Fetal mouse alveolar type II cells in culture express several type II cell characteristics found in vivo, together with major histocompatibility antigens.

Alveolar type II cells were isolated from fetal mouse lung by differential adherence and obtained in monolayer culture. Cultures display a high degree of purity as shown by histochemical and immunocytochemical staining procedures. Seventy-five percent of cells stained positive with specific anti-lavage serum mouse (SALS-M), an antiserum specific for (pre)alveolar type II cells of the mouse, and osmiophilic bodies were present in 82% of cells. These and other characteristics of type II cells in culture correspond to those of alveolar type II cells in fetal mouse lung. The pattern of reactivity of these cells with various anti-cytokeratin antibodies is described, and we show that, in contrast to rat type II cells, they do not exhibit alkaline phosphatase activity. Identity of the type II cell cultures was shown by their specific phospholipid composition and surfactant protein A (SP-A) content. The fetal alveolar type II cells in culture were found to synthesize and express class I but not class II major histocompatibility complex (MHC) antigens. The possibility to culture fetal alveolar type II cells of the mouse and the availability of genetically well-defined inbred and transgenic mouse strains opens ways to study the genetics of type II cell differentiation and function. Also, the in vitro availability of alveolar type II cells, the progenitor cells of mouse lung tumors, will enable us to study in vitro several of the processes involved in lung tumorigenesis in the mouse.

Identification and expression of the Tla region gene T11b and its Qa-like product.

In spite of the large number of class I genes in the Qa-Tla region of the H-2 complex, only few membrane-bound Qa and TL Ag have been identified. We show that one of the Qa-Tla region genes, the T11b gene, is transcribed in lymphoid cells, lymphoma cell lines, teratocarcinoma cell lines, and L cells transfected with the cloned T11b gene. The T11b gene potentially encodes a polypeptide with normal class I characteristics. The product as present at the cell surface of L cells transfected with the cloned T11b gene, is a sialylated protein of m.w. 41,000, associated with beta 2-microglobulin. This T11b Ag shares epitopes with H-2K and H-2D molecules of various haplotypes and with Qa-2 molecules, but has distinct biochemical properties. RFLP analysis revealed that the T11b gene is found in mice of the Tlab and Tlaf haplotype. Genes homologous to, but distinct from, T11b (allelic or duplicated) are present in all Tla haplotypes tested.

High-resolution one-dimensional isoelectric focusing of mouse MHC class I antigens. Identification of natural and experimentally induced class I antigens.

A method is described for a biochemical comparison of mouse class I antigens utilizing antisera with a monomorphic pattern of reaction and high-resolution one-dimensional isoelectric focusing (1D-IEF). The most commonly occurring and studied H-2K and D alleles were identified in a comparison of over 40 mouse strains. By comparing H-2 mutant mouse strains, cell lines transfected with defined class I genes, or mice transgenic for a mouse class I gene and H-2 recombinant mouse strains, unambiguous identification of class I alleles was possible. The complex pattern presented by H-2-heterozygous mice was readily resolved into the contribution by the individual parental alleles. The H-2b bm series of mutants was analyzed, and for those mutants where a charge difference was predicted based on their known sequence, a change in isoelectric point (IEP) was indeed observed. Based on analysis by IEF, the bm8 mutant may contain (an) amino acid substitution(s) in addition to those reported. The present method further appears useful in elucidating defects in class I antigen synthesis and post-translational modifications, as these cannot be easily characterized when using surface staining with monoclonal antibodies (mAbs).

Molecular basis of Qa-11 antigen and paradoxical Qa-gene expression in an H-2 recombinant.

The Qa-11 Ag expressed in certain strains with the B2-microglobulin-b allele, apparently maps into the Tla region as well as into the Qa-2 region. Moreover Qa-11 has been shown to be biochemically indistinguishable from Qa-2. Genetic complementation studies combining the right Qa and Tla regions failed to lead to Qa-11 expression. To elucidate the molecular basis of this apparent paradox, we examined the expression of Qa-11 on products of transfected Q-region class I genes. Immunochemical analysis has shown that the Qa-11 Ag is expressed on class I molecules encoded by the Q7 gene from both C57BL/10 (Q7b) and BALB/c (Q7d), but not on the protein product of the Q9 gene isolated from the C57BL/10 strain (Q9b). Inasmuch as the predicted protein products of the Q7b and Q9b genes would differ at a single amino acid, a residue critical for Qa-11 expression has been identified. Based on these results it is proposed that among the beta-2-mb strains, the Qa-11+/Qa-2+ mice are likely to express at least the Q7 gene, whereas Qa-11-/Qa-2+ mice express only Q9. In support of this model, the Qa-2+/Q-11- recombinant B6.K2, essential for the apparent mapping of Qa-11 into the Tla region, expresses only Q9 but not Q7 encoded molecules on the cell surface, and only Q9 and no processed Q7 mRNA is detected in the cytoplasm. This expression pattern in B6.K2 cannot be explained on the basis of a single crossing-over event.

Qa-12--a novel determinant of activated T and B lymphocytes.

Investigation of the antigenic phenotype of activated lymphocytes using the broadly cross-reactive mAb 6.3.4 revealed two phenotypic alterations as compared with the resting lymphocytes. The Qa specificity Qa-m208 disappears after lectin activation of Qa-m208-positive T lymphocytes. Analysis of Q7 and Q9 transfectants expressing the Qa-2 polypeptides shows that Qa-m208 is an epitope of the Qa-2 antigen. Because the Qa-2 antigens are still expressed on T lymphoblasts which have lost Qa-m208, changes of the Qa-2 molecules occur and result in the loss of certain epitopes. The second phenotypic change that we observed is the appearance of a novel specificity, Qa-12. Its expression is induced by lymphocyte activation and it is expressed on lymphoblasts of both T and B cell origin. The presence of this novel non-ubiquitous antigenic specificity is determined by the Tla region.

Genetics of experimental lupus nephritis: non-H-2 factors determine susceptibility for renal involvement in murine chronic graft-versus-host disease.

Chronic graft-versus-host disease (GvHD) was induced in (C57BL/10 x DBA/2)F1 and (B10.S x DBA/2)F1 hybrids by injection of DBA/2 lymphocytes. All of the animals developed GvHD. Renal disease and proteinuria occurred in all of the (C57BL/10 x DBA/2)F1 hybrids, but only in 54% of the (B10.S x DBA/2)F1. The type of renal lesion was similar in all diseased animals of both strains, i.e., immune complex glomerulonephritis (ICGN) with deposition of antibodies and complement in glomeruli. To find out whether H-2 haplotype or other factors, such as non-H-2 linked genes, determine the susceptibility for renal involvement in GvHD, we produced (B10 x B10.S)F1 x DBA/2 mice, determined their H-2 genotype serologically, and separated them into H-2b/d and H-2s/d groups. These two groups did not differ with respect to susceptibility to renal disease in the course of GvHD, which indicates that H-2 is not the decisive genetic factor. We conclude that factors not linked with H-2 exert a major influence on susceptibility to GvHD-related renal disease in these mice.

Correlation between quantitative expression of H-2K, H-2D and MuLV antigens on spontaneous AKR lymphomas.

Thymocytes of leukemic AKR mice were analysed with a fluorescence-activated cell sorter (FACS) using monoclonal antibodies (MAbs) reactive with H-2Kk and H-2Dk and conventional hetero-antibodies against MuLV gp-70 antigens. Comparison was made with thymocytes of the low-leukemia H-2k strain C3H, as well as with thymocytes of young AKR mice, late preleukemic AKR mice (6-9 months old) and mitogen-stimulated thymocytes of young AKR mice. Expression of H-2 antigens increased on cells of the majority of tumors, on thymocytes of some late preleukemic mice and on mitogen-stimulated thymocytes. An increase in H-2K and H-2D antigens was noted: imbalance of the K/D ratio in favor of H-2D was observed mostly in tumors with relatively lower total amounts of H-2K and D antigens; ratio shifts in favor of H-2K were also found, mostly in tumors with relatively higher amounts of H-2K and D antigens. Increased expression of MuLV antigens was found on cells of all tumors and on thymocytes of several late preleukemic mice. We show that in primary spontaneous AKR leukemias, in spite of large individual differences, the expression of high amounts of MuLV gp70 is not random, but is associated with high expression of H-2K and H-2D antigens. The same phenomenon is seen in thymocytes of preleukemic mice, but in mitogen-stimulated thymus cells increase of H-2 expression is not accompanied by increase of MuLV gp70.

H-2 class I antigen expression on mouse teratocarcinoma cell lines.

Immunity against PCC3 teratocarcinoma cells (129, H-2b) was induced in allogeneic (C3H, H-2k) mice by preimmunization with L cells (C3H, H-2k) expressing cosmid-introduced Kb or Db genes, but not with nontransfected L cells. In addition, the growth of PCC3 cells in sublethally irradiated (C3H X B6-H-2bm1)F1 and (C3H X B6-H-2bm13)F1 mice bearing the Kbm1 and Dbm13 mutations, respectively, was either prevented, stopped, or delayed in comparison with the (C3H X B6)F1 (k X b) mice, which failed to reject the PCC3 cells. The teratocarcinoma line OC15S was exceptional because it reacted specifically with Kb- and Db-specific (but not Ib-specific) alloantisera, and because Kb- and Db-specific antibodies could be absorbed by OC15S cells. The subpopulation of OC15S cells bearing the ECMA-7 antigen characteristic for embryonic carcinoma (EC) cells was isolated by the fluorescence-activated cell sorter and was shown to react specifically with Kb- and Db-specific antisera. These experiments show that teratocarcinoma cells express antigens similar or identical to the K- and D-region products of differentiated cells. The lack of expression of class I antigens is thus neither a condition nor a consequence of the pluripotentiality of the EC cells. The exact nature of the major histocompatibility complex antigens on EC cells has yet to be established using the methods of molecular biology and biochemistry.

A new determinant, Qa-m208, detected on T lymphocytes and transfected L cells by a Kb-specific monoclonal antibody.

A Kb-specific monoclonal antibody, 6.3.4, defining a new class I specificity, m208, reacts with some K and D region products and with a Qa determinant present on T lymphocytes but not detectable on thymocytes and B lymphocytes. The strain distribution of reactions indicates that this determinant is controlled by the Qa-Tla region, and shows no concordance with the strain distribution pattern of any of the known Qa antigens. The antibody reacts also with L cells expressing a cloned H-2b class I gene mapping in the Qa-Tla region.

Qualitative and quantitative aspects of anti-H-2Ld sera.

Two anti-H-2Ld sera were analyzed, BALB/c-H-2dm2 anti-BALB/cBy and (C3H X BALB/c-H-2dm2)F1 anti-BALB/cHe, the latter also containing anti-Qa antibodies. Their reaction patterns were compared with an anti-Qa serum (C3H X BALB/cBy)F1 anti-BALB/cHe. Four H-2 specificities could be detected by the anti-H-2Ld sera, two already known (H-2.64, H-2.65) and two new specificities (H-2.81, H-2.82). According to their reaction pattern H-2.64, H-2.81, and H-2.82 can be regarded as members of the H-2.28 family of specificities. A quantitative difference in the expression of these H-2 specificities exists in different haplotypes. The cells of the strain against which the sera were made (BALB/cHe and BALB/cBy, respectively) did not give the highest titers with the antisera and had a relatively low absorbing capacity. The H-2dx haplotype carries two new specificities of the H-2.28 family, namely, H-2.81 and H-2.82. Lysostrip tests showed that the antibodies against those specificities cap the H-2.1-positive H-2Ddx molecules, suggesting that these molecules may react with both anti-H-2.1-like and anti-H-2.28-like antibodies. The H-2 specificities detected by the BALB/c-H-2dm2 anti-BALB/cBy serum were detected also in liver, kidney, spleen, heart, and lung tissue. New information on the strain distribution of Qa-2 was obtained from the experiments and a quantitative difference in Qa-2 antigens between H-2 congenic strains was observed as well. The H-2b strains react with these antibodies with higher titers than the strains carrying the H-2d haplotype.

Molecular heterogeneity of H-2 antigens.

Since the discovery of the H-2Ld molecule (Lemonnier et al. 1975) we have demonstrated that several K and D region alleles produce more than one type of H-2 molecules. Two of four different molecules were distinguished in the products of different alleles. Some of these molecules are products of different genes (H-2D, H-2L), in other instances the evidence for distinct genes is not available. Some of the different molecules produced by the same region might be modified products of the same gene. In the instances where no information implicating different genes is available, we use a neutral terminology which does not presume a genetic difference: H-2K1d and H-2K2d, H-2D1k, H-2D2k, H-2D1dx, H-2D2dx, H-2L1d, H-2L2d, etc. Immunoprecipitation experiments with some anti-H-2L and anti-Qa-2 sera revealed proteins with the apparent molecular weight of 41,000. We designate these antigens provisionally Lq and Qx, respectively. The Lq protein is polymorphic and it is at least partly under the control of H-2L-linked genes since it is absent from BALB/c-H-2dm2 cells. Since we have never seen the 41,000 proteins in precipitates of H-2K or H-2D antigens, it appears that whatever the origin of these molecules, they reveal some features common to products of L and Qa region. The basic relationship of H-2 K, D, L antigens is revealed also by the shared antigenic specificities between these H-2 molecules which we demonstrate using anti-H-2.28 sera. In summary, our results show that the class I antigens in each haplotype represent a family of several distinct but antigenically related molecules. The specificities of the H-2.28 family are the strongest allotype common to different H-2 K, D, and L molecules. Recent direct demonstration of several different genes in the Dd region (Steinmetz et al. 1981) provides evidence for the genetic complexity of H-2 genes which may be underlying basis of the molecular heterogeneity of H-2 antigens discussed here.