Biomedical subjects
Publications and source records attributed to J J Cebra.
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It has proven difficult to evaluate the functional potential of germinal center (GC) B cells, including those from Peyer's patches (PP), by either in vivo or in vitro methods. Thus, rather than assess secreted Ig product as an indicator of functional potential we have instead sought to detect mRNAs related to the various Ig heavy chains in GC B cells from PP by in situ hybridization. We have found that the GCs of PP contain the vast majority of B cells with easily detectable levels of mRNA alpha. These levels are intermediate between those of small resting B cells and plasmablasts. When PP B cells are enriched for cells bearing GC markers, approximately 50% contain mRNA mu and 40% mRNA alpha. Similar enrichment for sIgA+ B cells gave 50% of cells with easily detectable mRNA alpha and few if any positive for mRNA mu. The sizes of these mRNAs were similar to those encoding the membrane and secretory form of mu and alpha chains. No C alpha germ-line transcripts could be detected by Northern analyses using a probe for sequences 5' to the alpha switch regions. Finally, GC and sIgA+ cells from PP also showed the absence of a portion of their genomic DNA for CH genes 5' of C alpha. Thus, it seems likely that most of the GC cells expressing mRNA alpha have undergone conventional VDJ recombination to C alpha at the DNA level in order to switch to the expression of IgA. Our findings reflect the extraordinary preference for switching to IgA by GC cells in PP.
Lymphoid tissue fragment cultures were established to analyze the differentiative processes among B cells in Peyer's patches (PP) and peripheral lymph nodes (PLN), especially those in germinal centers. PP cultures from both conventionally reared mice and formerly germ-free mice colonized with Morganella morganii could be maintained for greater than 12 days with continued B-cell division, especially among cells binding high levels of peanut agglutinin, a characteristic of germinal center cells. PLN cultures from conventionally reared mice injected with a heat-killed vaccine of M. morganii could be maintained for the same amount of time. Over this period, PP cultures continued to secrete immunoglobulin A (IgA) as well as smaller amounts of IgM. PP cultures from formerly germ-free mice colonized with M. morganii showed net increases of IgA antiphosphocholine (anti-PC) antibodies with avidities as high as those of the prototypic T15 monoclonal antibody. Similar PLN fragment cultures from conventionally reared mice given footpad injections of M. morganii showed net increases of IgM and IgG anti-PC antibodies in the culture fluid. Thus, although M. morganii stimulated lymphoid tissues in vivo to produce an anti-PC response in vitro when given by either the oral or the parenteral route, the antibody isotypes differed between PP and PLN fragment cultures. Fragment culturing may offer a complementary and simpler way to detect a local secretory IgA response than does either measuring IgA antibody in secretions or detecting IgA antibody in the cytoplasm of plasma cells in the lamina propria of gastrointestinal or respiratory tissue.
CD8 lymphocyte population heterogeneity has been examined by using reovirus serotype 1, strain Lang (reovirus 1/L) as a model infection. We have previously reported that gut mucosal infection with reovirus stimulates the appearance of virus-specific cytotoxic T cell precursors (pCTL) in Peyer's patches (PP). The effectors that mediate reovirus-specific cytotoxicity were found to express both the Thy-1 and CD8 Ag and were MHC-restricted in their recognition of reovirus Ag. To further characterize the virus-specific precursor and effector cells we have analyzed PP cells for the expression of a novel surface Ag (termed germinal center and T cell Ag (GCT)) found on germinal center B cells and a subpopulation of CD8+ T cells. Gut mucosal infection with reovirus 1/L is capable of increasing the proportion of GCT+ CD8+ T cells in PP. Positive selection as well as depletion of GCT+ cells has demonstrated that pCTL can express this Ag, and depletion experiments have demonstrated that effector CTL express the GCT Ag. Thus, a subpopulation of GCT+ cells have been identified as Ag-specific precursor and effector CTL. These observations indicate that the expression of the GCT Ag may provide a means to identify recently stimulated pCTL or effector CTL in gut mucosal tissues.
Adult severe combined immunodeficient (SCID) mice can be infected by the oral route with reovirus, and a systemic infection can be established. Infectious virus is recovered from all internal organs, and the mice die in 4-6 wk. Chronic, discrete inflammatory lesions appear in the liver of infected mice, and are associated with hepatocytes containing demonstrable levels of viral antigen. The adoptive transfer of Peyer's patch (PP) cells from congenic mice before infection protects the SCID mice against disease and death. Immune donor PP cells can be distinguished from nonimmune cells by their ability to contain and resolve infection by 1 wk after challenge.
Microcultures of helper T (Th) cells and a few appropriately primed murine B cells can be used to detect cognate T-B interactions which lead to clonal production of IgM, IgG1, and IgE. However, IgG2, IgG3, and IgA are very rarely expressed. We have found that the addition of dendritic cells to such cultures creates an extremely supportive environment for clones expressing IgA with other isotypes, as well as clones expressing only detectable IgA. Typically, 400 dendritic cells were added to 3000 conalbumin-specific Th cells (D10.G4.1) and 30 hapten-specific Peyer's patch (PP) B cells with antigen in 15 microliters. The response was antigen dependent and clonal. Almost half of the clones expressed only non-IgM isotypes, 43% expressed some IgA, and 14% expressed some IgG3; isotype diversity increased over time. Dendritic cells from PP and spleen were found to be equally supportive, and allowed the number of T cells required in microculture to be decreased from 3000 to 400. However, T cell proliferation was not required for the supportive effect of dendritic cells. Surface IgD-bearing cells were also found to switch to IgA production in microculture as judged by their generating clones expressing IgM along with IgA and other isotypes. Again, IgA was usually expressed only in the presence of dendritic cells. The mechanism may involve dendritic cell-induced T cell activation and/or dendritic cell factors, and is under investigation.
The role of passively acquired immunity to reovirus-induced meningoencephalitis in neonatal mice was examined. It was determined that female mice were capable of conferring protection against viral infection and meningoencephalitis in neonates depending on the route by which the dams were immunized and the serotype of the immunizing virus. Female mice immunized with homotypic virus via the oral route developed the most potent response. Infected neonates born and nursed by these females developed no signs of disease, and no virus was recoverable from their small intestines, livers, or brains following infection. Neonates born to females immunized with homotypic virus by the subcutaneous route manifested no evidence of meningoencephalitis or virus dissemination, yet virus was recovered from neonatal intestines. Mice immunized with heterotypic virus by either the subcutaneous or the oral route also conferred protection against disease; however, virus was recovered in small intestines and livers of infected neonates. Based on results from foster-nursing experiments, it appears that factors obtained both during suckling and by transplacental transfer contribute to protection. Passive transfer of reovirus-immune mouse serum also protected neonates from disease. These results demonstrate that passive immune mechanisms can mediate the protection of neonates against reovirus infection and provide further evidence of the importance of the mucosal immune response in protection against pathogens that invade the host via mucosal tissues.
IgA from human milk and colostrum is a substrate for a galactosyltransferase also present in milk and colostrum. The secreted IgA that serves as the best acceptor for the transferase activity is the IgA that fails to bind readily to jacalin lectin. Upon becoming galactosylated by the transferase, however, the IgA shows an increased affinity for jacalin. Glycosidase and electrophoretic results indicate that the transferred galactose is beta-linked to the alpha-chain of the IgA. The IgA:transferase activity can be purified by gel filtration and cation exchange chromatographies, as well as by affinity chromatography on Sepharose derivatized with UDP or IgM. The enzyme has an apparent Mr of about 64 kDa, is prevalent in both milk and colostrum, but has a sixfold higher sp act in colostrum.
Reovirus, a virus that contains neither an envelope nor glycosylated polypeptides, has been found to induce virus-specific, major histocompatibility complex (MHC) class I antigen restricted, cytotoxic T lymphocyte (CTL) responses. The cytotoxic T cells require in vitro stimulation in the presence of virus to phenotypically express cytotoxic activity. Utilizing reovirus types 1 and 3, the CTLs derived from mice infected with one serotype can lyse target cells infected with a second serotype of reovirus. In addition, lymphocytes primed in vivo with one serotype develop into fully functional CTLs during in vitro stimulation with the other serotype of reovirus. Therefore, these results suggest that reovirus induced CTLs are virus, but not serotype specific. Common determinants shared by reovirus polypeptides from reovirus types 1 and 3 are most likely the stimuli for the majority of CTLs responses to reovirus.
Populations of murine B cells enriched for fluorescein (FLU)- or phosphocholine (PC)-binding cells stimulated with LPS, or FLU- or PC-LPS at low density in 10 microliter cultures form clones of cells that secrete antibodies. Antibody isotypes were determined by radioimmunoassay and their avidities were determined relative to standard, monoclonal antibodies by hapten inhibition using a radioimmunoassay. These analyses further characterize the development of B cell clones in microcultures and reveal that differing culturing conditions stimulate qualitatively different B cell populations to divide and differentiate. Without filler cells, isotype switching is rare. Co-culturing B cells with 10(5) (CBA/N x BALB/c) F1 male thymocyte filler cells leads to IgG and/or IgA antibody secretion by 15-20% of cultures; antibodies from clones that switch isotypes are exclusively of high avidity. IgM is almost always present as one clonal product; pre-switched cells rarely score in microcultures. Without filler cells, a high percentage of antibodies from FLU-LPS stimulated, FLU-binding cells are of high avidity (60%). However, clonotypes of lower avidity dominate with mitogenic culture conditions, 100 micrograms/ml LPS or with thymocytes. PC-binding cells are less sensitive to these mitogenic effects. Antibodies produced by PC-specific clones have a more restricted pattern of avidities and resemble in quality anti-PC antibodies produced in vivo.
Antibody isotype expression by B cell clones was analyzed using in vitro microcultures containing low numbers of hapten-gelatin-enriched B cells and higher numbers of hemocyanin-specific helper T cell lines or clones. Twenty-eight to sixty-three percent of clones grown in microculture with haptenated hemocyanin and T cells from established lines expressed IgG and/or IgA isotypes in random mixtures, almost always accompanied by IgM. Helper T cells from hemocyanin-specific clones also supported the expression of non-IgM isotypes by the B cell clones, suggesting that a single specificity of T cell can provide sufficient growth and differentiation factors for the display of isotype switching. A positive correlation between the antibody output of clones and the expression of non-IgM isotypes indicated that the switching process may be associated with cell division. Although memory B cells that give clones expressing IgG and/or IgA in the absence of IgM are also enriched on haptenated gelatin, they are not stimulable under conditions of this microculture assay.
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IgA memory B cells have been operationally defined as precursors that give rise to clones exclusively secreting IgA antibodies upon antigen stimulation in a T-cell dependent splenic fragment culture. B lymphocytes that are sIgA+ account for a small fraction of Peyer's patch lymphocytes, but these can be clearly divided into two subsets. One subset contains the majority of sIgA+ B cells and most of these are in S, G2, or M phase of the cell cycle. These cells are germinal center B cells, as defined by being S kappa low and peanut agglutinin (PNA)high, and contain most of the mRNA alpha. Though these germinal center cells may contain the majority of sIgA+ B cells and may contain precursors for memory cells, preplasma cells, or both, they do not appear to be immediately responsive to stimulation by antigen. Rather, the S kappa high, PNAlow subset of sIgA+ B cells, most of which are in G0 or G1 and have only low levels of mRNA alpha appear to contain most of the clonal precursors that are committed to IgA, i.e., the functional memory cells that give rise to clones exclusively secreting IgA upon stimulation with thymus-dependent antigen in the presence of T cells. There is also a population of Peyer's patch B cells that neither bears detectable sIgA nor has mRNA alpha detectable by cytoplasmic dot blotting but contains a small proportion of the functional IgA memory cells.
In this report we have shown that reovirus 1/L is an effective mucosal immunogen capable of generating a cytotoxic T cell (CTL) and associated helper T cell response to the nominal antigens associated with reovirus 1/L. The effectors that mediate reovirus-specific cytotoxicity are Thy-1+, Lyt-2+, and major histocompatibility complex (MHC)-restricted in their recognition of reovirus antigens, and can therefore be classified as CTLs. Frequency analysis of precursor CTLs occurring in Peyer's patches (PP) and peripheral lymph nodes (PLN) 6 d and 6 mo after intraduodenal stimulation have demonstrated that a persistent gradient of precursors is established, with higher frequencies present in PP. The generation of a CTL response in PP may be important in preferentially repopulating mucosal tissues with effector CTLs that could result in the local containment of infections in the gut. We also found that reovirus 1/L generates a virus-specific B cell response that is dominated by IgA memory cells after intraduodenal immunization. We hypothesize that the efficacy of reovirus 1/L at stimulating T and B cells in the gut mucosa is related to its ability to selectively enter PP via microfold (M) cells after enteric application. In this study we have also demonstrated that PP cells, upon in vitro culture and unrelated to prior reovirus priming, can generate natural killer-like (NK) cytotoxic activity. This may be an in vitro correlate of the in vivo generation of effectors that may populate mucosal tissues (i.e., the intestinal epithelium) with NK-like effector cells.
A modified splenic fragment assay was used to assess the role of antigen-specific helper T cells in B cell isotype expression. Limiting numbers of carrier-specific helper T cells from lines or clones were injected along with a source of B cells into lethally irradiated unprimed recipients. The incidence of lodging of the T cell lines in recipient spleens at 18 h was determined by autoradiography to be 1.5 to 4.3% of the injected cells. These T cells were necessary and sufficient for the generation of T-dependent B cell responses within splenic fragments cultured in vitro with specific antigen. A comparison of isotypic responses from splenic and Peyer's patch B cells generated with the same T cell population revealed that a high proportion of the response from Peyer's patch B cells consisted of IgA antibody exclusively (46-57%) while the percentage of such responses from splenic B cells was much lower (7-10%). Thus, the isotype pattern of the response reflected the B cell source. Experiments in which cloned hemocyanin-specific T cells provided help to T-depleted spleen cells within splenic fragments from athymic recipients indicated that a single specificity of helper T cell is both necessary and sufficient to support the generation of antibody responses consisting of multiple isotypes. Isotype-specific T cells do not appear to be required in this system.
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