C4d deposition is associated with immune cells infiltrating in kidney allograft glomerulitis and peritubular capillaritis

Figures & data

Table 1. Demographic date and clinical characteristics of patients.

Group	C4d^+ (n = 26)	C4d^− (n = 25)
Age (year ± SD)	38.9 ± 10.4	42.7 ± 8.7
Gender (F/M)	(14/12)	(15/10)
Primary renal disease
Chronic glomerulonephritis	21 (80.7%)	22 (88.0%)
Hypertension nephropathy	0	0
Gout nephropathy	1(3.8%)	1 (4.0%)
SLE	4 (15.4%)	2 (8.0%)
Cold ischemia time (h)	7.8 ± 3.3	8.1 ± 2.3
Warm ischemia time (min)	6.3 ± 1.4	6.0 ± 1.6
HLA mismatches	3.7 + 1.4	3.9 + 0.7
CDC (%)	3.1 + 1.1	3.2 + 1.5
PRA > 10% pretransplantation	4	1
Time post-transplantation (day)	12.0 + 8.8	9.8 + 7.6
Creatinine concentration on  biopsy time (μmol/L)	465 + 165	382 + 218
Immunosuppression protocol
Pred + CsA + Aza	0	0
Pred + CsA + MMF	7	9
Pred + Tac + MMF	17	15
Pred + Rapa + MMF	2	1
Banff 05 classification
IA	15	14
IB	2	1
IIA	9	10

Notes: All p > 0.05, C4d⁺ versus C4d⁻; CDC, complement-dependent-cytotoxic; PRA, panel reactive antibody.

Figure 1. Example of the glomerulitis and immunostaining for two markers (×400, DAB stain × 400): (A) glomerulitis with HE stain (arrow); (B) CD3-positive controls; (C) CD3-negative controls; (D) CD3-positive T lymphocytes in the glomerulus (arrow); (E) glomerulitis with PAS stain (arrow); (F) CD68-positive controls. (G) CD68-negative controls; (H) CD68-positive T lymphocytes in the glomerulus (arrow).

Figure 2. Example of the two PTCitis scores immunostaining for two markers (PAS stain × 1000, DAB stain × 1000). (A) Capillaritis with a PTCitis-score 1, i.e., max 3–4 luminal inflammatory cells in PTC (arrow). (B) Capillaritis with a PTCitis-score 2, i.e., max 5–10 luminal inflammatory cells in PTC (arrow). (C) CD3-positive T lymphocytes in the PTC (arrow). (D) CD68-positive macrophages in the PTC (arrow).

Figure 3. Immunostaining for C4d (DAB stain × 1000). (A) Negative C4d stain in PTC (arrow). (B) Minimal C4d stain/positive in PTC (arrow), i.e., C4d deposition in < 10% PTCs. (C) Focal C4d stain/positive in PTC (arrow), i.e., C4d deposition in 10–50% PTCs. (D) Diffuse C4d stain/positive in PTC (arrow), i.e., C4d deposition in > 50% PTCs.

Table 2. Quantitative analysis of biopsy findings for C4d⁺ and C4d⁻ groups.

Variable	C4d^+	C4d^−	p-Value
Mean no. of macrophages+T cells/glomerulus	17.79 ± 7.70	8.17 ± 3.80	<0.0001
Mean no. of macrophages/glomerulus	13.73 ± 7.03	2.57 ± 1.22	<0.0001
Mean no. of T cells/glomerulus	4.05 ± 2.60	5.60 ± 2.81	0.023
Mean no. of macrophages+T cells/PTC	5.64 ± 2.06	3.68 ± 1.52	<0.0001
Mean no. of macrophages/PTC	4.36 ± 1.85	2.26 ± 1.64	0.001
Mean no. of T cells/PTC	1.29 ± 0.52	2.01 ± 1.02	0.031
Mean no. of granzyme B-positive T cells/glomerulus	3.37 ± 2.34	4.27 ± 2.41	0.141
Mean no. of granzyme B-positive T cells/PTC	1.01 ± 0.58	1.98 ± 0.96	0.231
Mean no. of Foxp3-positive T cells/glomerulus	0	0
Mean no. of Foxp3-positive T cells/PTC	0	0

Figure 4. Immunostaining for granzyme-B (DAB stain × 1000). (A) Granzyme B-positive controls (arrow). (B) Granzyme B-negative controls (arrow). (C) Granzyme B-positive T lymphocytes in the glomerulus (arrow). (D) Granzyme B-positive T lymphocytes in the PTC (arrow).

Figure 5. Immunostaining for Foxp3 (DAB stain × 1000). (A) Foxp3-positive controls. (B) Foxp3-negative controls (arrow). (C) Foxp3-negative T lymphocytes in the glomerulus (arrow). (D) Foxp3-negative T lymphocytes in the PTC (arrow).

Table 3. Quantitative analysis of biopsy findings for the diffuse C4d and focal C4d groups.

Variable	Diffuse C4d^+	Focal C4d^−	p-Value
Mean no. of macrophages+T cells/glomerulus	23.25 ± 4.71	14.49 ± 6.86	0.001
Mean no. of macrophages/glomerulus	19.62 ± 4.97	9.11 ± 4.48	<0.0001
Mean no. of T cells/glomerulus	3.64 ± 1.50	4.38 ± 3.24	0.74
Mean no. of macrophages+T cells/PTC	7.53 ± 2.38	4.32 ± 1.89	<0.0001
Mean no. of Macrophages/PTC	5.89 ± 2.03	2.67 ± 1.73	0.001
Mean no. of T cells/PTC	2.97 ± 0.43	2.36 ± 0.98	0.53
Mean no. of granzyme B-positive T cells/glomerulus	2.99 ± 1.36	4.57 ± 2.21	0.215
Mean no. of granzyme B-positive T cells/PTC	1.31 ± 0.45	2.04 ± 0.76	0.182
Mean no. of Foxp3-positive T cells/glomerulus	0	0
Mean no. of Foxp3-positive T cells/PTC	0	0