Enhanced antigen presentation using human Fc gamma receptor (monocyte/macrophage)-specific immunogens.

A major new challenge for vaccine development is to target APC such as monocytes and macrophages for efficient Ag processing and presentation. It has been shown that Fc gamma R-mediated uptake of Ag-antibody complexes can enhance Ag presentation by myeloid cells at least 100-fold, and directing Ag to Fc gamma R in mice brings about a substantial increase in the effectiveness of immunization while eliminating the requirement for adjuvant. It has not been determined which of the three subclasses of human Fc gamma R on myeloid cells (Fc gamma RI, Fc gamma RII, or Fc gamma RIII) function to enhance Ag presentation. We have targeted our Ag (TT) to each of the three subclasses of human Fc gamma R on monocytes using Fc gamma R subclass-specific mAb-TT conjugates, and have measured TT presentation by monitoring T cell proliferation in response to TT. In addition, we have examined enhanced Ag presentation mediated by a human IgG1 (HIgG1) anti-TT mAb. All anti-Fc gamma R-TT conjugates enhanced Ag presentation. HIgG1 anti-TT, in monomeric form, enhanced Ag presentation through Fc gamma RI only. Anti-Fc gamma RI-Ag conjugates appear to be optimal for application as vaccines. They are monocyte/macrophage-specific, are very efficiently processed and presented, and enhance Ag presentation despite occupation of Fc gamma RI with HIgG.     

Role of ICAM-1 in antigen presentation demonstrated by ICAM-1 defective mutants

We report a methodology for selecting APC with mutations that have impaired their ability to present Ag to T cells. A20 B lymphoblastoid cells were mutagenized and then repeatedly cocultured with murine T-T hybridomas in the presence of specific Ag. During these cocultures, the T-T hybridomas kill the competent APC, allowing the outgrowth of inactive variants. Two variants, A20.M1 and A20.M2, were isolated and studied in detail. These variants are impaired in their ability to present multiple Ag to T cells. This defect is also observed for the presentation of processing independent peptides by fixed APC indicating that a lesion exists in a post-Ag processing step. The level of expression of MHC molecules is unaffected and the functional defect in the APC is not localized to a particular MHC molecule. In contrast, these mutants were found to have a selective decrease in the expression of the murine homolog of ICAM-1, and the residual ability of these cells to present Ag was not blocked by anti-ICAM-1 mAb. Conversely, Ag presentation by the wild-type A20 is inhibited by anti-ICAM-1 mAb. Similarly, anti-LFA-1 mAb inhibited the response of T cells to Ag presented by the wild-type A20 to a much greater degree than by the mutant cells, indicating that LFA-1 is involved in interaction of T cells with the former, but not latter, APC. In the apparent absence of a contribution of LFA-1 to the T cell-APC interaction, either as a result of mAb blocking or the disruption of the APC membrane, the mutant and wild-type APC have a similar level of Ag-presenting activity. Reconstitution of ICAM-1 expression in these mutants by transfection with murine ICAM-1 cDNA fully restores their ability to present Ag. Together these results demonstrate that a murine ICAM-1 homolog is expressed on A20 B cells, where it functions as a major cell interaction molecule. The degree of functional impairment in these mutant APC gives insight into the contribution of cell interaction molecules to efficient Ag presentation and T cell-B cell interaction. Finally, these results also demonstrate the feasibility of selecting APC with mutations affecting Ag presentation.     

Intracellular interference with antigen presentation

Murine peritoneal macrophages were cultured with heat-killed Listeria monocytogenes organisms and then with the protein hen egg white lysozyme. Hen egg lysozyme is well known to need intracellular processing for presentation to T cells. The presentation to T cells of lysozyme was affected despite no reduction in the amount taken up or catabolized by the macrophage. This interference with Ag presentation was not found if the macrophages were cultured with lysozyme before the Listeria pulse. The interference with Ag presentation induced by Listeria was found for a second Ag (conalbumin). Uptake of Listeria did not affect the presentation of the lysozyme peptide 46-61, indicating that MHC class II molecules were available at the macrophage surface. Other materials that are retained in the macrophages affected presentation of lysozyme but not of the processed peptide. These included SRBC, dextran, sucrose, cellobiose, polyvinyl pyrrolidone, sodium dextran sulfate, Ficoll, and polyethylene glycol. Except for SRBC, which were not tested, the remaining molecules did not interfere with presentation of 46-61 by formaldehyde-fixed macrophages, an indication that they did not affect the peptide interaction with class II molecules. Finally, uptake of latex beads did not affect presentation of lysozyme. We conclude that retention in the macrophage of a variety of soluble or particulate molecules can interfere with the intracellular events that result in the creation of an immunogenic determinant. This interference is independent of the catabolism of the Ag or of the availability of class II molecules to bind peptides.     

Human T cell clones present antigen

Two human T cells clones are described which react with influenza virus hemagglutinin type H3 and synthetic peptides of H3 when presented by PBMC APC. Both T cell clones also responded to peptide Ag in the absence of additional APC suggesting that T cells can simultaneously present and respond to Ag. T cell clones could only present peptide Ag and not an appropriate strain of inactivated whole influenza virus thus indicating an inability to process Ag conventionally. Peptide presentation by T cells was dose dependent, restricted by MHC class II Ag and was dependent on the number of Ag presenting T cells per culture. Experiments with nested peptides showed that the same epitope was recognized in the presence and absence of PBMC APC. No Ag or IL-2 from the propagation procedure was carried over into assays and two-color fluorescence-activated cell sorter analysis of each clone detected no contaminating cells with the phenotype of monocytes, macrophages or B cells; in each T cell clone, all cells expressing MHC class II Ag co-expressed CD3. These date therefore provide strong evidence that human T cell clones can simultaneously present and respond to appropriate forms of Ag.     

Protein synthesis in antigen processing

Recent studies indicate that Ag pass through a chloroquine-sensitive intracellular pathway in accessory cells before they are recognized by class II-restricted T cells. Our results indicate that this is also true for insulin. Unexpectedly, we find that protein synthesis is required for optimal accessory cell-dependent processing of insulin and other proteins by adherent macrophages. Treatment of APC with inhibitors of protein synthesis, before and during exposure to Ag, inhibits their subsequent ability to activate murine T cell hybridomas. Experiments are described which suggest that this effect is localized to intracellular processing of Ag rather than uptake or presentation, per se. Inhibition is reversible, and is not observed in special situations where intracellular processing of Ag is not required. A distinct lag period is required for inhibition of processing after inhibition of macrophage protein synthesis. One possible interpretation is that protein synthesis is necessary for maintenance of a labile protein crucial for intracellular processing of Ag. Alternatively, the susceptibility of processing to inhibitors of protein synthesis may reflect an obligate intracellular association of Ag and newly synthesized class II histocompatibility molecules.     

Inhibition by brefeldin A of the specific B cell antigen presentation to MHC class II-restricted T cells

We have shown previously that specific Ag presentation is prevented by the inhibition of protein synthesis but nonspecific presentation is not. In the present paper, Ag presentation by Ag-specific B cells was examined for sensitivity to brefeldin A (BFA), which blocks protein export from the endoplasmic reticulum. A20-HL B lymphoma expressing surface receptors specific for TNP was used as a B cell, and TNP-OVA was used as a specific Ag. The presence of BFA during pulsing of A20-HL cells with TNP-OVA inhibited the ability of the pulsed cells to stimulate 42-6A T cell clone, specific for OVA323-339 and Iad. The inhibition was not due to nonspecific toxicity of BFA, because the presence of BFA during pulsing of A20-HL cells with OVA323-339 did not affect their APC function. Ag binding to the receptor on A20-HL cells and internalization by the cells were observed in the presence of BFA. Thus, BFA might inhibit intracellular processing of specific Ag or intracellular complex formation of antigenic peptide from specific Ag with MHC class II molecules. Nonspecific Ag presentation by A20-HL cells, however, was resistant to BFA. A20-HL cells pulsed with OVA in the presence of BFA, even after fixation, could stimulate 42-6A cells to produce IL-2, although the IL-2 production was lower than that induced by A20-HL cells pulsed in the absence of BFA. These results suggest that the processing pathways for specific Ag and nonspecific Ag are different from each other, at least partly, in A20-HL cells.     

Functional abolition of monocyte HLA-DR by aldehyde treatment. A novel approach to studies of class II restriction elements in antigen presentation

The effect of low concentration aldehyde treatment (0.0012 to 0.005%) on the expression of HLA-DR Ag by human monocytes was investigated. This treatment was shown to selectively abolish the expression of HLA-DR determinants defined by a monomorphic mAb (YE2.36) in a rosette assay. The expression of class I MHC Ag and Fc gamma R remained unaffected. As a result, the presentation of the recall Ag tetanus toxoid and streptokinase-streptodornase (SK-SD) to freshly isolated autologous T cells and T cell clones was completely inhibited. Increasing the concentration of aldehyde to 0.05% consistently produced partial restoration of Ag-presenting capacity. Low dose aldehyde treatment did not affect monocyte viability or membrane turnover. Thus, aldehyde-treated monocytes produced a second generation of HLA-DR and expression was almost completely restored to normal after 24 h of culture. The presentation of monocyte class II Ag as alloantigens was also inhibited by low dose aldehyde treatment but inhibition was much less marked when monocytes were aldehyde treated at 2 h rather than at 24 h of culture. This is consistent with the reexpression of HLA-DR which occurred readily in the first 24 h of culture and much less readily thereafter. Low dose aldehyde treatment did not affect Ag uptake and processing. However, monocytes which had been pulsed with Ag, aldehyde-treated to abolish HLA-DR, and then cultured to allow regeneration of HLA-DR could present Ag only when given a second Ag pulse, suggesting that once the association between microbial Ag and HLA-DR had formed the Ag was not then free to reassociate with novel HLA-DR. Low dose aldehyde treatment did not affect monocyte IL-1 production, neither did it inhibit the detection of HLA-DR by soluble mAb in FACS analysis. These results are consistent with the view that low dose aldehyde treatment disrupts the tertiary structure of human Ia molecules such that allostimulatory determinants and restriction elements for exogenous Ag are rendered inaccessible to T lymphocyte receptors and to cell-bound anti-DR mAb in the rosette assay, although DR determinants may remain accessible to soluble mAb.     

Characterization of cell surface molecules involved in the recognition of antigen-presenting cells by cloned helper T-cell lines

The recognition of antigen-presenting cells (APCs) by T helper (TH) cells occurs in an antigen (Ag)-specific, MHC-restricted manner. Recent evidence, however, suggests that other interaction molecules may also be involved in TH:APC interaction in addition to the T-cell receptor (Ti) and class II or la antigens. We chose, therefore, to examine the role of various interaction molecules (Ia, Ti, L3T4, and LFA-1) in Ag presentation using several TH clones with distinct recognition patterns (self-Ia, self-Ia/Ag, and allogenic Ia). We describe here the use of a rapid clustering assay to study the initial binding events that occur between TH cells and APCs of various types. In all combinations of TH cells and APCs, conjugate formation was both Ag-specific and MHC-restricted. Moreover, with one exception cell clustering was prevented by the addition of monoclonal antibodies (mAb) against either the T-cell receptor or class II MHC molecules. In contrast, mAb to L3T4 and LFA-1 generally failed to inhibit cluster formation even though T-cell proliferation was profoundly inhibited. The relative importance of these interaction molecules in conjugate formation appeared to depend on the APC type as well as on the T-cell clone used. The implications of these findings for the mechanisms of Ag presentation and T-cell activation are discussed.     

Tolerance induction in T helper (Th1) cells by thymic macrophages.

Most macrophages in the peripheral tissues present Ag optimally to a variety of functionally distinct Th cells. Although thymic macrophages have been implicated in deleting autoreactive thymocytes, their role in influencing the functional capacities of mature T cells is not clear. We have established a normal untransformed macrophage cell line, named TMC, from the mouse thymus. The TMC line presents protein Ag to an IL-4-producing Th2 type Th clone after IFN-gamma treatment as evidence by T cell proliferation and the release of IL-3 and IL-4. However, these thymic macrophages are inefficient at stimulating a well characterized cytochrome C-specific IL-2-producing Th1 clone, A.E7. Ag presentation by TMC results in the production of IL-3 but not IL-2 production or proliferation of A.E7 cells. This selective Ag presentation defect to Th1 cells is corrected by the addition of live but not fixed allogeneic irradiated spleen cells, suggesting that the thymic macrophages lack the expression of costimulatory activity required for Th1 activation. This is further demonstrated by the failure of live thymic macrophages to provide costimulatory activity to A.E7 cells stimulated with fixed spleen cells plus the antigenic peptide 81-104. Exposure of A.E7 cells to paraformaldehyde-treated TMC in the presence of 81-104 peptide induces specific hyporesponsiveness, anergy. These data demonstrate that thymic macrophages can have a profound influence on the response of selected T cells to Ag. Furthermore, the nature of the T cell stimulus is also critical because Th1 and Th2 cells responded equally well to the T cell mitogen, Con A, and a bacterial superantigen presented by the thymic macrophages.     

Colocalization of Fc gamma RI-targeted antigen with class I MHC: implications for antigen processing

The high-affinity receptor for IgG (CD64 or FcgammaRI) is constitutively expressed exclusively on professional APCs (monocytes, macrophages, and dendritic cells). When Ag is targeted specifically to FcgammaRI, Ag presentation is markedly enhanced, although the mechanism of this enhancement is unknown. In an effort to elucidate the pathways involved in FcgammaRI targeting, we developed a model targeted Ag using enhanced green fluorescent protein (eGFP). This molecule, wH22xeGFP, consists of the entire humanized anti-FcgammaRI mAb H22 with eGFP genetically fused to the C-terminal end of each CH3 domain. wH22xeGFP binds within the ligand-binding region by its Fc end, as well as outside the ligand-binding region by its Fab ends, thereby cross-linking FcgammaRI. Confocal microscopy studies revealed that wH22xeGFP was rapidly internalized by the high-FcgammaRI-expressing cell line U937 10.6, but did not associate with intracellular proteins Rab4, Rab5a, or Lamp-1, suggesting that the targeted fusion protein was not localized in early endosomes, recycling vesicles, or lysosomes. Interestingly, wH22xeGFP was found colocalized with intracellular MHC class I, suggesting that FcgammaRI-targeted Ags may converge upon a class I processing pathway. These data are in agreement with studies in the mouse showing that FcgammaRI targeting can lead to Ag-specific activation of cytotoxic T cells. Data obtained from these studies should lead to a better understanding of how Ags targeted to FcgammaRI are processed and under what conditions they lead to presentation of antigenic peptides in MHC class I, as a foundation for the use of FcgammaRI-targeted Ags as vaccines.     

Regulation of B cell receptor-mediated MHC class II antigen processing by FcgammaRIIB1

The processing and presentation of Ag by Ag-specific B cells is highly efficient due to the dual function of the B cell Ag receptor (BCR) in both signaling for enhanced processing and endocytosing bound Ag. The BCR for IgG (FcgammaRIIB1) is a potent negative coreceptor of the BCR that blocks Ag-induced B cell proliferation. Here we investigate the influence of the FcgammaRIIB1 on BCR-mediated Ag processing and show that coligating the FcgammaRIIB1 and the BCR negatively regulates both BCR signaling for enhanced Ag processing and BCR-mediated Ag internalization. Treatment of splenic B cells with F(ab')2 anti-Ig significantly enhances APC function compared with the effect of whole anti-Ig; however, whole anti-Ig treatment is effective when binding to the FcgammaRIIB1 was blocked by a FcgammaRII-specific mAb. Processing and presentation of Ag covalently coupled to anti-Ig were significantly decreased compared with Ag coupled to F(ab')2anti-Ig; however, the processing of the two Ag-Ab conjugates was similar in cells that did not express FcgammaRIIB1 and in splenic B cells treated with a FcgammaRII-specific mAb to block Fc binding. Internalization of monovalent Ag by B cells was reduced in the presence of whole anti-Ig as compared with F(ab')2 anti-Ig, but the internalized Ag was correctly targeted to the class II peptide loading compartment. Taken together, these results indicate that the FcgammaRIIB1 is a negative regulator of the BCR-mediated Ag-processing function.     

Cloned Listeria monocytogenes specific non-MHC-restricted Lyt-2+ T cells with cytolytic and protective activity

Mice were infected with Listeria monocytogenes and Lyt-2+ T cell clones capable of lysing Ag-primed bone marrow macrophages were established. In accordance with earlier findings obtained at the population level, some T cell clones were identified which lysed bone marrow macrophages of different MHC type provided the relevant Ag was present. This unusual target cell recognition was further analyzed using a T3+, L3T4-, Lyt-2+, F23+, KJ16+ T cell clone, designated L-28. Target cell lysis by this clone was Ag specific, apparently non-MHC restricted. In contrast, YAC cells and P815 cells were not lysed by clone L-28. However, lysis of irrelevant targets could be induced by anti-T3, F23, or KJ16 mAb. Furthermore, Ag-specific lysis was blocked by anti-Lyt-2 mAb and by F(ab)2 fragments of F23 mAb. In addition to its cytolytic activity, clone L-28 produced IFN-gamma after co-stimulation with accessory cells, Ag, and rIL-2 and conferred significant protection on recipient mice when given together with rIL-2. These data suggest that non-MHC-restricted Lyt-2+ killer cells generated during listeriosis are cytolytic T lymphocytes that interact with their target Ag via the T cell receptor/T3 complex and the Lyt-2 molecule and, furthermore, that these cells play a role in anti-listerial resistance. The possible relevance of IFN-gamma secretion and target cell lysis for antibacterial protection is discussed.     

Macrophages present pinocytosed exogenous antigen via MHC class I whereas antigen ingested by receptor-mediated endocytosis is presented via MHC class II

Macrophages present exogenous Ag either via MHC class I or MHC class II molecules. We investigated whether the mode of hemagglutinin (HA) uptake influences the class of MHC molecule by which this Ag is presented. Normally, HA is ingested by receptor-mediated endocytosis, but this may be switched to macropinocytosis and pinocytosis by adding phorbol esters to the cells. This switch resulted in altered intracellular routing of ingested Ag and a transition from Ag presentation via MHC class II molecules to presentation via MHC class I molecules. Similarly, inhibition of receptor-mediated HA endocytosis, by treating the cells with the HA receptor destroying enzyme neuraminidase, abrogated Ag presentation via MHC class II molecules and induced presentation via MHC class I molecules. If, however, under these conditions, receptor-mediated uptake of HA was restored, by virtue of HA/anti-HA Ab interaction and subsequent uptake of HA via the Fc receptor, presentation via MHC class II was restored as well, whereas presentation of HA via MHC class I molecules was no longer detectable. We conclude that in macrophages the mode of Ag uptake is decisive in determining via which class of MHC molecules Ag is presented: pinocytosis and macropinocytosis produce exclusive presentation of exogenous Ag via MHC class I molecules whereas receptor-mediated endocytosis leads exclusively to presentation via class II molecules.     

Characterization of antigen processing and presentation by resting B lymphocytes

The production of antibody to a thymus-dependent Ag requires cooperation between the B cell and an Ag-specific Th cell. MHC restriction of this interaction implies that the Th cell recognizes Ag on the B cell surface in the context of MHC molecules and that the Ag-specific B cell gets help by acting as an APC for the Th cell. However, a number of studies have suggested that normal resting B cells are ineffective as APC, implying that the B cell must leave the resting state before it can interact specifically with a Th cell. Other studies, including our own with rabbit globulin-specific mouse T cell lines and hybridomas, show that certain T cell lines can be efficiently stimulated by normal resting B cells. One possible explanation for the above contradiction is that our B cells have become activated before presentation. Here we show that presentation by size-selected small B cells is not the result of nonspecific activation signals generated by the T cells or components of the medium. Also, although LPS activation does increase the efficiency of presentation by small B cells, use of large cells in place of small cells or preincubation of resting B cells with mitogenic doses of anti-Ig does not. Another possibility that we considered was that small B cells are unable to process Ag and that we had selected T cell lines that were capable of recognizing native Ag on the B cell surface. In the majority of cases, experiments with B cell lines and macrophages have shown that Ag presentation requires Ag processing, a sequence of events that includes internalization of Ag into an acid compartment, denaturation or digestion of Ag into fragments, and its return to the cell surface in the context of class II MHC molecules. The experiments reported here show that our T cell lines require an Ag processing step and that small resting B cells, like other APC, process Ag before presenting it to T cells. Specifically, we show that an incubation of 2 to 4 h is required after the Ag pulse before Ag presentation becomes resistant to irradiation. Shortly after the pulse, the Ag enters a pronase-resistant compartment. Although efficient Ag presentation requires initial binding to membrane Ig, Ag is no longer associated with membrane Ig at the time of presentation and is not presented in its intact form, because removal of membrane Ig by goat anti-Ig blocks presentation before but not after the Ag pulse.     

The neonatal FcR-mediated presentation of immune-complexed antigen is associated with endosomal and phagosomal pH and antigen stability in macrophages and dendritic cells

The FcγRs found on macrophages (Ms) and dendritic cells (DCs) efficiently facilitate the presentation or cross-presentation of immune-complexed Ags to T cells. We found that the MHC class I-related neonatal FcR for IgG (FcRn) in both Ms and DCs failed to have a strong effect on the cross-presentation of immune complex (IC) OVA Ag to CD8(+) T cells. Interestingly, endosomal FcRn enhanced the presentation of the monomeric OVA-IC to CD4(+) T cells robustly, whereas FcRn in phagosomes exerted distinctive effects on Ag presentation between Ms and DCs. The presentation of phagocytosed OVA-ICs to CD4(+) T cells was considerably enhanced on wild-type versus FcRn-deficient Ms, but was not affected in FcRn-deficient DCs. This functional discrepancy was associated with the dependence of IgG-FcRn binding in an acidic pH. Following phagocytosis, the phagosomal pH dropped rapidly to <6.5 in Ms but remained in the neutral range in DCs. This disparity in pH determined the rate of degradation of phagocytosed ICs. Thus, our findings reveal that FcRn expression has a different effect on Ag processing and presentation of ICs to CD4(+) T cells in the endosomal versus phagosomal compartments of Ms versus DCs.     

The extracellular domains of MHC class II molecules determine their processing requirements for antigen presentation

We have evaluated the relative contributions of the extracellular and cytoplasmic domains of MHC class II molecules in determining the Ag-processing requirements for class II-restricted Ag presentation to T cells. Hybrid genes were constructed to encode a heterodimeric I-Ak molecule in which the extracellular portion of the molecule resembled wild type I-Ak but where the connecting stalk, transmembrane and cytoplasmic domains of both the alpha- and beta-chain were derived from the class I molecule H-2Dd. Mutant I-Ak molecules were expressed as heterodimeric membrane glycoproteins reactive with mAb specific for wild type I-Ak. Fibroblast and B lymphoma cells expressing either wild type or mutant I-Ak molecules were able to process and present hen egg lysozyme (HEL) and conalbumin to Ag-specific, I-Ak-restricted, T cell hybridomas or clones. The mutant-expressing cells presented native and peptide Ag less efficiently than the wild type-expressing cells, suggesting that the disparity in presentation efficiency was not due to a difference in Ag processing. CD4 interaction was intact on the mutant I-Ak molecules. Presentation of native Ag by mutant and wild type-I-Ak-expressing cells was abolished by preincubation with chloroquine, or after paraformaldehyde fixation. After transfection of a cDNA encoding the gene for HEL, neither mutant nor wild type-I-Ak-expressing cells presented endogenously synthesized HEL to a specific T hybrid. Newly synthesized mutant I-Ak molecules were associated with invariant chain. These data demonstrate the ability of hybrid class II molecules to associate intracellularly with invariant chain and degraded foreign Ag in a conventional class II-restricted processing pathway indicating that the extracellular domains of class II molecules play a dominant role in controlling these Ag-processing requirements.     

Cationization of protein antigens. IV. Increased antigen uptake by antigen-presenting cells

Cationization of BSA generates a molecule that mounts antibody responses of increased magnitude and duration and induces T cell proliferation at concentrations 500 times less than native BSA (nBSA). To explain the alteration in immunogenic properties of this Ag, the uptake of nBSA and cationized BSA (cBSA) by splenic APC has been investigated. T cell proliferation assays were conducted with nBSA and cBSA preparations with varying degrees of substitution. An inverse correlation between the degree of cationization and the amounts of Ag needed for optimal T cell reactivity was observed. To determine whether affinity for APC resulted in an increased uptake of cBSA, splenic APC were incubated with nBSA or cBSA for varying amounts of time. Comparisons were made at each time point between untreated Ag-pulsed APC (Ag uptake) and paraformaldehyde-fixed Ag-pulsed APC (processed Ag). Proliferation of T cells primed with nBSA or cBSA increased in proportion to the amount of time of APC exposure to high concentrations of nBSA, first appearing after a 2-h pulse and peaking at 8 h. Conversely, untreated APC needed only a 30-min cBSA exposure to induce either nBSA- or cBSA-primed T cell proliferation, indicating a rapid uptake of cBSA. Comparisons with proliferation induced by paraformaldehyde-fixed cBSA APC indicate that nBSA T cells recognize a lag phase-processed form of cBSA, whereas a majority of cBSA T cells recognize either a rapidly processed form of cBSA, or a membrane-processed cBSA molecule without a classical lag phase processing event. When monensin was used as an inhibitor of fluid phase pinocytosis in splenic APC, the presentation of nBSA was inhibited by 85%, but the presentation of cBSA was inhibited by only 20%. These results imply that nBSA enters the cell by fluid phase pinocytosis, whereas cBSA enters by a nonspecific adsorptive mechanism. The different modes of cellular entry for the two molecules, nBSA and cBSA, resulting in a rapid uptake of cBSA, may have important ramifications on T cell activation and immunoregulation.     

HIV-1 transmission and function of virus-infected monocytes/macrophages.

Monocyte/macrophages (MM) were isolated from HIV-1 seronegative individuals, infected with HIV-1 and examined for their ability to infect autologous T lymphocytes with and without concomitant presentation of exogenous Ag. HIV-1-infected MM presented tetanus toxin (TT) and streptokinase to T cells (as measured by [3H]thymidine incorporation) comparable to presentation by uninfected MM. In these studies, it was observed that HIV-1-infected MM without additional exogenous Ag stimulated autologous T lymphocytes, however, to a lesser degree than with TT and streptokinase. Virus production in T cells appeared to be relative to the degree of stimulation with the highest levels of stimulation and infection observed when T cells were exposed to HIV-1-infected TT-presenting MM. Studies were carried out to examine some of the restricting elements in MM-mediated infection of T lymphocytes with and without TT presentation. Antibodies to CD4, as well as soluble immunopurified gp120, blocked cell-mediated infection indicating that infection of T cells was through the CD4 molecule as has been demonstrated with cell-free virus. In addition, soluble gp120 inhibited Ag presentation by HIV-1-infected and uninfected MM. mAb to MHC class II Ag HLA-DR and -DP blocked T cell infection by HIV-1-infected MM with and without presentation of TT. No effect was observed with mAb to MHC class I Ag. These results indicate that virus transmission to T lymphocytes can be mediated by HIV-1-infected MM and that these cells maintain their function as APC. Activation of T cells appears to be important in the process of T cell infection in this system inasmuch as antibodies that block Ag presentation and thus a T cell proliferative signal inhibit infection.     

Recognition of a shared human prostate cancer-associated antigen by nonclassical MHC-restricted CD8+ T cells

To identify prostate cancer-associated Ags, tumor-reactive T lymphocytes were generated using iterative stimulations of PBMC from a prostate cancer patient with an autologous IFN-gamma-treated carcinoma cell line in the presence of IL-2. A CD8+ T cell line and TCR alphabeta+ T cell clone were isolated that secreted IFN-gamma and TNF-alpha in response to autologous prostate cancer cells but not to autologous fibroblasts or lymphoblastoid cells. However, these T cells recognized several normal and malignant prostate epithelial cell lines without evidence of shared classical HLA molecules. The T cell line and clone also recognized colon cancers, but not melanomas, sarcomas, or lymphomas, suggesting recognition of a shared epithelium-associated Ag presented by nonclassical MHC or MHC-like molecules. Although Ag recognition by T cells was inhibited by mAb against CD8 and the TCR complex (anti-TCR alphabeta, CD3, Vbeta12), it was not inhibited by mAb directed against MHC class Ia or MHC class II molecules. Neither target expression of CD1 molecules nor HLA-G correlated with T cell recognition, but beta2-microglobulin expression was essential. Ag expression was diminished by brefeldin A, lactacystin, and cycloheximide, but not by chloroquine, consistent with an endogenous/cytosolic Ag processed through the classical class I pathway. These results suggest that prostate cancer and colon cancer cells can process and present a shared peptidic Ag to TCR alphabeta+ T cells via a nonclassical MHC I-like molecule yet to be defined.