Induction of classical and nonclassical MHC-I on mouse brain astrocytes by Japanese encephalitis virus

Infection with Flaviviruses upregulates the cell surface expression of MHC-I, MHC-II, ICAM-1 (CD54), VCAM-1 (CD106) and TAP proteins. Although all these studies have been confirmed using West Nile virus and other Flaviviruses, there are few reports that have examined the effects of Japanese encephalitis virus (JEV) infection directly on nonclassical and classical MHC expression in astrocytes. We show in this report that JEV infection of mouse brain astrocytes results in induction of the nonclassical MHC Class Ib genes, H-2T23, H-2Q4 and H-2T10 in addition to MHC-I, Type I (alpha/beta) IFNs, TAP-1, TAP-2, Tapasin, LMP-2, LMP-7 and LMP-10 but not IFNgamma, CD80, CD86 and MHC-II genes. The increased cell surface expression of these antigens as well as induction of the genes mentioned above as measured by RT-PCR suggests that JEV infection may lead to the induction of classical MHC Class Ia as well as nonclassical MHC Class Ib molecules.     

Nonclassical MHC-I and Japanese encephalitis virus infection: induction of H-2Q4, H-2T23 and H-2T10

Nonclassical MHC Class 1b antigens differ from classical MHC class 1a antigens in having a restricted polymorphism as well as varied surface expression in different cell types. They have been hypothesized to play a role in bridging adaptive and innate immune responses. We examined the effects of JEV infection on the expression of classical MHC class 1a and nonclassical MHC class 1b genes in five different cell lines. Among the nonclassical genes, H-2Q4 was induced in H-6 hepatoma, primary astrocytes, mouse embryo fibroblasts, L929 and 3T3 cells. H-2T23 and H-2T10 genes were not induced in H-6 and 3T3, respectively, but were induced in the other cell lines examined. Both H-2Q4 encoded Qb1 and H-2T23 encoded Qa-1(b) antigens were induced on the cell surface upon JEV infection in primary astrocytes and mouse embryonic fibroblasts. Classical MHC-I genes and the genes associated with antigen presentation such as Tap1, Tap2, Tapasin, Lmp2, Lmp7 and Lmp10 as well as type 1 (alpha/beta) IFNs were induced in all cell lines. However, IFNgamma was not induced. Further, induction of H-2Q4 and H-2T23 by JEV was independent of NF-kappaB but type 1 IFN dependent while H-2T10 was dependent on NF-kappaB and type 1 IFN independent. Thus, while classical MHC genes were induced by JEV in all cell lines tested despite high levels of constitutive expression in L929 and 3T3, nonclassical genes were not inducible in all cell lines tested and involved different mechanisms of induction.     

Differential effect of transporter Tap 2 gene introduction into RMA-S cells on viral antigen processing

The protein products of the Tap (Transporter associated with antigen processing) 1 and 2 genes are presumed to deliver peptides across the endoplasmic reticulum (ER) for assembly with major histocompatibility complex (MHC) class I molecules. The antigen processing-defective cell line RMA-S (H-2^b) has a premature stop in the Tap 2 gene and probably therefore fails to deliver peptides into the ER, which leads to a low level of cell surface MHC class I molecules. Transfection of a Tap 2 gene restores to RMA-S both MHC class I molecule expression and the ability to present influenza viral antigens. We investigated the ability of RMA-S cells transfected with a Tap 2 gene to process and present alloantigens, Sendai and Rauscher viral antigens to allogeneic and virus-specific cytotoxic T lymphocytes. We found that allogeneic peptides as well as Rauscher and Sendai viral peptides can be processed and presented by RMA-S but at reduced levels. Transfection of a Tap 2 gene of mouse (BALB/c, H-2^d) or rat origin into RMA-S increased the presentation of Sendai viral antigens and partially restored the presentation of allogeneic antigens. The already low level of Rauscher viral peptides presented by RMA-S is not elevated by transfection of either Tap 2 gene into RMA-S. This indicates a differential effect of transfection of a Tap 2 gene of rat or allogeneic mouse origin into RMA-S on viral antigen processing.     

Peptide-receptive class I major histocompatibility complex molecules on TAP-deficient and wild-type cells and their roles in the processing of exogenous antigens.

These studies addressed the nature and origin of peptide-receptive class I major histocompatibility complex (MHC-I) molecules used to present exogenous antigens. Peptide-receptive Kb molecules in transporter for antigen presentation (TAP)1-/- and TAP1+/+ macrophages were quantitated by exposing cells to exogenous ovalbumin (OVA)(257-264) peptide and then measuring OVA(257-264):Kb complexes with a T hybridoma assay or flow cytometry (using a complex-specific antibody). Relative to TAP1+/+ cells, TAP1-/- cells had decreased levels of pre-existing cell-surface peptide-receptive MHC-I molecules at 37 degrees. With continued exposure of viable cells to peptide, however, TAP1-/- and TAP1+/+ cells formed similar levels of OVA(257-264):Kb complexes, suggesting that nascent labile MHC-I molecules were captured and stabilized by exogenous peptide. Brefeldin A inhibited generation of OVA(257-264):Kb complexes on TAP1-/- (but not TAP1+/+) cells at 37 degrees, confirming the importance of a flux of unstable nascent MHC-I molecules in TAP1-/- cells at 37 degrees. In contrast, at 26 degrees both TAP1-/- and TAP1+/+ cells expressed brefeldin A-resistant, peptide-receptive MHC-I molecules at similar levels. Alternate MHC-I processing of exogenous particulate antigen correlated with ability to present exogenous peptide. Thus, processing was brefeldin A-sensitive with TAP1-/- macrophages at 37 degrees, but brefeldin A-resistant with TAP1+/+ cells at 37 degrees, as well as with TAP1+/+ or TAP1-/- cells at 26 degrees. We conclude that alternate MHC-I antigen processing normally utilizes pre-existing MHC-I molecules, but TAP1-/- cells at 37 degrees mainly use nascent MHC-I molecules, because of a lack of pre-existing, stable, peptide-receptive MHC-I molecules. The results support a vacuolar processing mechanism with binding of peptides to MHC-I molecules in post-Golgi compartments or on the cell surface.     

MHC class I molecules, structure and function

MHC class I molecules (MHC-I) are cell surface recognition elements expressed on virtually all somatic cells. These molecules sample peptides generated within the cell and signal the cell's physiological state to effector cells of the immune system, both T lymphocytes and natural killer (NK) cells. In addition, molecules structurally related to MHC-I, collectively known as MHC-Ib, are more specialized and, in some cases, interact with more limited subsets of lymphoid cells. Using the recently determined structure of the classical MHC-I molecule, H-2Dd, as a paradigm for structure and function, we review other MHC-I and MHC-Ib molecules, with an emphasis on how the same basic structural fold is employed by classical MHC-I molecules to bind specific peptides and T cell receptors, and is exploited by the MHC-Ib molecules in more stringent molecular interactions. It is instructive that structurally related molecules have evolved to perform a number of unique and distinct functions in immune and non-immune recognition.     

Defective assembly of class I major histocompatibility complex molecules in an embryonic cell line.

Developmentally regulated expression of the products of the major histocompatibility complex (MHC) is thought to play a key role in maternal tolerance of the fetal allograft. Here we analyze a cell line (EE2H3), derived from early post-implantation-stage mouse embryos, that is defective for MHC class I assembly. To follow expression of a single well-defined class I product, we introduced the H-2Dd gene under control of the human beta-actin promoter. We found that the transfected EE2H3 cells expressed abundant levels of H-2Dd heavy chains and beta 2-microglobulin protein, but only small amounts of H-2Dd surface protein. Surface expression was rescued by the addition of an appropriate antigenic peptide, or by culturing the cells at low temperature. The phenotype exhibited by EE2H3 is thus remarkably similar to that described for class I-negative somatic cell variants selected using antibodies and complement. However, a striking difference was that surface expression in H-2Dd-transfected EE2H3 cells was markedly enhanced in response to treatment with interferon. Thus, we have identified a novel class I assembly-defective cell line. Considering that EE2H3 was established from primary cultures of mouse embryo cells without immunoselection, and is therefore likely to represent a cell population normally present in post-implantation-stage embryos, these findings raise the possibility that expression of class I surface antigens during early development may in part be controlled post-translationally at the level of MHC class I assembly.     

Major histocompatibility complex antigens in v-Ki-ras transformed cells: the different antigens are expressed and induced by interferons independently of one another and of the anti-viral state.

A series of cell lines, which differed in their expression of class I (H-2K) or II (I-A) major histocompatibility complex (MHC) antigens, was derived from a line of C3H/He (H-2k) mouse embryo fibroblasts transformed by the v-Ki-ras oncogene. These were prepared by fluorescence-activated cell sorting of cells stained for these antigens either without interferon (IFN) treatment or after induction of antigen expression by either IFN-alpha beta or IFN-gamma, selecting for low or high staining. Cells selected for low (undetectable) constitutive H-2Kk expression were still strongly inducible by either IFN; cells selected for high constitutive expression were induced by IFN to express still higher levels. In all cell lines induction of H-2Kk with one IFN type was paralleled by induction with the other. Expression of H-2Kk appeared largely independent of H-2Dk; in lines which were selected for low H-2Kk expression (constitutive or induced), H-2Dk expression was not much reduced, and lines selected for high H-2Kk expression showed only modest augmentation of H-2Dk. Varying inducibility of I-Ak by IFN-gamma was not closely paralleled by H-2K inducibility by either IFN-alpha beta or -gamma, with again only weak correlation of high and low expression of H2-Kk and I-Ak. On the other hand, expression of I-Ak seemed to be correlated with I-Ek. None of these variable effects could be attributed to differing sensitivity to IFN-alpha beta or -gamma since all the lines showed about the same sensitivity to the anti-viral effects of IFN.     

Differential reactivity of residual CD8+ T lymphocytes in TAP1 and β2-microglobulin mutant mice

TAP1 -/- and β2-microglobulin (β2m) -/- mice (H-2^b background) express very low levels of major histocompatibility complex (MHC) class I molecules on the cell surface. Consequently these mice have low numbers of mature CD8⁺ T lymphocytes. However, TAP1 -/- mice have significantly higher numbers of CD8⁺ T cells than β2m -/- mice. Alloreactive CD8⁺ cytotoxic T lymphocyte (CTL) responses were also stronger in TAP1 -/- mice than in β2m -/- mice. Alloreactive CTL generated in TAP1 -/- and β2m -/- mice cross-react with H-2^b-expressing cells. Surprisingly, such cross-reactivity was stronger with alloreactive CTL from β2m -/- mice than with similar cells from TAP1 -/- mice. The β2m -/- mice also responded more strongly when primed with and tested against cells expressing normal levels of H-2^b MHC class I molecules. Such H-2^b-reactive CD8⁺ CTL from β2m -/- mice but not from TAP1 -/- mice also reacted with TAP1 -/- and TAP2-deficient RMA-S cells. In contrast, H-2^b-reactive CD8⁺ CTL from neither β2m -/- mice nor TAP1 -/- mice killed β2m -/- cells. In line with these results, β2m -/- mice also responded when primed and tested against TAP1 -/- cells. We conclude that the reactivity of residual CD8⁺ T cells differs between TAP1 -/- and β2m -/- mice. The MHC class I-deficient phenotype of TAP1 -/- and β2m -/- mice is not equivalent: class I expression differs between the two mouse lines with regard to quality as well as quantity. We propose that the differences observed in numbers of CD8⁺ T cells, their ability to react with alloantigens and their cross-reactivity with normal H-2^b class I are caused by differences in the expression of MHC class I ligands on selecting cells in the thymus.     

Hybrid resistance modulates the inflammatory process induced by lymphocytic choriomeningitis virus-immune T cells.

The magnitude of the meningitis occurring 72 hr after adoptive transfer of 8-day lymphocytic choriomeningitis (LCMV)-immune effector T cells from cyclophosphamide-treated or untreated donors into cyclophosphamide-suppressed, LCMV-infected recipients is modified by the hybrid histocompatibility (Hh) effect. Evidence of inhibition in (H-2Kd Dd X H-2Kb Db)F1, as compared with H-2Kb Db, recipient mice is found for H-2Kd Db, H-2Kd Db and (H-2Kk Db X H-2Kb Db)F1, but not for (H-2Kk Dk X H-2Kb Db)F1 or H-2Kb Dd, LCMV-immune donor populations. The effect is cell dose-dependent, and the inflammatory process is 2.5-10 times lower in F1 than in recipient mice of parental type. These data indicate that the Hh effect is not directed solely at precursor populations, or at T cells which bear idiotypes reactive to MHC glycoproteins expressed in the F1 recipients. The inflammatory process initiated by fully-functional, LCMV-immune T cells is inhibited in the same way. The fact that the levels of natural killer cell activity in the virus-infected, cyclophosphamide-treated recipients are not much higher than those in normal mice indicates that the operation of hybrid resistance in LCM is not likely to be a special case, and should be taken into account in all T cell transfer systems.     

Analysis of the extracellular processing of HIV-1 gp160-derived peptides using monoclonal antibodies specific to H-2Dd molecule complexed with p18-I10 peptide

The immunodominant peptide of human immunodeficiency virus 1 gp 160 for murine cytotoxic T cells of H-2d haplotype, has been originally identified as a 15 amino acid residue peptide P18IIIB (RIQRGPGRAFVTIGK) (Takahashi et al., 1988). Further studies have indicated that a more active form of the peptide is generated by removal of the C-terminal dipeptide by angiotensin-I-converting enzyme (ACE), and additional detailed studies have shown that the actual immunodominant peptide is a decamer P18-I10 (RGPGRAFVTI) (Kozlowski et al., 1993). The effect of proteolytic processing on the antigenicity of P18IIIB peptide and its analogs was investigated by functional T cell assays based on the ability of T cell receptor (TCR) to recognize a specific major histocompatibility complex class I (MHC-I)/peptide complex. Recently we described a new monoclonal antibody (MAb) KP15 directed against the MHC-I molecule H-2Dd complexed with the 10-mer peptide P18-I10. Using this MAb, the cell surface H-2Dd/P18-I10 complex can be easily detected by flow cytometry (Polakova et al., 2000). Here we examined whether peptides longer than P18-I10 decamer form H-2Dd complexes recognized by KP15 MAb. Further we also analyzed how the ACE processing of P18IIIB-related peptides of different length affects their ability to form complexes with H-2Dd recognized by MAb KP15. These experiments confirmed that the ACE digestion of 15-mer peptide P18IIIB is the most effective in the production of a peptide capable of forming complex with H-2Dd recognized by KP15 MAb. The ACE digestion of longer peptides (16-mer to 19-mer) did not produce a significant quantity of peptides, capable of forming H-2Dd complexes recognizable with by MAb KP15. Peptides shorter than P18IIIB (13-mer to 10-mer), notably the optimally sized P18-I10 peptide lost most of their capacity to form H-2Dd complexes recognized by KP15 MAb. Our results show that the extracellular processing of MHC-I-restricted peptides, which cannot be overlooked in designing peptide-based vaccines, can be also studied by as simple and rapid assay as flow cytometry, provided MAbs specific to a particular MHC-I/peptide complex are available.     

Quantification by flow cytofluorimetry of HLA class I molecules at the surface of murine cells transformed by cloned HLA genes

A method allowing quantitative analysis of the expression of foreign antigens at the surface of transformed cells is described. Aminoethyl-Sephadex G-25 beads labelled with different amounts of fluorescein isothiocyanate (FITC) were used to calibrate an Epics V cell sorter. The quantity of FITC molecules bound per bead was found to be a linear function of relative fluorescence intensity expressed by channel number for intermediate and high levels of fluorescence and a non-linear function for low levels. The lowest limit of detectable fluorescence was 3400 FITC molecules bound per bead. Using FITC-conjugated monoclonal antibodies (m.Ab.) the number of HLA class I molecules expressed at the surface of murine LMTK- (H-2Kk) cells transfected by cloned HLA class I genes was estimated and compared with the amount expressed on human B (Raji) and T (MOLT 4) lymphoblastoid reference cells. Murine transformed cells expressed approximately 3 times less HLA class I determinants per surface unit (micrometer 2) than Raji cells and 1.4 times less than MOLT 4 cells. Similar results were obtained by Scatchard analysis of the same populations using radiolabelled m.Ab. A detailed analysis of fluorescent cells showed that 5-20% of transformed cells expressed less than 33,000 HLA class I molecules/cell whereas most MOLT 4 cells exhibited at least 280,000 molecules/cell and the majority of Raji cells more than 700,000 molecules/cell. The expression of foreign antigen did not reduce the amount of H-2Kk molecules at the surface of transformed cells (mean of 350,000 molecules/cell).     

Assembly of tapasin-associated MHC class I in the absence of the transporter associated with antigen processing (TAP)

The assembly of MHC class I molecules is regulated by a multi-protein complex in the endoplasmic reticules (ER) termed the loading complex. Tapasin is suggested to be one of the molecules forming this complex on the basis of its interaction with both the transporter associated with antigen processing (TAP) and MHC class I molecules. To address whether TAP is indispensable for the processing of the assembly of tapasin-associated MHC class I molecules, we studied the association of MHC class I molecules with tapasin, the assembly of tapasin-associated MHC class I with peptides and the peptide-mediated dissociation of MHC class I from tapasin in TAP-mutant T2 cells. In the absence of TAP, MHC class I heavy chain and beta(2)-microglobulin dimers were found to be properly associated with tapasin. The stable MHC class I dimer was required for its association with tapasin in the ER. In the absence of TAP, tapasin retained MHC class I molecules much longer in the ER than in the presence of TAP. This low off-rate of MHC class I from tapasin was due to the absence of high-affinity peptides in the ER of TAP-mutant cells but not to the absence of TAP per se. The introduction of peptides into permeabilized microsomes of TAP-mutant cells led to effective loading of the peptides onto tapasin-associated MHC class I and to the subsequent dissociation of MHC class I from tapasin. These results demonstrate that regulation of the assembly of tapasin-associated MHC class I is independent of the interaction of tapasin with TAP, but is dependent upon the peptides transported by TAP.     

beta 2-Microglobulin from serum associates with several class I antigens expressed on the surface of mouse L-cells

Bovine beta 2-microglobulin (beta 2-m) present in fetal calf serum (FCS) is able to replace endogenous beta 2-m associated with several class I antigens from human and mouse cells maintained in culture [Bernabeu et al. (1984) Nature, Lond. 308, 642-645]. Here we show that human HLA-A2 and HLA-B7, as well as mouse H-2Ld and H-2Dd heavy chains expressed after gene transfer in mouse L-cells, associate to a large extent with bovine beta 2-m. We also demonstrate that bovine beta 2-m associated with the endogenous H-2Kk/Dk heavy chains generates an antibody response when L-cells are injected into syngeneic C3H mice.     

A monoclonal antibody recognizes a subset of the H-2Dd mouse major class I antigens

Binding studies and competition experiments have shown that a monoclonal antibody (mAb) named 28-8-6 recognizes only 5 to 10% of the cell surface Dd molecules. The molecules detected by 28-8-6 mAb appear to be genuine H-2Dd antigens on the basis of their MW and isolectric points. In addition, the detectability of the subset of cell surface Dd molecules by 28-8-6 does not depend on their degree of glycosylation nor on the presence of mouse beta-2-microglobulin. Several interpretations are discussed. mAb 28-8-6 might detect a particular conformation or a particular chemical derivatization of otherwise normal H-2Dd molecules. Also, because the epitope recognized by 28-8-6 lies close to the peptide binding site, it is possible that mAb 28-8-6 recognizes a subset of Dd molecules bearing a certain category of self peptides.     

Mutant MHC class I molecules define interactions between components of the peptide-loading complex

Class I histocompatibility molecules, consisting of a heavy chain, beta2-microglobulin and peptide, are assembled in the endoplasmic reticulum (ER) with the assistance of several molecular chaperones and accessory proteins. Peptide binding occurs when assembling class I molecules associate with a loading complex consisting of the transporter associated with antigen processing (TAP) peptide transporter, tapasin, ERp57 and calreticulin (CRT)/calnexin. To assess the physical organization of this complex, we generated a series of mutants in the murine H-2Dd heavy chain and assessed their association with components of the complex. Seven mutations, clustered between amino acids 122 and 136 in the heavy chain alpha2 domain plus one mutation at position 222 in the alpha3 domain, resulted in loss of interaction with tapasin. Association with TAP was always lost simultaneously, supporting the view that tapasin acts as an obligatory bridge between class I molecules and TAP. Compared with previous studies on the HLA-A2 molecule, some differences in points of tapasin interaction were observed. Failure of the H-2Dd mutants to bind tapasin resulted in low cell-surface expression and altered intracellular transport. Most mutants retained a substantial degree of peptide loading, consistent with the view that although tapasin may promote peptide binding to class I, it is not required. A surprising observation was that all mutants lacking tapasin interaction retained normal association with CRT. This contrasts with previous observations on other class I molecules and, combined with differences in tapasin interaction, suggests that the organization of the ER peptide-loading complex can vary depending on the specific class I molecule examined.     

Inhibition of Serine-Peptidase Activity Enhances the Generation of a Survivin-Derived HLA-A2-Presented CTL Epitope in Colon-Carcinoma Cells

Cytotoxic T lymphocytes eliminate tumor cells expressing antigenic peptides in the context of MHC-I molecules. Peptides are generated during protein degradation by the proteasome and resulting products, surviving cytosolic amino-peptidases activity, may be presented by MHC-I molecules. The MHC-I processing pathway is altered in a large number of malignancies and modulation of antigen generation is one strategy employed by cells to evade immune control. In this study we analyzed the generation and presentation of a survivin-derived CTL epitope in HLA-A2-positive colon-carcinoma cells. Although all cell lines expressed the anti-apoptotic protein survivin, some tumors were poorly recognized by ELTLGEFLKL (ELT)-specific CTL cultures. The expression of MHC-I or TAP molecules was similar in all cell lines suggesting that tumors not recognized by CTLs may present defects in the generation of the ELT-epitope which could be due either to lack of generation or to subsequent degradation of the epitope. The cells were analyzed for the expression and the activity of extra-proteasomal peptidases. A significant overexpression and higher activity of TPPII was observed in colon-carcinoma cells which are not killed by ELT-specific CTLs, suggesting a possible role of TPPII in the degradation of the ELT-epitope. To confirm the role of TPPII in the degradation of the ELT-peptide, we showed that treatment of colon-carcinoma cells with a TPPII inhibitor resulted in a dose-dependent increased sensitivity to ELT-specific CTLs. These results suggest that TPPII is involved in degradation of the ELT-peptide, and its overexpression may contribute to the immune escape of colon-carcinoma cells.     

Aberrant MHC antigens in a sarcoma virus-induced mouse tumour

From a series of mouse sarcomata, newly induced by Rous sarcoma virus (RSV), the DOH cell line was shown to lack expression of syngeneic H-2Kd and Dk antigens (Noll et al., 1986). It unexpectedly displayed determinants specific for H-2Kk molecules. Interferon treatment of DOH stimulated the expression of H-2Kk determinants and also the display of some, but not all, determinants of the syngeneic H-2Kd molecules. H-2Dk expression was not stimulated. Southern blot hybridization of genomic DNA digests from DOH cells confirmed the identity of the H-2K region with that from syngeneic C3H.OH liver cells, but also showed changes in the pattern of restriction fragments that contain class I genes from the D and Qa regions. These results suggest that aberrant MHC class I molecules that carry H-2Kd- and H-2Kk-like determinants are expressed by DOH sarcoma cells. These molecules may act as target antigens for tumour-specific cytotoxic T cells elicited by injection of DOH cells into syngeneic mice.     

Ecto-F1-ATPase and MHC-class I close association on cell membranes

Subunits of the mitochondrial ATP synthase complex are expressed on the surface of tumors, bind the TCR of human Vgamma9/Vdelta2 lymphocytes and promote their cytotoxicity. Present experiments show that detection of the complex (called ecto-F1-ATPase) at the cell surface by immunofluorescence correlates with low MHC-class I antigen expression. Strikingly, the alpha and beta chains of ecto-F1-ATPase are detected in membrane protein precipitates from immunofluorescence-negative cells, suggesting that ATPase epitopes are masked. Removal of beta2-microglobulin by mild acid treatment so that most surface MHC-I molecules become free heavy chains reveals F1-ATPase epitopes on MHC-I+ cell lines. Ecto-F1-ATPase is detected by immunofluorescence on primary fibroblasts which express moderate levels of MHC-I antigens. Up-regulation of MHC-I on these cells following IFN-gamma and/or TNF-alpha treatment induces a dose-dependent disappearance of F1-ATPase epitopes. Finally, biotinylated F1-ATPase cell surface components co-immunoprecipitate with MHC-I molecules confirming the association of both complexes on Raji cells. Confocal microscopy analysis of MHC-I and ecto-F1-ATPase beta chain expression on HepG2 cells shows a co-localization of both complexes in punctate membrane domains. This demonstrates that the TCR target F1-ATPase is in close contact with MHC-I antigens which are known to control Vgamma9/Vdelta2 T cell activity through binding to natural killer inhibitory receptors.     

Restoration of H-2b expression and processing of endogenous antigens in the MHC class I pathway by fusion of a lymphoma mutant to L cells of the H-2k haplotype

The RMA-S lymphoma mutant cannot process and present antigens to H-2-restricted cytotoxic T lymphocytes. It synthesizes major histocompatibility complex class I heavy (H-2KbDb) and light beta 2-microglobulin (beta 2mb) chains of normal size and charge, but only a fraction of these assemble and reach the cell surface. As a first step investigating the genetic defect of this line, we have fused it to a L cell fibroblast line (H-2KkDk/beta 2ma). The fusion restored H-2Kb, Db and beta 2mb expression as well as the ability to process and present internally derived (minor histocompatibility and influenza virus nucleoprotein) antigens in RMA-S. This shows that the mutation(s) responsible for the phenotype of RMA-S is (are) not located within the MHC class I heavy and light chain genes. Other cellular factors, derived from the L cell fusion partner, can control antigen processing and transport of MHC class I molecules. These findings are discussed in relation to the observation that assembly and transport of MHC class I molecules can be induced in the mutant by H-2b-restricted peptides. The recessive nature of the defect and its independence of MHC class I genes in the mutant has important implications for future transfection studies, of this and similar mutants, aiming at establishing cells containing non-assembled MHC class I molecules of different alleles and identifying the gene(s) controlling processing of endogenous antigens.