The dataset from administration of single or combined immunomodulation agents to modulate anti-FVIII antibody responses in FVIII plasmid or protein primed hemophilia A mice

Hemophilia A mice with pre-existing inhibitory antibodies against factor VIII (FVIII) were treated with single agents, AMD3100 and GCS-F, respectively. Inhibitor titers in treated mice and control HemA inhibitors mice were followed over time. Total B cells and plasma cells (PCs) were characterized by flow cytometry. HemA inhibitor mice were then treated with a combination regimen of IL-2/IL-2mAb complexes plus rapamycin and AMD3100. Finally, HemA inhibitor mice were treated with a new combination therapy using include IL-2/IL-2mAb complexes + Anti-CD20+AMD3100+G-CSF. The timeline of combination therapy was illustrated. Inhibitor titers following treatment in FVIII plasmid or protein induced inhibitor mice were evaluated overtime. A representative figure and gating strategies to characterize the subsets of Treg cells and B cells are presented. Please see http://dx.doi.org/10.1016/j.cellimm.2016.01.005 [1] for interpretation and discussion of these data and results.


Specification
Animals were immunized to generate inhibitory antibodies

Experimental features
Groups of animals were treated with plasmid mediated gene therapy or protein replacement therapy followed by immunomodulation Data source location

Seattle, USA
Data accessibility Data is with this article

Value of the data
This is the first attempt to use stem cell mobilizing agents to modulate pre-existing anti-FVIII antibodies.
Using combination therapy with Treg expansion agents IL-2/IL-2mAb complexes and rapamycin illustrates the importance of Treg cells not only in prevention of anti-FVIII antibody production but also in modulating pre-existing antibody responses.
Detailed analysis of changes in subsets of T and B cells can help elucidate the mechanisms of these immunomodulation agents with single or combination treatment.

Data
Hemophilia A (HemA) mice were treated with either FVIII plasmid or protein to induce production of pre-existing inhibitory antibodies. These HemA inhibitor mice were then treated with either single agent or combination treatment. Immune responses against FVIII and FVIII activities were followed over time. Subsets of B and T cells were analyzed using flow cytometry. First, HemA inhibitor mice were treated with a single cycle (200 μg/mouse/day for 10 days) of AMD3100. HemA inhibitor mice were used as controls. Antibody titers in treated and control mice were shown in Fig. 1A and B. Staining results of total B and CXCR4 þ plasma cells (PCs) were shown in Fig. 1C and D. Next, we explored the single cycle treatment of G-CSF (250 μg/kg/day for 5-days) in HemA inhibitor mice. Antibody titers and staining results of total B and PCs are shown in Fig. 2. Subsequently, we treated HemA mice with a combination regimen (IL-2/IL-2mAb complexesþrapamycinþAMD3100) for 4 weeks. Antibody titers in treated and control mice were shown in Fig. 3A and B. Peripheral blood mononuclear cells (PBMCs) in HemA mice were collected on weeks 2 and 3 following combination treatment and staining of CD4 þ CD25 þ Foxp3 þ and CD4 þ Foxp3 þ Helios þ Tregs, and Treg activation markers were carried out and analyzed by flow Cytometry (Fig. 4). In addition, total B cell (%; Fig. 5A), PCs (%; Fig. 5B) Transitional B cells (%; Fig. 5C) and CXCR4 þ PCs (%; Fig. 5D) were investigated using flow cytometry analysis. Furthermore, a new combination treatment was administered into HemA inhibitor mice. Fig. 6 demonstrates the combination treatment timeline for one cycle per every two weeks. The combination treatment include IL-2/IL-2mAb complexes þ Anti-CD20þAMD3100þ G-CSF. Inhibitor titers in FVIII plasmid primed HemA mice with pre-existing inhibitors following combination treatment and control HemA inhibitor mice over 16 weeks were shown in Fig. 7. A representative figure and gating strategies  for characterizing CD4 þ CD25 þ Foxp3 þ , CD4 þ Foxp3 þ Helios þ Tregs, and B cell populations (total B, transitional B, PCs and CXCR4 þ PCs) in peripheral blood in treated HemA inhibitor mice was shown (Fig. 8). Inhibitor titers in FVIII protein primed HemA mice with pre-existing inhibitors following Fig. 3. Inhibitor titers were reduced during the treatment with IL-2/IL-2mAb complexesþ rapamycin þ AMD3100 in FVIIIplasmid treated HemA mice with pre-existing inhibitors. FVIII primed inhibitor mice (n¼ 8) were treated with IL-2/IL-2mAb complexesþ rapamycinþ AMD3100 for 4 weeks. (A) The inhibitor titers were reduced during the treatment period and sustained for 2 more weeks, however the titers returned to higher levels afterwards compared to persistently high levels of inhibitor titers in the inhibitor only mice (n¼ 4) (B). Data shown is representative of two independent experiments. Fig. 4. Effects of Immunomodulation on CD4 þ CD25 þ Foxp3 þ and CD4 þ Foxp3 þ Helios þ Tregs, and Treg activation markers in peripheral blood in treated HemA inhibitor mice. PBMC were collected on weeks 2 and 3 after combination treatment (IL-2/IL-2mAb complexes þrapamycin þAMD3100) in HemA inhibitor mice (n ¼ 8), the percentage (%) of gated cells of CD4 þ CD25 þ Foxp3 þ Tregs (A) and Helios þ Tregs (B) were investigated in each mouse group. MFI levels of Treg activation markers CD25 þ (C), GITR þ (D) and CTLA4 þ (E) were also investigated at different time points after treatment. Data shown is representative of two independent experiments. combination treatment over 18 weeks are shown in Fig. 9. The summary and interpretation of the data and results were described in Ref [1].

Mice
All mice were maintained at a specific pathogen-free (SPF) facility according to the National Institutes of Health guidelines for animal care and the guidelines of Seattle Children's Research   6. The combination treatment timeline for one cycle per two weeks. The combined regimens were given as shown in the timeline plotted. One cycle treatment includes two weeks long (14-days) period. For each cycle, IL-2/IL-2mAb complexes were given on Mon, Tue and Wed every week, G-CSF was administered everyday for 6-days in the first week, Anti-CD20 was given 3-days on Monday, Tuesday and Wednesday in the first week, and AMD3100 was administered everyday for 10-days within the 14-days treatment period.
Institute. HemA mice in a 129/SV Â C57BL/6 mixed genetic background were used at the age of 6-8 weeks.

Administration of single or combined immunomodulating agents into mice
Immunomodulation agents with indicated dosages were administered into mice according to schedules specified in experiments. Blood samples were taken from the retro-orbital plexus at serial time points.

Flow cytometry and antibodies
Cell suspensions of peripheral blood and spleens of each treated mouse group were prepared according to standard protocols. Cell suspensions were stained for FACS analysis using appropriate antibodies. Samples were analyzed on an LSRII flow cytometer (Becton Dickinson, Palo Alto, CA) and data were analyzed using FlowJo software (Tree Star, Ashland, OR).

FVIII activities and inhibitor titers assays
Peripheral blood samples were taken from the experimental mice and collected in a 3.8% sodium citrate solution. FVIII activities were evaluated from the activated partial thromboplastin time (APTT) by a modified clotting assay using FVIII deficient plasma and reagents. FVIII activities were calculated from a standard curve generated with serially diluted normal human pooled plasma. Anti-FVIII antibody titers were measured by Bethesda assay.

Acknowledgment
This work was supported in part by a R01 grant (R01 HL69049) from NIH-NHLBI and by a special project grant from Bayer Hemophilia Foundation to CHM. We also thank Bayer (Whippany, NJ) for providing recombinant FVIII (Kogenate s ) and Dr. Chérie Butt at Biogen Idec (Weston, MA) for providing anti-mouse CD20 for our experiments. Fig. 9. Inhibitor titers in FVIII protein primed HemA mice with pre-existing inhibitors following combination treatment. Two groups of HemA mice were treated separately with different combination regimens 2-weeks per cycle for 3 cycles: (A) IL-2/IL-2mAb complexesþ anti-CD20 þ AMD3100 þG-CSF (n¼ 4), (B) Anti-CD20 þ AMD3100þG-CSF (n¼ 5), and (C) Inhibitor mice only (n¼5) as the control group. Peripheral blood was collected at different time points following the combination treatment. Anti-FVIII antibody titers were assessed by Bethesda assay over time. Each symbol represents data obtained from an individual mouse. Data shown is representative of two independent experiments.