Nicole A. Mifsud

Human Leukocyte Antigen Class I-Restricted Activation of CD8+ T Cells Provides the Immunogenetic Basis of a Systemic Drug Hypersensitivity

The basis for strong immunogenetic associations between particular human leukocyte antigen (HLA) class I allotypes and inflammatory conditions like Behçets disease (HLA-B51) and ankylosing spondylitis (HLA-B27) remain mysterious. Recently, however, even stronger HLA associations are reported in drug hypersensitivities to the reverse-transcriptase inhibitor abacavir (HLA-B57), the gout prophylactic allopurinol (HLA-B58), and the antiepileptic carbamazepine (HLA-B*1502), providing a defined disease trigger and suggesting a general mechanism for these associations. We show that systemic reactions to abacavir were driven by drug-specific activation of cytokine-producing, cytotoxic CD8+ T cells. Recognition of abacavir required the transporter associated with antigen presentation and tapasin, was fixation sensitive, and was uniquely restricted by HLA-B*5701 and not closely related HLA allotypes with polymorphisms in the antigen-binding cleft. Hence, the strong association of HLA-B*5701 with abacavir hypersensitivity reflects specificity through creation of a unique ligand as well as HLA-restricted antigen presentation, suggesting a basis for the strong HLA class I-association with certain inflammatory disorders.

A Naturally Selected Dimorphism within the HLA-B44 Supertype Alters Class I Structure, Peptide Repertoire, and T Cell Recognition

HLA-B*4402 and B*4403 are naturally occurring MHC class I alleles that are both found at a high frequency in all human populations, and yet they only differ by one residue on the α2 helix (B*4402 Asp156→B*4403 Leu156). CTLs discriminate between HLA-B*4402 and B*4403, and these allotypes stimulate strong mutual allogeneic responses reflecting their known barrier to hemopoeitic stem cell transplantation. Although HLA-B*4402 and B*4403 share >95% of their peptide repertoire, B*4403 presents more unique peptides than B*4402, consistent with the stronger T cell alloreactivity observed toward B*4403 compared with B*4402. Crystal structures of B*4402 and B*4403 show how the polymorphism at position 156 is completely buried and yet alters both the peptide and the heavy chain conformation, relaxing ligand selection by B*4403 compared with B*4402. Thus, the polymorphism between HLA-B*4402 and B*4403 modifies both peptide repertoire and T cell recognition, and is reflected in the paradoxically powerful alloreactivity that occurs across this “minimal” mismatch. The findings suggest that these closely related class I genes are maintained in diverse human populations through their differential impact on the selection of peptide ligands and the T cell repertoire.

Natural HLA class I polymorphism controls the pathway of antigen presentation and susceptibility to viral evasion

HLA class I polymorphism creates diversity in epitope specificity and T cell repertoire. We show that HLA polymorphism also controls the choice of Ag presentation pathway. A single amino acid polymorphism that distinguishes HLA-B*4402 (Asp116) from B*4405 (Tyr116) permits B*4405 to constitutively acquire peptides without any detectable incorporation into the transporter associated with Ag presentation (TAP)-associated peptide loading complex even under conditions of extreme peptide starvation. This mode of peptide capture is less susceptible to viral interference than the conventional loading pathway used by HLA-B*4402 that involves assembly of class I molecules within the peptide loading complex. Thus, B*4402 and B*4405 are at opposite extremes of a natural spectrum in HLA class I dependence on the PLC for Ag presentation. These findings unveil a new layer of MHC polymorphism that affects the generic pathway of Ag loading, revealing an unsuspected evolutionary trade-off in selection for optimal HLA class I loading versus effective pathogen evasion.

Linking CMV Serostatus to Episodes of CMV Reactivation Following Lung Transplantation by Measuring CMV-Specific CD8+ T-Cell Immunity

CMV‐specific immunity was assessed in a longitudinal cohort of 39 lung transplant recipients (LTR) who were followed prospectively from the time of transplant using a novel assay. At the time of surveillance bronchoscopy, CMV‐specific CD8+ T‐cell responses were assessed in the peripheral blood, using the QuantiFERON®‐CMV assay, which measures IFN‐γ‐secreting T cells following stimulation with CMV peptides. In total, 297 samples were collected from 39 LTR (CMV D+/R−, n = 8; D+/R+, n = 18; D−/R+, n = 6; D−/R−, n = 7). CMV‐specific T‐cell immunity was not detected in any of the CMV D−/R− LTR. In CMV seropositive LTR levels of CMV immunity were lowest early posttransplant and increased thereafter. While levels of CMV‐specific immunity varied between LTR, measurements at any one time point did not predict episodes of CMV reactivation. In CMV mismatched (D+/R−) LTR, primary CMV immunity was not observed during the period of antiviral prophylaxis, but typically developed during episodes of CMV reactivation. Measuring CMV‐specific CD8+ T‐cell function with the QuantiFERON®‐CMV assay provides insights into the interrelationship between CMV immunity and CMV reactivation in individual LTR. A better understanding of these dynamics may allow the opportunity to individualize antiviral prophylaxis in the future.

Cell‐mediated rejection results in allograft loss after liver cell transplantation

Liver cell transplantation in humans has been impeded by invariable loss of the graft. It is unclear whether graft loss is due to an immune response against donor hepatocytes. Transplantation with ABO‐matched liver cells was performed in a patient with Crigler‐Najjar type 1. After successful engraftment, there was a gradual loss of graft function. Solid‐phase enzyme immunoassay testing and cell‐complement cytotoxicity assays detecting preformed antibodies directed toward class I and/or class II human leukocyte antigen (HLA) molecules were negative. In contrast, a striking host alloresponse to either the HLA‐B39 or C7 antigen was found, suggesting that a vigorous response to a defined mismatched HLA antigen contributed to graft loss in our patient. This study provides evidence that a T‐cell–mediated immune mechanism could be responsible for human liver cell transplant graft loss. This finding warrants confirmation in future liver cell transplants in humans. Liver Transpl 14:688–694, 2008.

Striking Immunodominance Hierarchy of Naturally Occurring CD8+ and CD4+ T Cell Responses to Tumor Antigen NY-ESO-1

Immunodominance has been well-demonstrated in many antiviral and antibacterial systems, but much less so in the setting of immune responses against cancer. Tumor Ag-specific CD8+ T cells keep cancer cells in check via immunosurveillance and shape tumor development through immunoediting. Because most tumor Ags are self Ags, the breadth and depth of antitumor immune responses have not been well-appreciated. To design and develop antitumor vaccines, it is important to understand the immunodominance hierarchy and its underlying mechanisms, and to identify the most immunodominant tumor Ag-specific T cells. We have comprehensively analyzed spontaneous cellular immune responses of one individual and show that multiple tumor Ags are targeted by the patient’s immune system, especially the “cancer-testis” tumor Ag NY-ESO-1. The pattern of anti-NY-ESO-1 T cell responses in this patient closely resembles the classical broad yet hierarchical antiviral immunity and was confirmed in a second subject.

A novel cis‐AB variant allele arising from a nucleotide substitution A796C in the B transferase gene

A novel cis-AB variant allele arising from a nucleotide substitution A796C in the B transferase gene Cis-AB is a rare phenotype of the ABO blood group system.1 Serologically, it is characterized by the presence of A, weakened B, and elevated H antigens on the RBCs. A nonautoanti-B is usually detected in the serum. Of the studies that have reported the nucleotide (nt) sequence of the cisAB allele, all have demonstrated an A allele bearing the G803C nt substitution characteristic of the B allele.2-4 This nt substitution results in the amino acid (aa) substitution, Gly268→Ala, at one of two critical positions for determining A/B-transferase specificity, with the other position being aa 266 encoded by nt 796.5 Here we report the identification of a novel cis-AB allele* that appears to have arisen from a single nt substitution at position 796 in the B-transferase gene. The propositus, a 37-year-old Vietnamese man, presented during routine blood grouping and screening for related-donor organ matching. By serologic typing, the propositus typed as A2Bweak with elevated H antigen on his RBCs, while a non-autoanti-B and a weak anti-A1 were detected in his serum. The father of the propositus also demonstrated this phenotype. Transfersase studies could not be conducted. The three siblings of the propositus all typed as group O. Thus, it was deduced that the A2Bweak phenotype of the father arose from a cis-AB allele that had been passed on to the propositus. To identify the genetic basis of this observation, molecular analysis was performed on all direct family members using PCR-sequence-specific oligonucleotide (SSO),6 PCR-restriction fragment length polymorphism (RFLP),7 and sequence analysis (the latter performed by two independent laboratories: Sequence Facility, Department of Microbiology, Monash University, Clayton, VIC, Australia; and Flinders University Automated DNA Sequencing Facility, Flinders Medical Centre, Bedford Park, SA, Australia). The ABO genotype of the mother of the propositus was found to be BO1v, and all the siblings of the propositus were O1vO1v. By PCR-RFLP, the propositus demonstrated B-allele specificity at nt positions 526 and 703, but not at 796, and O1v-allele specificity at nt positions 646 and 771. Sequence analysis of the region spanning nt 411-892 of exon 7 of both the propositus and the father confirmed the B-allele specificity at nt positions 526, 657, 703, and 803, but it also revealed a cytosine residue at nt 796, which is indicative of an A allele (Fig. 1). A final genotype of cis-ABvar/O1v was assigned to both persons: the nomenclature cis-ABvar is proposed to distinguish this variation from the gene configuration previously identified.2-4 Figure 2 shows the pedigree of the family. The data suggest that the cis-AB allele described in this study arose from a point mutation at nt 796 of a seemingly normal B allele. In this case, the mutation involved the substitution of the B-transferase-specific adenosine residue with an A-transferase-specific cytosine residue. An A796C substitution results in the aa substitution Met266→Leu, which changes the sugar donor specificity at that site to an A-transferase and could give rise to a bifunctional transferase, as is suggested by the serologic findings presented in this study. Other molecular mechanisms, such as recombination between A and B alleles, may also provide an ex* Footnote: The nucleotide sequence of this allele has been listed on GenBank, Accession Number AF062487. Fig. 1. The nt sequence analysis at and around nt 796 of exon 7 of the cis-ABvar allele. Cytosine (C) residue at nt 796 indicated by arrow. “N” at nt 803 signifies the presence of two nts, cytosine and guanosine, which are indicative of a B allele and an O1v allele, respectively.

Recognition of Distinct Cross-Reactive Virus-Specific CD8+ T Cells Reveals a Unique TCR Signature in a Clinical Setting

Human CMV still remains problematic in immunocompromised patients, particularly after solid organ transplantation. CMV primary disease and reactivation greatly increase the risks associated with incidences of chronic allograft rejection and decreased survival in transplant recipients. But whether this is due to direct viral effects, indirect viral effects including cross-reactive antiviral T cell immunopathology, or a combination of both remains undetermined. In this article, we report the novel TCR signature of cross-reactive HLA-A*02:01 (A2) CMV (NLVPMVATV [NLV])–specific CD8+ T cells recognizing a specific array of HLA-B27 alleles using technical advancements that combine both IFN-γ secretion and multiplex nested RT-PCR for determining paired CDR3α/β sequences from a single cell. This study represents the first evidence, to our knowledge, of the same A2-restricted cross-reactive NLV-specific TCR-α/β signature (TRAV3TRAJ31_TRBV12-4TRBJ1-1) in two genetically distinct individuals. Longitudinal posttransplant monitoring of a lung transplant recipient (A2, CMV seropositive) who received a HLA-B27 bilateral lung allograft showed a dynamic expansion of the cross-reactive NLV-specific TCR repertoire before CMV reactivation. After resolution of the active viral infection, the frequency of cross-reactive NLV-specific CD8+ T cells reduced to previremia levels, thereby demonstrating immune modulation of the T cell repertoire due to antigenic pressure. The dynamic changes in TCR repertoire, at a time when CMV reactivation was subclinical, illustrates that prospective monitoring in susceptible patients can reveal nuances in immune profiles that may be clinically relevant.

Influenza A Infection Enhances Cross-Priming of CD8+ T Cells to Cell-Associated Antigens in a TLR7- and Type I IFN-Dependent Fashion

The initiation of antitumor immunity relies on dendritic cells (DCs) to cross-present cell-associated tumor Ag to CD8+ T cells (TCD8+) due to a lack of costimulatory molecules on tumor cells. Innate danger signals have been demonstrated to enhance cross-priming of TCD8+ to soluble as well as virally encoded Ags; however, their effect on enhancing TCD8+ cross-priming to cell genome-encoded Ags remains unknown. Furthermore, influenza A virus (IAV) has not been shown to enhance antitumor immunity. Using influenza-infected allogeneic cell lines, we show in this study that TCD8+ responses to cell-associated Ags can be dramatically enhanced due to enhanced TCD8+ expansion. This enhanced cross-priming in part involves TLR7- but not TLR3-mediated sensing of IAV and is entirely dependent on MyD88 and IFN signaling pathways. We also showed that the inflammasome-induced IL-1 and IFN-γ did not play a role in enhancing cross-priming in our system. We further demonstrated in our ex vivo system that CD8+ DCs are the only APCs able to prime TCR-transgenic TCD8+. Importantly, plasmacytoid DCs and CD8− DCs were both able to enhance such priming when provided in coculture. These observations suggest that IAV infection of tumor cells may facilitate improved cross-presentation of tumor Ags and may be used to augment clinical vaccine efficacy.

Immunodominance Hierarchies and Gender Bias in Direct TCD8‐Cell Alloreactivity

Allogeneic solid organ transplantation often occurs across multiple donor–recipient HLA mismatches with consequent risk of allograft rejection. However, there is growing evidence that not all HLA mismatches are equivalent in their stimulation of allogeneic T cells making it important to determine which of these might be more significant as predictors of allograft rejection. To this end, we used defined antigen‐presenting cell (APC) transfectants expressing single MHC‐I allotypes as target cells that could discriminate the relative contribution of individual mismatched MHC‐I allotypes to direct T‐cell alloreactivity. We demonstrate remarkably reproducible patterns of immunodominance in reactivity across mismatched MHC‐I allotypes. These patterns are HLA context‐dependent, partly reflecting alloantigenic competition in responder cell responses. In strong alloresponses, we also observed an increased percentage of alloreactive TCD8 cells in female responders, regardless of the stimulator gender, highlighting HLA‐independent factors in the potency of the alloresponse. This approach provides a potential measure of specific alloreactive T cells that could be used in clinical practice for selection of donors, assessment of posttransplant outcomes, modulation of immunosuppression and detection of rejection episodes.