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 CD45RB^lo, CD27^lo and CD122^hi (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 β₂-microglobulin (β₂m)-deficient BALB/c mice with OVA. β₂m-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 β₂m-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.

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.

Repeated immunization with OVA can also lead to autoimmune tissue injury and the production of autoantibodies reactive against IgG, Sm and dsDNA (Figure 3 and Figure S2A). Serum immune complex (IC), proteinuria, and the deposition of IC and OVA in the kidney were noted in mice immunized 12× with OVA (Figure 3A). Typical diffuse proliferative glomerular lesions were seen in the kidney, and these glomeruli were infiltrated with CD8⁺ T cells. These observations resemble the clinical features observed in lupus patients, who typically exhibit an increase in CD8⁺ T cells in the peripheral blood and infiltration of CD8⁺ T cells in kidney [12], [13]. Immunization of mice 12× with OVA led to re-expression of the V(D)J recombinase complex and enlargement of the spleen (Figure S4A), and an increase in anti-dsDNA antibody, which is uniquely linked to autoimmune tissue injury in lupus nephritis [14] (Figure S2A). Pathological findings included diffuse membranous (wire-loop) and/or proliferative glomerulonephritis in the kidney (Figure 3A), infiltration of plasma cells around hepatic bile ducts (Figure S4B), enlarged lymphoid follicles with marked germinal center in spleen (Figure S4B), occasional lymphocyte infiltration into the salivary glands (data not shown), lymphoid cell infiltration into the thyroid, and perivascular infiltration of neutrophils and macrophages into the skin dermis of the auricle (Figure S4B). The lupus band test, diagnostic of SLE, was positive in the skin at the epidermal-dermal junction (Figure 3B).

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 β₂m-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.

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.

Since CTL appear to play a rather passive role in autoimmunity, we next studied whether or not aiCD4⁺ T cell help is required for the induction of autoimmune tissue injury. Since anti-CD4 treatment almost abrogates generation of IFNγ-producing CD8⁺ T cell and autoimmune tissue injury in OVA-immunized BALB/c mice (Figure S8), to test whether this CD4⁺ T cell-mediated help is mediated by aiT cells or antigen-specific T cells, we have transferred CD4⁺ T cells from mice immunized 12× with KLH into CD4⁺ T-depleted BALB/c mice immunized 8× with OVA (Figure 4D). Because full-matured IFNγ⁺ CTLs do not develop with less than 8× immunization with OVA (Figure S9), this experiment can test the ability of aiCD4⁺ T cells that have undergone TCR revision to promote the maturation of OVA-specific CTL. The result showed that both autoimmune tissue injury and OVA-specific IFNγ⁺CD8⁺ T cells arose in these mice after transfer, indicating that aiCD4⁺ T cells with de novo TCR revision are required for the maturation of CD8⁺ T cell and autoimmune tissue injury (Figure 4D).

This study was approved by the Institutional Animal Care and Use Committee and carried out according to the Kobe University Animal Experimental Regulations.

APC (allophycocyanin)-conjugated antibody against CD4 (RM4-5), and PE-conjugated antibodies against CD62L (MEL-14), CD69 (H1.2F3) and were purchased from BioLegend (San Diego, CA); FITC-conjugated antibodies against CD44 (IM7.8.1) and DO 11.10 clonotypic TCR (KJ1-26) and PE-conjugated rat IgG1 isotype control from CALTAG Laboratories (Burlingame, CA); PE-Cy5 (phycoerythrin-cyanin 5)-conjugated antibody against CD8α (53-6.7), PE-conjugated antibodies against Vβ8 TCR (F23.1) and IFNγ (XMG1.2) from BD PharMingen (San Diego, CA).

Animal studies with BALB/c female mice (Japan SLC, Inc., Hamamatsu, Japan) and DO11.10 TCR transgenic mice [19] (Jackson Laboratory, Bar Harbor, ME), β₂m-deficient mice [3] and rag1/gfp knock-in mice [6] of BALB/c background were performed with the approval of the Institutional Review Board. Mice (8 weeks-old) were immunized with 25 µg SEB (Toxin Technologies, Sarasota, FL), 500 µg OVA (grade V; Sigma, St. Louis, MO), 100 µg KLH (Sigma) or PBS by means of i.p. injection every 5 d.

Frozen sections of kidney and dermis were stained for C3, IgG or OVA using goat anti-C3 (Bethyl laboratories, Inc., Montgomery, TX) and Alexa Fluor 488-conjugated anti-goat IgG antibodies (Molecular Probes, Eugene, OR), Alexa Fluor 594-conjugated anti-mouse IgG antibody (Molecular Probes), or rabbit anti-OVA antibody (Sigma). For CD8 or IFNγ staining, paraffin-embedded sections of kidney were stained with rat antibodies against CD8α (53-6.7; BD PharMingen) or IFNγ (R4-6A2; BD PharMingen), followed by reaction with VECTASTAIN Elite ABC rat IgG kit (Vector, Burlingame, CA).

To detect intracellular IFNγ, cells (1×10⁶/ml) were stimulated with 50 ng/ml phorbol myristate acetate (PMA; Sigma) and 500 ng/ml ionomycin (Sigma) in the presence of brefeldin A (10 µg/ml; Sigma). After 4 h, cells were stained with anti-CD8 antibody, followed by fixation with 2% formaldehyde, permeabilization with 0.5% saponin (Sigma) and stained for IFNγ.

For adoptive cell transfer, B, T, CD4⁺ T and CD8⁺ T cells were isolated from spleens to >90% purity using MACS beads (Miltenyi Biotec, Germany). The cells were transferred into naïve BALB/c or β₂m-deficient mice via i.p. (5×10⁶/mouse) or i.v. (2.5×10⁷/mouse) injection. The recipients received a single i.p. injection of 25 µg SEB or 500 µg OVA 24 h after cell transfer, and sera, urine and organ of recipients were studied 2 weeks afterwards.

BALB/c mice were injected i.p. with 200 µg anti-CD4 antibody (GK1.5; BioLegend) to deplete CD4⁺ T cell 24 h after immunization 8× with OVA. Four days later, CD4⁺ T cells from mice immunized 12× with KLH were transferred to the CD4⁺ T-depleted mice. The recipient mice received a single i.p. injection of 100 µg KLH 24 h after the cell transfer.

Sera were assayed for anti-Sm antibody using Sm antigen (ImmunoVision, Springdale, AR), RF (Shibayagi Co., Gunma, Japan), RF for galactose-deficient IgG (Eisai Co., Ltd., Tokyo, Japan) and anti-dsDNA antibody using dsDNA (Worthington Biochemical Co., Lakewood, NJ) after digestion by S1 nuclease (Promega, Madison, WI). Serum IC was detected using goat anti-C3 antibody (Bethyl Lab.).

cDNAs from thymocytes and CD4⁺ splenocytes were subjected to PCR amplification using Cβ- and Vβ8-specific primers. Amplified products were subjected to run-off reactions using three fluorophore-labeled Jβ primers, Jβ1.1, Jβ1.3 and Jβ2.4, and analyzed by GeneScan software (Perkin-Elmer Applied Biosystems, Emeryville, CA) [4].

Total RNA was reversely transcribed to cDNA and amplified by PCR [23]. The products were fractionated by electrophoresis and transferred to nylon membranes (Roche Diagnostics, Mannheim, Germany). The membranes were hybridized to fluorescein end-labeled probes and visualized by alkaline phosphatase (ALP)-labeled anti-fluorescein antibody and Gene Images CDP-Star chemiluminescence reaction (Amersham Pharmacia Biotech, Piscataway, NJ). The primers and probes were: 5′-CCAAGCTGCAGACATTCTAGCACTC-3′ (forward), 5′-CAACATCTGCCTTCACGTCGATCC-3′ (reverse) and 5′-AACATGGCTGCCTCCTTGCCGTCTACCCT-3′ (probe) for RAG1 [24]; 5′-CACATCCACAAGCAGGAAGTACAC-3′ (forward), 5′-GGTTCAGGGACATCTCCTACTAAG-3′ (reverse) and 5′-GCAATCTTCTCTAAAGATTCCTGCTACCT-3′ (probe) for RAG2 [24]; 5′-GAACAACTCGAAGAGCCTTCC-3′ (forward), 5′-CAAGGGCATCCGTGAATAGTTG-3′ (reverse) and 5′-ATTCGGTCACCCACATTGTGGCAGAGAAC-3′ (probe) for TdT; 5′-CAACTGGGTCATGCTTCTCC-3′ (forward), 5′-TGGCTGTCGAAGATTCCC-3′ (reverse) and 5′-CCGTCTCTGGCTCCACCCATCACACTGCT-3′ (probe) for pTα.

DNA (1 µg) was ligated to 20 µM BW linker using T4 ligase (Takara Bio Inc., Shiga, Japan) [25]. Primary PCR was performed using 200 ng ligated DNA, BW-1HR primer (5′-CCGGGAGATCTGAATTCGTGT-3′) [24], primer specific for 3′ flanking sequence of TCRAV2 (5′-AGATGATACAGAGACAAAATGTGAGC-3′) and 2 U of AmpliTaq Gold DNA polymerase (Applied Biosystems, Foster City, CA). A second PCR was performed using 1 µl of the first PCR product (diluted 1/100), BW-1HR, and nested primer specific for 3′ flanking sequence of TCRAV2 (5′-TATTGTGGATGCTAACAAGTGCTTTC-3′). Amplified DNA was transferred to membranes and visualized using fluorescein end-labeled probe specific for TCRAV2 (5′-TAACATAAGAATGCACCGCTTACACC-3′) and ALP-labeled anti-fluorescein antibody. Primers for control TCRAC region were amplified using the primers 5′-CAGAACCCAGAACCTGCTGTG-3′ and 5′-ACGTGGCATCACAGGGAA-3′. Nomenclature of the TCRA gene segments was according to the ImMunoGeneTics (IMGT) database (http://imgt.cines.fr).

OVA-reactive CD8⁺ T cells were isolated from spleens of DO 11.10 mice using MACS beads (Miltenyi Biotec). CD11c⁺ DCs (4×10⁵/well) were isolated using MACS beads (Miltenyi Biotec) and incubated with 1 mg/ml OVA for 3 h, then co-cultured with DO11.10 CD8⁺ T (KJ1-26⁺CD8⁺) cells (2×10⁵/well) for 24 h, and the expression of CD69 on DO11.10 CD8⁺ T cells was examined. IL-2 and IFNγ in culture supernatants were measured by ELISA (Biosource, Camarillo, CA).

To inhibit cross-presentation, mice were immunized in vivo with 250 µg of chloroquine (Sigma) 3 h prior to immunization with 500 µg OVA or PBS every 5 d. Presence of autoantibodies was analyzed 2 d after each immunization, and proteinuria and IFNγ⁺CD8⁺ T cells were examined 9 d after the final immunization.

Statistical analyses were performed using Student's t test, and the data are expressed as the mean ± SD.