Generation of H-2-reactive T cell lines that bear the 5936 idiotype(s)

The present experiments showed 1) that it was possible to produce mouse T cell lines against MHC determinants with a relatively high success rate by stimulation of purified T cells with allogeneic cells in the presence of irradiated syngeneic spleen cells; 2) that these lines could be led to react against selected H-2 specificities; 3) that only T cell lines established from Ig-1b allotype mice contained 5936-Id+ T cells (5936-Idiotypes are defined by an antiserum against B6 anti-CBA IgG produced in rabbit no 5936, which was tolerant to mouse gamma-globulin); and 4) that antigenic determinants coded by IAk genes induce the 5936-Idiotype(s). The latter data are in accordance with the 5936-idiotype characteristics of primary MLC T blasts. All T cell lines contained both specific MLC-responding cells and cytolytic cells. However, studies on the functional capacity of 5936-Id+ T cells from both primary MLC and the T cell lines showed that neither MLC-responding cells nor cytolytic cells directed against H-2Kk, IAk, or H-2Dk were 5936-Id+. Thus, 5936-Id+ T cells may be regulator cells induced by IAk antigens.     

Receptor diversity of insulin-specific T cell lines from C57BL (H-2b) mice

To characterize the T cell receptor repertoire in an immune response in which the Ia and nominal antigenic determinants are defined and limited, we have cloned and sequenced the expressed receptors from four independent, beef insulin-specific T cell lines from C57BL mice. Each of these lines responded to beef but not to the pork insulin, thus defining the nominal antigenic determinant recognized. Furthermore, each of these lines could only be presented antigen by B6 but not mutant B6.C-H-2bm12 antigen-presenting cells, thus defining the requisite Ia recognition or antigen-association site. In spite of this functional similarity in ligand specificity, each of these T cell lines was found to use different V alpha and V beta gene segments. Moreover, structural comparisons of implied protein sequences of each of these receptors showed no stretches of conserved amino acid residues that could be implicated in ligand interaction. However, the V alpha genes used by these four clones appeared considerably more homologous to each other than were their V beta genes.     

Stability of surface H-2K(b), H-2D(b), and peptide-receptive H-2K(b) on splenocytes.

We have used flow cytometry to study the stability and peptide-binding capability of MHC class I (MHC-I) on the surface of normal C57BL/6 mouse T lymphoblasts. The MHC-I molecules on each cell are nearly evenly divided into two populations with mean half-life values of approximately 1 and 20 h. Our observations suggest that members of the later contain peptide bound with medium to high affinity. Cell surface MHC-I molecules capable of binding exogenous peptide (thus, "peptide-receptive") belong almost entirely to the less stable population. Before exogenous peptide can bind, MHC-I must undergo a change, probably loss of a very low affinity peptide. For MHC-I-K(b), we found that the maximum rate for binding of exogenous peptide corresponds to a t(1/2) value of 12 min. To maintain the 50:50 steady-state distribution of long- vs short-lived MHC-I molecules on the cell surface, approximately 20 short-lived molecules must be exported to the cell surface for each long-lived molecule.     

Variation in p30-related proteins in gross virus-induced tumor cell lines derived from H-2 congenic mice

The distribution of DNA topoisomers in intracellular simian virus 40 DNA was analyzed by gel electrophoresis. The results suggested that DNA extracted from 70S chromatin had a different superhelical density distribution as compared with the DNA obtained from virions or virion assembly intermediates. The heterogeneity of simian virus 40 viral DNA superhelical density at a late time after infection was partly due to increased virion production and partly due to the intrinsic heterogeneity of the superhelical density of DNA extracted from virions. Using two-dimensional gel electrophoretic analysis we also showed that simian virus 40 DNA templates used for DNA replication have a higher average superhelical density than the bulk of intracellular viral DNA.     

CD8(+) T cells from mice transnuclear for a TCR that recognizes a single H-2K(b)-restricted MHV68 epitope derived from gB-ORF8 help control infection

To study the CD8(+) T cell response against a mouse γ-herpes virus, we generated K(b)-MHV-68-ORF8(604-612)RAG(-/-) CD8(+) T cell receptor transnuclear (TN) mice as a source of virus-specific CD8(+) T cells. K(b)-ORF8-Tet(+) CD8(+) T cells, expanded in the course of a resolving MHV-68 infection, served as a source of nucleus donors. Various in vivo and ex vivo assay criteria demonstrated the fine specificity and functionality of TN cells. TN cells proliferated extensively in response to viral infection, helped control viral burden, and exhibited a phenotype similar to that of endogenous K(b)-ORF8-Tet(+) cells. When compared to OT-1 cells, TN cells displayed distinct properties in response to lymphopenia and cognate antigen stimulation, which may be attributable to the affinity of the TCR expressed by the TN cells. The availability of MHV-68-specific CD8(+) TCR TN mice provides a new tool for investigating aspects of host-pathogen interactions unique to γ-herpes viruses.     

Establishment and immunocharacterization of an immortalized pancreatic cell line derived from the H-2Kb-tsA58 transgenic mouse

Summary This study describes the establishment and characterization of an immortalized cell line derived from the pancreas of an adult H-2K^b-tsA58 transgenic mouse. These cells, designated IMPAN for IMmortalized PANcreatic cells, displayed a cobblestone appearance typical of confluent epithelial cells and a distinct polarity in the organization of their cytoplasmic organelles. Immunocytochemical studies revealed that all IMPAN cells stained positively for a wide range of markers characteristic of pancreatic acinar cells, namely the secretory products α-amylase, chymotrypsinogen, DNAse, the lectinlike secretory protein PAP (pancreatitis associated protein), and the zymogen granule membrane proteins GP-2 and gp300. They also stained positively for carbonic anhydrase II and cytokeratin 19, two proteins characteristic of pancreatic duct cells, as well as for rab3A, a small GTP-binding protein specifically localized in pancreatic islet cells. No reactivity was ever obtained with insulin antibodies. Taken together, these results show that the IMPAN cells exhibit a phenotype comparable to exocrine pancreatic acinar cells. However the expression of some proteins more specific to duct and islet cells make them similar to in vivo or in vitro growing acinar cells. The cell line should be a valuable model to study the mechanisms of growth, differentiation, and transformation of the exocrine pancreatic acinar cell.     

Expression of granulosa cell-specific genes and induction of apoptosis in conditionally immortalized granulosa cell lines established from H-2Kb-tsA58 transgenic mice

Granulosa cell lines have been established from H-2Kb-tsA58 transgenic mice. Using immunocytochemistry, significant amounts of insulin-like growth factor-I (IGF-I) were found in all cell lines investigated, whereas estrogen and progesterone receptor expression could be detected in only some of the lines. All cell lines showed low basal production of the gonadal steroids estradiol and progesterone. The genes for the ovarian paracrine regulators IGF-I and basic fibroblast growth factor were expressed, as well as the genes for anti-Müllerian hormone and for the P450 side-chain cleavage enzyme (P450scc). Expression of P450scc could be shown to be up-regulated in the cell lines under conditions mimicking the hormonal environment of the luteinizing granulosa cells in vivo. Inactivation of the temperature-sensitive SV40 T antigen by a shift of the cell lines to the nonpermissive temperature of 39.5 degrees C led to massive induction of apoptosis in several cell lines. These cell lines will allow a detailed study of the mechanisms regulating the expression of granulosa cell-specific functions, as well as the induction of granulosa cell apoptosis.     

Generation and characterization of a conditionally immortalized lung clara cell line from the H-2Kb-tsA58 transgenic mouse

The Clara cell is believed to be the progenitor of the peripheral airway epithelium, and it produces the surfactant proteins SP-A and SP-B, in addition to the 10-kDa Clara cell secretory protein (CCSP or CC10). To date, attempts to develop Clara cell lines have been unsuccessful. Most such attempts have involved the in vitro insertion of a transforming viral oncogene. We have reported previously the characterization of a differentiated conditionally immortalized murine lung Type II epithelial cell line, T7, from the H-2Kb-tsA58 transgenic mouse. We have also used this mouse model to derive Clara cell lines. In this model, the need for in vitro gene insertion is circumvented by the creation of a transgene, in which the large tumor antigen of a temperature-sensitive strain (tsA58) of the simian virus 40 (SV40) is fused with the major histocompatibility complex promoter H-2Kb. The promoter is active in a wide range of tissues and is induced by interferons (IFN). From the lungs of animals harboring the hybrid construct, we isolated and characterized Clara cells. The cells contain dense secretory granules and mitochondria typical of Clara cells, and express SP-A, SP-B, SP-D, and the Clara cell secretory protein, CC10. Withdrawal of the IFN and elevation of the incubation temperature permit normal cell differentiation similar to that of Clara cells in vivo. This cell line should be very useful for the investigation of normal Clara cell function and gene expression.     

Generation of an immortal differentiated lung type-II epithelial cell line from the adult H-2KbtsA58 transgenic mouse

Summary This paper describes a new fully differentiated Type-II alveolar epithelial cell line designated T₇, derived from transgenic H-2K^b-tsA58 mice, capable of being passaged as an immortalized cloned cell line in culture. H-2K^b-tsA58 mice harbor a temperature-sensitive (ts) mutant of the simian virus 40 (SV40) large tumor antigen (T antigen) under the control of the γ-interferon (INF)-inducible mouse major histocompatibility complex H-2K^b promoter. When cultured under permissive conditions (33°C and in the presence of γ-INF) cells isolated from H-2K^b-tsA58 mice express the large T antigen, which drives the cells to proliferate. However, upon withdrawal of the γ-INF and transfer of the cells to a higher temperature (39°C), T antigen expression is turned off, the cells stop proliferating and differentiate. The T₇ cell line is a clonal cell line originally derived from a Type-II cell-rich fraction isolated from lungs of H-2K^b-tsA58 mice. The T₇ cells form confluent monolayers, and have a polarized epithelial cell morphology with tight junctions and apical microvilli. In addition, the T₇ cells have distinct cytoplasmic lamellar bodies, which become more numerous and pronounced when the cells are grown under nonpermissive conditions. The T₇ cells synthesize and secrete phosphatidylcholine and the three surfactant proteins, SP-A, SP-B, and SP-C. The T₇ cell line is unique in that it is the first non-tumor-derived Type-II cell line capable of synthesizing and secreting the major components of surfactant. Based on the criteria studied, the T₇ cell line is phenotypically very similar to normal Type-II cells. The T₇ cell line, therefore, should prove a valuable experimental system to advance the study of the cell biology/physiology of surfactant metabolism and secretion as well as serve as a model for other studies of Type-II cell physiology.     

H-2-associated specificity of virus-immune cytotoxic T cells fromH-2 mutant and wild-type mice: M523 (H-2K ka ) and M505(H-2K bd ) do,M504 (H-2D da ) and M506(H-2K fa ) do not crossreact with wild-typeH-2K orH-2D

B10.A(3R) (H-2K ^b ) mice infected with lymphocytic choriomeningitis virus (LCMV) or vaccinia virus generate cytotoxic T cells capable of specifically lysing virus-infected macrophage target cells fromH-2K ^b mutant mice M505 (H-2K ^bd ), and vice versa. Similarly, virus-immune B10.A(4R) (H-2K ^k ) T cells specifically lyse infected targets from M523 (H-2K ^ka ), and vice versa. In contrast, virus-specific cytotoxic T cells from neither M504 (H-2D ^da ) and B10.A(5R) (H-2D ^d ) nor M506 (H-2K ^fa ) and B10.M(11R) (H-2K ^f ) mutually crossreact at the cytotoxic effector-cell level. As far as tested, the crossreactivity patterns between wild-type and mutantK orD specificities are identical for LCMV- and vaccinia virus-immune spleen cells. Although this finding is no proof for either the altered self nor the dual recognition concept of T-cell recognition, it may be compatible with the latter model.     

Generation and characterization of cloned T helper cell lines for anti-DNA responses in NZB.H-2bm12 mice.

We have previously demonstrated that the introduction of the bm12 mutation into NZB mice results in animals that spontaneously produce high titer IgG autoantibodies to dsDNA. The observation that NZB.H-2bm12 develop lupus although NZB.H-2b control mice do not, provides a unique system to study the role of Th cells in the production of antibodies to dsDNA. We have isolated, in the absence of a known stimulating autoantigen, a series of seven autoreactive T cell clones that provide help in vitro for the production of IgG anti-dsDNA antibodies by syngeneic B cells. The data on these seven cloned T cell lines was compared to two cloned T cell lines specific for keyhole limpet hemocyanin. The seven cloned T cell lines, coined clones 19D, 23G, 410F, 410H, C1, C15, and C52 all show significant help in vitro for production of IgM and IgG antibodies to ssDNA and dsDNA; antibody levels increased 7- to 30-fold compared to cultures without T cells. Clones C1, C15, and C52 were furthered studied and were shown to provide help for IgM antihistone and anti-OVA responses but provided significantly less help for IgG antibodies. In contrast, keyhole limpet hemocyanin-specific cloned T cell lines TK2 and TK5 provided help for IgM antibodies to ssDNA, dsDNA, and histone, but failed to significantly increase IgG antibodies to ssDNA, dsDNA, or histone. The cloned T cell lines were restricted to H-2bm12 and proliferated only in response to APC from NZB.H-2bm12 and B6.C-H-2bm12 but not NZB.H-2b or NZB.H-2d mice; their in vitro helper activity was inhibited by antibodies to class II. All cloned T cell lines expressed Thy-1, CD5, and TCR-alpha/beta. Three of the seven clones used TCR-V beta 4. However, the V beta expression of the four remaining autoreactive T cell clones could not be determined. All of the autoreactive cloned T cell lines produce significant IL-4 but no detectable IL-2 or IFN-gamma. We believe that HPLC-purified peptides eluted from I-Abm12 molecules from APC can potentially provide insight on the putative autoantigen.     

An allosteric mechanism controls antigen presentation by the H-2K(b) complex.

The mechanism of assembly/dissociation of a recombinant water-soluble class I major histocompatibility complex (MHC) H-2Kb molecule was studied by a real-time fluorescence resonance energy transfer method. Like the H-2Kd ternary complex [Gakamsky et al. (1996) Biochemistry 35, 14841-14848], the interactions among the heavy chain, beta2-microglobulin (beta2m), and antigenic peptides were found to be controlled by an allosteric mechanism. Association of the heavy chain with beta2m increased peptide binding rate constants by more than 2 orders of magnitude and enhanced affinity of the heavy-chain molecule for peptides. Interaction of peptides with the heavy-chain binding site, in turn, increased markedly the affinity of the heavy chain for beta2m. Binding of peptide variants of the ovalbumin sequence (257-264) to the heavy chain/beta2m heterodimer was found to be a biphasic reaction. The fast phase was a second-order process with nearly the same rate constants as those of binding of peptides derived from the influenza virus nucleoprotein 147-155 to the H-2Kd heavy chain/beta2m heterodimer [(3.0 +/- 1.0) x 10(-6) M-1 s-1 at 37 degrees C]. The slow phase was a result of both the ternary complex assembly from the "free" heavy chain, beta2m, and peptide as well as an intramolecular conformational transition within the heavy chain/beta2m heterodimer to a peptide binding conformation. Biexponential kinetics of peptide or beta2m dissociation from the ternary complex were observed. They suggest that it can exist in two conformations. The rate constants of beta2m dissociation from the H-2Kb ternary complex were, in the limits of experimental accuracy, independent of the structure of the bound peptide, though their affinities differed by an order of magnitude. Dissociation of peptides from the Kb heavy chain was always faster than from the ternary complexes, yet the heavy chain/peptide complexes were considerably more stable compared with their Kd/nucleoprotein peptide counterparts.     

The H-2KbtsA58 transgenic mouse: A new tool for the rapid generation of novel cell lines

The ability to generate expanded populations of individual cell types able to undergo normal differentiationin vitro andin vivo is of critical importance in the investigation of the mechanisms that underly differentiation and in studies on the use of cell transplantation to repair damaged tissues. This review discusses the development of a strain of transgenic mice that allows the direct derivation of conditionally immortal cell lines from a variety of tissues, simply by dissociation of the tissue of interest and growth of cells in appropriate conditions. In these mice the tsA58 mutant of SV40 large T antigen is controlled by the interferon-inducible Class I antigen promoter. Cells can be grown for extended periodsin vitro simply by growing them at 33°C in the presence of interferon, while still retaining the capacity to undergo normal differentiationin vivo andin vitro. In addition, it appears that cell lines expressing mutant phenotypes can readily be generated by preparing cultures from appropriate offspring of matings between H-2K^btsA58 transgenic mice and mutant mice of interest.     

The structure of H-2K(b) and K(bm8) complexed to a herpes simplex virus determinant: evidence for a conformational switch that governs T cell repertoire selection and viral resistance

Polymorphism within the MHC not only affects peptide specificity but also has a critical influence on the T cell repertoire; for example, the CD8 T cell response toward an immunodominant HSV glycoprotein B peptide is more diverse and of higher avidity in H-2(bm8) compared with H-2(b) mice. We have examined the basis for the selection of these distinct antiviral T cell repertoires by comparing the high-resolution structures of K(b) and K(bm8), in complex with cognate peptide Ag. Although K(b) and K(bm8) differ by four residues within the Ag-binding cleft, the most striking difference in the two structures was the disparate conformation adopted by the shared residue, Arg(62). The altered dynamics of Arg(62), coupled with a small rigid-body movement in the alpha(1) helix encompassing this residue, correlated with biased Valpha usage in the B6 mice. Moreover, an analysis of all known TCR/MHC complexes reveals that Arg(62) invariably interacts with the TCR CDR1alpha loop. Accordingly, Arg(62) appears to function as a conformational switch that may govern T cell selection and protective immunity.