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We have previously demonstrated that mycobacterial lipoproteins engage TLR2 on human CD4(+) T cells and upregulate TCR-triggered IFN-γ secretion and cell proliferation in vitro. Here we examined the role of CD4(+) T-cell-expressed TLR2 in Mycobacterium tuberculosis (MTB) Ag-specific T-cell priming and in protection against MTB infection in vivo. Like their human counterparts, mouse CD4(+) T cells express TLR2 and respond to TLR2 costimulation in vitro. This Th1-like response was observed in the context of both polyclonal and Ag-specific TCR stimulation. To evaluate the role of T-cell TLR2 in priming of CD4(+) T cells in vivo, naive MTB Ag85B-specific TCR transgenic CD4(+) T cells (P25 TCR-Tg) were adoptively transferred into Tlr2(-/-) recipient C57BL/6 mice that were then immunized with Ag85B and with or without TLR2 ligand Pam3 Cys-SKKKK. TLR2 engagement during priming resulted in increased numbers of IFN-γ-secreting P25 TCR-Tg T cells 1 week after immunization. P25 TCR-Tg T cells stimulated in vitro via TCR and TLR2 conferred more protection than T cells stimulated via TCR alone when adoptively transferred before MTB infection. Our findings indicate that TLR2 engagement on CD4(+) T cells increases MTB Ag-specific responses and may contribute to protection against MTB infection.

Inflammatory bowel disease (IBD) is characterized by damage to the gut mucosa and systemic inflammation. We sought to evaluate the role of chronic inflammation on circulating T-cell activation in human subjects with Crohn's disease and ulcerative colitis. We studied 54 patients with IBD and 28 healthy controls. T-cell activation and cycling were assessed in whole blood samples by flow cytometry. Levels of lipopolysaccharide (LPS) were measured in serum by Limulus amoebocyte lysate assay, and plasma levels of inflammatory markers and LPS-binding proteins were measured by ELISA. The proportions of circulating CD4(+) and CD8(+) T lymphocytes in cycle (Ki67(+) ) are increased in patients with IBD compared with these proportions in controls. CD8(+) T cells from patients with IBD are also enriched for cells that expressed CD38 and HLA-DR, and proportions of these cells are related to plasma levels of interleukin-6 and C-reactive protein in these patients. Intracellular interleukin-2 and interferon-γ levels were elevated in resting and polyclonally activated CD4(+) and CD8(+) T cells in patients with IBD when compared with levels from healthy controls. Surprisingly, we did not find increased levels of LPS in the serum of patients with IBD. We did, however, find a signature of recent microbial translocation, as levels of LPS-binding protein are increased in the plasma of patients with IBD compared with plasma levels in healthy controls; LPS-binding protein levels are also directly related to proportions of CD38 HLA-DR-expressing CD4(+) and CD8(+) T cells. Local damage to the gastrointestinal tract in IBD may result in systemic inflammation and T-cell activation.

To examine heterogeneity in dendritic cell (DC) antigen presentation function, murine splenic DCs were separated into CD4+ and CD8+ populations and assessed for the ability to process and present particulate antigen to CD4+ and CD8+ T cells. CD4+ and CD8+ DCs both processed exogenous particulate antigen, but CD8+ DCs were much more efficient than CD4+ DCs for both major histocompatibility complex (MHC) class II antigen presentation and MHC class I cross-presentation. While antigen processing efficiency contributed to the superior antigen presentation function of CD8+ DCs, our studies also revealed an important contribution of CD24. CD8+ DCs were also more efficient than CD4+ DCs in inducing naïve T cells to acquire certain effector T-cell functions, for example generation of cytotoxic CD8+ T cells and interferon (IFN)-gamma-producing CD4+ T cells. In summary, CD8+ DCs are particularly potent antigen-presenting cells that express critical costimulators and efficiently process exogenous antigen for presentation by both MHC class I and II molecules.

We demonstrated that an epitope from the recombinant protective antigen (rPA) of Bacillus anthracis was presented by mature major histocompatibility complex class II (MHC-II) molecules, whereas an epitope from the recombinant virulent (rV) antigen of Yersinia pestis was presented by newly synthesized MHC-II. We addressed which endosomal compartments were involved in the antigen processing of each epitope. Bone-marrow-derived macrophages were subjected to subcellular fractionation; fractions were analysed for the expression of endosomal markers and used as a source of enzyme activity for the processing of rPA and rV antigens. The rPA epitope was productively processed by dense lysosomal fractions and light membrane fractions expressing early endosomal markers Rab5 and early endosomal antigen-1 as well as markers of antigen-presenting compartments (MHC-II, DM, DO and Ii chain). In contrast, the rV epitope was productively processed only by dense fractions with lysosomal activity. No productive antigen-processing activity was associated with fractions of intermediate density expressing Rab7 and Rab9, characteristic of late endosomes. The data suggest that endosomal compartments expressing Rab5 guanosine triphosphatase can productively process protein antigens for presentation by mature MHC class II molecules.

MHC class II heterozygotes form haplotype-mismatched heterodimers (combining α and β chains of different alleles). Transfected L-cells expressing AαdAβd or AαdAβb presented exogenous OVA(323–339) peptide to T cells with similar high efficiency, while AαbAβb was less efficient and AαbAβd was ineffective. In contrast, AαdAβb greatly exceeded AαdAβd in processing of intact OVA for presentation of OVA(323–339); AαbAβb was even less efficient and AαbAβd was ineffective. Of macrophages from C57BL/6 (H-2b), DBA/2 (H-2d) and B6D2F1 (H-2b×d)mice, B6D2F1 macrophages had highest I-A expression and efficiency for OVA processing or presentation of exogenous OVA(323–339) peptide. Blocking antibodies specific for I-A chains showed that OVA processing by B6D2F1 macrophages primarily involved haplotype-mismatched AαdAβb heterodimers, whereas AαdAβd and AαbAβb contributed more to presentation of exogenous OVA(323–339) peptide. OVA(323–339):I-A complexes were formed from OVA within 10 min with B6D2F1 macrophages but not until 20 min with C57BL/6 or DBA/2 macrophages, and OVA processing was more resistant to inhibition of late endocytic function by hypothermia (18°C) in B6D2F1 than C57BL/6 or DBA/2 macrophages. These results indicate that AαdAβb haplotype-mismatched heterodimers may contribute to antigen processing in early endocytic compartments.

Mycobacterium tuberculosis (Mtb) cell wall glycolipid mannose‐capped lipoarabinomannan (ManLAM) inhibits CD4+ T‐cell activation by inhibiting proximal T‐cell receptor (TCR) signaling when activated by anti‐CD3. To understand the impact of ManLAM on CD4+ T‐cell function when both the TCR–CD3 complex and major costimulator CD28 are engaged, we performed label‐free quantitative MS and network analysis. Mixed‐effect model analysis of peptide intensity identified 149 unique peptides representing 131 proteins that were differentially regulated by ManLAM in anti‐CD3‐ and anti‐CD28‐activated CD4+ T cells. Crosstalker, a novel network analysis tool identified dysregulated translation, TCA cycle, and RNA metabolism network modules. PCNA, Akt, mTOR, and UBC were found to be bridge node proteins connecting these modules of dysregulated proteins. Altered PCNA expression and cell cycle analysis showed arrest at the G2M phase. Western blot confirmed that ManLAM inhibited Akt and mTOR phosphorylation, and decreased expression of deubiquitinating enzymes Usp9x and Otub1. Decreased NF‐κB phosphorylation suggested interference with CD28 signaling through inhibition of the Usp9x‐Akt‐mTOR pathway. Thus, ManLAM induced global changes in the CD4+ T‐cell proteome by affecting Akt‐mTOR signaling, resulting in broad functional impairment of CD4+ T‐cell activation beyond inhibition of proximal TCR–CD3 signaling.

Some tumor cells have deficits in class I MHC antigen processing, suggesting that T cells exert selective pressure on tumor cells. Previous studies have not revealed increased tumor incidence in mice with deficits in T-cell immunity, including mice lacking TAP1 (a subunit of the transporter for antigen presentation) or LMP2 (a regulated subunit of the 20S proteasome). The incidence of spontaneous tumors in these mice, however, is too low to assess differences in host resistance to tumors. To increase tumor incidence and better assess the role of systemic expression of TAP1 and LMP2 in responses to tumors, TAP1–/– and LMP2–/– mice were bred with p53–/– mice to create TAP1–/–p53–/– and LMP2–/–p53–/– double knockout mice. Lymphomas and sarcomas (malignant fibrous histiocytoma and angiosarcoma) occurred with high incidence in all p53-deficient populations. Tumor incidence and death rate were similar in TAP1–/–p53–/– mice and closely matched control TAP1+/+p53–/– mice. Tumor incidence and death rate were slightly accelerated in LMP2–/–p53–/– mice relative to control LMP2+/+p53–/– mice, but the biological significance of this difference was unclear. The relative incidence of lymphomas vs. sarcomas was not significantly altered by variation in TAP1 or LMP2. In conclusion, systemic absence of TAP1 did not alter tumor incidence, while absence of LMP2 was associated with only a slight acceleration of tumor incidence of uncertain significance. These observations are consistent with other evidence that normal T-cell responses do not effectively limit tumorigenesis. Even though T cells can attack some tumor cells, the ability of tumors to alter their immunogenicity and evade T-cell surveillance may render the native immune system ineffective at providing a rate-limiting barrier to tumorigenesis and preventing cancer. © 2001 Wiley-Liss, Inc.

Processing of exogenous antigens and microbes involves contributions by multiple different endocytic and phagocytic compartments. During the processing of soluble antigens, different endocytic compartments have been demonstrated to use distinct antigen-processing mechanisms and to process distinct sets of antigenic epitopes. Processing of particulate and microbial antigens involves phagocytosis and functions contributed by phagocytic compartments. Recent data from our laboratory demonstrate that phagosomes containing antigen-conjugated latex beads are fully competent class II MHC (MHC-II) antigen-processing organelles, which generate peptide:MHC-II complexes. In addition, phagocytosed antigen enters an alternate class I MHC (MHC-I) processing pathway that results in loading of peptides derived from exogenous antigens onto MHC-I molecules, in contrast to the cytosolic antigen source utilized by the conventional MHC-I antigen-processing pathway. Antigen processing and other immune response mechanisms may be activated or inhibited by microbial components to the benefit of either the host or the pathogen. For example, antigen processing and T-cell responses (e.g. Th1 vs Th2 differentiation) are modulated by multiple distinct microbial components, including lipopolysaccharide, cholera toxin, heat labile enterotoxin of Escherichia coli, DNA containing CpG motifs (found in prokaryotic and invertebrate DNA but not mammalian DNA) and components of Mycobacterium tuberculosis.

Antigen processing and presentation experiments can be done with a wide variety of antigen-presenting cells (APCs). Most experiments will use one of the “professional” APC types: dendritic cells (DCs), macrophages, and B lymphocytes. Other types of cells may be used for antigen presentation in some circumstances. Each type of professional APC has an important antigen-presentation function, but the different APC types contribute to different aspects of the immune response. Therefore, selection of an APC type for study must include consideration of the stage or aspect of immune response that is to be modeled in the experiment. An important technical distinction for some types of experiments is whether the APCs are adherent or nonadherent, since this dictates the procedures that must be used to wash the cells as the medium is changed. Curr. Protoc. Immunol. 88:16.2.1-16.2.18. © 2010 by John Wiley & Sons, Inc. Keywords: antigen presenting cells; antigen processing; antigen presentation; T lymphocyte; T hybridoma cells

CpG oligodeoxynucleotides (ODNs) and single stranded RNAs (ssRNA) signal through TLR9 and TLR7, respectively, to induce type-I IFN (IFNαβ) and IFNαβ-dependent MHC-I cross-presentation of exogenous ...

The external surface of the cornea and adjacent epidermis of larvae in representative developmental stages and of adult frogs, Rana pipiens, was studied by scanning electron microscopy. Surface cells are polygonal, usually hexagonal, in outline and covered with microprojections. During larval development prior to metamorphic stages, neither eyelids nor Harderian glands have developed; microprojections on the corneal surface are high and branched, and cell boundaries are elevated. On the anterior portion of the cornea and on the epidermis near the eye, the surface pattern is less dense, and ciliated cells are present. During metamorphic stages, corneal cell boundaries become less prominent and the pattern of microprojections more variable and markedly different from that of larvae of earlier stages. Corneal cells have a spongy appearance, are covered by a coating material, or are characterized as light or dark based on their brightness and surface texture. As eyelids develop in metamorphic stages XX-XXI, the numbers of ciliated cells increase dramatically, both on the corneal surface and on the edges of the developing lids. In later metamorphic stages XXII-XXV, lids and Harderian glands become well-developed, and cilia are no longer observed. The adjacent epidermal surface becomes devoid of cilia but perforated by openings of cutaneous glands. Its spongy appearance is similar to that of both the cornea and neighboring epidermis of the mature frog. Changes in corneal surface features are probably metamorphic events associated with development of lids and Harderian glands and a shift from an aqueous to an air environment.

Using MAb and monovalent Fab probes and saponin permeabilization we have demonstrated that PEC and TA3 B lymphoma-hybridoma cells contain a significant intracellular pool of Ia. At least in TA3 cells, this intracellular pool was independent of protein synthesis. In PEC, adherence caused redistribution of Ia with disappearance of the intracellular pool. Endocytosis of Ia occurred in both TA3 and PEC, and internalized Ia reached a plateau level corresponding in size to the total intracellular Ia pool revealed by saponin treatment. These results suggest that intracellular Ia is largely in a recycling pool derived from the plasma membrane by endocytosis. Subcellular fractionation studies suggest that Ia processing occurs in endosomes similar to those involved in transferrin processing. Antigen processing by TA3 cells was found to be unaffected by cycloheximide. In contrast, antigen processing by adherent PEC was markedly inhibited by cycloheximide, despite the fact that they maintained surface Ia and were still capable of presenting antigen peptides. This suggests that an important intracellular Ia processing step or antigen processing step was blocked in these cells. Adherent PEC may contain less recycling Ia, making protein synthesis the major source for intracellular Ia and the availability of intracellular Ia sensitive to cycloheximide. Alternatively, the inhibition of antigen processing by cycloheximide in PEC may reflect depletion of enzymes or other factors involved in antigen processing. Proteins and polysaccharides may interfere with the events that result in the formation of an immunogenic Ia-peptide complex. We had previously documented that peptides compete for the binding site of Ia molecules. We discussed here a second form of interference by polysaccharides and microbial products. These materials did not compete or interfere with the binding and presentation of processed peptides by Ia. Rather, their presence inside the macrophage inhibited MHC-dependent presentation of immunogenic proteins by inhibiting intracellular steps in antigen processing. This intracellular interference with antigen presentation can be of major importance in the presentation of complex mixtures of protein and carbohydrates.