Human adult tonsil xenotransplantation into SCID mice for studying human immune responses and B cell lymphomagenesis.

OBJECTIVE: To generate a human-mouse xenochimeric model where human cells remain clustered in the animal to optimize their interactions and recovery. MATERIALS AND METHODS: Severe combined immune deficient mice (SCID) were xenografted subcutaneously with human adult tonsil pieces (hu-ton-SCID mice). Such animals were: (a) compared with those receiving tonsil cells in suspension, and (b) immunized with de novo and recall antigens. RESULTS: Human tonsil pieces survived a long period of time in SCID mice, while polyclonal human T- and B-lymphocytes persisted in close vicinity within the implantation area; however, little or no graft-versus-host disease was detectable. Not surprisingly, local development of lymphoproliferative disease was often observed in animals receiving lymphoid implants from donors previously infected by the Epstein-Barr virus. One month after surgery, higher serum levels of human IgG were found in SCID mice transplanted with tonsil pieces (2x10(7) cells/animal) than in animals injected with 5x10(7) tonsil cells in suspension (1.9 vs. 0.3 mg/mL, p < 0.002). Importantly, the production of human IgG in hu-ton-SCID mice remained polyclonal for at least 6 months and was linked to the presence of cells within the implants. Immunization of hu-ton-SCID mice with hepatitis B core, a de novo antigen, did not produce a significant IgG immune response; however immunization with tetanus toxoid (TT), a thymus-dependent recall antigen, yielded high (> 700-fold increase in anti-TT IgG levels) and long-lasting (> 6 months) secondary immune responses. CONCLUSION: The hu-ton-SCID mouse xenochimeric model described in this report may improve our understanding of human lymphoid cell interactions, secondary immune responses, and lymphomagenesis.

Identification of clonally expanded T cells in rheumatoid arthritis using a sequence enrichment nuclease assay.

Identification of expanded clones engaged in immune and autoimmune responses is still imperfect, since they are often diluted by irrelevant cells expressing diverse specificities. To efficiently characterize T cell receptors expressed by clonally expanded lymphocytes in rheumatoid arthritis (RA) and other inflammatory conditions, we developed an assay system, termed sequence enrichment nuclease assay (SENA). Key elements of SENA are the efficiency of heat-denatured DNA strand reassociation, which increases exponentially with concentration, and the elimination of unhybridized sequences by single-strand-specific DNase. T cell clonal expansions were identified primarily in synovial fluids, but also in peripheral blood of RA patients. Synovial fluids had more prominent expansions in the CD8 than the CD4 subset, whereas clonal expansions in the CD4 subset predominated among peripheral blood lymphocytes. Dominant clones exhibited diverse sequences with no clear conservation of junctional motifs, although the same amino acid sequence was identified in two patients. In most instances, dominant clones in the blood were discordant to those in the corresponding synovial fluid, suggesting local stimulation or preferential sequestration of T cells displaying particular specifities.

Fas (CD95) participates in peripheral T cell deletion and associated apoptosis in vivo.

Following exposure to some types of antigen (superantigens), responsive T cells expand and then decline in numbers, a phenomenon that has been called 'peripheral deletion'. This process may play a role in limiting autoimmune reactions and in the maintenance of immune homeostasis. Here we describe experiments on peripheral deletion in mice carrying the lpr/lpr defect, which has been shown to be due to defective production of the CD95/Fas molecule. Young lpr/lpr mice with no apparent immunologic abnormalities display a defect in bacterial superantigen-induced peripheral deletion. Apoptotic death of the expanded T cell population associated with such peripheral deletion. Apoptotic death of the expanded T cell population associated with such peripheral deletion in normal animals is dramatically reduced in the mutant mice. Further, the levels of Fas on responding cells in normal mice increases and decreases together with increases and decreases in cell numbers, suggesting that cells with the highest levels of Fas are preferentially deleted. These observations are consistent with the known ability of CD95 to transduce a signal leading to apoptosis, and they implicate this signal transduction pathway in peripheral deletion. In contrast, bacterial superantigen-induced deletion of thymocytes appears to be fully functional in these mice, and thus Fas/APO-1 does not appear to be required for this process. Further, antibody ligation of the TCR on activated T cells from normal or young lpr/lpr mice can induce apoptosis and therefore under some circumstances this phenomenon is not dependent upon CD95/Fas. Thus, to avoid autoreactivity and ensure immune homeostasis, several different apoptotic mechanisms exist in peripheral T lymphocytes, only some of which involve Fas.(ABSTRACT TRUNCATED AT 250 WORDS)

V beta gene repertoire in the aging mouse: a developmental perspective.

To define age-associated alterations in the immune system at the molecular level, we have analyzed TCR V beta gene expression patterns at the fetal, neonatal, adult, and advanced ages of mice. In contrast to V gamma and VH genes, V beta genes rearranged without any preference related to their chromosomal organization. Endogenous superantigen-mediated clonal deletions were registered for the first time at the neonatal stage, presumably reflecting the late developmental appearance of these molecules. Such deletions, once established, were maintained throughout life with little, if any, leakage in this process. Furthermore, bone marrow transplantation and other studies indicated that an involuted thymus maintained its capacity to perform both its functions, i.e. positive and negative selection. Although overall V beta repertoires showed remarkable stability with advanced age, modifications in expression levels for some V beta, particularly those associated with the CD8 subset and presumably reflecting antigenic stimulation, were recorded. Mice with lupus and early-life thymic involution were fully capable of deleting endogenous superantigen-reactive V beta clones, and even lupus mice with a genetic defect in the apoptosis-promoting Fas gene were normal in this regard. The results indicate that, aside from some anticipated clonal expansions induced by antigenic stimulation, age-associated alterations in immune functions are not caused by any profound changes in the overall TCR repertoire.

V beta gene repertoires in aging mice.

To determine whether aging and thymic involution are associated with defects in intrathymic T cell selection or clonal instabilities, we compared transcript levels for 18 V beta genes in thymic and splenic T cells of young (2 month), adult (12 month), and old (20 month) mice of various Mls and MHC haplotypes (C57BL/6, BALB/c, and DBA/2). The results showed that the unselected thymic V beta repertoires remain stable throughout life, despite severe reduction in cellularity in the thymus of aged mice. Similarly, splenic CD4 and CD8 V beta repertoires showed no significant alterations, and no leakage to the periphery of endogenous superantigen-reactive V beta clones was observed with age, even in irradiated and bone marrow-reconstituted old mice. Responses in vitro to bacterial superantigens were undiminished with age but, significantly, some of these superantigens expanded V beta clones that are cross-reactive with endogenous superantigens and are normally partially (V beta 11 and -12) or severely (V beta 3.1) deleted in BALB/c mice. In the course of these studies, several previously unrecognized reactivities of V beta with staphylococcal toxins were also revealed.

Poly(Glu60Ala30Tyr10) (GAT)-induced IgG monoclonal antibodies cross-react with various self and non-self antigens through the complementarity determining regions. Comparison with IgM monoclonal polyreactive natural antibodies.

Previous studies have shown that the antibodies of the preimmune repertoire are able to bind to various auto- and xenoantigens including chemical haptens. Sequence analysis of two such murine monoclonal IgM natural autoantibodies showed that they are encoded by unmutated germ-line variable regions of the light and heavy chain (V alpha and VH) genes which were also found in various murine immune responses, like phenyl-oxazolone, dinitrophenyl, arsonate, phosphorylcholine and influenza virus hemagglutinin. These data raised the question as to whether induced antibodies possessing germ-line sequence are also able to react with autoantigens. To study this problem, anti-poly(Glu60Ala30Tyr10) (GAT) and anti-alprenolol (Alp) monoclonal antibodies, carrying similar VH and V alpha genes and the same IgG1 isotype, were examined for their capacity to react with several self and non-self antigens. The results showed that: (a) the anti-GAT antibodies tested reacted with different autoantigens, such as murine tubulin, actin and myosin as well as trinitrophenyl (TNP) and bovine serum albumin. Similarly, one of the anti-Alp showed weak reactivities for myosin, DNA, actin and TNP; (b) in contrast two other anti-Alp antibodies did not react with any of the tested antigens. Since the major differences between the oligoreactive anti-GAT and the monoreactive anti-Alp antibodies are in the complementarity determining regions (CDR) our results suggest that the observed cross-reactions are mediated by hypervariable loops. Sequence comparison of these antibodies indicate a possible correlation between cross-reactivity and the presence of aromatic and charged amino acids in the CDR.