Volume 16, Issue 3, Pages 403-415 (March 2002) CD40L Blockade Prevents Autoimmune Diabetes by Induction of Bitypic NK/DC Regulatory Cells  Dirk Homann, Angelika Jahreis, Tom Wolfe, Anna Hughes, Bryan Coon, Marianne J.B van Stipdonk, Kiley R Prilliman, Stephen P Schoenberger, Matthias G von Herrath  Immunity  Volume 16, Issue 3, Pages 403-415 (March 2002) DOI: 10.1016/S1074-7613(02)00290-X

Figure 1 Prevention of Type 1 Diabetes by Costimulation Blockade (A) Therapeutic efficacy of aCD40L and aB7 costimulation blockade. Incidence of diabetes is plotted as a function of the frequency of antibody administration. While one or more injections of aCD40L antibody (100 μg) effectively prevented diabetes in LCMV-infected RIP-GP mice, five to seven treatments per week for a total of 2 weeks were required using aB7.1 and/or aB7.2 antibodies (100 μg). (B) Temporal susceptibility to therapeutic CD40L blockade. A single-dose treatment (100 μg) of aCD40L blocking antibody prevents virus-induced autoimmune diabetes when administered during a therapeutic window comprising days 3–10 after LCMV infection. (C) Groups of RIP-GP and -NP mice were infected with LCMV and treated with different regimens of aCD40L, aB7.1, aB7.2, or control antibodies (100 μg/injection). Blood glucose values (BGV) were monitored twice weekly and onset of diabetes (defined as 2 consecutive BGV of >250 mg/dl) was recorded. Combined data was from two to three individual experiments per group. d, day; ND, not determined. Immunity 2002 16, 403-415DOI: (10.1016/S1074-7613(02)00290-X)

Figure 2 Reduction of T Cell Infiltration into Islets by Costimulation Blockade Immunohistochemistry was performed on pancreatic sections prepared 14 days after LCMV infection and antibody treatment of RIP-GP mice. Results for CD8+ T cell stains are shown. (A) No antibody treatment, diabetic. (B) aB7.1 treatment daily d0-14 post-LCMV, not diabetic. (C) aB7.2 treatment daily d0-14 post-LCMV, not diabetic. (D) aCD40L treatment d4 post-LCMV, not diabetic. Immunity 2002 16, 403-415DOI: (10.1016/S1074-7613(02)00290-X)

Figure 3 Analysis of Primary and Secondary LCMV-Specific T Cell Responses in the Presence and Absence of Costimulation Blockade (A) Eight days after infection with LCMV and treatment with aCD40L or hamster Ig (d3), primary CTL responses were evaluated on infected (closed symbols) and uninfected (open symbols) target cells. (B) Spleen cells obtained before (left panel), 8 days (middle panels), or 41 days (right panels) after LCMV infection and indicated antibody treatment (day 3) were stimulated for 7 hr with GP33 and subsequently stained for CD8 and intracellular IFNγ and TNFα. Dot blots are gated on CD8+ T cells. (C) Frequencies of LCMV GP61- and NP309-specific CD4+ T cells in spleen 8 days after infection as determined by intracellular cytokine staining (black bars, aCD40L; gray bars, hamster Ig). (D) Functional avidities of specific CD8+ and CD4+ T cells were determined by plotting the frequencies of detectable IFNγ-producing T cells as a function of the peptide concentration used for 5 hr in vitro restimulation. Avidities for GP33-specific CD8+ T cells were 1.98 ± 0.17 × 10−10 M for hamster Ig-treated (open circles) and 2.00 ± 0.29 × 10−10 M for aCD40L-treated (closed circles) animals; avidities for GP61-specific CD4+ T cells were 7.57 ± 2.81 × 10−8 M for hamster Ig- and 8.12 ± 1.26 × 10−8 M for aCD40L-treated animals. (E) Frequencies of epitope-specific memory T cells in spleen and pancreas draining lymph node (PDLN) evaluated 41 days after infection and antibody treatment by intracellular cytokine staining (black bars, aCD40L; gray bars, hamster Ig). (F) Secondary CTL activity determined 4 months after infection and restimulation culture under suboptimal conditions (see Experimental Procedures) demonstrated impaired CTL expansion in all cultures from aCD40L- and aB7-treated mice. Specific killing at E:T ratio of 5:1 is shown. (G) Concomitant determination of pCTL frequencies under optimal culture conditions demonstrated reduced frequencies in aCD40L-treated mice (black bars) as compared to control mice (gray bars) (left bars, p = 0.0004). Similar differences were observed in mice 69 days after treatment with adoptive transfer using cells from infected and aCD40L-treated (black bars) or hamster Ig-treated (gray bars) donors. All data are from representative experiments involving at least three mice per group. Values represent mean ±1 SE. Immunity 2002 16, 403-415DOI: (10.1016/S1074-7613(02)00290-X)

Figure 4 Termination of Diabetes Progression by aCD40L-Conditioned Bitypic NK/DC Regulatory Cells (A) Experimental design: RIP-GP recipients were infected with LCMV to initiate diabetes. In parallel, RIP-GP donors were infected (LCMV, VV, or mock infection) and subsequently treated with a single dose of aCD40L or hamster Ig. Four to six days after infection, single-cell suspensions were prepared from donor spleens and transferred i.v. into prediabetic recipients. In some experiments, donor cells were depleted of or enriched for defined cell subpopulations prior to adoptive transfer. Diabetes onset in recipients was monitored twice per week. (B) Diabetes protection by costimulation blockade can be transferred by immunotherapy. Adoptive transfers of unfractioned spleen cells from LCMV-infected/aCD40L-treated donors prevented diabetes in 8/10 recipients. In contrast, cells from donors that received only aCD40L treatment or LCMV challenge failed to protect prediabetic recipients. Similarly, cells from donors infected with a heterologous virus (VV) and treated with aCD40L had no therapeutic effect. n, number of mice treated with 2–4 × 107 cells per transfer. (C) Protection is mediated by cells expressing CD11c and NK1.1. In vitro depletion of indicated cell populations from LCMV-infected/aCD40L-treated donors demonstrated that protection was conferred by DC (CD11c+) and/or NK cells (NK1.1+) but not T or B lymphocytes. (D) Transfer of 106 CD11c+ cells enriched from spleens of LCMV-infected/aCD40L-treated donors according to standard DC purification protocols prevented diabetes onset. Direct ex vivo fractionating into DX5+ and DX5− CD11c+ cells showed further that protection was mediated by NK/DC “hybrid” cells coexpressing signature NK (DX5) and DC (CD11c) markers (∼2 × 105 cells per transfer). Immunity 2002 16, 403-415DOI: (10.1016/S1074-7613(02)00290-X)

Figure 5 Kinetics of NK/DC Expansion and Activation (A) Spleen cells of uninfected (d0) and LCMV-infected (d5) B6 mice were stained for CD11c, DX5, and NK1.1. Values in histograms indicate percentage of NK1.1+ cells among indicated CD11c/DX5 subsets. (B) Expansion and contraction of NK/DC populations in spleen following LCMV infection. (C and D) NK/DC cells (CD11c+/DX5+) increase CD11c and DX5 expression, while CD11c+/DX5− dendritic cells downregulate CD11c and CD11c−/DX5+ NK cells maintain unchanged levels of DX5 expression. The geometric mean of fluorescence intensity (GMFI) of indicated subsets is depicted as a function of time after infection. Immunity 2002 16, 403-415DOI: (10.1016/S1074-7613(02)00290-X)

Figure 6 Phenotypic and Functional Properties of NK/DC Regulatory Cells (A) Phenotypic analysis of DC and NK cells. Spleen cells from LCMV-infected and aCD40L-treated RIP-GP mice were analyzed for expression of CD11c, DX5, and indicated additional surface markers. Marker expression on live cells in three distinct subsets (CD11c+/DX5+, black bars; CD11c+/DX5−, gray bars; CD11c−/DX5+, white bars) is displayed. Similar results were obtained using hamster Ig-treated RIP-GP mice or B6 mice (data not shown). (B) Differential effector functions of DC-NK subsets. DX5+ and DX5− CD11c+ cells were purified 5 days after LCMV infection and tested for ex vivo NK activity. Killing of uninfected RMAS and YAC cells by DX5+ (black bars) and DX5− (gray bars) CD11c+ cells at an E:T ratio of 100:1 is shown. Cytokine production by indicated DC-NK subsets among unfractioned splenocytes was determined after 5 hr in vitro activation (CD11c+/DX5+, black bars; CD11c+/DX5−, gray bars; CD11c−/DX5+, white bars). (C) APC function of DC and NK/DC cells. Purified DX5+ and DX5− CD11c+ cells (d5 after LCMV) were cultured overnight in the presence or absence of ovalbumin (OVA), and the capacity for antigen processing and presentation was subsequently determined by coculture with the KbOVA257-specific B3Z T cell hybridoma transfected with a NFAT-lacZ reporter construct. Negative and positive controls were OVA-loaded EL4 thymoma cells and OVA257 peptide-pulsed EL4 cells, respectively. Values represent SEM of stimulator:B3Z cocultures at 1:2 ratios assayed in triplicate. Compared to OVA-loaded EL4 stimulators (negative control), B3Z responses were significantly higher in the presence of OVA and DX5− (p < 0.0001) or DX5+ (p = 0.0002) stimulators. Immunity 2002 16, 403-415DOI: (10.1016/S1074-7613(02)00290-X)