Serological detection of H-2K- and H-2D-gene products

Using the fluorescence-activated cell sorter (FACS II), we have analyzed the expression of H-2K- and H-2D-gene products on the membrane of various cellular components of the murine immune system. Using this serological technique we show a basic difference between T and B lymphocytes. Whereas all cellular components analyzed — hydrocortisone-resistant thymocytes, splenic T and B lymphocytes, macrophages and bone-marrow cells — expressed H-2K-subregion-encoded alloantigens at a high density, it seems that the high density expression of H-2D-encoded alloantigens is restricted mainly to B cells and to macrophages. Hydrocortisone-resistant thymocytes, splenic T lymphocytes and bone-marrow cells, on the other hand, showed significant expression of the H-2D alloantigens only at low membrane density. These results, then, provide evidence for the existence of an imbalance in serologically detectable expression of H-2K- and H-2D-region-gene products on the cell membrane of various cells comprising the murine immune system.Abbreviations usedin this paper DTH delayed type hypersensitivity - FCS fetal calf serum - FITC fluorescein isothiocyanate - HrT hydrocortisone-resistant thymocytes - Ig immunoglobulins P. De Baetselier is an EMBO and Euratom postdoctoral fellow     

Serological analysis of H-2 and Ia molecules with monoclonal antibodies

Five H-2 and seven Ia monoclonal antibodies were tested against a panel of 43 independentH-2 haplotypes (11 of laboratory-mouse and 32 of wild-mouse origin), 33 recombinantH-2 haplotypes, and up to 74 wild mice. All the antibodies gave negative reactions in the PVP hemagglutination tests; all, however, gave positive reaction with some members of the panel in the dye-exclusion cytotoxic test. Four of the antibodies (H-2.m2,Ia.m2, Ia.m5 and Ia.m7) reacted identically to conventional antibodies detecting determinants H-2.2., and Ia-1.2, Ia-1.15, and Ia-5.7, respectively (this statement does not apply to wild mice in which minor differences in reactivity patterns of the corresponding antibodies were found: the reproducibility of these differences, however, could not be checked by absorption). Five other antibodies (H-2.m5, H-2.m3, H-2.m4, Ia.ml, and Ia.m6) had very similar though not identical reactivity patterns to conventional antibodies detecting determinants H-2.5, H-2.11, H-2.25, Ia-1.2, and Ia-1.19, respectively. The last three monoclonal antibodies (H-2.ml, Ia.m3, and Ia.m4) had a reactivity pattern that did not match those of any known conventional antibodies. The near identity or great similarity of many monoclonal and conventional antibodies indicates that the cleanest of the conventional antisera are truly monospecific, and gives credence to the H-2 serology as defined by conventional antibodies. The serological analysis of monoclonal antibodies supports the true existence of private and public determinants, and reveals that the H-2 and Ia determinants are complex, even when the antibody is simple.     

Spontaneous autocytotoxicity against an unexpected H-2 d haplotype in MRL/lpr (H-2 k) autoimmune disease-prone mice

The MRL/lpr (H-2 ^k) inbred strain, a model for the autoimmune disease systemic lupus erythematosus, differs from the healthy inbred strain MRL +/+ (H-2 ^k) by only 0.1 % of its genome. Southern blot analysis using class I and class II probes confirmed the H-2 ^k genotype of both strains. Among the Ia^k-positive peritoneal cells, cells with an unexpected expression of Ia^d specificities were detected in a radioimmunoassay using several monoclonal antibodies and one conventional antiserum. This was only found in aged (6- to 9-month-old) mice both in the MRL/lpr strain (32 % Ia^d-positive mice) and in the MRL +/+ strain (42% Ia^d-positive mice). Furthermore, 24% of aged MRL/lpr mice exhibited strong spontaneous cytotoxic T lymphocyte (CTL) activities against P815 (H-2 ^d) target cells, and 57% had a weaker but still detectable level of cytotoxicity. In contrast, such a CTL activity has never been found in the MRL +/+ strain. These results suggest that the anti-H-2^d d CTL plays a role in the onset of the autoimmune process in MRL/lpr mice.     

Serological detection of H-2K- and H-2D-gene products. I. Principal difference between T and B lymphocytes in expression of H-2D-encoded alloantigens

Using the fluorescence-activated cell sorter (FACS II), we have analyzed the expression of H-2K- and H-2D-gene products on the membrane of various cellular components of the murine immune system. Using this serological technique we show a basic difference between T and B lymphocytes. Whereas all cellular components analyzed--hydrocortisone-resistant thymocytes, splenic T and B lymphocytes, macrophages and bone-marrow cells--expressed H-2K-subregion-encoded alloantigens at a high density, it seems that the high density expression of H-2D-encoded alloantigens is restricted mainly to B cells and to macrophages. Hydrocortisone-resistant thymocytes, splenic T lymphocytes and bone-marrow cells, on the other hand, showed significant expression of the H-2D alloantigens only at low membrane density. These results, then, provide evidence for the existence of an imbalance in serologically detectable expression of H-2K- and H-2D-region-gene products on the cell membrane of various cells comprising the murine immune system.     

Specificity of the mouse cytotoxic T lymphocyte response to adenovirus 5. E1A is immunodominant in H-2b, but not in H-2d or H-2k mice

The Ag specificity and MHC restriction of the CTL response to adenovirus 5 (Ad5) in three strains of mice, C57BL/10 (H-2b), BALB/c (H-2d), and C3H/HeJ (H-2k), were tested. Polyclonal Ad5-specific CTL were prepared by priming mice in vivo with live Ad5 virus followed by secondary in vitro stimulation of the spleen cells with virus-infected syngeneic cells. The Ad5-specific CTL were Db restricted in C57BL/10 and Kk restricted in C3H/HeJ. In BALB/c mice both Kd- and Dd/Ld-restricted CTL were detected. The polyclonal Ad5-specific CTL response in C57BL/10 mice is directed exclusively against the products of the E1A region, which comprises only 5% of the Ad5 genome. In BALB/c mice E1A is at best a very minor target Ag and in C3H/HeJ mice E1A is not recognized at all. Using the H-2 congenic mouse strains B10.BR (H-2k) and C3H.SW (H-2b) it was shown that the immunodominance of E1A is H-2 dependent. The 19-kDa glycoprotein encoded in the E3 region of Ad5, which binds to class I MHC in the endoplasmic reticulum and prevents its translocation to the cell surface, does not affect the specificity of the CTL response in C57BL/10 mice toward E1A. However, it affects the MHC restriction of the Ad5-specific response in BALB/c mice, selectively inhibiting generation of Kd-restricted CTL.     

The multi-locus H-2D w16 region has an organization distinct from the D d region

Genomic DNA blot analyses using probes derived from the BALB/c 3 flanking region of the L ^d gene (L ^d 3 fl-C) and from near the BALB/c D3 ^d gene (50.2A) indicate that the B10.GAA37 mouse strain has a multi-locus D (D ^w16) region distinct from the five-gene organization observed in the D ^d and D ^q regions. To isolate the D ^w16 region class I genes, a genomic B10. GAA37-EMBL3 library was generated and screened with probes that preferentially hybridize to K and D region class I genes. Hybridization analyses of the isolated clones with L ^d derived oligonucleotide probes suggested that one of the clones contained the L ^w16 gene, whereas several other clones contained the L ^w16 gene. The sequence of the D ^w16 gene is most similar to that of the D ^p gene, particularly in the 3 half. Furthermore, the L ^w16 gene is quite similar in the 5 half and virtually identical in the 3 half to the L ^d gene, indicating that L ^w16, but not D ^w16, is a member of the L ^d gene family. Collectively, these data suggest that, through a D region recombination event, the novel D ^w16 region may have been assembled from primordial counterparts of the D ^p and L ^d genes.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M60774-M60776, and M62759.     

F1 hybrid anti-parental H-2k cell-mediated lympholysis. I. Stimulator and target determinants controlled by the H-2K region.

H-2k-heterozygous F1 hybrid mouse spleen cells cultured with irradiated H-2k-homozygous stimulator cells generated specific anti-parent cytolytic effectors. The parental antigenic determinants recognized by responder cells during induction (afferent arm) and by effector cells during cytolysis (efferent arm) were coded for, or regulated by, the H-2K-Hh3 region of the MHC, according to recombinant analysis. There were no detectable influences by other linked or unlinked genes on the phenotypic expression of parental antigens; however, the anti-parent responsiveness was modulated by background genes of responder cells. These experiments establish that the K end of H-2 controls determinants of F1 anti-parental H-2k CML, like the D end controls those of F1 anti-parental H-2b CML, thus confirming the basic symmetry of the H-2 complex. The relationship of this primary in vitro cell-mediated response with natural in vivo resistance to parental and allogeneic bone marrow grafts is discussed.     

Nucleotide sequence of the BALB/c H-2T region gene,T3 d

The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M75875.     

Murine infection by vesicular stomatitis virus: initial characterization of the H-2d system.

BALB/c mice and congenic H-2Ld-deficient BALB/c-H-2dm2 (dm2) mice were experimentally infected intranasally with isolates of vesicular stomatitis virus (VSV). The survival of infected hosts, viral replication in lungs and brains, and histopathologic in the two mouse strains were compared. In both strains of mice, mortality occurred during the period 7 to 10 days postinfection. However, dm2 mice were relatively resistant to lethal infections. Viral replication occurred at low levels in the lungs of both strains and did not evoke significant pathologic changes. In contrast, viral replication in the brains was much greater; in the BALB/c strain, this was accompanied by more frequent and more severe pathologic changes. In general, mice surviving at day 10 had effectively cleared virus from central nervous system but not respiratory sites. Evidence is presented that viral replication occurs first in the nasal cavity and is transmitted both to the lungs and to the olfactory bulb where focal cytopathology occurs. Virus enters the ventricles, causing encephalitis; necrosis occurs around the ventricles and in the lumbosacral region of the spinal cord. Necrotic lesions were accompanied by mononuclear infiltration. Mice immunized with virus of the same serotype or with a vaccinia virus hybrid encoding the VSV glycoprotein were protected from lethal infection; in contrast, mice immunized with heterotypic virus were susceptible to challenge.     

Teratocarcinoma transplantation antigens are encoded in the H-2 region

Evidence is presented for the existence of teratocarcinoma transplantation antigens (Gt) encoded within the H-2 complex and present also on adult tissues. It has not been possible to separate these Gt loci from H-2 by recombination, and Gt factors map to each end of the H-2 complex. Previous reports indicating separation of all Gt loci from H-2 are reinterpreted. One class of such apparent recombinants has been shown to result from the outgrowth of tumor variants in mice of resistant genotype.Aspects of this work were presented at the 10th Cold Spring Harbor Conference on Cell Proliferation, September 1982.     

Antigen-specific helper T cells recognize determinants encoded in the H-2D region of the H-2 gene complex

Observations have frequently been interpreted as showing that the helper T cells which collaborate with alloantigen-specific cytotoxic T-cell precursors can only recognize antigens encoded in the I region of the H-2 gene complex. An experimental system is described here that allows analysis of the recognition repertoire of these helper cells. CBA helper T-cell precursors can be primed in vitro to antigens encoded in the H-2 ^b gene complex. These helpers can then be tested for the existence of a subset of helper cells which recognize antigens encoded in the D region of H-2 ^b haplotype. CBA thymocytes were used as a source of cytotoxic T-cell precursors that respond poorly in the absence of exogeneous helper activity. The source of alloantigen was varied by using irradiated spleen cells from various (BALB/c × recombinant)F₁ hybrid mice as stimulator cells. When the stimulator cell bears BALB/c determinants recognized by the cytotoxic T-cell precursor and also bears only the D region antigens of the H-2 ^b haplotype, an anti-BALB/c cytotoxic response is generated only if the anti-H-2^b helper population contains cells able to recognize H-2D^b. A positive cytotoxic response was obtained, indicating that helper cells are not limited to recognition of I region antigens and can efficiently recognize antigens encoded in the D region of the H-2 gene complex. This was confirmed by the demonstration of helpers specific for H-2D^d. We were unable to detect any evidence for Ia-restricted recognition of the H-2D alloantigens, suggesting that, as for cytotoxic T lymphocytes (CTL), helper cell recognition of class I alloantigens is an unrestricted event.     

Immunochemical characterization of molecules identified in human thymic epithelium by anti-thymulin monoclonal antibodies

Four high molecular weight molecules (ranging from 59 to 48 kd) were evidenced in the human thymic epithelium, after one and two dimensional immunoblot analyses, using monoclonal antibodies directed against synthetic thymulin. One of these immunoreactive proteins might correspond to the intra-thymic precursor of the hormone.     

Functional characterization of the H-2Dp gene product

A clone containing the D ^p gene was used to transform L cells. The D^p product expressed was identified by two-dimensional gel electrophoreis and flow cytometry. The D^p product expressed by the L cells was recognized by D^P-specific flow cytometry. The D^p product expressed by the L cells was recognized by D^p-specific but not K^p-specific killer T cells. This killing was inhibited by monoclonal antibodies specific for D^p but not K^p or K^k antigens. Similarly, lymphocytic choriomeningitis virus (LCMV) killer T cells from B 10.P mice were able to kill LCMV-infected L12a cells, but not LCMV-infected Ltk⁺. Again only D^p monoclonal antibodies could inhibit this killing.