Repeated immunization with antigen causes systemic autoimmunity in mice otherwise not prone to spontaneous autoimmune diseases. Overstimulation of CD4 + T cells led to the development of autoantibody-inducing CD4 + T (aiCD4 + T) cell which had undergone T cell receptor (TCR) revision and was capable of inducing autoantibodies. The aiCD4 + T cell was induced by de novo TCR revision but not by cross-reaction, and subsequently overstimulated CD8 + T cells, driving them to become antigen-specific cytotoxic T lymphocytes (CTL). These CTLs could be further matured by antigen cross-presentation, after which they caused autoimmune tissue injury akin to systemic lupus erythematosus (SLE).

Funding: This work is supported by the Global Center of Excellence (GCOE) Program grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and the Japan Science and Technology Organization. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Since ‘clonal selection theory of immunity’ of F. Macfarlane Burnet and subsequent molecular biological discoveries on V(D)J recombination and the diversity and individuality of immune response, how autoimmunity arises remains unclear. Apart from the term ‘autoimmunity’ which is now ready-made, in the present study, we tried to see the pathogenesis of autoimmunity from different angle and test the integrity of immune ‘system’. The method we have chosen was to stimulate the system maximally by antigen to the levels far beyond its steady-state just like testing the capability of automobile. In a perfectly reproducible experiments in which the mice not prone to autoimmune diseases were immunized repeatedly with antigen, we have unexpectedly and surprisingly discovered that overstimulation of immune system beyond its self-organized criticality inevitably leads to systemic autoimmunity. Subsequent detailed molecular analyses revealed in the first that autoantibodies are induced not by cross reaction to antigen but by de novo T cell receptor (TCR) revision. Second, final maturation of effector cytotoxic T lymphocyte (CTL) via antigen cross-presentation is sine qua non for generating autoimmune tissue injury. Most importantly, we now show that autoimmunity arises not from ‘autoimmunity’, but as a natural consequence of normal immune response when stimulated maximally beyond system's self-organized criticality.

Results

Induction of Autoantibodies Consistent with the common observation that T cells become anergic after strong stimulation with antigen [1], we observed that 2× immunization with staphylococcus enterotoxin B (SEB) caused SEB-reactive Vβ8+CD4+ T cells from BALB/c mice to become anergized. However, these cells recovered from anergy to divide and produce IL-2 after further immunization 8× with SEB (Figure S1A). This was accompanied by the induction of autoantibodies, including IgG- and IgM-rheumatoid factor (RF), anti-Sm antibody, and in particular, RF reactive against galactose-deficient IgG, typically found in human autoimmunity [2] (Figure 1A). Autoantibodies can also be induced by other conventional antigens, including ovalbumin (OVA) or keyhole limpet hemocyanin (KLH) () as long as immunizing antigen is correctly presented to T cells (Figure S1B). CD4+ T cells of repeatedly-immunized mice become fully matured, expressing CD45RBlo, CD27lo and CD122hi (data not shown), and these primed CD4+ T cells can confer RF generation in naïve recipients following adoptive transfer (Figure 1B). The induction of autoantibodies is independent of CD8+ T cells or MHC class I-restricted antigen presentation for the following reasons. First, both RF and anti-dsDNA antibody can be consistently induced upon repeated immunization of β 2 -microglobulin (β 2 m)-deficient BALB/c mice with OVA. β 2 m-deficient mice are deficient in CD8+ T cells, which are reduced to <0.8% of splenic T cells [3] (Figure S3). Second, the ability to induce autoantibodies was transferable from OVA-immunized BALB/c mice to β 2 m-deficient mice solely via CD4+ T cells (Figure 1C). Thus, CD4+ T cells from repeatedly-immunized mice acquire the ability to induce autoantibodies. We refer to these as autoantibody-inducing CD4+ T (aiCD4+ T) cells in this communication. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 1. Induction of autoantibodies and proteinuria. BALB/c mice were repeatedly injected i.p. with 25 µg SEB, 500 µg OVA or PBS every 5 d. (A) Serum IgG- and IgM-RFs, anti-galactose-deficient IgG and anti-Sm antibodies were measured using ELISA. The arbitrary unit (AU) of 1.0 is equivalent to the titer obtained from sera of prototypic autoimmune MRL/lpr mice. Data from each mouse are connected by dotted lines. (B) Adoptive transfer of splenic B, T, CD4+ T or CD8+ T cells of SEB-, OVA- or PBS-immunized BALB/c mice into naïve BALB/c mice. The recipient mice were given single i.p. injection of 25 µg SEB or 500 µg OVA 24 h after cell transfer, and autoantibodies were measured 2 weeks later. (C) Adoptive transfer of cells from OVA-immunized BALB/c mice into β 2 m-deficient mice. https://doi.org/10.1371/journal.pone.0008382.g001

Mechanism of Autoantibody Induction To further clarify the characteristics of aiCD4+ T cells, we examined their TCR repertoire by spectratyping of their complementarity determining region 3 (CDR3) [4]. Combinatorial assessment of Vβ and Jβ showed that the CDR3 length profiles of CD4+ splenocytes in mice immunized either 8× with PBS or 2× with SEB fit a normal Gaussian curve, typical of a diverse and unbiased TCR repertoire (Figure 2A). However, splenocytes, but not thymocytes, from mice immunized 8× with SEB showed skewed length profiles, suggesting that TCR revision was in progress at periphery of the spleen. Genes encoding components of the V(D)J recombinase complex were specifically re-expressed in mice immunized 8× with SEB, including the recombination-activating genes 1 and 2 (RAG1/2), terminal deoxynucleotidyl transferase (TdT) and surrogate TCRα chain (pTα) [5] (Figure 2B). The RAG1 gene is expressed in vivo after immunization 8× with SEB in rag1/gfp knock-in mice [6] (Figure 2C). In these mice, serum RF was increased in conjunction with an increase of GFP-expressing Vβ8+CD4+ T cells in the spleen. To directly prove that V(D)J recombination took place at the periphery in spleen, we used ligation-mediated PCR (LM-PCR) to detect blunt-end DNA fragments harboring a rearranged coding V region flanked by recombination signal sequences (RSS) [7], [8]. We identified rearranged intermediates corresponding to the TCRα variable region 2 (TCRAV2) in the splenocytes of mice immunized 8× with SEB (Figure 2D). These findings indicate that repeated immunization with conventional antigen can induce the generation of aiCD4+ T cells which have undergone TCR revision and are capable of stimulating B cells [9]. This observation is in line with previous findings showing that such somatic mutations occur often in lymphocytes, a process which is considered to be a major stochastic element in the pathogenesis of autoimmunity [10], [11]. Thus, overstimulation of CD4+ T cells by repeated immunization with antigen and induction of full maturation inevitably leads to the generation of aiCD4+ T cells which have undergone TCR revision and are capable of inducing autoantibodies. Importantly, the present study shows that such aiCD4+ T cells are induced by de novo TCR revision but not by cross-reaction to antigen. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 2. TCR revision upon repeated immunization with antigen. (A) TCR CDR3 length profiles of mice immunized 8× with PBS, 2× or 8× with SEB. TCR repertoire of splenic CD4+ T cell was skewed only after immunization 8× with SEB. (B) Expression of V(D)J recombinase complex and related molecules in the spleen of PBS- or SEB-injected BALB/c mice. (C) GFP+ cells in the Vβ8+CD4+ T population of rag1/gfp knock-in mice. IgG-RF as induced in rag1/gfp knock-in mice after immunization 8× with SEB (lower left). The GFP+ T cell fraction was also increased among Vβ8+CD4+ T cells (mean ± SD, 4–5 mice/group). (D) TCRα chain revision in the spleen of mice immunized 8× with SEB was determined by LM-PCR detection of dsDNA breaks at the RSS flanking the TCRAV2, with PCR-amplified TCRα constant region (TCRAC) as a DNA quality control. https://doi.org/10.1371/journal.pone.0008382.g002

Mechanism of Autoimmune Tissue Injury It has been shown previously that IFNγ is increased in association with autoimmune tissue injury [15]–[17]. Consistent with this, we found that the number of IFNγ+CD8+ T cells, but not regulatory T or T helper 17 cells, was increased following immunization 12× with OVA (Figure 4A and data not shown). We also observed an expansion of IFNγ-producing effector/memory CD8+ T cells, which are necessary for adaptive immunity [18] (Figure 4A). These IFNγ-producing CD8+ T cells were observed to have infiltrated into OVA-deposited glomeruli of OVA-immunized mice (Figure 3A). CD8+ T cells are required for tissue injury based on the following observations. First, the transfer of CD8+ T cells can induce renal lesions in mice (Figure 4B), as well as the generation of new IFNγ+CD8+ T cells in the spleens of recipient mice following cell transfer (Figure S5). Second, autoimmune tissue injury is not induced by the transfer of CD8+ T cells from OVA-immunized wild-type mice into β 2 m-deficient mice (Figure 1C). And finally, CD8+ T cell transfer must be accompanied by at least a 1× booster immunization with OVA to induce autoimmune tissue injury in the recipient mice (Figure S6). The findings indicate that full-matured, IFNγ-producing effector CD8+ T cells are required for the induction of autoimmune tissue injury, provided that the relevant antigen is correctly presented on the target organs. These are well-established characteristic of CTL and not novel. We show, however, that (i) CTL is induced through an immune, but not ‘autoimmune’, process, and that (ii) autoimmune tissue injury inevitably occurs when CD8+ T cells are overstimulated to become matured effector CTLs. The latter means that regardless of how CTL is induced, the consequence of CTL over-induction is immune tissue injury. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 4. Expansion of CD8+ T cells and antigen cross-presentation. (A) Spleen cells stimulated with 50 ng/ml phorbol myristate acetate (PMA) and 500 ng/ml ionomycin for 4 h in the presence of brefeldin A (10 µg/ml) and stained for intracellular IFNγ (upper). Subsets of CD8+ T cells categorized into naïve (CD44lowCD62Lhigh), effector (CD44highCD62Llow), and memory (CD44highCD62Lhigh) fractions (middle). Flow cytometry of IFNγ+ cells within naïve or effector/memory CD8+ T cell populations. Spleen cells were separated into naïve (CD44low) and effector/memory (CD44high) cells using CD44 MACS beads, and IFNγ+ cells within the CD8+ T population was evaluated (lower). (B) Adoptive transfer of splenocytes of OVA-immunized BALB/c mice into naïve recipients. The recipients were injected with 500 µg OVA 24 h after cell transfer, and proteinuria examined 2 weeks later. (C) Cross-presentation of OVA to CD8+ T cells. Splenic CD11c+ DC from OVA-immunized or control mice were incubated in the presence (OVA(+)) or absence (OVA(−)) of 1 mg/ml OVA with or without chloroquine (CQ) (20 µg/ml) for 3 h, followed by a co-culture with KJ1-26+CD8+ T cells of DO11.10 transgenic mice for 24 h to examine surface expression of CD69 (upper). Inhibition of cross-presentation in vivo by administration of 250 µg CQ per mouse 3 h prior to immunization with OVA or PBS. IFNγ+CD8+ T cells (middle), autoantibodies and proteinuria (lower) after 12× immunization. (D) Requirement of autoantibody-inducing CD4+ T cells for CD8+ T cell-mediated autoimmune tissue injury. BALB/c mice were immunized 12× with KLH, and CD4+ T cells were isolated using MACS beads. Cells were transferred into the anti-CD4 antibody-treated recipient mice immunized 8× with OVA. Percent matured CTL, i.e., IFNγ+CD8+ T cells, and proteinuria were measured 2 weeks after booster immunization 1× with KLH. https://doi.org/10.1371/journal.pone.0008382.g004

Antigen Cross-Presentation We next show that antigen cross-presentation is required for the induction of CTL and tissue injury. To test this, we co-cultured OVA-pulsed dendritic cells (DC) from mice immunized 12× with OVA together with T cells from OVA-TCR transgenic DO11.10 mice exclusively expressing OVA-reactive TCR [19]. We show that OVA-reactive DO11.10 CD8+ T cells are activated upon co-culture with OVA-pulsed DCs (Figure 4C and Figure S7). Further, autoimmune tissue injury and the increase in IFNγ+CD8+ T cells, but not of autoantibody generation, were both abrogated by adding chloroquine (CQ), an inhibitor of antigen cross-presentation (Figure 4C). This indicates that antigen cross-presentation is required for the expansion of IFNγ-producing CD8+ T cells and autoimmune tissue injury.