Blockade of both B7-H4 and CTLA-4 co-signaling pathways enhances mouse islet allograft survival

Figures & data

Figure 1. Prolongation of allograft survival in streptozotocin-induced diabetic recipient C57BL/6 mice treated with CTLA-4.Ig, transplanted with Ad-B7-H4–transduced BALB/c mouse islets, and combined therapy. (A) Blood glucose levels in the recipients with control and CTLA-4.Ig treatment. A significantly longer period of time of euglycemia is observed in recipient C57BL/6 (B6) mice treated with CTLA-4.Ig (red line) compared with mice treated with control (black line) protein. (B) Significantly longer period of time of euglycemia in B6 mice transplanted with Ad-B7-H4–transduced islets (green line), compared with that treated with Ad-LacZ control (gray lines). (C) Blood glucose levels in B6 mice transplanted with Ad-B7-H4–transduced islets plus CTLA-4.Ig treatment (blue line) were plotted. (D) Kaplan-–Meier graft survival curve is derived from blood glucose data. Monotherapy with either CTLA-4.Ig or Ad-B7-H4 resulted in prolongation of islet allograft survival (p = 0.001, CTLA-4 vs. control; p = 0.0001, Ad-B7-H4 vs. Ad-LacZ). Combination of these two treatments resulted in further prolongation compared with either monotherapy (p = 0.03 combined vs. CTLA-4.Ig; p = 0.05 vs. Ad-B7-H4 by the log-rank test), n = 12 in each group. There were four, four, and eight mice in group 2, 4 and 5 received the nephrectomy if they survived more than 90 d after transplantation. Mice in group G2, G4 and G5 survived a significant longer period of time compared with two control groups (on an average of 43.75, 54.67, and 77.75 vs.14.83 and 14.5 d, respectively). (E) Intraperitoneal glucose tolerance test (IPGTT) in C57BL/6 recipients with mono- or combined therapies 30 d post-transplant were compared with wild type control mice without transplants (n = 6). Data are expressed as means ± SD, and there is no significant difference between therapy and control groups.

Figure 2. CTLA-4.Ig treatment resulted in reduced IL-2 receptor CD25 activation on allogeneic CD4⁺ subsets and generated immunological ignorance in the renal lymph node. (A) Single cells harvested from renal lymph nodes of C57BL/6 (B6) mice treated with CTLA-4.Ig, Ad-B7-H4, and combined therapy (compared with controls) at 12 d post-transplant were subjected to FACS analysis. The percentage of CD25⁺CD4⁺ is shown as means, each dot represents one mouse (n = 10 in each group). Monotherapy with CTLA-4.Ig alone significantly reduced the expression of IL-2 receptor CD25 on CD4⁺ subsets (p = 0.001, G1 vs. G2). (B) Single cells harvested from renal lymph nodes of C57BL/6 (B6, H-2b) mice treated with CTLA-4.Ig vs. the control group were co-cultured with γ-irradiated (at 3000 rad) stimulators from donor-specific BALB/c (H-2d) at a responder/stimulator (R/S) ratio of 1:2 for 3 d in a MLR assay. Cell proliferation was calculated as mean counts per minute (cpm) ± SE of triplicate culture wells. The response of lymphocytes from CTLA-4.Ig therapy was significantly lower than those from control mice (p = 0.01 control vs. CTLA-4.Ig). A similar hyporesponsiveness was detected in response to third-party antigen CBA (H-2k) stimulation (p = 0.04). (C) IL-2 production from the above co-culture was examined using ELISPOT. Decreased spots were observed in the CTLA-4.Ig–treated group compared with controls in response to either donor-specific BALB/c or third party CBA antigen (p = 0.002). The results represented three independent experiments. One star (*) indicates p ≤ 0.05. Two stars (**) indicate p < 0.01. Three stars (***) indicate p < 0.001.

Figure 3. CTLA-4.Ig treatment resulted in reduced expression of CD69 on CD19 subsets and donor-specific alloantibody production. Single cells harvested from renal lymph nodes of C57BL/6 (B6) mice treated with CTLA-4.Ig, Ad-B7-H4, and combined therapy (compared with controls) 12 d post-transplant were subjected to FACS analysis and alloantibody detection. (A) Representative density plots are shown for CD69⁺ and CD19⁺ subsets. (B) The percentage of CD69⁺CD19⁺ is plotted as means, each dot representing one mouse (n = 10 in each group). Monotherapy with CTLA-4.Ig alone significantly reduced the expression of CD69 on CD19⁺ subsets (p = 0.002). Combined therapy exhibited a similar decline in CD69⁺CD19⁺ expression (p = 0.002, G5 vs. G1; p = 0.02, vs. G3). Donor-specific alloantibody IgM (C) or IgG (D) was detected using cellular ELISA, as described in Methods and expressed as mean ± SD. A significantly decreased production of IgM was detected in monotherapy with CTLA-4.Ig and in combined therapy (p = 0.001, G1 vs. G2; p = 0.05, G5 vs. G1; p = 0.03 vs. G3). A similar decline in IgG production was also observed. Data represented three independent experiments. One star (*) indicates p ≤ 0.05. Two stars (**) indicate p < 0.01. Three stars (***) indicate p < 0.001.

Figure 4. Immune infiltrates in the allograft are reduced after CTLA-4.Ig treatment. (A) Representative slides of grafts stained for CD4⁺, CD8⁺ or CD19⁺ cells are shown at 200 × magnification from five groups. (B) Quantification of CD4⁺, CD8⁺ and CD19⁺ cells showed low infiltrates in CTLA-4.Ig treated group. At least 10 sections were counted blindly in each group and expressed as number of cells per field. Absolute numbers of individual cell subsets CD4⁺ (C), CD8⁺ (D), CD19⁺ (E) were calculated. (F) Percentage of CD69⁺ cells in the CD8⁺ subset. One star (*) indicates p ≤ 0.05. Two stars (**) indicate p < 0.01. Three stars (***) indicate p < 0.001.

Figure 5. Distinct phenotypes of allograft are observed after CTLA-4.Ig and/or Ad-B7-H4 treatment. (A) Representative density plots of IFN-γ⁺ on CD4⁺. (B) Quantification data of IFN-γ⁺ expression in the CD4⁺ subset. Relative expression of RNA using real-time PCR was detected for IFN-γ, Gzmb, EOMES, and T-bet from islet allografts after CTLA-4.Ig monotherapy (C), Ad-B7-H4 monotherapy (D), and combined therapy with CTLA-4.Ig plus Ad-B7-H4 (E and F) are shown. GAPDH was used as loading control. Each dot represents one mouse. Each group consisted of at least six recipients, and data represent the mean ± SE of three independent experiments. One star (*) indicates p ≤ 0.05. Two stars (**) indicate p < 0.01. Three stars (***) indicate p < 0.001.

Figure 6. Enhanced β-cell function was observed in the allograft of recipients treated with CTLA-4.Ig or Ad-B7-H4. β-cell functionwas assessed by immunohistochemistry and IPGTT 12 d post-transplant. (A) Representative sections were stained for H&E, CD45 and insulin from each treatment group. (B) Isletitis was scored according to staining for H&E, CD45 plus insulin. Sections were randomly selected and blindly scored using six to eight animals per group. Isletitis was graded by Yoon’s method. Grade: 0, normal islets; 1, mononuclear infiltration, largely in the periphery, in less than 25% of the islet; 2, 25 to 50% of islet showing mononuclear infiltration; 3, over 50% of islet showing mononuclear infiltration; and 4, small, retracted islet with few mononuclear cells. (C) IPGTT was performed in 5 experimental groups. Treatment with CTLA-4.Ig and/or Ad-B7-H4 significantly improved β-cell function (p < 0.001 vs. controls by ANOVA).

Figure 7. Allografts from long-term surviving recipients treated with CTLA-4.Ig and/or Ad-B7-H4 exhibit distinct expression patterns. β-cell function, infiltrates and subsets of infiltrates were assessed from long-term surviving mice 90 d post-transplant. (A) Representative sections were stained for H&E, CD45 plus insulin, CD4, CD8, and Foxp3 from each treatment group. (B) Isletitis was scored according to staining for H&E, CD45 plus insulin. Sections were randomly selected and blindly scored using four to eight animals per group. Isletitis was graded by Yoon’s method (see Fig. 6). (C) Expression of CD4, CD8, and Foxp3 was calculated according to the numbers of positive-staining cells per field, and the field was randomly selected and blindly scored. Treatment with CTLA-4.Ig exhibited minimal infiltrates (p < 0.001 vs. Ad-B7-H4 by ANOVA). A high number of Foxp3⁺ cells was found in Ad-B7-H4 and combined groups (p < 0.001 vs. CTLA-4.Ig by ANOVA). n = 6–10 islet allografts per group.

Table 1. Primer pairs for real-time PCR

Gene	Sense	Anti-Sense
EOMES	GGC AAA GCG GAC AAT AAC AT	AGC CTC GGT TGG TAT TTG TG
Gzmb	TCG ACC CTA CAT GGC CTT AC	GAG CAG CAG TCG GCA CAA AG
IFN-γ	ACT GGC AAA AGG ATG GTG AC	TGA GCT CAT TGA ATG CTT GG
T-bet	CCT GGA CCC AAC TGT CAA CT	AAC TGT GTT CCC GAG GTG TC