|
|
Blood, Vol. 113, Issue 20, 5002-5009, May 14, 2009
Bone marrow deficient in IFN- signaling selectively reverses GVHD-associated immunosuppression and enhances a tumor-specific GVT effect
Blood Capitini et al.
113: 5002
Supplemental materials for: Capitini et al
IL-7 administration
Recombinant human IL-7 (Peprotech, Rocky Hill, NJ) was reconstituted with sterile deionized water and resuspended in buffer containing 5% sucrose and 0.1% human serum albumin in PBS and administered at a dose of 5 µg/day intraperitoneally daily for 28 days beginning on day 14 following BMT. Flow cytometric analysis
Spleens were harvested on day+42 and flow cytometric-based enumeration of lymphocytes was accomplished using a FACSCalibur (Becton Dickinson, San Jose, CA) equipped with CellQuest software version 5.2.1. Briefly, 1 × 106 freshly isolated, erythrocyte-depleted splenocytes were treated with anti-FcγIII/II receptors monoclonal antibody (clone 2.4G2) and then stained at 4°C for 20 minutes with a monoclonal antibody cocktail containing NK1.1-FITC, B220−PE, CD4-PerCP Cy5.5 and CD8–APC (BD Pharmingen, San Jose, CA), then washed in FACS buffer (Phosphate buffered salt solution with 0.2% human serum albumin and 0.1% sodium azide).
Files in this Data Supplement:
- Figure S1. Using an alternative donor strategy, C3H.SW→B6 × C3H.SW T-cell–depleted BMT also decreased vaccine responses to class I and II H-Y dominant antigens, but enhanced class I subdominant antigen responses (JPG, 32.2 KB)
-
DLI doses of 1 × 106 (low), 5 × 106 (moderate) or 10 × 106 (high) cells was given on days +14 and 28. ELISPOT analysis of CD8+ and CD4+ T cells to the H-Y antigens were assessed in thymus-bearing recipients on day +42 post-BMT, 6–7 mice/group, ** = p < 0.05, *** p < 0.01.
- Figure S2. Adjuvant IL-7 administration does not improve HY-vaccine responses after allogeneic BMT (JPG, 22.9 KB)
-
A DLI dose of 1 × 106 cells was given with or without IL-7 to thymectomized BMT recipients on days +14 and 28. IL-7 was given daily on days +14-42. ELISPOT analysis of CD8+ and CD4+ T cells to H-Y antigens were assessed on day +42 post-BMT, 10 mice/group, no significance noted between groups.
- Figure S3. Absence of IFNγ signaling on donor marrow does not abrogate GVHD in the setting of a T-cell–replete graft, and does not act as the dominant mechanism in the presence of wild type marrow (JPG, 33.5 KB)
-
(A) B6 or IFNγR1−∕− bone marrow was administered to lethally irradiated F1 recipients along with 2 × 106 purified splenic T cells, and weights were recorded twice weekly, 5 mice/group. (B) All groups were given T-cell–depleted marrow on day 0, followed by 20 × 106 B6 lymph node cells on days +14 and 28. 50:50 mix represented an equal combination of wild type and IFNγR1−∕− bone marrow used on day 0 to see if the abrogation of GVHD by IFNγR1−∕− bone marrow was a dominant effect, 5–6 mice/group.
|
|
