Enteric immunization reveals a T cell network for IgA responses and suggests that humans possess a common mucosal immune system

The local IgA response is a result of two related events, the induction of sensitized T and B cells in gut- or b ronchial-associated lymphoreticular tissues (GALT or BALT) and the final differentiation of IgA plasma cells in mucosal tissues where IgA is produced and transported to become secretory IgA (S-IgA) antibodies into external secretions. Oral administration of various types of antigens/vaccines may result in two types of response, i.e., S-IgA antibodies at mucosa and systemic unresponsiveness to antigen, a state termed oral tolerance. Regulatory T cells in GALT help account for both S-IgA responses and oral tolerance and thus serve to fine tune responses to orally encountered antigens. Studies in animal models and humans have shown that oral administration of antigens sensitize lymphoid cells in GALT which subsequently home to mucosa and result in S-IgA responses in several external secretions. The significant promise of oral vaccines for prevention of microbial diseases including neisserial diseases is discussed.     

Streptococcal serotype carbohydrate represents a novel class of type 2 antigen which is T-independent

Our studies reported here, fully characterize two unique type 2 antigens trinitrophenol (TNP)-M1 serotype carbohydrates (TNP-M1 g and TNP-M1 c) derived from streptococci, which fail to induce antibody responses in xid or neonatal mouse splenic cultures. These antigens generate brisk responses in normal spleen and Peyer's patch cell cultures of xid mice, all of which suggest that responses are elicited in the Lyb-3+, 5+ B subpopulation. The antibody responses to TNP-M1 g (and TNP-M1 c) are not dependent upon T cells. Furthermore, TNP-M1 carbohydrates induce anti-TNP plaque-forming (PFC) responses in cultures of small, resting splenic B cell populations without an added T cell requirement. Thus two categories of type 2 antigens are distinguished, one which requires T cells or derived factors, e.g., TNP-Ficoll, and a second TNP-carbohydrate antigen TNP-M1 that does not. Studies of the mitogenic and polyclonal B cell activation properties of M1 carbohydrates indicated that B cell proliferation is induced in both xid (Lyb-3-, 5-) and normal (Lyb-3-, 5- and Lyb-3+, 5+) splenic B cell subpopulations, but that differentiation to IgM synthesis fails to occur in the Lyb-3-, 5- B cell subpopulation. Thus M1 carbohydrates are unique probes that allow the selective induction of proliferation and differentiation of mature B cells that are presumably Lyb-3+, 5+. Because the M1 serotype carbohydrates induce polyclonal IgM synthesis and antigen-specific responses in only the mature B cell population in the absence of T cells, whereas TNP-Ficoll and other type 2 antigens require T cells or their derived factors, the Lyb-3+, 5+ B cell subpopulation may consist of a T cell-dependent and a T cell-independent compartment for responses to different carbohydrate type 2 antigens.     

Murine bone marrow IgA responses to orally administered sheep erythrocytes

Specific immunization protocols have been established for the induction of murine bone marrow IgA responses to the T cell-dependent (TD) antigen sheep red blood cells (SRBC). Systemic immunization, either i.p. or i.v., followed by a second injection, induced splenic IgM and IgG responses and a bone marrow IgM response. No significant IgA responses were observed in either lymphoid tissue compartment. Oral immunization with SRBC by gastric intubation for 2 days, followed 1 wk later by an i.p. injection of SRBC resulted in a splenic IgA plaque-forming cell (PFC) response, but did not elicit a bone marrow IgA response. Repeated daily gastric intubation of SRBC to C3H/HeN and C3H/HeJ mice led to the previously reported pattern of systemic unresponsiveness in C3H/HeN mice and good anamnestic type IgM, IgG, and IgA splenic anti-SRBC PFC responses in the C3H/HeJ strain upon parenteral challenge. Oral administration of SRBC for 14 days to C3H/HeN mice, followed by systemic SRBC challenge, resulted in diminished splenic PFC responses of all isotypes, whereas gastric intubation of SRBC for 28 days led to complete systemic unresponsiveness to antigen in C3H/HeN mice. Interestingly, the repeated oral administration of SRBC resulted in significant bone marrow IgA PFC responses upon i.p. challenge in both C3H/HeN and C3H/HeJ mouse strains. The bone marrow IgA responses were clearly dependent upon chronic oral exposure to SRBC, because gastric intubation with SRBC for 2 consecutive days/wk for 10 wk also induced bone marrow and splenic IgA anti-SRBC PFC responses in C3H/HeN mice. These results suggest that memory B cells reside in the bone marrow of orally immunized mice and can yield anamnestic-type responses to challenge with the inducing antigen. The memory cells may arise in the Peyer's patches of the gut and migrate to the bone marrow. The possibility that the bone marrow is a component of the common mucosal immune system in mammals is suggested by this study.     

Relationship between immune system and gram-negative bacteria. II. Natural killer cytotoxicity of Salmonella minnesota Rb 345-unbound human peripheral blood lymphocytes

Spontaneous binding of human peripheral blood lymphocytes (PBL) to bacteria represents a promising approach for the characterization of lymphocyte subsets mediating different functions. In the light of previous findings on the high degree of spontaneous adherence of S. minnesota Rb cells to PBL, we have evaluated the natural killer (NK) cytotoxicity of PBL subpopulations that fail to bind to Rb bacteria. The S. minnesota Rb-unbound cell fraction exhibits higher levels of cytotoxic capacity, which is related to a more elevated frequency of active NK cells, as determined in an agarose-single cell cytotoxic assay. Moreover, the cytotoxic activity of the unbound fraction is additionally boosted by interferon-alpha pretreatment. The effector cells bear Fc gamma receptors that are involved in NK cell lysis, because a decrease of NK activity is observed after immune complex modulation of the receptors. Finally, these cells, which display a high percentage (approximately 70%) of typical large granular lymphocyte morphology, express HNK-1, T10, T8, and M1 antigens, and to a lesser extent T3 and T4 antigens. These data indicate a selective enrichment of NK cells in the S. minnesota Rb-unbound fraction.     

Monoclonal antibodies to Salmonella lipopolysaccharide: anti-O-polysaccharide antibodies protect C3H mice against challenge with virulent Salmonella typhimurium

The present investigation reports the production of monoclonal antibodies to antigenic determinants of the O-polysaccharide of Salmonella typhimurium lipopolysaccharide (LPS), and assesses the effectiveness of these antibodies in protecting C3H mice against the lethal effects of Salmonella infection. Hybridomas were generated by fusing spleen cells from (BALB/c X A/J)F1 (CAF1) mice hyperimmunized by i.v. injection with acetone-killed S. typhimurium SR-11 with X63-Ag8.653 murine myeloma cells. Hybridomas producing antibodies reactive with S. typhimurium SR-11 whole cells were subcloned, and seven of the resulting clones as well as one previously described clone were selected for use in the studies reported here. Monoclonal antibodies from these eight clones were of the IgG1 (1), IgG3 (6), or IgM (1) isotype and were specific for the O-polysaccharide region of Salmonella LPS, reacting with LPS from smooth S. typhimurium SR-11 and LT-2, but not with LPS from rough S. minnesota R60 (Ra), R345 (Rb), or R595 (Re). The effectiveness of each monoclonal antibody in protecting C3H/HeN and C3H/HeJ mice against the lethal effects of Salmonella infection was evaluated by comparing the median length of survival of groups of mice given antibody by i.p. injection before i.p. challenge with virulent S. typhimurium SR-11 to that of animals that received no antibody. Three out of eight monoclonal anti-O-polysaccharide antibodies, ST-1 (IgM), 10-5-47 (IgG3), and 10-5-6 (IgG3), provided significant (p less than 0.01) protection to C3H/HeN mice challenged with approximately 10(4) LD100 of Salmonella. Only antibodies ST-1 and 10-5-6, however, extended the median length of survival of C3H/HeJ mice beyond that of infected controls. Mouse antiserum prepared against S. typhimurium SR-11 was equally protective in C3H/HeJ mice. In an attempt to understand the contribution of antibody specificity to the relative differences in the protective capacities of the monoclonal antibodies, their reactivities with several Salmonella reference strains of different classical serotypes were examined. Although some differences in reactivity against the different strains were apparent, this approach was not adequate for defining the fine specificity of these monoclonal antibodies. The results of this study provide evidence that monoclonal antibodies with specificity to the O-polysaccharide region of Salmonella LPS can protect C3H mice against challenge with the homologous bacterial strain.     

A 145-base pair DNA sequence that positions itself precisely and asymmetrically on the nucleosome core.

A 145-bp DNA sequence, cloned from Escherichia coli, was reconstituted into nucleosome core particles by a number of methods. The behaviour of the resulting complex upon sucrose gradient sedimentation and nucleoprotein gel electrophoresis closely resembled that of control bulk nucleosome core particles. DNase I digestion of the 32P-end-labelled complex revealed the 10-bp periodicity of cleavages expected for DNA bound on a histone surface. The narrow cleavage sites observed (1 bp wide) imply that the sequence occupies a single preferred position on the nucleosome core, accurate to the level of single base pairs. By relating the digestion pattern observed to the pattern of site protection found for random sequence nucleosomes, the DNA position was found to be offset by 17 bp from that in the normal core particle. A number of experiments argue against the involvement of length or end effects and suggest that it is some feature of the DNA sequence itself that determines this precise positioning of DNA on the nucleosome.     

Partial IgA-deficiency with increased Th2-type cytokines in TGF-beta 1 knockout mice.

Though it has been shown that TGF-beta 1 directs B cells to switch to IgA in vitro, no studies have assessed TGF-beta 1 effects on mucosal vs systemic immunity in vivo. When the B cell functions of TGF-beta 1 gene-disrupted (TGF-beta 1-/-) mice were analyzed, significantly decreased IgA levels and increased IgG and IgM levels in serum and external secretions were observed. Further, analysis of Ab forming cells (AFC) isolated from both mucosal and systemic lymphoid tissue showed elevated IgM, IgG, and IgE, with decreased IgA AFC. A lack of IgA-committed B cells was seen in TGF-beta 1-/- mice, especially in the gastrointestinal (GI) tract. Splenic T cells triggered via the TCR expressed elevated Th2-type cytokines and, consistent with this observation, a 31-fold increase in serum IgE was seen in TGF-beta 1-/- mice. Thus, uncontrolled B cell responses, which include elevated IgE levels, a lack of antiinflammatory IgA, and an excess of complement-binding IgG and IgM Abs, will promote inflammation at mucosal surfaces in TGF-beta 1-/- mice and likely contribute to pulmonary and GI tract lesions, ultimately leading to the early death of these mice.