutively or can be induced to do so with interferon gamma (IFNgamma). We have previously demonstrated that peptide-specific CD4(+) T cells proliferate in response to HLA-class-II-antigen-mediated peptide presentation by melanoma cells in vitro and produce interleukin-10 (IL-10) and (IFNgamma). We asked whether the responding T cells kill the tumor cells and, if so, whether direct cell contact was required. METHODS: Two HLA class II(+) melanoma cell lines derived from metastases were co-cultured with a human CD4(+) T cell clone specific for influenza hemagglutinin peptide (HA). T cells, melanoma, and HA were co-cultured for 48 h. Melanoma cells with and without HA and/or T cells served as controls. After 36 h, the medium was removed for cytokine analysis by enzyme-linked immunosorbent assay (ELISA). Twelve hours later non-adherent cells were washed away and the adherent melanoma cells were trypsinized and counted. Dual-chamber culture plates were used to determine whether cell contact and/or exposure to cytokine were required for tumor cell death. RESULTS: Melanoma cell counts were over 80% lower in wells containing T cells than in wells with melanoma and peptide alone (P < 0.05). ELISA of supernatants revealed production of IFNgamma and IL-10 by the responding T cells. Direct T cell contact with tumor cells was not required for tumor cell death, as melanoma cells were killed when they shared medium but had no contact with T cells responding to peptide presentation by HLA-class-II-antigen-positive melanoma cells in a separate chamber. Blocking antibody to IFNgamma but not IL-10 prevented melanoma cell death at levels of cytokine similar to that present in co-culture assays. CONCLUSIONS: Peptide-specific CD4(+) T cells kill melanoma cells in vitro when they recognize peptide presented by the tumor cell in the context of HLA class II antigen. Direct cell contact is not required, suggesting that it is a cytokine-mediated event. Immunotherapy, using primed CD4(+) T cells and peptide, may be beneficial in patients whose tumors express HLA class II antigens or can be induced to do so with IFNgamma.

Cytokine production by CD4+ T-cells responding to antigen presentation by melanoma cells.

Melanoma cells are unusual because, unlike most epithelial tumours, constitutive expression of HLA class II antigens is common. We have previously demonstrated that a peptide-specific CD4+ T-cell clone proliferates briskly in response to peptide and HLA class II expressing melanoma cell lines derived from metastases. Here we demonstrate that these CD4+ T-cells secrete large amounts of interferon-gamma (IFNgamma) and interleukin-10 (IL10), and insignificant quantities of IL2 or IL4, in response to peptide presentation by both melanoma and autologous B-cells. T-cells produced more IL10 when responding to peptide presentation by melanoma cells compared with B-cells, and less IFNgamma (P<0.01). Addition of IL12 did not alter the cytokines produced but increased the T-cell production of both, especially the production of IL10 in response to peptide presentation by melanoma cells. Our data suggest that differential cytokine production by CD4+ T-cells in response to peptide presentation by HLA class II expressing tumour cells may contribute to tolerance to tumour antigens.

In vitro human Th-cell responses to a recombinant hepatitis C virus antigen: failure in IL-2 production despite proliferation.

Hepatitis C Virus (HCV) causes chronic infection in 80-90% of those exposed and persists despite evidence of immune recognition. To understand the immunological basis of this phenomenon, we have synthesized a non structural (NS) protein that is critical to HCV infection and replication, NS3, and used it to study in vitro helper T-cell responses from infected individuals. Strong proliferative responses were generated by peripheral T-cells isolated from a subset of chronically infected patients, but not by normal, non-infected controls. Interestingly, though gamma-interferon (gammaIfn) and IL-10 were both secreted in response to stimulation by NS3 antigen, IL-2 was not. In contrast, IL-2 was secreted in response to influenza virus vaccine antigen. Lack of IL-2 induction was confirmed by a failure to amplify IL-2 mRNA upon NS3 antigen stimulation, whereas IL-4, IL-15, and gammaIfn mRNA were seen as early as 24 h. The predominance of IL-4 and IL-10 and the lack of IL-2 suggests that in vitro responses to at least some HCV antigens are biased towards a Th2 phenotype, which may be conducive to viral persistence.

Identification of antigenic escape variants in an immunodominant epitope of hepatitis C virus.

Numerous investigators have postulated that one mechanism by which hepatitis C virus (HCV) may evade the immune system is through the formation of escape mutants. This hypothesis is based largely on the observed mutability of the viral genome resulting in evolution of diverse quasispecies over the course of infection. That such diversification is a product of viral RNA polymerase infidelity, immune-driven selection or a combination of the two processes has not been addressed. We have examined sequence variability in a specific segment of HCV RNA encoding a known immunodominant region of the viral helicase, amino acids 358-375 of the non-structural 3 protein. Using sequence-specific oligonucleotide probe hybridization and automated DNA sequencing, we report a high frequency of mutations, essentially all of which result in amino acid replacements. To assess the biological impact of such mutations, corresponding chemically synthesized peptides were compared to wild-type peptide in T cell proliferation assays. We observed that a sizeable fraction of such peptides stimulated attenuated or negligible levels of proliferation by peripheral T cells from a chronically infected patient. This observation is consistent with expectations for immune-mediated selection of escape variants at the epitope level. We postulate that such a mechanism may be important in the immunopathogenesis of HCV infections.

Mutations in immunodominant T cell epitopes derived from the nonstructural 3 protein of hepatitis C virus have the potential for generating escape variants that may have important consequences for T cell recognition.

One of the most disturbing features of hepatitis C virus (HCV) is its long-term persistence in the host. One hypothesis to explain this phenomenon is that HCV escapes immune recognition through its intrinsic hypermutability. To determine whether immunodominant T cell epitopes derived from HCV nonstructural 3 (NS3) protein might be subject to sequence variations leading to escape mutants, we examined sequence variations of one IL-2-producing epitope, NS3358-375, and one IL-10-producing epitope, NS3505-521. By PCR amplification, cloning, and sequencing, we observed significant sequence variations in the two epitopes, although the selection intensity for each epitope was different. For NS3358-375, more variants were observed, and for NS3505-521, fewer mutations were observed. Moreover, functional studies revealed that three NS3358-375 and one NS3505-521 variants failed to stimulate T cell proliferation, and two other NS3358-375 and NS3505-521 variants weakly stimulated T cell responses. Our results are consistent with immune selection of viral variants at the epitope level, which may enable HCV to evade host defenses over time.

Functionally distinct T-cell epitopes within the hepatitis C virus non-structural 3 protein.

Clearance of Hepatitis C Virus (HCV) infection is an uncommon phenomenon. To understand the mechanism of viral persistence despite active cellular and humoral responses, we examined the in vitro cytokine response of PBMC from an HCV sero-positive, asymptomatic individual to recombinant intact antigen and sixty-nine overlapping peptides of the HCV non-structural (NS) 3 protein. Whereas, intact antigen induced strong proliferation and significant levels of gammaIFN and IL-10, little or no IL-2 was produced. Only 7% of peptides induced IL-2, which also coincided with their ability to stimulate proliferation. In contrast, 38% of the peptides induced gammaIFN while 35% induced IL-10. All IL-2 stimulating peptides also induced significant levels of gammaIFN and among these, a peptide corresponding to residues 358-375 was the strongest. In addition, 16% of the peptides induced both gammaIFN and IL-10. Exogenous recombinant IL-10 inhibited proliferation and IL-2 induction in response to peptide 358-375. Furthermore, neutralization of IL-10 with an anti-IL-10 antibody resulted in enhanced IL-2 production in response to recombinant NS3 protein. We suggest that IL-10 inducing epitopes within HCV NS3 may thus down-regulate IL-2 dependent T-cell responses.

A site for CD4 binding in the beta 1 domain of the MHC class II protein HLA-DR1.

Using a lymphocyte binding assay, we have previously demonstrated that the CD4 protein can mediate cell adhesion by direct interaction with MHC class II molecules. In this report, we have used this assay to test whether synthetic peptides, corresponding to DR beta sequences, could inhibit CD4-class II adhesion. A peptide derived from sequences within the beta1 domain (DR beta 41-55), as well as two peptides derived from sequences within the beta 2 domain (DR beta 121-135 and DR beta 141-155), were shown to inhibit CD4-class II adhesion. Inasmuch as a site for CD4 binding in the beta 2 domain had been previously documented, these studies were designed to investigate the role of the beta 1 domain as an additional site of interaction with CD4. Sixteen site-specific mutations were engineered within the beta1 domain of DR beta 1*0101. Several mutations were shown to disrupt CD4-dependent T cell activation. Based on these results, we propose a model for the molecular interaction of CD4 with MHC class II proteins in which both the beta 1 and beta 2 domains of class II interact with the two amino-terminal Ig-like domains of CD4.

The association of HLA-DR15 and intermediate uveitis.

PURPOSE: To evaluate the association between human leukocyte antigen (HLA-DR15) specificity and intermediate uveitis. METHODS: Eighteen patients diagnosed with intermediate uveitis underwent HLA-DR15 serotyping. Additionally, DNA-based phenotyping for a specific HLA-DR15 allele was performed in four patients. The clinical features of HLA-DR15-positive intermediate uveitis were compared with those of HLA-DR15-negative intermediate uveitis. RESULTS: Thirteen of 18 patients (72%) were positive for HLA-DR15. The frequency of the HLA-DR15 specificity in intermediate uveitis patients was significantly higher than in the control subjects (relative risk, 6.36; P < .001). Each of four patients tested carried the specific allele, DR beta 1*1501, which has been associated with multiple sclerosis. In the HLA-DR15-positive group were four patients (31%) with coexisting multiple sclerosis or optic neuritis, one patient with coexisting narcolepsy, and three patients (23%) with a family history of multiple sclerosis. Retinal periphlebitis, especially if bilateral, was a frequent ophthalmoscopic finding in HLA-DR15-positive intermediate uveitis. CONCLUSIONS: This study identifies a significant association between intermediate uveitis and the HLA-DR15 specificity. Patients who are HLA-DR15-positive and have intermediate uveitis may have systemic findings of another HLA-DR15-related disorder. Intermediate uveitis may belong to a constellation of HLA-DR15-related disorders, which includes multiple sclerosis, optic neuritis, and narcolepsy.

MHC class II-mediated antigen presentation by melanoma cells.

Constitutive expression of major histocompatibility complex (MHC) class II molecules is normally restricted to professional antigen-presenting cells (APCs) of the immune system, although it also occurs frequently in melanoma. Clinical evidence suggesting that MHC class II expression by melanoma is associated with tumor progression led us to postulate a role for MHC class II-mediated antigen presentation in this disease. First, we investigated whether melanoma cells derived from metastases can process antigen and/or present peptide vi MHC class II molecules to a peptide-specific CD4+ T-cell clone. In all cell lines tested, melanoma cells were able to process antigen and present peptide efficiently to CD4+ T cells, resulting in T-cell proliferation increased 5-26-fold over controls. Next, we found that CD28-mediated costimulation was not required, because blocking with CTLA-4Ig had no effect on the T-cell response to either melanoma or B cells as APCs. In contrast, blocking CD54 (ICAM-1) resulted in a decrease in proliferation in response to peptide presentation by melanoma but not B cells. These data demonstrate that MHC class II molecules on melanoma cells are functional and that antigen-processing pathways are intact. In addition, CD54 seems to play a significant role in peptide presentation by melanoma.

Diverse usage of human T-cell receptor gene segments in HLA-DR1 allospecific T-cell clones.

T-cell recognition of alloantigen involves both the MHC molecule and its associated peptide ligand. To understand the relationship between the specificity of alloantigen recognition and the structure of TCR molecules, we have investigated TCR gene utilization by sequencing TCR genes from well-defined allospecific T-lymphocyte clones. Alloreactive TLC consisted of a panel of clones primed to recognize DR1-related alloantigens. Our sequencing results revealed extensively diverse, but nonrandom, usage of TCR AV and BV gene segments and essentially no conservation in CDR3 or junctional sequences. Such observations are consistent with allospecific TCR that interact with MHC molecules on a generic level while recognizing specific peptides. They also reduce potential enthusiasm for anti-TCR therapy in allograft rejection.

T cell recognition of MHC class II-associated peptides is independent of peptide affinity for MHC and sodium dodecyl sulfate stability of the peptide/MHC complex. Effects of conservative amino acid substitutions at anchor position 1 of influenza matrix protein19-31.

T cells recognize peptide fragments of Ags bound to MHC-encoded molecules. Pockets in the MHC peptide-binding groove accommodate a limited set of amino acid side chains present at anchor positions in peptide; however, the functional significance of accommodation of different side chains at an anchor position in peptide is not clear. A panel of T cell clones was evaluated to test the effect of conservative amino acid substitution at a primary peptide anchor position. Results of T cell stimulation studies were correlated with two well studied characteristics of the peptide/MHC complex, which are the affinity of peptide binding to MHC and the stability of the resulting complex upon PAGE in the presence of SDS. We found that formation of a functional complex required neither high affinity peptide binding nor SDS stability. Furthermore, T cell clones differed in their ability to recognize individual peptide variants, suggesting that some structural aspect of the peptide/MHC complex is influenced by interactions between peptide anchor residues and MHC pockets.

Differential activation through the TCR-CD3 complex affects the requirement for costimulation of human T cells.

Although it is well established that T cells require at least two activation signals, the coordination of primary signaling through the TCR-CD3 complex with costimulatory signals through accessory molecules is incompletely understood. To mimic the signal provided by natural ligand for TCR, we used eight anti-TCR V-region-specific mAbs as well as two anti-TCR-CD3 mAbs, OKT3 and T10B9, to stimulate human peripheral blood T cells in the presence or absence of accessory cells. With accessory cells, only OKT3 in soluble form stimulated T cells, but when mABs were immobilized on plastic, all except the V alpha mAb, F1, induced proliferation. This result suggests that the signaling qualities of TCR V-region-specific mAb may differ from OKT3, which activates through CD3 epsilon. To address this issue, the costimulatory requirements of two V beta-specific mAbs, 1C1 and OT145, were also compared with OKT3 and T10B9 using T cells depleted of accessory cells. The V beta-specific mAb and T10B9 could only be complemented by costimulation through the CD28 molecules, whereas OKT3 was able to synergize with mAb directed not only at CD28, but also CD2 and CD11a. Furthermore, mAb specific for CD80 was able to block activation of T cells in the presence of accessory cells when V beta-specific mAbs were used to activate, whereas anti-CD80 had no effect on activation of T cells by immobilized OKT3. Thus the nature of the signal received through the TCR-CD3 complex, whether TCR alpha beta or CD3 epsilon, may determine the qualitative requirements for costimulation.