Adaptive Tolerance Can Be Induced Independently of Regulatory Activity

<div><p>(A) Intracellular staining with antibodies to <i>foxp3</i> (left panels) or an isotype-matched control (right panels) on naive 5C.C7 T cells (middle two panels) or antigen-adapted T cells (bottom two panels). Positive control staining in a polyclonal population of B10.A T cells marks approximately 19% of the CD4⁺ T cells (top two panels).</p> <p>(B) A new cohort of naive 5C.C7 T cells expands similarly in B10.A, PCC^+/+, CD3ε^−/− hosts (open squares) even if they already harbor expanded 5C.C7 T cells (solid squares). Newly transferred 5C.C7 T cells were distinguished from the previous population using congenic markers (Ly5.1).</p> <p>(C) Congenically marked Ly5.1^+/+,5C.C7 T cells that expanded in a naive B10.A, PCC^+/+, CD3ε^−/− host (open squares) or a PCC^+/+, CD3ε^−/− host that had been injected with Ly5.2 ^+/+,5C.C7 T cells 28 d in advance (solid squares) were purified by FACS sorting (Ly5.1+, CD4+) and restimulated in vitro to measure IFNγ secretion.</p></div

Polyclonal B-Cell Activation and Autoantibody Production following the Induction of T Cell Adaptive Tolerance

<div><p>(A–G) Total serum antibody levels of individual isotypes, measured by sandwich ELISA at different days after T cell transfer (solid circles). Open circles at time 0 represent serum measurements from normal mice, without T cell transfer. *Serum samples where the levels of the isotype was below detection [for two, four, three, three, and six samples each in (B), (C), (D), (E), and (F)]. Data in (A–E) are combined from two experiments between 4 and 60 d and two additional experiments at later time points, with two to five mice per experimental group. Three additional experiments between days 7 and 120 gave similar results. Data in (F) and (G) are from two separate experiments with three mice per time point. Serum IgA levels were below our limit of detection after the day 3 time point as indicated (*). Solid triangles in (G) represent measurements from solubilized fecal pellets. Note that the <i>y</i>-axes are scaled differently for (A), (F), and (G) to accommodate different absolute amounts of IgM, IgE, and IgA.</p> <p>(H) Increased circulating immune complexes in the serum after T cell transfer, demonstrated by a C1q binding ELISA. Each data point represents an individual mouse, whose sera were all sampled on the same day after receiving T cells at various prior times (<i>x</i>-axis).</p> <p>(I) Titers of anti-DNA antibodies in the sera of 16 individual T cell transfer recipients sampled in two separate experiments. Optical density values obtained from isotype specific ELISAs for IgM (solid circles), IgG2b (solid squares), and IgG2a (solid triangles) are shown. Empty circles (IgM), squares (IgG2b), and triangles (IgG2a) on day 0 represent sera obtained from three normal mice, assayed in the two experiments.</p> <p>(J–O) Sera from mPCC,CD3ε^−/− mice were assayed for anticellular antibodies of the IgM (green) and IgG (red) isotypes, by hybridizing with frozen tester sections of B10.A,Rag2^−/− kidneys. Numbers in the inset denote days after T cell transfer. (O) shows an enlarged area from (N) to illustrate speckled nuclear staining. (J) was probed with serum from a normal mPCC,CD3ε^−/− mouse without T cell transfer. Similar sera samples from mice 4 d (<i>n</i> = 3) or 7 d (<i>n</i> = 2) after T cell transfer did not show measurable IgG or IgM anti cellular antibodies. However, all mice tested after 28 d (<i>n</i> = 22) scored positive for both IgG and IgM antibodies to various extents.</p></div

Autoantibodies and Arthritis Do Not Develop in the Presence of Polyclonal Endogenous T Cells

<div><p>(A–C) 3 million Ly5.1⁺,5C.C7 T cells were transferred into mPCC^+/+ (T cell–replete) mice that had an intact repertoire of polyclonal T cells. Serum antibody titers were measured at various days after transfer. Total IgG1 (A), IgG2a (B), and IgG2b (C) levels are shown (filled circles). Inverted open triangles represent titers before T cell transfer. Data are pooled from two separate experiments with two to five mice per time point. Similar results were obtained in two other experiments.</p> <p>(D–F) Serum from T cell–replete (F) or T cell–deficient (E) PCC transgenic mice were used to stain tester B10.A,Rag2^−/− kidney sections at 1:100 dilution. Staining with control serum from a normal B10.A mouse is shown in (D).</p> <p>(G) Development of arthritis scored in the T-replete mPCC transgenic, 5C.C7 transfer recipients.</p></div

Poor Differentiation and Survival Are Superimposed on Adaptive Tolerance in the Presence of Endogenous T Cells

<div><p>(A) The total recovery of Ly5.1⁺ 5C.C7 T cells from lymph nodes and spleen of recipient mice, various days after transfer into a T cell–replete (mPCC⁺,CD3ε⁺; open squares) host are compared with the recovery from a T cell–deficient (mPCC⁺,CD3ε^−/− ; filled squares) transfer recipient.</p> <p>(B) Carboxy fluorescein succinimidyl ester profiles of transferred Ly5.1⁺ 5C.C7 T cells, 3 d after transfer into a lymphopenic (top) or T cell–replete (bottom) PCC transgenic host.</p> <p>(C) Ly5.1⁺,5C.C7 T cells, recovered 9 d after transfer to a T cell–replete PCC transgenic (red line) or T cell–deficient PCC transgenic (blue line) host, were loaded with Indo-1 and stimulated as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040340#s4" target="_blank">Materials and Methods</a>. The influx of calcium, measured as the ratio of violet to blue fluorescence, is compared to that of naïve 5C.C7 T cells (green line).</p> <p>(D) IL-2 or (E) IFN-γ secretion from sorted Ly5.1⁺,5C.C7 T cells restimulated in vitro with 10 μM MCC peptide and irradiated syngeneic splenocytes for 48 h, 15 d after residence in a T cell–replete (gray bars) or T cell–deficient (black bars) PCC transgenic hosts. Naïve 5C.C7 T cells (white bars) were used for comparison.</p> <p>(F) Ex vivo IFNγ secretion by transferred T cells recovered 4 d after transfer to a T cell–replete (right) or T cell–deficient (left) PCC transgenic host. FACS plots are gated on CD4+, 7AAD-ve cells in the lymphocyte area.</p></div

Adoptively Transferred T Cells Induce Arthritic Pathology in Recipient Mice

<div><p>(A) X-ray radiographs of fore limbs and (B) and hind limbs comparing mPCC,CD3ε^−/− mice without a T cell transfer (left panels) and one 86 d after (right panels).</p> <p>(C–E) Hindlimbs from mPCC,CD3ε^−/− mice were sectioned and stained by HEMATOXYLIN AND EOSIN before (C) T cell transfer and 14 d (D) or 60 d (E) afterward. Lower panels show magnification of the joint regions from the top panels, to illustrate cellular infiltrates in the tibiotarsal region.</p> <p>(F) Clinical scores quantitate increased limb pathology in transfer recipients on a scale of 0 to 56. Bars represent arithmetic means in each group.</p></div

Cytokine and CD40L Dependence of Help Rendered by Adaptively Tolerant T Cells

<div><p>(A–C) Serum from mice that received T cells 8 d before were assayed for total immunoglobulins as described. T cells were from 5C.C7 TCR transgenic mice (white bars) or similar mice also deficient in IFN-γ (black bars) or IL-4 (gray bars). Host mice were mPCC,CD3ε^−/− (left three bars in each graph) or IL-21 receptor–deficient mPCC,CD3ε^−/− mice (right three bars).</p> <p>(D–F) B cells were infused into mPCC,Rag2^−/− mice that harbored stably adapted 5C.C7 T cells. The blocking anti-CD40L antibody MR1 (black bars) or a control hamster antibody (white bars) was administered at the same time and on every other subsequent day for 13 d, before assaying serum for antibody titers.</p> <p>(G, H) Similar experiments (as in D–F) were carried out, but the blocking antibody treatment was terminated at 6 d. Twenty-two days afterward, serum IgG2a (G) and IgG2b (H) levels were measured in the MR1 treated (black bars) or control (white bars) mice.</p></div

Stable Adaptive Tolerance Does Not Abrogate the Ability of T Cells to Help Autoreactive B Cells

<div><p>(A–E) Purified splenic B cells (25 million) were transferred into mPCC,Rag2^−/− mice that received 5C.C7 T cells 48 d earlier. The amount of serum antibody of each isotype 8, 14, and 21 d after the transfer of PCC transgenic B cells (triangles) or antigen-free B cells (circles) is plotted. Solid triangles and open circles are measurements from mice that received 5C.C7 T cells 48 d before the B-cell transfer. Open inverted triangles are measurements from mPCC,Rag2^−/− mice that did not receive T cells. Two other experiments, including one using FACS-sorted B cells, showed similar results.</p> <p>(F–H) Antinuclear antibodies in the serum of mPCC,Rag2^−/− mice 30 d after B-cell transfer (H). Three other mice from the same experiment also showed anticellular reactivity. Similarly stained tester sections using serum from an mPCC,CD3ε^−/− mouse that received 5C.C7 T cells 60 d earlier (G) or a control B10.A mouse (F) are provided for comparison.</p> <p>(I) Arthritis development between 21 and 82 d after the transfer of mPCC⁺ B cells (triangles) into mPCC,Rag2^−/− mice bearing adoptively tolerant 5C.C7 T cells. Arthritis in mPCC,CD3ε^−/− mice 60 and 82 d after receiving naive 5C.C7 T cells (squares: C 60, C 82) are scored for comparison.</p></div