With remarkable prescience, Bretscher and Cohn1 in 1970 postulated that the induction of humoral antibody formation to hapten-carrier antigens required the recognition of 2 signals: recognition of the hapten by B cells and associative carrier recognition by thymus-derived T cells. They further postulated that paralysis of antibody formation occurred when only antigen recognition by B cells occurred without the associative carrier recognition, or when the carrier-specific T cells were paralyzed. Over the next 47 years, we have observed an evolution of the 2-signal theory of immune activation (reviewed in Baxter and Hodgkin and Turner et al2,3); with Lafferty and Cunningham’s clarification (1975) of the role of a MHC-expressing stimulating cell-derived second signal, Jenkins and Schwartz’s (1987) demonstration of experimentally induced T cell anergy in the absence of signal 2, Janeway’s theory (in 1989) of a second signal triggered in antigen-presenting cells by microbial products through Toll-like receptors, and Matzinger's danger hypotheses (1994) showing that antigen-presenting cell activation is induced by host-cell damage and recognition of “danger” signals and the recognition of antigens in the absence of danger resulting in T cell tolerance.
Consistent with the 2-signal model, the current paradigm states that B cells activated by antigen follow 1 of 3 pathways: (i) they proliferate and differentiate to give rise to a productive humoral response, (ii) they acquire an anergic state, or (iii) they are deleted. The study by Turner et al3 examines the consequence of antigen recognition by B cells in the absence or presence of T cells, by focusing on the impact of antigen persistence showing a fourth possible outcome of B cells exposed transiently to antigen. Using an elegant, 2-step exposure of B cell receptor-transgenic (BCR-Tg) B cells to antigen (Figure 1), they demonstrated that transient (5 minutes) antigen exposure together with antigen-specific T cell help was sufficient to induce the full development of an antibody response and memory B cells. In the presence of persistent antigen in vivo, the B cell response was enhanced, albeit only modestly. Thus transient exposure to soluble antigen was sufficient to prime the full B cell differentiation over a broad range of antigen doses and at lower antigen affinity, underscoring the sensitivity of B cells to signal 1.
Despite the many conceptual advances and the identification of the nature of the second costimulatory signals, many questions remain unclear in fully understanding the mechanisms that underlie T and B cell activation versus tolerance.
Things became more interesting when the experiment was performed in the absence of T cell help or in T cell–deficient hosts. B cells transiently exposed to antigen and then transferred in hosts that received repeated (0, 12, and 24 hours postadoptive transfer [AdT]) intravenously administered antigen were significantly depleted by 30 hours post-AdT compared with B cells that were not reexposed to antigen in vivo. These observations are consistent with the 2-signal model where B cells exposed to persistent antigen in the absence of T cell help resulted in B cell tolerance.4,5 However, when B cells were only transiently exposed to antigen and then adaptively transferred into recipients without T cell help or into T cell–deficient hosts, B cell numbers were comparable with naive (nonantigen-exposed) B cells maintained in vivo. These antigen-exposed B cells migrated to the T-B border and upregulated CCR7 and downregulated IgM. However, by 24 hours post-AdT, B cells had migrated away from the T-B border, downregulated CCR7, and upregulated IgM. Furthermore, B cells transiently exposed to antigen-upregulated CD86 and presented antigen:I-Ab complexes that peaked at 12 hours post-AdT returning to baseline by 72 hours post-AdT. This process was paralleled by the loss in their ability to engage with antigen-specific T cells 24 to 48 hours after antigen exposure. Most intriguingly, transiently antigen-exposed B cells were fully capable of entering into a productive immune response when reexposed in vivo, 3 or 5 days later, to antigen in the presence of T cells help. These observations demonstrate that B cells exposed to antigen without T cell help are not deleted or anergic, but rather that antigen encounters are innocuous with B cells retaining their ability to respond productively upon antigen encounter (Figure 1).
There are some caveats of this experimental model that are worth highlighting: the readout of B cell fate was completely dependent on 2 BCR-Tg B cells (MD4 or HyHEL10) specific for hen egg lysozyme or duck egg lysozyme that were adoptively transferred in large numbers (1.5-4.5 × 107/recipient). Moreover, T cell help was provided by AdT TCR-Tg OTII cells (5 × 105/recipient) activated by immunization with ovalbumin emulsified in complete Freund’s antigen. Amplified numbers and affinities of transgenic T and B cells may induce cell fates that differ from endogenous cells.6,7 Furthermore, follow-up durations for key experiments were relatively short (<5 days). Indeed, it is possible that with time, transiently, antigen-exposed B cells will be deleted gradually or anergized.
Despite these limitations, these observations may have relevance to transplant recipients. For example, with the ability of B cells to engage with antigens and the capacity to receive T cell help for 24 to 48 hours improve their chances for encountering the rare antigen-specific T cell at the T-B interface. If these B cells do not receive T cell help, they can still undergo repeated rounds of antigen exposure while preserving their ability to become fully functional upon T cell help. Because immunosuppression severely curtails T cell help, the ability of pathogen-specific B cells to return to baseline while becoming fully functional once T cell help becomes available may be critical for immunosuppressed patients in developing protective immunity after immunosuppression is reduced.8
These findings also underscore the importance of antigen persistence for B cells to become anergic or deleted. In solid organ transplant recipients, the allograft is a persistent source of antigen yet antibody-mediated rejection is one of the major causes of graft loss, raising the question on why B cell anergy or deletion does not occur. There are several possible explanations, for example, under conventional immunosuppression, the incomplete suppression of T cell help prevents alloreactive B cells from undergoing deletion or anergy. Alternatively, memory B cells have relaxed restimulation requirements as a result of epigenetics, expression of high-affinity BCR and costimulatory molecules,9 and may therefore be more resistant to anergy and deletion compared with naive B cells. Indeed, the frequency of memory B cells in the peripheral blood of humans has been shown to increase with age.10 Thus, additional investigations are necessary to define the susceptibility to anergy or deletion of memory compared with naive alloreactive B cell in the presence of solid organ allografts. Solving the conundrum of inducing anergy or deletion of memory B cells, in addition to memory T cells, is likely to be key to achieving successful transplantation tolerance.
1. Bretscher P, Cohn M. A theory of self-nonself discrimination. Science
2. Baxter AG, Hodgkin PD. Activation rules: the two-signal theories of immune activation. Nat Rev Immunol
3. Turner JS, Marthi M, Benet ZL, et al. Transiently antigen-primed B cells return to naive-like state in absence of T-cell help. Nat Commun
4. Getahun A, Beavers NA, Larson SR, et al. Continuous inhibitory signaling by both SHP-1 and SHIP-1 pathways is required to maintain unresponsiveness of anergic B cells. J Exp Med
5. Reed JH, Jackson J, Christ D, et al. Clonal redemption of autoantibodies by somatic hypermutation away from self-reactivity during human immunization. J Exp Med
6. Hataye J, Moon JJ, Khoruts A, et al. Naive and memory CD4+ T cell survival controlled by clonal abundance. Science
7. Shlomchik MJ. Sites and stages of autoreactive B cell activation and regulation. Immunity
8. Acott P, Babel N. BK virus replication following kidney transplant: does the choice of immunosuppressive regimen influence outcomes? Ann Transplant
9. Phan TG, Tangye SG. Memory B cells: total recall. Curr Opin Immunol
10. Morbach H, Eichhorn EM, Liese JG, et al. Reference values for B cell subpopulations from infancy to adulthood. Clin Exp Immunol