Endocytosis and de novo expression of major histocompatibility complex encoded class I molecules: kinetic and ultrastructural studies

The endocytic pathway and expression of the major histocompatibility complex encoded class I molecule H-2Kk was investigated in murine fibroblasts. Internalization of H-2K molecules did not occur constitutively. Endocytosis of the molecules was induced by addition of multivalent ligands such as rabbit anti-mouse immunoglobulin serum or protein A-bearing liposomes to cells pretreated with anti-H-2Kk antibodies. The complete removal of H-2K molecules took about 5 h at 37 degrees C and was not inhibited by the lysosomotropic agent NH4Cl or the protein synthesis inhibitor cycloheximide. When targeted liposomes that contained carboxyfluorescein at a self-quenched concentration were directed against H-2K molecules, the cells became highly fluorescent after 30 min: a consequence of carboxyfluorescein release from the liposomes. This process was inhibited by NH4Cl but not by cycloheximide, suggesting internalization of H-2K molecules into acidic intracellular compartments. The endocytic pathway of liposomes directed against H-2K molecules and the subcellular compartments involved in this process were investigated with targeted liposomes containing horseradish peroxidase. By electron microscopy, the endocytic process was shown to start very rapidly (1-2 min) and involved uncoated cell surface invaginations. The cytoplasmic uncoated vesicles fused together into larger vacuoles containing concentrated liposomes and by 1 h, liposomes began to be destroyed in lysosomal compartments. Within 4 h, 90% of liposomes were lysed inside the cell. The fate of radiolabeled anti-H-2K antibody was also investigated. Degradation of the antibody occurred only when cross-linked with a second layer of antibody, beginning after 2 h and becoming more pronounced after 20 h of incubation. The original cell surface abundance of H-2K molecules was reestablished after 5 to 7 h. During this time neither NH4Cl nor cycloheximide had any effect on the cell surface expression of the molecule. However, after a second cycle of internalization, cells incubated with cycloheximide no longer expressed these molecules. These results suggested that H-2K molecules were not recycled back to the surface after internalization but were degraded in lysosomal compartments together with their ligand. Preexisting molecules, already present in intracellular pools, were expressed to replace them. By immunoprecipitation of metabolically labeled intracellular and surface H-2K molecules, we observed an intracellular pool of H-2K of about 70 to 80% of the total cellular H-2K.     

Dual parameter, quantitative cytofluorometric analysis of endogenous H-2Kk and foreign HLA class I molecules expressed at the surface of murine transformed L cells

By using a calibrated dual laser cell sorter and monoclonal antibodies directly conjugated to fluorescein and rhodamine and specific for H-2Kk and HLA class I antigens, quantitative cytofluorometric analysis was performed on individual HLA-A3 or -CW3 transformed mouse L cells (H-2k). More than 80% of these cells expressed both HLA class I and H-2Kk molecules. Their respective levels of expression were calculated: a mean of 4 X 10(5) HLA class I and 2.3 X 10(5) H-2Kk molecules per single cell. Quantitative comparison with control untransformed L cells and double fluorescence contour maps showed a positive correlation between the levels of expression of HLA class I and H-2Kk molecules suggesting that expression of foreign class I molecules did not occur at the expense of the endogenous H-2k product.     

Differential induction of H-2K vs H-2D class I major histocompatibility complex antigen expression by murine recombinant interferon-gamma

The results presented here indicate that recombinant murine interferon-gamma can cause a dramatic differential induction of two distinct class I MHC molecules. Thus, IFN-gamma treatment of the murine leukemia virus (MuLV)-induced AKR SL3 tumor, a cell line that normally expresses moderate levels of class I MHC antigens, resulted in a large increase in H-2Dk expression, but no change or a slight decrease in H-2Kk expression as measured by cytofluorography. Explanations of the selective enhancement of Dk expression based on increased Fc receptor display or differential kinetics of induction were ruled out. The phenomenon was observed over a wide range of doses of IFN-gamma and with two different monoclonal antibodies to Kk, the latter finding making it unlikely that an altered form of the Kk molecule was induced. The same differential induction of the Dk antigen was observed for the LBRM.5A4 tumor cell line. Because LBRM.5A4 is also MuLV+ but of congenic B10.BR (H-2k) origin, these results were consistent with the possibility that such differential induction was associated with the H-2k haplotype and/or MuLV. The implications of these results, as a possible mechanism of tumor cell escape from an immune surveillance system monitored by class I MHC-restricted T cells and as a useful model system to dissect the mechanism of IFN-gamma induction of class I MHC antigens, are discussed.     

Defining cytolytic T lymphocyte recognition of chemically modified self. I. Response to trinitrophenyl-H-2Kk

We used purified class I antigen incorporated into liposomes to examine the response of secondary cytolytic T lymphocytes (CTL) to chemically modified self. By generating the secondary response in the presence of T cell helper factor, the level of CTL response was limited by CTL recognition of added antigen rather than by helper cell generation of lymphokines. We found a strong secondary response against chemically modified self when spleen cells from trinitrophenyl (TNP)-primed C3H/HeJ mice were stimulated with a) TNP-modified liposomes containing H-2Kk, b) liposomes containing H-2Kk purified from TNP-modified RDM-4 (H-2k) cells, or c) liposomes containing the limited trypsin proteolysis product of H-2Kk that had been directly modified with TNP. In contrast, we were not able to generate a significant CTL response with unmodified H-2Kk incorporated into vesicles along with TNP-modified membrane components lacking H-2Kk. These results suggest that TNP-modified H-2Kk is a major antigenic site recognized by CTL from C3H/HeJ mice after priming against TNP-modified self.     

Promotion of tumor growth by transfecting antisense DNA to suppress endogenous H-2Kk MHC gene expression in AKR mouse thymoma

Many AKR spontaneous thymomas are reported to express different amounts of the major histocompatibility complex class I H-2Kk molecules. Moreover, H-2Kk-deficient AKR tumor cells are found to be more malignant when compared to tumor cells that express abundant levels of the H-2Kk molecules. To corroborate further the role of H-2Kk in tumorigenesis of AKR leukemia, we have, in this study, expressed antisense H-2Kk RNA in a high-H-2Kk-expressing and poorly tumorigenic AKR thymoma cell line 369. The down-regulation of H-2Kk molecules in the transfected 369 clones rendered them more tumorigenic in syngeneic AKR/J mice. The increase in oncogenicity correlates well with a concomitant reduction in their susceptibility to tumor-specific cytotoxic T lymphocytes in vitro. These results suggest the relevance of H-2Kk molecules in the immune surveillance of AKR tumors.     

"Physiological interaction" does not explain the H-2 requirement for recognition of virus-infected cells by cytotoxic T cells.

B10.A (H-2Kk, H-2Dd) ectromelia-immune T cells from secondary responses in vitro were protent killers of both infected L929 (H-2Kk H-2Dk) and infected P-815 (H-2Kd, H-2Db) target cells. Specific competition with unlabelled targets showed that two separate T cell subsets were responsible for lysis of infected L929 and infected P-815 cells. One hypothesis to account for this (a form of "physiological interaction") is that T cells which kill one target e.g. infected L929) display only one out of two possible self-complementary recognition structures, in this example the H-2Kk alloantigen, not H-2Dd, whereas T cells that lyse infected P-815 targets display only H-2Dd, not H-2Kk. This hypothesis was tested and seems untenable because of the following results: A.TH (H-2Ks, H-2Dd) ectromelia-immune, secondary cytotoxic T cells which killed infected SJL/J (H-2Ks, H-2Ds) targets were themselves inactivated by pre-incubation with SJL/J cytotoxic T cells generated in one-way mixed lymphocyte reaction (MLR) against BALB/c (H-2Kd, H-2Dd). A.TL (H-2Ks, H-2Dd) ectromelia-immune secondary cytotoxic T cells which killed infected BALB/c targets were themselves inactivated by BALB/c cytotoxic T cells generated in MLR against SJL/J. Thus, virus-immune T cells which lyse infected targets by virtue of shared H-2K are also displaying H-2D alloantigen, and vice versa.     

Introduction of the H-2Dk gene into a class I-negative tumor cell line confers interferon-gamma inducibility upon the silent endogenous H-2Kk gene.

Kgv cells do not constitutively express class I mRNA or protein. Interferon (IFN)-gamma, but not IFN-alpha/beta, induces H-2Dk expression. IFN does not induce H-2Kk expression. We examined constitutive and IFN-inducible class I expression on Kgv cells stably transfected with genomic clones of H-2Kk or H-2Dk and on somatic cell hybrid lines constructed between Kgv cells and constitutively class I-positive cells of a distinguishable H-2 haplotype. Our results suggest that both the lack of constitutive class I expression and the inability of IFN-alpha/beta to induce class I expression on Kgv cells are primarily due to cis-regulatory mechanisms. However, stable introduction of the H-2Dk gene into Kgv cells conferred IFN-gamma inducibility upon the silent endogenous H-2Kk gene. Therefore, the failure of IFN-gamma to induce H-2Kk expression on Kgv cells is due, at least in part, to a trans-regulatory mechanism.     

Endocytosis of MHC molecules by B cell-B lymphoma and B cell-T lymphoma hybrids

Different cells and different cell surface determinants of the same cells take up liposomes, bound to them via monoclonal antibodies, with variable efficiency. We have previously reported that T and B lymphocytes differ in the extent to which they take up liposomes bound to MHC class I molecules; T cells endocytose class I molecules rapidly, but B cells endocytose class I molecules much less efficiently, although their endocytosis of class II molecules is rapid. An approach toward understanding the molecular basis for this difference was made by evaluating the internalization patterns of somatic cell hybrids of B and T cells. Hybrid cells were constructed between LPS-stimulated purified B cell blasts from C57BL/6 mice (H-2b) and the HAT-sensitive AKR T lymphoma BW 5147 (H-2k). Hybrids between the BALB/c B lymphoma M12.4.1 (H-2d) and B cell LPS blasts from B10.BR (H-2k) mice were also evaluated. In all cases, for hybrid tumor cells, liposomes that were bound to class I molecules encoded by genes from the B cell donor were endocytosed as efficiently as liposomes bound to the class I molecules of the recipient lymphoid cell. T cell tumors efficiently internalized their own class I molecules and those donated by B cells; B cell tumors internalized liposomes that were bound to their own and the donor B cell class I molecules poorly. Thus, our results suggest that the internalization of an MHC molecule is not an intrinsic property of the molecule, but rather of the cell in which it is found.     

Clustering of antigenic determinants on H-2 molecules

The spatial relationship of individual antigenic determinants on H-2Kk and H-2Db molecules was investigated with seven different monoclonal anti-H-2Kk and seven anti-H-2Db antibodies. In these studies the binding of radiolabeled monoclonal anti-H-2 to target cells was competed by addition of various cold anti-H-2 antibodies. The results indicate that on both H-2Kk and H-2Db molecules the antigenic determinants are arranged in two spatially separated clusters. Thus, antibodies to determinants within a cluster show mutual inhibition of binding but do not block the binding of antibodies to the other cluster, and vice versa. Furthermore, in the case of H-2Db antigens it was observed that binding of antibodies to one cluster would considerably enhance the binding of antibodies to the other cluster. A preliminary Scatchard analysis indicated that the enhancing antibody did not alter the affinity of the radiolabeled antibody, but led to an increase of available binding sites on the cell membrane. In addition, binding inhibition studies revealed that the conventional private specificity H-2.2 of H-2Db consists of at least two independent sites on the molecule.     

Nonimmune thyroid destruction results from transgenic overexpression of an allogeneic major histocompatibility complex class I protein.

The overexpression of major histocompatibility complex (MHC) class I molecules in endocrine epithelial cells is an early feature of autoimmune thyroid disease and insulin-dependent diabetes mellitus, which may reflect a cellular response, e.g., to viruses or toxins. Evidence from a transgenic model in pancreatic beta cells suggests that MHC class I overexpression could play an independent role in endocrine cell destruction. We demonstrate in this study that the transgenic overexpression of an allogeneic MHC class I protein (H-2Kb) linked to the rat thyroglobulin promoter, in H-2Kk mice homozygous for the transgene, leads to thyrocyte atrophy, hypothyroidism, growth retardation, and death. Thyrocyte atrophy occurred in the absence of lymphocytic infiltration. Tolerance to allogeneic class I was revealed by the reduced ability of primed lymphocytes from transgenic mice to lyse H-2Kb target cells in vitro. This nonimmune form of thyrocyte destruction and hypothyroidism recapitulates the beta-cell destruction and diabetes that results from transgenic overexpression of MHC class I molecules in pancreatic beta cells. Thus, we conclude that overexpression of MHC class I molecules may be a general mechanism that directly impairs endocrine epithelial cell viability.     

Signal transduction through class I MHC by a monoclonal antibody that detects multiple murine and human class I molecules.

We have generated a hamster anti-mouse class I reactive mAb that is capable of activating T cells in the presence of the cofactor PMA, as assayed by both IFN-gamma production and cellular proliferation. This mAb detects an epitope present on the majority of murine class I molecules, with the known exceptions of H-2Kk and H-2Kq, and is therefore not beta 2-microglobulin-specific. It also recognizes multiple human class I molecules. The epitope recognized by this antibody maps to the class I alpha 1 domain. The activation properties of this mAb are not mediated exclusively through the glycosylphosphatidylinositol-linked Qa-2 molecule, as the antibody activates spleen cells from Qa-2 negative strains. Although class I molecules are not usually considered as activation Ag, these data demonstrate their potential for involvement in signal transduction.     

The mechanism of antigen presentation by dendritic cells and splenic adherent cells in the induction of an allogeneic cytotoxic T-lymphocyte response to H-2Kk liposomes

Induction of an allogeneic cytotoxic T-lymphocyte (CTL) response to purified alloantigen is partially dependent on uptake and processing of the class I alloantigen by antigen-presenting cells (APC) followed by recognition of the alloantigen and self Ia by helper T cells (TH). The activated TH provides the helper signal(s) to the alloantigen-specific CTL for proliferation and differentiation into an active effector CTL. The role of antigen processing and presentation of major histocompatibility complex alloantigens was examined and the ability of different types of APC to present purified H-2Kk liposomes was investigated. Splenic adherent cells (SAC), splenic dendritic cells (DC), and B-cell lymphoblastoid lines were all shown to be effective in the presentation of H-2Kk liposomes. The relative ability of these cells to serve as APC was determined to be DC greater than B-cell tumors greater than SAC. The role of processing of H-2Kk liposomes by SAC and DC was examined by investigating the effect of weak bases on pulsing of the APC. These experiments suggest that presentation of alloantigen by both SAC and DC involves a step which is sensitive to inhibition by weak bases. We examined whether the TH were activated by similar mechanisms when stimulated by the various APC. The functional involvement of the T-cell surface marker L3T4 was demonstrated in the induction of TH. In contrast, L3T4 was not involved in the subsequent generation of CTL since monoclonal antibody (MAb) specific for L3T4 was not effective in blocking CTL function in the presence of nonspecific T helper factor (THF). Similarly, Ia on the APC was shown to be involved in the stimulation of the TH pathway but not directly in the differentiation of the CTL. Thus, DC and B cells in addition to SAC can present H-2Kk to TH. The presentation of alloantigen by both cell types may involve an intracellular route as demonstrated by the blocking of the TH response by weak bases. Both Ia and L3T4 are required on the APC for induction of the TH response. The minimal requirements for activation of the CTL were H-2Kk liposomes and a source of THF.     

Somatic variation of H-2Kk expression and structure in a T-cell lymphoma: instability, stabilization, high production and structural mutation

In the heterozygous T lymphoma line LDHB, variants which have lost the expression of individual H-2 class I genes are spontaneously generated in vitro at a frequency of 10(-1)-10(-2). A cell line (HK13) in which the class I gene Kk is stably expressed (frequency of loss variants less than 10(-4) was selected from LDHB cells by fluorescence activated cell sorting. Further selection of HK13 cells for high Kk expression led to the isolation of the HK22 line which expresses twice as much Kk as HK13. From HK13 and HK22 cells, spontaneous structural variants of Kk having lost individual serological determinants of the Kk wild-type molecule, were isolated by fluorescence activated cell sorting. Such variants occur at a frequency of 10(-6)-10(-7) per cell per generation. The analysis of these variants indicates that they carry mutations in the Kk structural gene and that HK13 cells express a single Kk gene which appears to be duplicated in HK22 cells. We did not find evidence for the generation of variants expressing 'alien' class I products in the LDHB cell line. The instability of class I gene expression in LDHB cells and the transition to stable expression may represent steps of T-cell differentiation in the thymus.     

Cytolytic T cells recognize a chimeric MHC class I antigen expressed in influenza A infected transgenic mice

A chimeric H-2Kd/Kk gene, called pC31, contains the extracellular alpha 1 domain of Kd origin whereas the rest of the molecule is of Kk origin. Disruption of the syngeneic alpha 1-alpha 2 structure results in a total abrogation of the function of the C31 protein as a restriction element for H-2Kd and Kk restricted T cells during virus infection. In an attempt to obtain information on the functional polymorphism of MHC class I antigens as restriction elements, we have introduced the pC31 gene into the germ line of C3H/He mice (H-2k). The pC31 gene was transcribed in all tissues examined and the expression pattern paralleled the endogenous H-2Kk gene. However, the mRNA for the transgene was approximately 10-times more abundant, which was reflected in an elevated expression of the C31 protein in transgenic splenocytes. Most of the C31 antigen was found intracellularly. The C31 antigen could condition transgenic cytotoxic T lymphocytes in a specific manner during influenza A virus infection and functioned as the restricting element during T cell lysis of the infected cells. These results suggest that entire exons may be exchanged between MHC class I genes and that this exchange can generate novel and functional restriction elements.     

The E3/19K protein of adenovirus type 2 binds to the domains of histocompatibility antigens required for CTL recognition

The E3/19K protein of human adenovirus type 2 binds to HLA class I antigens and blocks their terminal glycosylation and cell surface expression. The nature of this interaction is non-covalent and involves neither disulfide bridges between the two molecules nor their carbohydrates. The murine H-2 Kd antigen associates with the E3/19K protein in a similar fashion to human HLA antigens whereas the allelic product H-2 Kk does not. Hybrid genes between the Kd and Kk alleles were constructed and their products were expressed in embryonic kidney cells together with the E3/19K protein. This allowed us to identify the alpha 1 and alpha 2 domains as the essential structures of the histocompatibility antigens for binding the viral protein. Interestingly, these domains are also crucial for T cell recognition. The implications for the evolution of adenoviruses and their ability to cause persistent infections are discussed.     

Interactions of immunoliposomes with target cells

We have covalently attached a monoclonal antibody (11-4.1) against the murine major histocompatibility antigen, H-2Kk, on the surface of liposomes. The interaction of these antibody-coated liposomes (immunoliposomes) with target cells, RDM-4 lymphoma (H-2Kk), was investigated. About 90% of the immunoliposomes taken up by target cells at 4 degrees C could be removed by a mild protease treatment of the cells, whereas only 30% of the uptake at 37 degrees C was labile to the same treatment. Furthermore, the uptake of immunoliposomes at 37 degrees C was inhibitable by cytochalasin B or by a combination of 2-deoxyglucose and NaN3. These results suggest that immunoliposome binding to the target cell surface is the primary uptake event at 4 degrees C and that the surface-bound liposomes are rapidly internalized by the cells at 37 degrees C, probably via an endocytic pathway. Studies with fluorescence microscopy of target cells treated with immunoliposomes containing carboxyfluorescein also supported this conclusion. If endocytosis is the mechanism by which immunoliposomes gain entry into target cells, the efficacy of a cytotoxic drug encapsulated would depend on the resistance of the drug to lysosomal inactivation and its ability to escape from the lysosomal system. Consistent with this notion, we observed that methotrexate encapsulated in liposomes bearing 11-4.1 antibody specifically inhibited deoxy[6-3H]uridine incorporation into DNA in target RDM-4 cells but not in P3-X63-Ag8 myeloma cells (H-2Kd) at the same doses. The observed cytotoxic effect of encapsulated methotrexate could be reversed by the treatment of cells with a lysosomotropic amine, chloroquine, which has been shown to increase the intralysosomal pH of mammalian cells. On the other hand, cytosine-beta-D-arabinofuranoside encapsulated in immunoliposomes showed no target-specific killing, probably because the drug is readily inactivated in the lysosomal system. These results are discussed in terms of the drug carrier potential of immunoliposomes.     

An H-2Ld hybrid molecule with a Qa-2 alpha-3 domain and phosphatidyl-inositol anchor is not recognized by H-2Ld-specific cytotoxic T lymphocytes

Ld/Q7d, a hybrid molecule consisting of alpha-1 and alpha-2 domains from H-2Ld and alpha-3 and carboxy-end components from Q7d, was expressed on the surface of CRL-3A rat liver cells. This molecule retained serologic H-2Ld epitopes. The Ag is attached to the cell membrane through a phosphatidyl-inositol linkage, characteristic of Qa-2 molecules. Both bulk cultured and cloned H-2Ld alloreactive CTL as well as H-2Ld restricted vesicular stomatitis virus-specific CTL lyse CRL-3A cells which express H-2Ld but show little or no lytic activity on cells which express the Ld/Q7d hybrid. These cells also fail to act as cold target competitors for alloreactive anti-H-2Ld CTL. However, cells expressing Ld/Q7d are not resistant to CTL mediated lysis because they can be killed in the presence of lectin. These data indicate that recognition of polymorphic class I CTL epitopes in the alpha-1 and alpha-2 domains are influenced by the structure of the carboxy-end of the molecule.     

Specificity of H-2 antigens expressed on mouse blastocysts

A highly sensitive cytotoxicity assay was used to detect H-2 antigens on mouse blastocyst stage embryos of the b, a, k, and d haplotypes. The assay was based on the principle that live embryos incorporate 3H-thymidine into DNA whereas embryos killed with antiserum and complement do not. The use of specific alloantisera showed that blastocysts of different haplotypes express different H-2 antigens. Thus, positive evidence was obtained for the expression of Kd and Dk molecules and negative evidence for the expression of Db, Kk, and Dd molecules. Evidence was also obtained that blastocysts express different H-2 antigens than those found on adult lymphocytes. Unexpected cross-reactions were found when some of the alloantisera were tested on blastocysts of different haplotypes. It is proposed that the aberrant expression of H-2 antigens on embryos might facilitate their escape from surveillance by the maternal immune system.