Assessment of Bet v 1-specific CD4+ T cell responses in allergic and nonallergic individuals using MHC class II peptide tetramers

In this study, we used HLA-DRB1*0101, DRB1*0401, and DRB1*1501 peptide tetramers combined with cytokine surface capture assays to characterize CD4(+) T cell responses against the immunodominant T cell epitope (peptide 141-155) from the major birch pollen allergen Bet v 1, in both healthy and allergic individuals. We could detect Bet v 1-specific T cells in the PBMC of 20 birch pollen allergic patients, but also in 9 of 9 healthy individuals tested. Analysis at a single-cell level revealed that allergen-specific CD4(+) T cells from healthy individuals secrete IFN-gamma and IL-10 in response to the allergen, whereas cells from allergic patients are bona fide Th2 cells (producing mostly IL-5, some IL-10, but no IFN-gamma), as corroborated by patterns of cytokines produced by T cell clones. A fraction of Bet v 1-specific cells isolated from healthy, but not allergic, individuals also expresses CTLA-4, glucocorticoid-induced TNF receptor, and Foxp 3, indicating that they represent regulatory T cells. In this model of seasonal exposure to allergen, we also demonstrate the tremendous dynamics of T cell responses in both allergic and nonallergic individuals during the peak pollen season, with an expansion of Bet v 1-specific precursors from 10(-6) to 10(-3) among circulating CD4(+) T lymphocytes. Allergy vaccines should be designed to recapitulate such naturally protective Th1/regulatory T cell responses observed in healthy individuals.     

ATGs help MHC class II,but inhibit MHC class I antigen presentation

We have recently shown that the LC3/Atg8 lipidation machinery of macroautophagy is involved in the internalization of MHC class I molecules. Decreased internalization in the absence of ATG5 or ATG7 leads to MHC class I surface stabilization on dendritic cells and macrophages, resulting in elevated CD8⁺ T cell responses during viral infections and improved immune control. Here, we discuss how the autophagic machinery supports MHC class II restricted antigen presentation, while compromising MHC class I presentation via internalization and degradation.     

The biosynthetic pathway of MHC class II but not class I molecules intersects the endocytic route

We studied the intracellular traffic and subcellular distribution of MHC class I and class II antigens in comparison with a recycling surface glycoprotein, the transferrin receptor (Tfr), in the human lymphoblastoid cell line JY. No internalization was detectable for class I molecules. Class II molecules were internalized but did not recycle. In contrast, Tfr was found to internalize and recycle. The biosynthetic pathway of class II molecules differ from that of class I molecules in that it shows a delay (1-3 hr) in transport from trans-Golgi to cell surface: here it intersects the endocytic route. Immunoelectron microscopy using anti-MHC antibodies revealed the existence of vesicular structures that were intensely labeled for class II molecules. It is proposed that at this site combination of class II molecules with processed antigen could occur.     

HIV-1-specific CD4+ T cells are detectable in most individuals with active HIV-1 infection, but decline with prolonged viral suppression

The role of HIV-1-specific CD4+ T-cell responses in controlling HIV-1 infection remains unclear. Previous work has suggested that such cells are eliminated in the early stages of infection in most subjects, and thus cannot substantially contribute to host defense against HIV-1. Here, using flow cytometric detection of antigen-induced intracellular cytokines, we show that significant frequencies of gag specific, T-helper-1 CD4+ memory T cells are detectable in most subjects with active/progressive HIV-1 infection (median frequency, 0.12% of memory subset; range, 0-0.66%). Median frequencies of these cells were considerably higher in nonprogressive HIV-1 disease (0.40%), but there was substantial overlap between the two groups (range of nonprogressors, 0.10-1.7%). Continuous HIV-1 suppression with anti-retroviral therapy was associated with a time-dependent reduction in median frequencies of gag-specific CD4+ memory T cells: 0.08% in subjects treated for 4-24 weeks, and 0.03% in subjects treated for 47-112 weeks. Thus, functional HIV-1-specific CD4+ T cells are commonly available for support of anti-HIV-1 effector responses in active disease, but their decline with anti-retroviral therapy indicates that immunologic participation in long-term HIV-1 control will probably require effective vaccination strategies.     

Marsupial MHC class II beta genes are not orthologous to the eutherian beta gene families

The major histocompatibility complex (MHC) class II DRB, DQB, DPB, and DOB gene clusters are shared by different eutherian orders. Such an orthologous relationship is not seen between the beta genes of birds and eutherians. A high degree of uncertainty surrounds the evolutionary relationship of marsupial class II beta sequences with eutherian beta gene families. In particular, it has been suggested that marsupials utilize the DRB gene cluster. A cDNA encoding an MHC class II beta molecule was isolated from a brushtail possum mesenteric lymph node cDNA library. This clone is most similar to Macropus rufogriseus DBB. Our analysis suggests that all known marsupial beta-chain genes, excluding DMB, fall into two separate clades, which are distinct from the eutherian DRB, DQB, DPB, or DOB gene clusters. We recommend that the DAB and DBB nomenclature be reinstated. DAB and DBB orthologs are not present in eutherians. It appears that the marsupial and eutherian lineages have retained different gene clusters following gene duplication events early in mammalian evolution.     

Local CD11c+ MHC class II- precursors generate lung dendritic cells during respiratory viral infection, but are depleted in the process

Increases in numbers of lung dendritic cells (DC) observed during respiratory viral infections are assumed to be due to recruitment from bone marrow precursors. No local production has been demonstrated. In this study, we isolated defined populations of murine lung cells based on CD11c and MHC class II (MHC II) expression. After culture for 12 days with GM-CSF, we analyzed cell numbers, DC surface markers, and Ag-presenting capacity. Only CD11c+ MHC II- cells from naive mice proliferated, yielding myeloid DC, which induced Ag-specific proliferation of naive T cells. After respiratory syncytial virus (RSV) infection, numbers of pulmonary CD11c+ MHC II- precursor cells were significantly reduced and DC could not be generated. Moreover, RSV infection prevented subsequent in vivo expansion of pulmonary DC in response to influenza infection or LPS treatment. These results provide direct evidence of local generation of fully functional myeloid DC in the lung from CD11c+ MHC II(-) precursor cells that are depleted by RSV infection, leading to an inability to expand lung DC numbers in response to subsequent viral infection or exposure to bacterial products. This depletion of local DC precursors in respiratory viral infections may be important in explaining complex interactions between multiple and intercurrent pulmonary infections.     

Human CD25+ regulatory T cells maintain immune tolerance to nickel in healthy, nonallergic individuals

We investigated the capacity of CD25(+) T regulatory cells (Treg) to modulate T cell responses to nickel, a common cause of allergic contact dermatitis. CD4(+) T cells isolated from the peripheral blood of six healthy, nonallergic individuals showed a limited capacity to proliferate in response to nickel in vitro, but responsiveness was strongly augmented (mean increment +/- SD, 240 +/- 60%) when cells were depleted of CD25(+) Treg. Although CD25(+) Treg were anergic to nickel, a small percentage up-regulated membrane CTLA-4 upon nickel exposure. CD25(+) Treg strongly and dose-dependently inhibited nickel-specific activation of CD25(-) T lymphocytes in coculture experiments in a cytokine-independent, but cell-to-cell contact-dependent, manner. Approximately 30% of circulating CD25(+) Treg expressed the cutaneous lymphocyte-associated Ag (CLA), and CLA(+)CD25(+) Treg were more efficient than CLA(-)CD25(+) cells in suppressing nickel responsiveness of CD25(-) T cells. The site of a negative patch test in response to nickel showed an infiltrate of CD4(+)CLA(+) cells and CD25(+) cells, which accounted for approximately 20% of the total T cells isolated from the tissue. Skin-derived T cells suppressed nickel-specific responses of peripheral blood CD25(-) T cells. In addition, 60 +/- 14% of peripheral blood CD25(+) Treg expressed the chemokine receptor CCR7 and strongly inhibited naive T cell activation in response to nickel. Finally, CD25(+) T cells isolated from peripheral blood of nickel-allergic patients showed a limited or absent capacity to suppress metal-specific CD4(+) and CD8(+) T cell responses. The results indicates that in healthy individuals CD25(+) Treg can control the activation of both naive and effector nickel-specific T cells.     

Invariant chain alters the malignant phenotype of MHC class II+ tumor cells.

T lymphocytes usually recognize endogenously encoded Ag in the context of MHC class I molecules, whereas exogenous Ag is usually presented by MHC class II molecules. In vitro studies in model systems suggest that presentation of endogenous Ag by class II molecules is inhibited by the association of class II with its invariant chain (Ii). In the present study we test this hypothesis in an in vivo system in which endogenously encoded tumor peptides are presented by tumor cell MHC class II molecules. In this system, transfection of syngeneic MHC class II genes (Aak and Abk) into a highly malignant, Ii negative, mouse tumor (SaI sarcoma) produces an immunogenic tumor (SaI/Ak) that is rejected by the autologous host. The class II+ transfectants also effectively immunize autologous A/J mice against a subsequent challenge of wild-type class II- tumor cells. We have hypothesized that the SaI/Ak transfectants induce protective immunity because they function as APC for endogenously synthesized tumor peptides, and thereby stimulate tumor-specific Th cells, by-passing the need for professional APC. To test the role of Ii as an inhibitor of presentation of endogenous peptides, SaI/Ak tumor cells were supertransfected with Ii gene (SaI/Ak/Ii cells), and the tumorigenicity of the resulting cells determined. Nine SaI/Ak/Ii clones were tested, and their malignancy compared with that of SaI/Ak and SaI cells. Seven of the nine class II+/Ii+ tumor cells are more malignant than class II+/Ii- tumor cells in autologous A/J mice. Expression of Ii therefore restores the malignant phenotype, presumably by preventing presentation of endogenously synthesized tumor peptides. Ii therefore regulates Ag presentation and can be a critical parameter for in vivo tumor immunity.     

Ultrasensitive detection and phenotyping of CD4+ T cells with optimized HLA class II tetramer staining

HLA class I tetramers have revolutionized the study of Ag-specific CD8+ T cell responses. Technical problems and the rarity of Ag-specific CD4+ Th cells have not allowed the potential of HLA class II tetramers to be fully realized. Here, we optimize HLA class II tetramer staining methods through the use of a comprehensive panel of HIV-, influenza-, CMV-, and tetanus toxoid-specific tetramers. We find rapid and efficient staining of DR1- and DR4-restricted CD4+ cell lines and clones and show that TCR internalization is not a requirement for immunological staining. We combine tetramer staining with magnetic bead enrichment to detect rare Ag-specific CD4+ T cells with frequencies as low as 1 in 250,000 (0.0004% of CD4+ cells) in human PBLs analyzed directly ex vivo. This ultrasensitive detection allowed phenotypic analysis of rare CD4+ T lymphocytes that had experienced diverse exposure to Ag during the course of viral infections. These cells would not be detectable with normal flow-cytometric techniques.     

Lack of detectable endocytosis of B lymphocyte MHC class II antigens using an antibody-independent technique

MHC class II complexes may intercept ingested foreign Ag as the nascent class II molecules exit the cell or as mature complexes recycle between the outer membrane and an internal compartment. To examine endocytosis of HLA-DR, we radiolabeled B lymphoblastoid cells, warmed the cells to allow internalization of membrane proteins, and then subjected viable cells to neuraminidase (NANAse)3 digestion. DR complexes were precipitated and analyzed by two-dimensional PAGE for shifts in isoelectric point signifying sensitivity to or protection from NANAse treatment. DR molecules were completely sensitive to the enzyme with or without specific antibody in the medium during the 37 degrees C incubation, suggesting that no detectable endocytosis had occurred. Control transferrin receptors, which readily internalize, showed marked protection. Assay sensitivity was measured by proportionate mixing of mock and NANAse-treated lysates; precipitated sialylated molecules were detectable at the 10% level. Monensin treatment to block recycling also failed to reveal any internally accumulated. NANAse-protected DR molecules. Measurements of turnover for surface-labeled DR complexes revealed a t 1/2 of approximately 36 h. We conclude that no large endocytically-derived pool of HLA-DR molecules exists within cells and suggest that the majority of mature DR molecules do not actively undergo internalization but reside at the cell surface after synthesis/transport for significantly long periods of time.     

CD8+ T cells are necessary for recognition of allelic, but not locus-mismatched or xeno-, HLA class I transplantation antigens

Although HLA transgenic mice (HLA TgM) could provide a powerful approach to investigate human MHC-specific T cell responsiveness, the extent to which these molecules are recognized by the mouse immune system remains unclear. We established TgM expressing HLA class I alleles A2, B7, or B27 in their fully native form (HLAnat) or as hybrid molecules (HLAhyb) of the HLA alpha1/alpha2 domains linked to the H-2Kb alpha3, transmembrane, and cytoplasmic domains (i.e., to maintain possible species-specific interactions). Comparison of each as xeno- (i.e., by non-TgM) vs allo- (i.e., by TgM carrying an alternate HLA allele) transplantation Ags revealed the following: 1) Although HLAhyb molecules induced stronger xeno-CD8+ T cell responses in vitro, additional effector mechanisms must be active in vivo because HLAnat skin grafts were rejected faster by non-TgM; 2) gene knockout recipients showed that xenorejection of HLAnat and, unexpectedly, HLAhyb grafts doesn't depend on CD8+ or CD4+ T cells or B cells; 3) each HLAhyb strain developed tolerance to "self" but rejected allele- (-B27 vs -B7) and locus- (-B vs -A) mismatched grafts, the former requiring CD8+ T cells, the latter by CD8+ T cell-independent mechanisms. The finding that recognition of xeno-HLAhyb does not require CD8+ T cells while recognition of the identical molecule in a strictly allo context does, demonstrates an alpha1/alpha2 domain-dependent difference in effector mechanism(s). Furthermore, the CD8+ T cell-independence of locus-mismatched rejection suggests the degree of similarity between self and non-self alpha1/alpha2 determines the effector mechanism(s) activated. The HLA Tg model provides a unique approach to characterize these mechanisms and develop tolerance protocols in the context of human transplantation Ags.     

A recombinant C-terminal fragment of staphylococcal enterotoxin A binds to human MHC class II products but does not activate T cells.

Binding of staphylococcal enterotoxin A (SEA) to MHC class II encoded proteins is a prerequisite for its subsequent activation of a large fraction of T lymphocytes through interaction with variable segments of the TCR-beta chain. We cloned SEA in Escherichia coli and produced four recombinant fragments covering both the N- and C-terminal regions. These fragments were used to analyze the interaction between SEA and the human MHC class II products. A C-terminal fragment of SEA, representing amino acids 107-233 bound to HLA-DR and HLA-DP but did not activate T cells. The three other fragments (amino acids 1-125, 1-179 and 126-233) neither bound to MHC class II Ag nor activated T cells. SEA apparently bind to HLA-DR and HLA-DP through its C-terminal part, whereas T cell activation is dependent on additional parts of the protein.     

Expression of MHC class I, MHC class II, and cancer germline antigens in neuroblastoma

Background: Neuroblastoma is the most common solid extracranial tumor in childhood, still with poor survival rates for metastatic disease. Neuroblastoma cells are of neuroectodermal origin and express a number of cancer germline (CG) antigens. These CG antigens may represent a potential target for immunotherapy such as peptide-based vaccination strategies. Objective: The purpose of this study was to analyze the presence of MAGE-A1, MAGE-A3/A6, and NY-ESO-1 on an mRNA and protein level and to determine the expression of MHC class I and MHC class II antigens within the same tumor specimens. Methods: A total of 68 tumors were available for RT-PCR, and 19/68 tumors were available for immunohistochemical (IHC) analysis of MAGE-A1, MAGE-A3/A6, and NY-ESO-1. In parallel, the same tumors were stained with a panel of antibodies for MHC class I and MHC class II molecules. Results: Screening of 68 tumor specimens by RT-PCR revealed expression of MAGE-A1 in 44%, MAGE-A3/A6 in 21%, and NY-ESO-1 in 28% of cases. Immunohistochemistry for CG antigens of selected tumors showed good agreement between protein and gene expression. However, staining revealed a heterogeneous expression of CG antigens. None of the selected tumors showed MHC class I or MHC class II expression. Conclusions: mRNA expression of MAGE-A1, MAGE-A3/A6, and NY-ESO-1 is congruent with the protein expression as determined by immunohistochemistry. The heterogeneous CG-antigen expression and the lack of MHC class I and II molecules may have implications for T-cell–mediated immunotherapy in neuroblastoma.     

Novel regulation of MHC class II function in B cells

The presence of post-translational regulation of MHC class II (MHC II) under physiological conditions has been demonstrated recently in dendritic cells (DCs) that potently function as antigen-presenting cells (APCs). Here, we report that MARCH-I, an E3 ubiquitin ligase, plays a pivotal role in the post-translational regulation of MHC II in B cells. MARCH-I expression was particularly high in B cells, and the forced expression of MARCH-I induced the ubiquitination of MHC II. In B cells from MARCH-I-deficient mice (MARCH-I KO), the half-life of surface MHC II was prolonged and the ubiquitinated form of MHC II completely disappeared. In addition, MARCH-I-deficient B cells highly expressed exogenous antigen-loaded MHC II on their surface and showed high ability to present exogenous antigens. These results suggest that the function of MHC II in B cells is regulated through ubiquitination by MARCH-I.