Increased proportion of T regs in cell cycle.

<p>Groups of 5 FVB/N mice were primed IP with 2×10⁷ 3604 or FVB/N splenocytes. Four weeks later the mice were exposed to 550 cGy TBI and 11 days later spleens were harvested. (A) Representative plots of splenocytes from unirradiated and irradiated mice stained for expression of CD4 or CD8 and Foxp3 and Ki-67. (B) The percentage of Ki-67+ cells among CD4+Foxp3+ and CD4+Foxp3<b>−</b> cell populations, showing naïve and D^d-primed mice with and without irradiation. For each treatment group, a significantly greater proportion of the CD4+Foxp3+ cells express Ki-67 (p<0.001) than do the CD4+Foxp3<b>−</b> cell population.</p

In vivo depletion with anti-CD4 or anti-CD25 ablates RITMS.

<p>One day after (A,B), or 2 wk before (C,D), 550 cGy TBI, groups of 6 D^d primed (2×10⁷ 3604 spleen cells IP 3 wks prior to TBI) or naïve (equal vol. PBS IP) FVB mice were given a single IP injection of 80 µg of anti-CD4 (GK1.5) or anti-CD25 (PC61) or control rat IgG. Mice in the “untreated” group received no irradiation or antibody treatment. All groups were engrafted with 3604 (D^d) tail skin 4 weeks after irradiation. At 10 d after mAb treatment CD25 and CD4 depletion were >90%. Similar results were obtained in two analogous depletion experiments, one of which used the rat IgG2a GL113 as an isotype control for PC61.</p

Rejection of allogeneic D^d skin grafts by naïve and D^d primed FVB (H-2^q) mice.

<p>In vivo depletion of CD4 and/or CD8 T cells. On days −6, −5, −1 and +5, GK1.5 (150 µg) mAb and the 53.6.7 (200 µg) mAb were injected IP into adult thymectomized FVB/N mice.</p>*<p>Significant difference from control.</p

Allograft survival is prolonged in alloantigen primed, but not naïve mice, several weeks following γ-irradiation.

<p>A. Groups of 6 FVB mice were immunized IP with 2×10⁷ 3604 (D^d) splenocytes, and 3 wks later given a 550 cGy dose of total body γ radiation (TBI). Mice were engrafted with 3604 tail skin grafts at either 1 or 4 wks following irradiation. B. Radiation induced T memory suppression (RITMS) develops between 2 and 3 weeks following γ-irradiation of immunized mice. Experiment designed as in A, except that separate groups of mice received allogeneic tail skin grafts at 1 or 2 or 3 or 4 weeks post-irradiation (1 wk vs. 3 wks: p<0.01, 2 wks vs. 3 wks. p<0.05, 1 or 2 wks vs. 4 wks, p<0.01, 1 vs. 2 wks, p<0.05). C. Suppression persists after eventual allograft rejection in RITMS mice. Mice from the experiment described in Fig. 1B were given a second identical ipsilateral allograft after the initial graft had rejected (77 d after the initial engraftment). Time to graft rejection is shown for individual mice. One mouse identified as “C” had 30% of the original allograft remaining. This original graft rejected after 149 days. The experiment was terminated after 100 d of observation of the second allograft.</p

Expression of Foxp3, CD8 and skin-homing chemokine receptor genes in allogeneic skin grafts and lymph nodes.

<p>D^d primed (<b>P</b>) or unprimed (naïve, <b>N</b>) mice were treated with 550 cGy of γ irradiation (<b>I</b>) and subsequently received 3604 tail skin grafts (<b>G</b>) 21 days after irradiation. Allografts and LN were harvested at 16 days post-engraftment, when (data not shown) primed and unirradiated primed mice had rejected their allografts. Fold increase and ratio of fold increase were compared for Foxp3 and CD8 mRNA for skin (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039092#pone-0039092-g006" target="_blank">Figure 6A</a>), and lymph nodes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039092#pone-0039092-g006" target="_blank">Figure 6B</a>), and for chemokine receptor expression in allografted skin (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039092#pone-0039092-g006" target="_blank">Figure 6C</a>) by real-time PCR (n = 4−6/group) relative to control untouched skin (<b>C</b>). Statistical significance between groups is indicated in each graph and as assessed by One Way ANOVA and Tukey’s multiple comparison test.</p

Specificity of RITMS.

<p>Groups of 10 mice were primed I.P. with 2×10⁷ syngeneic (FVB), multiple minor allogeneic (DBA/1), or MHC-D^d allogeneic (3604) spleen cells. γ radiation (550 cGy) was given 5 wks later, and mice were engrafted ipsilaterally with both DBA/1 and D^d disparate skin after an additional 6 wks.</p

ITI treatment regimen which includes rituximab, methotrexate and intravenous immunoglobulin (IVIG).

<p>This short course of ITI regimen (5 weeks) needs to be started together with the first dose of ERT. IVIG is administered on a monthly basis for a period of 5–6 months.</p

Representative Western gel blot showing CRIM negative status of four patients (lanes 3–6).

<p>Lane 1- protein magic marker; lane 8 -CRIM negative control cell line; lane 10 - normal human fibroblast (NHF) control; Lanes 2, 7 and 9 - left empty. 20 ug of skin fibroblast cell protein extract was loaded for each patient lane and 2.5 ug protein was loaded for NHF. Western blot was probed with anti-GAA antibody and ß-Actin was used as a protein loading control.</p

Clinical parameters.

<p>Clinical parameters.</p

An algorithm for the management of cross-reactive immunologic material (CRIM)-negative (CN) infantile Pompe disease patients.

<p>^*Institutional review board (IRB) approved study (NCT01665326; <a href="http://www.clinicaltrials.gov" target="_blank">www.clinicaltrials.gov</a>) for rapid determination of CRIM status and long-term follow-up of response to treatment and ITI in Pompe disease. ^†CN status determination from an established CRIM negative mutation database, which allows prediction of CN status in more than 90% cases <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067052#pone.0067052-Bali1" target="_blank">[15]</a>. ^‡ITI regimen is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067052#pone-0067052-g002" target="_blank">Figure 2</a>. ^§Based on the literature antibody titers sustained at ≥6,400 results in a suboptimal therapeutic response to ERT. For that reason, 6,400 was used a cutoff for further intervention <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067052#pone.0067052-Banugaria1" target="_blank">[9]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067052#pone.0067052-Abbott1" target="_blank">[19]</a>. **Based on the half-life of rituximab, CD19% recovery is typically noted around 5 months. ^††The decision to repeat the same ITI regimen (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067052#pone-0067052-g003" target="_blank">figure 3</a>) or to administer ITI with a plasma-cell-targeting agent <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067052#pone.0067052-Banugaria4" target="_blank">[20]</a> should be based on multiple factors including, but not limited to, patients clinical status, CD19% and Fc_γ receptor polymorphism. ^‡‡ITI regimen with plasma cell targeting agent such as bortezomib has been described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067052#pone.0067052-Banugaria4" target="_blank">[20]</a>.</p