Characterization of Regulatory Dendritic Cells That Mitigate Acute Graft-versus-Host Disease in Older Mice Following Allogeneic Bone Marrow Transplantation

<div><p>Despite improvements in human leukocyte antigen matching and pharmacologic prophylaxis, acute graft-versus-host disease (GVHD) is often a fatal complication following hematopoietic stem cell transplant (HSCT). Older HSCT recipients experience significantly increased morbidity and mortality compared to young recipients. Prophylaxis with syngeneic regulatory dendritic cells (DCreg) in young bone marrow transplanted (BMT) mice has been shown to decrease GVHD-associated mortality. To evaluate this approach in older BMT recipients, young (3–4 months) and older (14–18 months) DCreg were generated using GM-CSF, IL-10, and TGFβ. Analysis of young versus older DCreg following culture revealed no differences in phenotype. The efficacy of DCreg treatment in older BMT mice was evaluated in a BALB/c→C57Bl/6 model of GVHD; on day 2 post-BMT (d +2), mice received syngeneic, age-matched DCreg. Although older DCreg-treated BMT mice showed decreased morbidity and mortality compared to untreated BMT mice (all of which died), there was a small but significant decrease in the survival of older DCreg-treated BMT mice (75% survival) compared to young DCreg-treated BMT mice (90% survival). To investigate differences between dendritic cells (DC) in young and older DCreg-treated BMT mice that may play a role in DCreg function <i>in vivo</i>, DC phenotypes were assessed following DCreg adoptive transfer. Transferred DCreg identified in older DCreg-treated BMT mice at d +3 showed significantly lower expression of PD-L1 and PIR B compared to DCreg from young DCreg-treated BMT mice. In addition, donor DC identified in d +21 DCreg-treated BMT mice displayed increased inhibitory molecule and decreased co-stimulatory molecule expression compared to d +3, suggesting induction of a regulatory phenotype on the donor DC. In conclusion, these data indicate DCreg treatment is effective in the modulation of GVHD in older BMT recipients and provide evidence for inhibitory pathways that DCreg and donor DC may utilize to induce and maintain tolerance to GVHD.</p> </div

DCreg treatment attenuates GVHD in young and older BMT mice.

<p>(A) Young and older survival and morbidity following BMT in B6 mice. Data are ± SEM. N = 3-5 mice/group. (B) Survival and morbidity of young and older BMT mice following DCreg treatment. Data are mean ± SEM. N ≥ 16 mice/group. * = p<0.05, ** = p<0.01, *** = p<0.001.</p

Regulatory dendritic cells express low levels of co-stimulatory molecules, but high levels of anti-inflammatory cytokines.

<p>Young BALB/c and B6 cDC and DCreg were stained for co-stimulatory (A) and inhibitory (B) cell surface molecules directly following culture. Live cells were gated and DC identified by gating on CD11c⁺ cells. (These cells were primarily CL II low.) rIg = rat IgG isotype control. Histograms are representative of N ≥ 4 independent experiments per group. (C) Cytokine concentrations in culture supernatants were measured by ELISA. ND, not detected. Data are mean ± SEM and represent 3 independent experiments.</p

Co-stimulatory and inhibitory molecule expression is altered on donor DC from young and old DCreg-treated BMT mice at d +3 vs. d +21.

<p>Young and older BMT mice were treated with GFP⁺ DCreg. DC populations were gated as described in Figure S4 identify donor DC (H-2K^d+) expression of (A) inhibitory and (B) co-stimulatory molecules. Data are mean ± SEM. N = 4-5 mice/group and represent two independent experiments. * = p<0.05, ** = p<0.01, *** = p<0.001.</p

Dendritic cell surface receptor expression and function in DCreg-treated BMT mice.

<p>Young and older BMT mice were treated with GFP⁺ DCreg. DC populations were gated as described in Figure S4 to distinguish between donor DC (H-2K^d+), recipient DC (GFP- H-2K^b+), and DCreg (GFP⁺ H-2K^b+). Co-stimulatory and inhibitory molecule expression on (A) donor DC, (B) recipient DC, and (C) DCreg on d +3 are compared to expression at the end of culture. Data are mean ± SEM. Young BMT mice were treated with PIR B-/- (D) or PD-L1-/- (E) DCreg and survival monitored for 3 wks. N > 4 mice/group and represent two independent experiments each for A-C, D, and E. * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001.</p

DCreg-treated BMT mice show reduced histologic evidence of GVHD.

<p>Young and older B6 mice were treated as described in Figure 3. Colon sections from (A) untreated B6 mice, (B) BMT mice, and (C) DCreg-treated BMT mice were stained with H & E. Original magnification was 20X (top panels) and 60X (lower panels). Tissues in (B) and (C) were obtained 1 wk post-BMT. Circles, necrosis/apoptotic bodies/cellular debris. Boxes, crypt degeneration/increased interstitial space. Representative images of 4-8 mice/group. (D) Total small intestine and colon histology scores of BMT mice 1 wk post-BMT, and DCreg-treated BMT mice 1 and 2 wk post-BMT were determined by adding individual organ scores as described in Methods. There were no statistical differences between young and older groups that received the same treatment. Data are mean ± SEM. N = 3-4 mice/group. * = p<0.05, ** = p<0.01, *** = p<0.001.</p

Acute GVHD induction in B6 mice.

<p>On d 0, lethally irradiated B6 mice received 5x10⁷ splenocytes and 1.5x10⁷ BM cells (BMT) from young BALB/c mice. 5x10⁶ age-matched DCreg were injected i.v. on d +2 and mice were subsequently euthanized at various time points for analysis.</p

Expression of surface receptors and production of cytokines is comparable between young and older DCreg.

<p>DC were identified as in Figure 1. (A) Mean fluorescence intensity (MFI) of cell surface molecules on young and older B6 DCreg following culture. Data are mean ± SEM. N = 4-5 mice/group. (B) Young and older B6 DCreg culture supernatants were assessed for production of cytokines by ELISA. Data are mean ± SEM and represent 3 independent experiments.</p

Operando Observation of Oxygenated Intermediates during CO Hydrogenation on Rh Single Crystals

The CO hydrogenation reaction over the Rh(111) and (211) surfaces has been investigated operando by X-ray photoelectron spectroscopy at a pressure of 150 mbar. Observations of the resting state of the catalyst give mechanistic insight into the selectivity of Rh for generating ethanol from CO hydrogenation. This study shows that the Rh(111) surface does not dissociate all CO molecules before hydrogenation of the O and C atoms, which allows methoxy and other both oxygenated and hydrogenated species to be visible in the photoelectron spectra

Operando Observation of Oxygenated Intermediates during CO Hydrogenation on Rh Single Crystals

The CO hydrogenation reaction over the Rh(111) and (211) surfaces has been investigated operando by X-ray photoelectron spectroscopy at a pressure of 150 mbar. Observations of the resting state of the catalyst give mechanistic insight into the selectivity of Rh for generating ethanol from CO hydrogenation. This study shows that the Rh(111) surface does not dissociate all CO molecules before hydrogenation of the O and C atoms, which allows methoxy and other both oxygenated and hydrogenated species to be visible in the photoelectron spectra