Analysis of the expression of peptide-major histocompatibility complexes using high affinity soluble divalent T cell receptors.

Understanding the regulation of cell surface expression of specific peptide-major histocompatibility complex (MHC) complexes is hindered by the lack of direct quantitative analyses of specific peptide-MHC complexes. We have developed a direct quantitative biochemical approach by engineering soluble divalent T cell receptor analogues (TCR-Ig) that have high affinity for their cognate peptide-MHC ligands. The generality of this approach was demonstrated by specific staining of peptide-pulsed cells with two different TCR-Ig complexes: one specific for the murine alloantigen 2C, and one specific for a viral peptide from human T lymphocyte virus-1 presented by human histocompatibility leukocyte antigens-A2. Further, using 2C TCR- Ig, a more detailed analysis of the interaction with cognate peptide-MHC complexes revealed several interesting findings. Soluble divalent 2C TCR-Ig detected significant changes in the level of specific antigenic-peptide MHC cell surface expression in cells treated with gamma-interferon (gamma-IFN). Interestingly, the effects of gamma-IFN on expression of specific peptide-MHC complexes recognized by 2C TCR-Ig were distinct from its effects on total H-2 Ld expression; thus, lower doses of gamma-IFN were required to increase expression of cell surface class I MHC complexes than were required for upregulation of expression of specific peptide-MHC complexes. Analysis of the binding of 2C TCR-Ig for specific peptide-MHC ligands unexpectedly revealed that the affinity of the 2C TCR-Ig for the naturally occurring alloreactive, putatively, negatively selecting, complex, dEV-8-H-2 Kbm3, is very low, weaker than 71 microM. The affinity of the 2C TCR for the other naturally occurring, negatively selecting, alloreactive complex, p2Ca-H-2 Ld, is approximately 1000-fold higher. Thus, negatively selecting peptide-MHC complexes do not necessarily have intrinsically high affinity for cognate TCR. These results, uniquely revealed by this analysis, indicate the importance of using high affinity biologically relevant cognates, such as soluble divalent TCR, in furthering our understanding of immune responses.

Enhanced in vivo sensitivity of in vitro interferon-treated B16 melanoma cells to CD8 cells and activated macrophages.

Mouse B16 melanoma cells maintained in vitro in the presence of interferon (IFN)-alpha become resistant to the in vitro antiproliferative effects of IFN-alpha. However, IFN-alpha-treated mice inoculated with these in vitro IFN-treated cells (B16 alpha res cells) have significantly increased life spans (ILS) and significantly higher cure rates than IFN-alpha-treated mice inoculated with B16 cells. This unexpectedly greater sensitivity of B16 alpha res cells to the in vivo antitumor effects of IFN-alpha was evaluated by in vivo cell depletion experiments. Depletion of either activated peritoneal macrophages or cytotoxic T lymphocytes (CTL) reduced the ILS of IFN-treated B16 alpha res-inoculated mice to a level comparable to that of IFN-treated B16-inoculated mice. Depletion of natural killer (NK) cells did not affect the ILS for IFN-treated B16 alpha res-inoculated mice. These studies indicate that activated macrophage and CD8 cell function, but not NK cell function, is important for the enhanced antitumor effects induced by IFN-alpha against B16 alpha res cells. Macrophage killing was unlikely to be mediated by TNF-alpha or IL-1 as B16 and B16 alpha res cells were equally sensitive to TNF-alpha and insensitive to IL-1 in vitro. Further, H-2K antigen expression is significantly more readily inducible on B16 alpha res cells than on B16 cells, consistent with enhanced CD8-mediated killing due to increased MHC class I antigen expression.

Augmenting major histocompatibility complex class I expression by murine tumors in vivo enhances antitumor immunity induced by an active immunotherapy strategy.

OBJECTIVE: Tumors down-regulate major histocompatibility complex class I expression, escaping recognition by the cellular immune response. We hypothesized that augmentation of tumor cell class I expression by interferon-gamma would enhance the cellular antitumor immune response and cure rate of an active immunotherapy strategy. METHODS: B16.F10 tumor cells were exposed to interferon-gamma in culture, and class I expression was quantified using flow cytometry. Syngeneic mice bearing established tumors were injected with interferon-gamma (5000 U, intraperitoneal), and class I expression was assessed using immunohistochemistry. Tumor-specific cytotoxic T lymphocytes were induced in mice by an intratumoral injection of AdCD40L (5 x 10(10) particles), an adenovirus gene transfer vector-based immunotherapy strategy previously demonstrated to augment cellular antitumor immunity. A conjugate-formation assay and the enzyme-linked immunospot assay were used to evaluate the binding and activation of cytotoxic T lymphocytes, respectively. Interferon-gamma was administered to tumor-bearing mice concomitantly with intratumoral AdCD40L. End points measured included the frequencies of cytotoxic T lymphocytes using the enzyme-linked immunospot assay, tumor size, and mouse survival. The role of class I expression was further evaluated by monoclonal antibody blockade in both in vitro and in vivo experiments. RESULTS: B16.F10 cells exposed to interferon-gamma expressed significantly more class I, both in vitro and in vivo, and were able to bind to and activate cytotoxic T lymphocytes more efficiently than untreated cells. Cytotoxic T-lymphocyte frequencies, tumor regression, and the cure rate induced by AdCD40L were augmented by the addition of a single dose of interferon-gamma in tumor-bearing mice. These in vitro and in vivo effects of interferon-gamma were attenuated by class I monoclonal antibody blockade. CONCLUSIONS: Up-regulation of class I expression using interferon-gamma enhances the cellular antitumor immune response and cure rate of AdCD40L, an active immunotherapy strategy. This approach may be useful for human tumors that lack class I expression.

TNF impairs in vivo and in vitro natural killer (NK) susceptibility of B16 melanoma cells.

Tumour necrosis factor alpha (TNF) is a multipotent cytokine which affects many biological properties of both normal and neoplastic cells. Here we show that treatment with TNF reduces B16-A melanoma cell susceptibility to normal and in vivo- and in vitro-activated NK cell-mediated killing. This resistance is associated with an enhancement of B16-A metastatic potential in normal syngeneic mice, but not in anti-asialo GM1-treated animals, further supporting the NK dependence of TNF-induced enhancement of metastatic ability. A significant increase of MHC class I expression on B16-A murine melanoma cells is observed after TNF treatment. In all these effects TNF interacts positively with interferon gamma (IFN gamma). Taken together, these results indicate that TNF treatment negatively affects the susceptibility of B16-A murine melanoma to NK effectors in vivo and in vitro. This decreased susceptibility may be related, at least in part, to enhanced expression of MHC class I antigens on tumour cells.

Surface projection of murine major histocompatibility determinants induced by hydrostatic pressure and cytokines.

Augmentation of surface presentation of the major histocompatibility complex (MHC) is a leading trend for preparation of tumor vaccines. Exposure of weakly immunogenic tumor cells, such as murine B16 melanoma, to hydrostatic pressure (P) in the presence of the membrane-impermeable protein crosslinker (CL) 2',3'-adenosine dialdehyde, was previously shown to induce a substantial increase in surface presentation of MHC molecules. When B-16 melanoma cells, used here as a model, were first treated for 72 h with interferon-gamma or tumor necrosis factor-alpha at concentrations of 10 and 100 units/ml, respectively, followed by application of pressure and cross-linking (PCL), the surface presentation of H2b molecules increased by 40% compared to treatment with cytokines alone, and by up to 1,700% when compared to treatment with PCL alone. Neither P nor CL alone enhanced the MHC presentation when cells were pretreated with these cytokines. The changes in MHC observed after the cytokine treatment were transient and decayed within several hours. However, the changes induced by the sequential treatment with cytokines and PCL were sustained for at least 96 h post-PCL which is of prime importance for immunogenic expression. A series of analogous experiments in the presence of cycloheximide indicated that approximately 50% of the observed PCL-induced increase in MHC projection originates from protein synthesis while the other 50% corresponds to passive translocation of MHC compartments. B16 melanoma cells, modified by the sequential treatment of cytokines and PCL, proved to be substantially more immunogenic by an in vitro sensitization assay than cells treated by either one of these treatments alone. These results may provide a guideline for the preparation of tumor vaccines which could be applied in immunotherapy treatment of cancer.

H2-M3wt-restricted, Listeria monocytogenes-specific CD8 T cells recognize a novel, hydrophobic, protease-resistant, periodate-sensitive antigen.

Mice infected with Listeria monocytogenes (LM) generate H2-M3wt-restricted CD8 effectors which recognize a heat-killed LM-associated antigen (HAA) presented by macrophages. To characterize HAA, we extracted a bioactive component from LM using SDS or NaOH. Extracted HAA aggregated in hydrophilic solvents but dissociated in the presence of SDS into a smaller subunit which migrated in Sephadex G-200 between chymotrypsinogen (25 kDa) and cytochrome c (12.5 kDa). HAA bioactivity and size was unaffected by proteinase K under conditions which degraded virtually all detectable protein. HAA was also unaffected by other proteases, RNase and DNase, but HAA bioactivity was destroyed by periodate, an agent that degrades carbohydrates. These studies demonstrate that H2-M3wt can present a hydrophobic, non-peptide, microbial antigen, probably glycolipid in origin, to CD8 T cells.

Identification of an intragenic interferon-stimulated response element sequence of the mouse class I major histocompatibility complex H-2Kb gene.

A 5'-enhancer-lacking construct of the mouse MHC class I H-2Kb gene is stimulated by interferons in a similar manner as a 5'-enhancer-containing H-2Kb gene. This ability is markedly reduced upon introducing a second deletion removing the first and second intron of the H-2Kb gene. Computational analysis of the deleted stretch reveals the presence of the interferon-stimulated response element (ISRE) in the middle of the second intron. The identified sequence exhibits typical ISRE characteristics in gel retardation experiments. A fragment containing the 5'-enhancer interferon responsive sequence competes effectively in the binding reactions. These results strongly suggest that the second intron of the H-2Kb gene participates in interferon stimulation via the identified ISRE sequence.

Differential expression and regulation of a human transgene, HLA-B27, in mouse placental and embryonic cell lines.

Unlike other somatic cells, human placental trophoblast cells do not express the highly polymorphic HLA-A and HLA-B human leukocyte major histocompatibility antigens that would stimulate maternal immunological rejection of the fetus. To investigate mechanisms underlying cell lineage-specific expression, cell lines were generated from homozygous matings of HLA-B27 transgenic mice. Trophoblast cell lines were generated from gestation day 10 placentas and fibroblasts were cultured from gestation day 13/14 embryos. Polymerase chain reaction (PCR) readily identified HLA-B DNA in transgenic trophoblastic cells but specific mRNA was of low abundance, being detectable by reverse transcriptase PCR but not by Northern blot hybridization. HLA-B-specific protein in/on the trophoblast cells was undetectable by cell enzyme-linked immunosorbent assay and the protein was not induced by exposing the trophoblastic cells to interferon-gamma (IFN-gamma). Restricted expression was specific for the HLA-B transgene and its antigen; IFN-gamma-inducible endogenous H-2Db class I antigens were detectable on the trophoblast cells. In contrast to the trophoblastic cells, HLA-B27 transgenic fibroblasts expressed IFN-gamma-inducible HLA class I antigens as well as H-2Db antigens. Thus, the mechanism(s) regulating expression of the polymorphic HLA-B antigen in trophoblastic cells is gene-specific, IFN-gamma-resistant and operative at the level of transcription or immediate post-transcription.

The mechanisms of peptide exchange and beta 2-microglobulin exchange on cell surface Ld and Kb molecules are noncooperative.

It has previously been shown that the presence of exogenous beta 2-microglobulin (beta 2m) can dramatically enhance the binding of exogenous peptide to cell surface class I MHC. However, the mechanism by which this enhancement takes place is unknown. Two models have been proposed to explain this phenomenon. In the first model, the exchange of peptide and the exchange of beta 2m are cooperative processes. In the alternative model, beta 2m stabilizes free class I heavy chains to increase the total number of peptide binding sites available. In this report, we have examined the relationship between peptide exchange and beta 2m exchange. Comparisons of Ld and Kb complexes formed with peptides possessing widely disparate affinities revealed a reciprocal correlation between the peptide off-rate and the rate of beta 2m exchange. This result indicates that peptide exchange and beta 2m exchange are noncooperative processes that may, in fact, antagonize one another. These findings provide the first demonstration of peptide-specific influences on the rate of beta 2m exchange and suggest that exogenous beta 2m promotes peptide binding by maintaining class I heavy chains in a peptide-receptive state.

Differential H-2 antigen expression in teratocarcinoma-fibroblast hybrids.

Embryo-derived teratocarcinoma cells, like early embryonic cells, do not express the classical MHC class I antigens. The mRNAs for both the H-2 alpha chain and beta2-microglobulin are also undetectable in these cells. We observed that upon fusion of H-2-negative mouse P19 teratocarcinoma cells (H-2k allotype) with H-2-positive embryonic fibroblasts of C57BL/6 origin (H-2b allotype), teratocarcinoma-like cell hybrids were obtained which express the H-2Kb antigen derived from the embryonic fibroblasts, but not the H-2KkDk antigens of the teratocarcinoma. This finding demonstrates that the teratocarcinoma H-2 genes do not respond to the positive regulatory factors present in the hybrids. The H-2k allele was not lost during fusion, as shown by its expression in retinoic-acid-differentiated hybrids treated with interferon-gamma (10 U/ml, 4 days). H-2KkDk antigen expression could also be induced in the undifferentiated hybrids by treating the cells with the protein synthesis inhibitor cycloheximide (1-10 mu g/ml, 18 h), but not with the demethylating agent 5-azacytidine (5 mu M, 2-4 days). These data suggest the presence of a labile, negative regulating protein factor which selectively prevents the expression of the teratocarcinoma-derived H-2 antigens. When the level of this factor(s) is reduced, the teratocarcinoma H-2 genes are capable of responding to the positive regulatory factors.

Inhibition of I-Ad-, but not Db-restricted peptide-induced thymic apoptosis by glucocorticoid receptor antagonist RU486 in T cell receptor transgenic mice.

Thymocytes differentiate by positive and negative selection of immature CD4+ CD8+ T cells. Negative selection occurs by default or by high-affinity recognition of peptides bound to proteins encoded by the major histocompatibility complex (MHC). MHC class I molecules are expressed on many different cell types, although at different levels, whereas MHC class II molecules are selectively expressed on thymic epithelial cells (TEC) and dendritic cells (DC). We investigated the role of the glucocorticoid receptor (GR) in thymic negative selection using the receptor antagonist RU486. Glucocorticoids (GC) are known to be potent inducers of apoptosis in CD4+ CD8+ thymocytes, and we have earlier shown that anti-CD3-induced thymic apoptosis can be blocked by RU486 in vivo. We now show that anti-CD3 induces thymic apoptosis in mice that have been adrenalectomized (ADX), and that RU486 inhibits anti-CD3 antibody-mediated thymocyte killing in newborn thymic organ cultures. Thymocyte apoptosis induced by ovalbumin peptide OVA323-339 treatment of mice transgenic for the DO11.10T cell receptor (TCR), which recognizes this peptide in the context of I-Ad, was found to be inhibited by RU486. These mice responded to peptide treatment by an extensive activation of the peripheral immune system, which became lethal in 60% of the mice when accompanied by simultaneous RU486 treatment. In contrast, RU486 had no effect on thymic apoptosis induced by the influenza A nucleoprotein NP366-374 peptide, recognized in context of Db, in F5 TCR transgenic mice. We interpret the results to demonstrate that different deletion systems operate in the thymus. We propose that endogenous GC may be important for negative selection by default and by high-affinity recognition of endogenous MHC-presented peptides on TEC.

Regulation of class I-restricted epitope processing by local or distal flanking sequence.

Influenza A/Puerto Rico/8/34 nucleoprotein (NP) contains an H-2Kd-restricted CD8+ T cell (T CD8+) epitope spanning amino acid residues 147-155. It was previously demonstrated that expression of NP147-155 and NP147-158 in isolation via "minigene"/recombinant vaccinia virus (vac) technology leads to sensitization of target cells for NP-specific killing while expression of 147-158 lacking the arginine at position 156 (termed here as 147-155TG) does not. The presentation block was overcome by placing this fragment into the context of full length NP. We show that addition of a single amino acid, Met159, to the C terminus of the blocked peptide (creating 147-155TGM) restores presentation. Presentation of 147-155TGM was not due to trimming in the exocytic compartment, consistent with severe limitations on C-terminal trimming activity in this location. Rescued presentation was also achieved when the blocked construct was extended in the N-terminal direction only, but in this case more than 55 amino acids of flanking sequence were required. The transition to presentation was abrupt, with 91-155TG and shorter constructs showing little or no detectable presentation and 90-155TG showing full level presentation. Presentation could not be attributed to acquisition of conventional targets for ubiquitination since mutation of all Lys residues, to which the ubiquitin moiety is conjugated, does not abrogate presentation. Rescued presentation was not inhibited by the peptide aldehyde N-acetyl-L-leucinyl-L-leucinal-L-norleucinal, suggesting that the added elements may be recruiting nonproteasomal activity. We have therefore identified and begun to characterize protease targeting of regulatory elements, both local and distal to an epitope, which strongly influence the ability of the epitope to be excised.

The effect of toxicokinetics on murine mercury-induced autoimmunity.

Mercury induces autoantibodies to the nucleolar protein fibrillarin (ANoA) in genetically susceptible (H-2AS) mouse strains. This study examines the importance of mercury toxicokinetics for the induction and strength (titer) of these autoantibodies. Female mice of the inbred strains A.SW and B10.S (H-2AS on the A and C57BL/10 genetic background, respectively) and A.TL and B10.TL (H-2Ak on the A and C57BL/10 background) were treated with 203HgCl2 in a dose of 1, 5, or 16 mg Hg/L drinking water for 56-70 days. Whole-body retention of 203Hg was monitored throughout the experimental period. Mercury accumulation in kidney, liver, heart, spleen, and brain was determined at end of the experiment when blood samples were also obtained for determination of ANoA. The drinking water consumption showed a limited variation between the strains and the dose groups. Therefore, intake of mercury did not vary much between the strains at a given dose level. The whole-body retention of mercury reached steady state after 4-5 weeks. In general, the B10.S and B10.TL strains showed a lower whole-body retention and deposition of mercury in the kidney and the liver, compared with the A.SW and A.TL strains given the same dose of mercury. The B10.S strain showed a threshold for induction of ANoA at 5 mg Hg/L, whereas ANoA were still seen in A.SW mice given 1 mg Hg/L. Taken together, this is compatible with a less efficient elimination of mercury in the A.SW and A.TL strains, which was also supported by the higher ratio between whole-body retention and intake of mercury in these strains. These findings indicate that genes residing outside the H-2 (MHC) complex play an important role for regulating mercury toxicokinetics, the A genes conferring higher accumulation of mercury in the body than the B10 genes. In mice congenic with regard to the susceptible H-2AS haplotype, a highly significant correlation (P<0.01) existed between on the one hand the whole-body retention and organ accumulation of mercury and on the other hand the titer of ANoA. We conclude that mercury toxicokinetics differs significantly among inbred mouse strains. The differences in toxicokinetics are regulated by non-H-2 genes and correlate with the autoimmune response in the genetically susceptible strains: quantitatively as the titer of the ANoA and qualitatively as different threshold doses for induction of ANoA by mercury.

Assembly of MHC class I molecules with biosynthesized endoplasmic reticulum-targeted peptides is inefficient in insect cells and can be enhanced by protease inhibitors.

To study the requirements for assembly of MHC class I molecules with antigenic peptides in the endoplasmic reticulum (ER), we studied Ag processing in insect cells. Insects lack a class I recognition system, and their cells therefore provide a "blank slate" for identifying the proteins that have evolved to facilitate assembly of class I molecules in vertebrate cells. H-2Kb heavy chain, mouse beta 2-microglobulin, and an ER-targeted version of a peptide corresponding to Ova(257-264) were expressed in insect cells using recombinant vaccinia viruses. Cell surface expression of Kb-OVA(257-264) complexes was quantitated using a recently described complex-specific mAb (25-D1.16). Relative to TAP-deficient human cells, insect cells expressed comparable levels of native, peptide-receptive cell surface Kb molecules, but generated cell surface Kb-OVA(257-264) complexes at least 20-fold less efficiently from ER-targeted peptides. The inefficient assembly of Kb-OVA(257-264) complexes in the ER of insect cells cannot be attributed solely to a requirement for human tapasin, since first, human cells lacking tapasin expressed endogenously synthesized Kb-OVA(257-264) complexes at levels comparable to tapasin-expressing cells, and second, vaccinia virus-mediated expression of human tapasin in insect cells did not detectably enhance the expression of Kb-OVA(257-264) complexes. The assembly of Kb-OVA(257-264) complexes could be greatly enhanced in insect but not human cells by a nonproteasomal protease inhibitor. These findings indicate that insect cells lack one or more factors required for the efficient assembly of class I-peptide complexes in vertebrate cells and are consistent with the idea that the missing component acts to protect antigenic peptides or their immediate precursors from degradation.