Disordered Toll-like receptor 2 responses in the pathogenesis of pulmonary sarcoidosis.

In this study, we hypothesized that the granulomatous disorder sarcoidosis is not caused by a single pathogen, but rather results from abnormal responses of Toll-like receptors (TLRs) to conserved bacterial elements. Unsorted bronchoalveolar lavage (BAL) cells from patients with suspected pulmonary sarcoidosis and healthy non-smoking control subjects were stimulated with representative ligands of TLR-2 (in both TLR-2/1 and TLR-2/6 heterodimers) and TLR-4. Responses were determined by assessing resulting production of tumour necrosis factor (TNF)-α and interleukin (IL)-6. BAL cells from patients in whom sarcoidosis was confirmed displayed increased cytokine responses to the TLR-2/1 ligand 19-kDa lipoprotein of Mycobacterium tuberculosis (LpqH) and decreased responses to the TLR-2/6 agonist fibroblast stimulating ligand-1 (FSL)-1. Subsequently, we evaluated the impact of TLR-2 gene deletion in a recently described murine model of T helper type 1 (Th1)-associated lung disease induced by heat-killed Propionibacterium acnes. As quantified by blinded scoring of lung pathology, P. acnes-induced granulomatous pulmonary inflammation was markedly attenuated in TLR-2(-/-) mice compared to wild-type C57BL/6 animals. The findings support a potential role for disordered TLR-2 responses in the pathogenesis of pulmonary sarcoidosis.

Distribution of productive antigen-processing activity for MHC class II presentation in macrophages.

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.

Effect of Pseudomonas-induced chronic lung inflammation on specific cytotoxic T-cell responses to adenoviral vectors in mice.

A mouse model of chronic Pseudomonas-induced bronchopulmonary inflammation that mimics chronic cystic fibrosis (CF) lung disease was employed to determine whether this inflammatory milieu influences immune responses to adenoviral vectors. Pseudomonas-infected and control mice were inoculated intranasally with a second-generation type 2 adenovirus (Ad2) vector (Ad2/betagal-2). After 3 weeks, serum and airway Ad2-specific antibodies and Ad2 vector-directed, cytotoxic T-lymphocyte (CTL) activity in splenocytes were measured. No differences in humoral immunity were observed between Pseudomonas-infected mice and controls. However, there was a two- to three-fold increase in Ad-specific CTL activity in the Pseudomonas-infected mice compared to control mice. MHC class I-dependent antigen presentation by antigen-presenting cells (APC) from lungs of Pseudomonas-infected mice was also significantly increased compared to APC from control mice, suggesting a mechanism that may contribute to increased Ad-specific CD8+ CTL responses. It was concluded that Ad-specific CTL activity is enhanced in the setting of pre-existing chronic Pseudomonas-induced lung inflammation similar to CF lung disease, and that increased antigen presentation via MHC class I in this setting may be one underlying mechanism. These findings underscore the importance of considering the influence of the disease milieu when evaluating modes of gene therapy for such diseases in animal models.

Surfactant protein D enhances bacterial antigen presentation by bone marrow-derived dendritic cells.

Surfactant protein (SP) D functions as a soluble pattern recognition molecule to mediate the clearance of pathogens by phagocytes in the innate immune response. We hypothesize that SP-D may also interact with dendritic cells, the most potent antigen presenting cell, to enhance uptake and presentation of bacterial antigens. Using mouse bone marrow-derived dendritic cells, we show that SP-D binds to immature dendritic cells in a dose-, carbohydrate-, and calcium-dependent manner, whereas SP-D binding to mature dendritic cells is reduced. SP-D also binds to Escherichia coli HB101 and enhances its association with dendritic cells. Additionally, SP-D enhances the antigen presentation of an ovalbumin fusion protein expressed in E. coli HB101 to ovalbumin-specific major histocompatibility complex class II T cell hybridomas. The enhancement of antigen presentation by SP-D is dose dependent and is not shared by other collectin-like proteins tested. These studies demonstrate that SP-D augments antigen presentation by dendritic cells and suggest that innate immune molecules such as SP-D may help initiate an adaptive immune response for the purpose of resolving an infection.

Processing of Mycobacterium tuberculosis antigen 85B involves intraphagosomal formation of peptide-major histocompatibility complex II complexes and is inhibited by live bacilli that decrease phagosome maturation.

Mycobacterium tuberculosis (MTB) inhibits phagosomal maturation to promote its survival inside macrophages. Control of MTB infection requires CD4 T cell responses and major histocompatibility complex (MHC) class II (MHC-II) processing of MTB antigens (Ags). To investigate phagosomal processing of MTB Ags, phagosomes containing heat-killed (HK) or live MTB were purified from interferon-gamma (IFN-gamma)-activated macrophages by differential centrifugation and Percoll density gradient subcellular fractionation. Flow organellometry and Western blot analysis showed that MTB phagosomes acquired lysosome-associated membrane protein-1 (LAMP-1), MHC-II, and H2-DM. T hybridoma cells were used to detect MTB Ag 85B(241-256)-I-A(b) complexes in isolated phagosomes and other subcellular fractions. These complexes appeared initially (within 20 min) in phagosomes and subsequently (>20 min) on the plasma membrane, but never within late endocytic compartments. Macrophages processed HK MTB more rapidly and efficiently than live MTB; phagosomes containing live MTB expressed fewer Ag 85B(241-256)-I-A(b) complexes than phagosomes containing HK MTB. This is the first study of bacterial Ag processing to directly show that peptide-MHC-II complexes are formed within phagosomes and not after export of bacterial Ags from phagosomes to endocytic Ag processing compartments. Live MTB can alter phagosome maturation and decrease MHC-II Ag processing, providing a mechanism for MTB to evade immune surveillance and enhance its survival within the host.

HIV-1 infection impairs cell cycle progression of CD4(+) T cells without affecting early activation responses.

Failure of CD4(+) T cells to proliferate in response to antigenic stimulation is a characteristic of HIV infection. Analysis of the proliferation defect has been hampered by an inability to identify CD4(+) cells with T cell receptor specificity for antigen. To focus only on cells that had been stimulated through the T cell receptor, CD4(+) T cells were stimulated with an anti-Vbeta3 Ab that activates approximately 3-5% of peripheral blood T cells. This approach revealed proliferation defects in cells from HIV-infected patients that were not appreciated using anti-CD3 Ab stimulation and provided the capacity to examine responses on a single cell basis. After anti-Vbeta3 Ab stimulation, CD4(+)Vbeta3(+) cells from HIV-infected patients demonstrated defects in expression of cell cycle-associated proteins, D-type cyclins, and cyclin A. However, the expression of early activation markers, CD69 and CD25, was not significantly impaired in cells from most patients. Thus, CD4(+) T cell proliferation failure in HIV disease is characterized by dysregulated activation that precludes cell cycle progression. This proliferation defect was most apparent in patients with diminished CD4(+) T cell numbers and higher plasma HIV RNA levels. CD4(+) T cell proliferation failure may be a key determinant of immune impairment in HIV disease.

Differential expression of interleukin-2 and gamma interferon in human immunodeficiency virus disease.

Subnormal T-cell production of interleukin-2 (IL-2) in human immunodeficiency virus (HIV) disease has been described; however, it is not clear whether failure to synthesize IL-2 represents a selective or global defect in T-cell cytokine production. We evaluated the intracellular production of gamma interferon (IFN-gamma) and IL-2 in CD4(+) cells that were stimulated with staphylococcal enterotoxin B or cytomegalovirus antigen. Strikingly, IFN-gamma and IL-2 are differentially regulated in T cells of HIV-infected patients such that the numbers of CD69(+) cells or IFN-gamma-positive cells that make IL-2 are proportionally decreased in CD4(+) T cells from HIV-infected patients. These findings demonstrate a selective defect in IL-2 production and suggest that enumeration of IFN-gamma-producing cells in response to T-cell receptor stimulation, while providing some estimate of antigen-reactive cell frequency, may not reflect or predict "normal" T-cell function in HIV-infected patients.

Neutrophils process exogenous bacteria via an alternate class I MHC processing pathway for presentation of peptides to T lymphocytes.

Peptides that are presented by class I MHC (MHC-I) molecules derive from cytosolic Ags processed via the conventional MHC-I pathway or exogenous Ags processed via alternate MHC-I processing mechanisms. Alternate MHC-I processing by macrophages and dendritic cells allows presentation of peptides from particulate Ags, including bacteria. Despite the established phagocytic activity of neutrophils, MHC-I processing and presentation of phagocytosed Ags by neutrophils has not been investigated. Murine neutrophils from peritoneal exudates were shown to express MHC-I molecules and tested for the ability to process HB101.Crl-OVA, Escherichia coli transfected to express a fusion protein containing the 257-264 epitope of OVA. Neutrophils were found to process HB101.Crl-OVA and present OVA(257-264)-K(b) complexes to CD8OVA T hybridoma cells via a pathway that was resistant to brefeldin A, an inhibitor of anterograde endoplasmic reticulum-Golgi transport, and lactacystin, a proteasome inhibitor. These results suggest that neutrophils process phagocytosed bacteria via a vacuolar alternate MHC-I pathway that does not involve cytosolic processing. In addition, neutrophils were found to secrete or "regurgitate" processed peptide that was subsequently presented by neighboring prefixed macrophages or dendritic cells. Thus, neutrophils may influence T cell responses to bacteria, either by directly presenting peptide-MHC-I complexes or by delivering peptides to other APCs for presentation. Hypothetically, neutrophils may directly present peptide to effector T cells in vivo at sites of inflammation, inducing cytokine production, whereas dendritic cells in receipt of neutrophil-derived antigenic peptides may migrate to lymphoid organs to initiate T cell responses.

CD4(+) and CD8(+) T cells kill intracellular Mycobacterium tuberculosis by a perforin and Fas/Fas ligand-independent mechanism.

Cytotoxic effector phenotype and function of MHC-restricted Mycobacterium tuberculosis (MTB)-reactive CD4(+) and CD8(+) T lymphocytes were analyzed from healthy tuberculin skin test-positive persons. After stimulation in vitro with MTB, both CD4(+) and CD8(+) T cells up-regulated mRNA expression for granzyme A and B, granulysin, perforin, and CD95L (Fas ligand). mRNA levels for these molecules were greater for resting CD8(+) than CD4(+) T cells. After MTB stimulation, mRNA levels were similar for both T cell subsets. Increased perforin and granulysin protein expression was confirmed in both in CD4(+) and CD8(+) T cells by flow cytometry. Both T cell subsets lysed MTB-infected monocytes. Biochemical inhibition of the granule exocytosis pathway in CD4(+) and CD8(+) T cells decreased cytolytic function by >90% in both T cell subsets. Ab blockade of the CD95-CD95L interaction decreased cytolytic function for both T cell populations by 25%. CD4(+) and CD8(+) T cells inhibited growth of intracellular MTB in autologous monocytes by 74% and 84%, respectively. However, inhibition of perforin activity, the CD95-CD95L interaction, or both CTL mechanisms did not affect CD4(+) and CD8(+) T cell mediated restriction of MTB growth. Thus, perforin and CD95-CD95L were not involved in CD4(+) and CD8(+) T cell mediated restriction of MTB growth.

Processing of exogenous antigens for presentation by class I MHC molecules involves post-Golgi peptide exchange influenced by peptide-MHC complex stability and acidic pH.

Vacuolar alternate class I MHC (MHC-I) Ag processing allows presentation of exogenous Ag by MHC-I molecules with binding of antigenic peptides to post-Golgi MHC-I molecules. We investigated the role of previously bound peptides and their dissociation in generating peptide-receptive MHC-I molecules. TAP1-knockout macrophages were incubated overnight with an initial exogenous peptide, producing a large cohort of peptide-K(b) complexes that could influence subsequent peptide dissociation/exchange. Initial incubation with FAPGNYPAL, KVVRFDKL, or RGYVYQGL enhanced rather than reduced subsequent binding and presentation of a readout peptide (SIINFEKL or FAPGNYPAL) to T cells. Thus, K(b) molecules may be stabilized by an initial (stabilizing) peptide, enhancing their ability to bind readout peptide and implicating peptide dissociation/exchange. In contrast, incubation with SIINFEKL as stabilizing peptide reduced presentation of readout peptide. SIINFEKL-K(b) complexes were more stable than other peptide-K(b) complexes, which may limit their contribution to peptide exchange. Stabilizing peptides (FAPGNYPAL, KVVRFDKL, or RGYVYQGL) enhanced alternate MHC-I processing of HB101.Crl-OVA (Escherichia coli expressing an OVA fusion protein), indicating that alternate MHC-I Ag processing involves peptide dissociation/exchange. Stabilizing peptide enhanced processing of HB101.Crl-OVA more than presentation of exogenous OVA peptide (SIINFEKL), suggesting that peptide dissociation/exchange may be enhanced in the acidic phagosomal processing environment. Furthermore, exposure of cells to acidic pH increased subsequent binding and presentation of readout peptide. Thus, peptide dissociation/exchange contributes to alternate MHC-I Ag processing and may be influenced by both stability of peptide-MHC-I complexes and pH.

Toll-like receptor 2-dependent inhibition of macrophage class II MHC expression and antigen processing by 19-kDa lipoprotein of Mycobacterium tuberculosis.

Mycobacterium tuberculosis (MTB) induces vigorous immune responses, yet persists inside macrophages, evading host immunity. MTB bacilli or lysate was found to inhibit macrophage expression of class II MHC (MHC-II) molecules and MHC-II Ag processing. This report characterizes and identifies a specific component of MTB that mediates these inhibitory effects. The inhibitor was extracted from MTB lysate with Triton X-114, isolated by gel electroelution, and identified with Abs to be MTB 19-kDa lipoprotein. Electroelution- or immunoaffinity-purified MTB 19-kDa lipoprotein inhibited MHC-II expression and processing of both soluble Ags and Ag 85B from intact MTB bacilli. Inhibition of MHC-II Ag processing by either MTB bacilli or purified MTB 19-kDa lipoprotein was dependent on Toll-like receptor (TLR) 2 and independent of TLR 4. Synthetic analogs of lipopeptides from Treponema pallidum also inhibited Ag processing. Despite the ability of MTB 19-kDa lipoprotein to activate microbicidal and innate immune functions early in infection, TLR 2-dependent inhibition of MHC-II expression and Ag processing by MTB 19-kDa lipoprotein during later phases of macrophage infection may prevent presentation of MTB Ags and decrease recognition by T cells. This mechanism may allow intracellular MTB to evade immune surveillance and maintain chronic infection.

Mycobacterium tuberculosis 19-kDa lipoprotein promotes neutrophil activation.

Certain microbial substances, e.g., LPS, can activate neutrophils or prime them to enhance their response to other activating agents, e.g., fMLP. We investigated the role of the Mycobacterium tuberculosis (MTB) 19-kDa lipoprotein in activation of human neutrophils. MTB 19-kDa lipoprotein initiated phenotypic changes characteristic of neutrophil activation, including down-regulation of CD62 ligand (L-selectin) and up-regulation of CD35 (CR1) and CD11b/CD18 (CR3, Mac-1). In addition, exposure of neutrophils to MTB 19-kDa lipoprotein enhanced the subsequent oxidative burst in response to fMLP as assessed by oxidation of dihydrorhodamine 123 (determined by flow cytometry). LPS also produced these effects with similar kinetics, but an oligodeoxynucleotide containing a CpG motif failed to induce any priming or activation response. Although the effects of LPS required the presence of serum, neutrophil activation by MTB 19-kDa lipoprotein occurred independently of serum factors, suggesting the involvement of different receptors and signaling mechanisms for LPS and MTB 19-kDa lipoprotein. Thus, MTB 19-kDa lipoprotein serves as a pathogen-associated molecular pattern that promotes neutrophil priming and activation.

Systemic deficits in transporter for antigen presentation (TAP)-1 or proteasome subunit LMP2 have little or no effect on tumor incidence.

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.

CpG DNA induces maturation of dendritic cells with distinct effects on nascent and recycling MHC-II antigen-processing mechanisms.

Murine bone marrow cultured with GM-CSF produced dendritic cells (DCs) expressing MHC class II (MHC-II) but little CD40, CD80, or CD86. Oligodeoxynucleotides (ODN) containing CpG motifs enhanced DC maturation, increased MHC-II expression, and induced high levels of CD40, CD80, and CD86. When added with Ag to DCs for 24 h, CpG ODN enhanced Ag processing, and the half-life of peptide:MHC-II complexes was increased. However, Ag processing was only transiently enhanced, and exposure of DCs to CpG ODN for 48 h blocked processing of hen egg lysozyme (HEL) to HEL(48-61):I-A(k) complexes. Processing of this epitope required newly synthesized MHC-II and was blocked by brefeldin A (BFA), suggesting that reduced MHC-II synthesis could explain decreased processing. Real-time quantitative PCR confirmed that CpG ODN decreased I-A(beta)(k) mRNA in DCs. In contrast, RNase(42-56):I-A(k) complexes were generated via a different processing mechanism that involved recycling MHC-II and was partially resistant to BFA. Processing of RNase(42-56):I-A(k) persisted, although at reduced levels, after CpG-induced maturation of DCs, and this residual processing by mature DCs was completely resistant to BFA. Changes in endocytosis, which was transiently enhanced and subsequently suppressed by CpG ODN, may affect Ag processing by both nascent and recycling MHC-II mechanisms. In summary, CpG ODN induce DC maturation, transiently increase Ag processing, and increase the half-life of peptide-MHC-II complexes to sustain subsequent presentation. Processing mechanisms that require nascent MHC-II are subsequently lost, but those that use recycling MHC-II persist even in fully mature DCs.

Phagosomes acquire nascent and recycling class II MHC molecules but primarily use nascent molecules in phagocytic antigen processing.

Phagosomes contain class II MHC (MHC-II) and form peptide:MHC-II complexes, but the source of phagosomal MHC-II molecules is uncertain. Phagosomes may acquire nascent MHC-II or preexisting, recycling MHC-II that may be internalized from the plasma membrane. Brefeldin A (BFA) was used to deplete nascent MHC-II in murine macrophages to determine the relative contributions of nascent and recycling MHC-II molecules to phagocytic Ag processing. In addition, biotinylation of cell-surface proteins was used to assess the transport of MHC-II from the cell surface to phagosomes. BFA inhibited macrophage processing of latex bead-conjugated Ag for presentation to T cells, suggesting that nascent MHC-II molecules are important in phagocytic Ag processing. Furthermore, detection of specific peptide:MHC-II complexes in isolated phagosomes confirmed that BFA decreased formation of peptide:MHC-II complexes within phagosomes. Both flow organellometry and Western blot analysis of purified phagosomes showed that about two-thirds of phagosomal MHC-II was nascent (depleted by 3 h prior treatment with BFA) and primarily derived from intracellular sites. About one-third of phagosomal MHC-II was preexisting and primarily derived from the plasma membrane. BFA had little effect on phagosomal H2-DM or the degradation of bead-associated Ag. Thus, inhibition of phagocytic Ag processing by BFA correlated with depletion of nascent MHC-II in phagosomes and occurred despite the persistent delivery of plasma membrane-derived recycling MHC-II molecules and other Ag-processing components to phagosomes. These observations suggest that phagosomal Ag processing depends primarily on nascent MHC-II molecules delivered from intracellular sites, e.g., endocytic compartments.

Mycobacterium tuberculosis inhibits MHC class II antigen processing in murine bone marrow macrophages.

Infection of murine bone-marrow-derived macrophages with viable Mycobacterium tuberculosis (MTB) H37Ra inhibited surface expression of MHC class II (MHC-II) molecules and processing of exogenous antigens for presentation to CD4(+) T hybridoma cells. The inhibition was not dependent on bacterial viability, since it was also produced by exposure to dead bacilli and MTB cytosol preparations, suggesting that it was initiated by a constitutively expressed bacterial component. Northern blot analysis demonstrated that MTB bacilli or cytosol decreased MHC-II mRNA, and immunoprecipitation of biosynthetically labeled molecules confirmed that MHC-II protein synthesis was diminished. Exposure to MTB or MTB cytosol also decreased expression of H2-DM, but H2-DM expression was still sufficient to catalyze conversion of MHC-II to SDS-stable dimers, a measure of MHC-II peptide loading. Thus, infection with MTB decreased both MHC-II and H2-DM expression, but diminished MHC-II synthesis provided the major limitation to antigen processing.

CpG oligodeoxynucleotides act as adjuvants for pneumococcal polysaccharide-protein conjugate vaccines and enhance antipolysaccharide immunoglobulin G2a (IgG2a) and IgG3 antibodies.

Pneumococcal polysaccharide-protein conjugate vaccines elicit antipolysaccharide antibodies, but multiple doses are required to achieve protective antibody levels in children. In addition, the immunogenicity of experimental multivalent pneumococcal conjugate vaccines varies with different polysaccharide serotypes. One strategy to improve these vaccines is to incorporate an adjuvant to enhance their immunogenicity. Synthetic oligodeoxynucleotides containing unmethylated CpG motifs (CpG ODN) are adjuvants that promote T-cell and T-dependent antibody responses to protein antigens, but it has been unclear whether CpG ODN can enhance polysaccharide-specific antibody responses. The present studies demonstrate significant adjuvant activity of CpG ODN for antibody responses against Streptococcus pneumoniae polysaccharide types 19F and 6B induced by conjugates of 19F and 6B with the protein carrier CRM(197). BALB/c ByJ mice were injected with 19F-CRM(197) or 6B-CRM(197) with or without CpG ODN, and sera were tested for anti-19F or anti-6B antibodies by enzyme-linked immunosorbent assay. The polysaccharide-specific antibody response to 19F-CRM(197) alone was predominantly of the immunoglobulin G1 (IgG1) and IgM isotypes, but addition of CpG ODN markedly increased geometric mean titers of total anti-19F antibody (23-fold), anti-19F IgG2a (26-fold), and anti-19F IgG3 (>246-fold). The polysaccharide-specific antibody response to 6B-CRM(197) alone consisted only of IgM, but addition of CpG ODN induced high titers of anti-6B IgG1 (>78-fold increase), anti-6B IgG2a (>54-fold increase), and anti-6B IgG3 (>3,162-fold increase). CpG ODN also increased anti-CRM(197) IgG2a and IgG3. Adjuvant effects were not observed with control non-CpG ODN. Thus, CpG ODN significantly enhance antipolysaccharide IgG responses (especially IgG2a and IgG3) induced by these glycoconjugate vaccines.

Deficiency of transporter for antigen presentation (TAP) in tumor cells allows evasion of immune surveillance and increases tumorigenesis.

Proteins involved in class I MHC (MHC-I) Ag processing, such as the TAP, are deficient in some human tumor cells. This suggests that antitumor responses by CD8 T cells provide selection pressure to favor outgrowth of cells with defective processing of tumor Ags. Nonetheless, this evidence is only correlative, and controlled in vivo experiments have been lacking to demonstrate that TAP deficiency promotes survival of tumor cells. To explore the role of Ag processing defects in tumor progression, matched panels of TAP1-positive and TAP1-negative tumor cell lines were generated from a parental transformed murine fibroblast line. Inoculation of C57BL/6 mice with TAP1-negative cells produced large and persistent tumors. In contrast, TAP1-positive cells did not generate lasting tumors, although small tumors were detected transiently and regressed spontaneously. Both TAP1-positive and TAP1-negative cells produced tumors in athymic mice, confirming that TAP-dependent differences in tumorigenicity were due to T cell-dependent immune responses. Inoculation of C57BL/6 mice with mixtures of TAP1-positive and TAP1-negative cells produced tumors composed exclusively of TAP1-negative cells, indicating in vivo selection for cells with TAP deficiency. Thus, loss of TAP function allows some tumor cells to avoid T cell-dependent elimination, resulting in selection for tumor cells with deficient Ag processing.