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Christopher C. Goodnow*, Srinivas Akkaraju, Sarah Townsend*, James I. Healy, Richard Cornall, Margaret Hibbs+, Ashley Dunn+, Kevin Otipoby#, Edward Clark#, Richard C. Glynne, David Mack$
* John Curtin School of Medical Research Canberra, Australia
Stanford University Stanford, CA
+ Ludwig Institute for Cancer Research Melbourne, Australia
# University of Washington Seattle, WA
$ Affyxmetrix Santa Clara, CA
Switchboards, Rheostats, and Ignition Triggers: How Antigen Receptors Signal Both Immunity and Tolerance in Peripheral Lymphocytes
Antigen receptors on mature circulating T and B lymphocytes convey signals that either promote an immune response or promote a state of actively acquired tolerance. To understand how genetic and environmental factors influence the balance between these two states, we are trying to elucidate the molecular “wiring” patterns that regulate fight or disarm decisions in lymphocytes. An understanding of this wiring is important to efforts to develop cancer vaccines, and will illuminate how survival and growth of lymphoma cells is controlled. Two models of peripheral immunity/tolerance decisions will be discussed here.
Peripheral tolerance in CD4 T cells. The first model involves transfer of naive CD4 T cells recognizing HEL antigen into mice expressing HEL either as a systemic autoantigen or as an organ-specific autoantigen on pancreatic islet b cells or thyroid epithelium. Confronted with a sudden and continuous stimulus from the systemic HEL, the CD4 T cells express activation markers, proliferate transiently, and then disappear presumably by activation-induced cell death. The same kind of abortive proliferation response is triggered when HEL antigen is presented by HEL-specific naive or tolerant B lymphocytes. By contrast, when the CD4 T cells are allowed to circulate in mice expressing organ-specific HEL they do not pass through a transient autoimmune response before becoming tolerant: they do not express activation markers, nor proliferate or disappear, but within 10 days the CD4 cells become functionally tolerant as assayed by their capacity to help an anti-HEL antibody response. We speculate that repetitive stimulation of the TCR with subactivating concentrations of HEL released from the organ-specific depots decreases the efficacy of signaling through some type of biochemical rheostat, as occurs in B cells (see below).
Peripheral tolerance in mature B cells. A detailed comparison has been made of BCR signaling and responses in HEL-specific B cells that have either been acutely stimulated with a weakly agonistic form of HEL (sHEL) as a foreign antigen or chronically stimulated with the same antigen expressed as a systemic autoantigen. The former promotes an immune response provided suitable costimuli are accompanying, whereas the latter promotes and reinforces a range of peripheral tolerance responses, through a suite of effects as follows (Figure 1): 1. Both acute and chronic stimulation with antigen triggers a rapid change in migratory tropism in the B cells, lowering their attraction for follicles and/or increasing their attraction for the outer T zone regions of lymph nodes and spleen. 2. Acute stimulation triggers rapid induction of B7.2 on the B cell and protects the cell from Fas-mediated elimination during interactions with CD4 T cells. These effects are not induced in chronically stimulated self-tolerant cells. By constitutively expressing B7.2 on self-tolerant cells, we find that display of this early response molecule is sufficient to block Fas mediated elimination by CD4 cells and induce autoantibody secretion. 3. Acute stimulation triggers cell cycle progression and mitosis in naive B cells, in concert with costimuli such as LPS or IL4. Chronic stimulation of tolerant B cells is unable to promote mitosis, but instead shortens B cell lifespan and promotes apoptosis by Fas-dependent and Fas-independent mechanisms.
By taking advantage of the relatively large number of naive and tolerant B cells that can be obtained from transgenic mice, we have begun to unravel how the same BCR receptor triggers such different outcomes (Figure 1). Biochemical analyses of signaling in these cells reveals that the BCR is acting partly like a switchboard: it connects to a broad range of signaling pathways in response to the acute immunogenic stimulus (NFAT, NFkB, JNK, and ERK), but connects only to a subset of these pathways (NFAT and ERK) in tolerogenic mode. These differential connections appear to reflect, in part, a molecular rheostat: the magnitude of the signal through the calcium pathway is much lower when the BCR signals in tolerogenic mode compared to immunogenic mode, and this is sufficient to differentially activate calcium responsive pathways.
It is not clear how antigen receptors function as rheostats or switchboards to differentially trigger immunity versus tolerance responses. We have genetically dissected one important feedback pathway that constitutes a key part of the BCR rheostat, involving phosphorylation of the co-receptor CD22 by the tyrosine kinase lyn, leading to recruitment of the tyrosine phosphatase SHP-1. Interestingly, each of the components of this feedback loop are limiting, so that partial loss of function mutations in the Lyn, Cd22, or Shp-1 genes have an intermediate phenotype and interact to produce a complex quantitative trait of B cell hyperactivity. These genes, and other molecules for tuning the BCR signal, are likely candidates for inherited susceptibility to systemic autoimmune diseases.
A new technology for measuring expression of thousands of genes in parallel, DNA microarrays produced by photolithography at Affymetrix Inc, has allowed us to begin linking the differences in BCR signaling in naive vs. tolerant cells to the different cellular responses they bring about. A DNA chip designed to measure mRNAs for 7000 “known” mouse genes was used to probe RNA from naive and tolerant B cells, and naïve cells stimulated in the presence of various pharmacological agents that selectively target different BCR signaling pathways. A set of acute vs. tolerant response genes has been able to be defined, and related to the different signaling pathways operating in the two opposite responses. Some of the genes identified are strong candidates for explaining the different responses triggered by antigen in naive vs. tolerant B cells.
References:
1. Cyster, J.G. & Goodnow, C.C. (1995) “Antigen-induced exclusion from follicles and anergy are separate and complementary processes that influence peripheral B cell fate.” Immunity 3, 691-701.
2. Cyster, J.G., Healy, J.I., Kishihara, K., Mak, T.W., Thomas, M.L. & Goodnow, C.C. (1996) “Regulation of B lymphocyte negative and positive selection by tyrosine phosphatase CD45.” Nature 381, 325-328.
3. Rathmell, J.C., Townsend, S.E., Xu, J.C., Flavell, R.A. & Goodnow, C.C. (1996) “Expansion or elimination of B cells in vivo: dual roles for CD40- and Fas (CD95)-ligands modulated by the B cell antigen receptor.” Cell 87, 319-329.
4. Healy, J.I., Dolmetsch, R.E., Timmerman, L.A., Cyster, J.G., Thomas, M.L., Crabtree, G.R., Lewis, R.S. & Goodnow, C.C. (1997) “Different nuclear signals are activated by the B cell receptor during positive versus negative signaling.” Immunity 6, 419-428.
5. Dolmetsch, R.E., Lewis, R.S., Goodnow, C.C. & Healy, J.I. (1997) “Differential activation of transcription factors induced by Ca2+ response amplitude and duration.” Nature 386, 855-858.
6. Akkaraju, S., Ho, W.Y., Leong, D., Canaan, K., Davis, M.M. & Goodnow, C.C. “Range of CD4 T cell tolerance: partial inactivation to organ-specific antigens allows thyroiditis or insulitis without tissue destruction.” Immunity 7, 255-271.
7. Cornall, R.J., Cyster, J.G., Hibbs, M.L., Dunn, A.R., Otipoby, K.L., Clark, E.A. & Goodnow, C.C. “Polygenic regulation of complex traits in autoimmunity: Lyn kinase, CD22, and SHP-1 phosphatase are limiting elements of a biochemical pathway that regulates BCR signaling and selection.” Immunity (in press).
8. Goodnow, C.C., Cyster, J.G., Hartley, S.B., Bell, S.E., Cooke, M.P., Healy, J.I., Akkaraju, S., Rathmell, J.C., Pogue, S.L. & Shokat, K.M. (1995) “Self-tolerance checkpoints in B lymphocyte development.” Adv. Immunol. 59, 279-368.
9. Goodnow, C.C. (1996) “Balancing immunity and tolerance: deleting and tuning lymphocyte repertoires.” Proc. Natl. Acad. Sci. USA 93, 2264-2271.
10. Healy, J.I. & Goodnow, C.C. “Positive versus negative signaling by lymphocyte antigen receptors.” Ann. Rev. Immunol. (in press).