TAP Deficiency Research Articles

Influenza A virus infection‐induced macroautophagy facilitates MHC class II‐restricted endogenous presentation of an immunodominant viral epitope

CD4+ T cells recognize peptides presented by major histocompatibility complex class II molecules (MHC-II). These peptides are generally derived from exogenous antigens. Macroautophagy has been reported to promote endogenous antigen presentation in viral infections. However, whether influenza A virus (IAV) infection-induced macroautophagy also leads to endogenous antigen presentation through MHC-II is still debated. In this study, we show that IAV infection leads to endogenous presentation of an immunodominant viral epitope NP311-325 by MHC-II to CD4+ T cells. Mechanistically, such MHC-II-restricted endogenous IAV antigen presentation requires denovo protein synthesis as it is inhibited by the protein synthesis inhibitor cycloheximide, and a functional ER-Golgi network as it is totally blocked by Brefeldin A. These results indicate that MHC-II-restricted endogenous IAV antigen presentation is dependent on denovo antigen and/or MHC-II synthesis, and transportation through the ER-Golgi network. Furthermore, such endogenous IAV antigen presentation by MHC-II is enhanced by TAP deficiency, indicating some antigenic peptides are of cytosolic origin. Most importantly, the bulk of such MHC-II-restricted endogenous IAV antigen presentation is blocked by autophagy inhibitors (3-MA and E64d) and deletion of autophagy-related genes, such as Beclin1 and Atg7. We have further demonstrated that in dendritic cells, IAV infection prevents autophagosome-lysosome fusion and promotes autophagosome fusion with MHC class II compartment (MIIC), which likely promotes endogenous IAV antigen presentation by MHC-II. Our results provide strong evidence that IAV infection-induced autophagosome formation facilitates endogenous IAV antigen presentation by MHC-II to CD4+ T cells. The implication for influenza vaccine design is discussed.

Optimization of the T2 HLA-A2 shift assay for testing of the biological activity of immunotherapies

Abstract T2 is a lymphoma-derived cell line that express low amounts of HLA-A2 on the cell surface due to TAP deficiency and can only present exogenous peptides. Binding of exogenous peptides to HLA-A2 stabilizes the HLA-A2- peptide complexes and can be detected using immunofluorescence staining. The T2 cell line is commonly used for identification and analysis of novel HLA-A2-restricted CTL epitopes in the assay known as T2 shift. In this study we evaluated parameters that may influence the T2 shift assay performance and considered new applications of this method for testing of the biological activity of peptide-based immunotherapies. Method optimization was performed using HLA-A2-restricted peptide SurA2.M alone and in the context of a peptide-based immunotherapy drug, DPX-Survivac. DPX-Survivac is a lipid nanoparticle-based formulation that contains five peptides aiming to elicit a CTL response against tumor cells presenting the survivin peptides on HLA class I molecules. Results indicated that performance of the T2 shift assay was highly sensitive to cell passaging intervals, pH variations, cell concentrations and β2-microglobulin concentrations. Discrimination of the T2 cells by either size or granularity did not affect the results for the T2 shift assay. We determined the linear response range for the SurA2.M peptide to be 30-110 μg/mL in the T2 shift assay. The T2 shift assay can detect SurA2.M within the DPX-Survivac matrix. In conclusion, we found that optimal culturing conditions of T2 cells are critical for the T2 shift assay reproducibility and linearity and should be carefully considered if the assay is to be used for quantitative or semi-quantitative evaluation of the peptide activity alone or as a part of an immunotherapeutic drug.

β2-Microglobulin deficiency causes a complex immunodeficiency of the innate and adaptive immune system

Most patients with MHC class I (MHC-I) deficiency carry genetic defects in transporter associated with antigen processing 1 (TAP1) or TAP2. The clinical presentation can vary, and about half of the patients have severe skin disease. Previously, one report described β2-microglobulin (β2m) deficiency as another monogenetic cause of MHC-I deficiency, but no further immunologic evaluation was performed. We sought to describe the molecular and immunologic features of β2m deficiency in 2 Turkish siblings with new diagnoses. Based on clinical and serologic findings, the genetic defect was detected by means of candidate gene analysis. The immunologic characterization comprises flow cytometry, ELISA, functional assays, and immunohistochemistry. Here we provide the first extensive clinical and immunologic description of β2m deficiency in 2 siblings. The sister had recurrent respiratory tract infections and severe skin disease, whereas the brother was fairly asymptomatic but had bronchiectasis. Not only polymorphic MHC-I but also the related CD1a, CD1b, CD1c, and neonatal Fc receptor molecules were absent from the surfaces of β2m-deficient cells. Absent neonatal Fc receptor surface expression led to low serum IgG and albumin levels in both siblings, whereas the heterozygous parents had normal results for all tested parameters except β2m mRNA (B2M) expression. Similar to TAP deficiency in the absence of a regular CD8 T-cell compartment, CD8(+) γδ T cells were strongly expanded. Natural killer cells were normal in number but not "licensed to kill." The clinical presentation of patients with β2m deficiency resembles that of patients with other forms of MHC-I deficiency, but because of the missing stabilizing effect of β2m on other members of the MHC-I family, the immunologic defect is more extensive than in patients with TAP deficiency.

Sculpting MHC class II–restricted self and non‐self peptidome by the class I Ag‐processing machinery and its impact on Th‐cell responses

It is generally assumed that the MHC class I antigen (Ag)-processing (CAP) machinery - which supplies peptides for presentation by class I molecules - plays no role in class II-restricted presentation of cytoplasmic Ags. In striking contrast to this assumption, we previously reported that proteasome inhibition, TAP deficiency or ERAAP deficiency led to dramatically altered T helper (Th)-cell responses to allograft (HY) and microbial (Listeria monocytogenes) Ags. Herein, we tested whether altered Ag processing and presentation, altered CD4(+) T-cell repertoire, or both underlay the above finding. We found that TAP deficiency and ERAAP deficiency dramatically altered the quality of class II-associated self peptides suggesting that the CAP machinery impacts class II-restricted Ag processing and presentation. Consistent with altered self peptidomes, the CD4(+) T-cell receptor repertoire of mice deficient in the CAP machinery substantially differed from that of WT animals resulting in altered CD4(+) T-cell Ag recognition patterns. These data suggest that TAP and ERAAP sculpt the class II-restricted peptidome, impacting the CD4(+) T-cell repertoire, and ultimately altering Th-cell responses. Together with our previous findings, these data suggest multiple CAP machinery components sequester or degrade MHC class II-restricted epitopes that would otherwise be capable of eliciting functional Th-cell responses.

Investigation of Natural Killer (NK) cells in TAP deficiency

Investigation of Natural Killer (NK) cells in TAP deficiency

TAP deficiency syndrome.

Bare lymphocyte syndrome (BLS) is characterized by a severe down-regulation of HLA class I and/or class II molecules. In type 1 BLS the defect is confined to HLA class I molecules, while in type 2 BLS HLA class II molecules are down-regulated [1]. Characterization of 22 patients with type 1 BLS over the last 22 years has revealed the existence of several clinically and immunologically distinct disease subsets [1–20]. In this review we will focus on a recently characterized group of patients with a distinct disease phenotype due to a defective TAP complex, the peptide transporter complex associated with antigen presentation [2–15]. We will describe clinical manifestations and immunological findings of patients suffering from TAP deficiency syndrome, and discuss the differential diagnosis and therapeutic options.

TAP deficiency syndrome: chronic rhinosinusitis and conductive hearing loss

Nose-ear-throat manifestations of immunodeficiency disorders represent a diagnostic challenge for clinicians as these diseases often constitute the initial sign for connective disorders or autoimmune disease. The history of chronic rhinosinusitis and conductive hearing loss is often non specific. Therefore attention to an HLA class I deficiency must be considered if the disease has not been diagnosed on routine examination. One of the syndromes is due to a defective TAP complex, the peptide transporter complex associated with antigen presentation. Herein, we report two sisters with TAP-deficiency. The treatment of choice for TAP-deficient patients is conservative.

Recurrent infections and bilateral uveitis in a patient with CD8 deficiency.

CD8 deficiency is a rare primary immunodeficiency with low or absent peripheral CD8 cells which results from TAP deficiency, Zap 70 deficiency and CD8 alpha gene mutation.We report a 14 year old female who presented with a history of recurrent pneumonia, bronchiectasis, otitis, severe varicella, herpetic lesions of mouth, bilateral uveitis, and cataract formation since the age of 8 years.She had growth failure, a huge spleen and moderate clubbing. In immunologic workup, humoral and phagocytic systems were normal. DTH response to candida, PPD and DT were negative but LTT response to PHA mitogen was normal. HLA typing showed normal class I expression. Flowcytometry of peripheral blood showed CD8: 0 to 2% (absolute count, 0-60 cells/mm3) with increased CD4/CD8 ratio on several occasions.Diagnosis of this patient cannot be HLA class I deficiency (TAP1 or TAP2), because class I expression had been normal. It is possible to be Zap -70 deficiency or CD8 alpha gene mutation. Bilateral uveitis in our patient was a unique presentation which might have resulted because of immune dysregulation in CID.

Inefficient protection of human TAP-deficient fibroblasts from autologous NK cell-mediated lysis by cytokines inducing HLA class I expression.

We studied HLA class I expression and susceptibility to lysis of activated autologous NK cells in normal and TAP-deficient fibroblasts. These cells were cultured in the presence or absence of cytokines known to increase the surface expression of HLA class I molecules. All the cytokines tested (IFN-alpha, IFN-gamma, TNF-alpha and IFN-gamma + TNF-alpha) increased the expression of HLA class I molecules on fibroblasts after 48-h culture, but on TAP-deficient cells this expression remained very low as compared to that of normal cells. In the presence of IFN-alpha, IFN-gamma or IFN-gamma + TNF-alpha, normal target cells became resistant to lysis by autologous NK cells, whereas this effect was much less pronounced in the case of TAP-deficient fibroblasts. Addition of an anti-HLA class I mAb to fibroblasts treated with cytokines increased lysis of normal but not of TAP-deficient cells. These results suggest that activated TAP-deficient NK cells are strongly cytotoxic to normal autologous cells and that these cells cannot be efficiently protected by cytokines inducing HLA class I expression. Thus, in human TAP deficiency, activated NK cells may contribute to the progressive lung degradation which characterizes the clinical course of these patients.

Human peptide transporter deficiency: importance of HLA-B in the presentation of TAP-independent EBV antigens.

Two siblings with a peptide TAP deficiency were recently described. Despite poor cell surface expression of HLA class I molecules, these patients were not unusually susceptible to viral infections. The majority of the cell surface-expressed class I molecules were HLA-B products as assessed by cytofluorometry and biochemical analysis. Analysis of two peptides eluted from the class I molecules expressed by TAP-deficient EBV B lymphoblastoid cell lines indicated that both were derived from cytosolic proteins and presented by HLA-B molecules. Peripheral alphabeta CD8+ T cells were present and their TCR repertoire was polyclonal. Most of the alphabeta CD8+ T cell clones studied (21 of 22) were nonreactive against cells expressing normal levels of the same HLA alleles as those of the TAP-deficient patients. However, it was possible to isolate one cytotoxic CD8+ alphabeta T cell clone recognizing the EBV protein LMP2 presented by HLA-B molecules on TAP-deficient cells. These observations suggest that in the TAP-deficient patients, CD8+ alphabeta T cells could mature and be recruited in immune responses to mediate HLA class I-restricted cytotoxic defense against viral infections. They also strengthen the physiologic importance of a TAP-independent processing pathway of the LMP2 protein, which was previously shown to contain several other TAP-independent epitopes.