Spatial differences in the presence of FOXP3+ and GranzymeB+ T cells between the intra- and extravascular compartments in renal allograft vasculopathy

Abstract

Background: Allograft vasculopathy (AV) and native atherosclerosis (NA) share the presence of a T-cell mediated inflammatory response, but differ in overall plaque morphology and growth rate. We studied the distribution and frequency of regulatory- and cytotoxic T cells in the arterial intima lesions in both conditions.

Methodology/principal findings: The study is based on vessels of 15 explanted human renal allografts with AV and 10 carotid artery plaques obtained at surgery. Distribution and frequency of cytotoxic- and regulatory T cells, as identified by the expression of Granzyme B (GrB) and FOXP3 was established in NA and AV. Furthermore, we compared the distribution of these cells in AV with the perivascular, interstitial renal tissue using immunohistochemistry. The total number of T cells was much higher in AV than in NA lesions (711±135 and 37±8 CD3/mm(2) respectively, p<0.005, mean, ± SEM). Total numbers of FOXP3(+) regulatory cells were also significantly increased in AV (36±10 and 0.9±0.3 FOXP3(+)/mm(2) p<0.05), but relative numbers, expressed as a percentage of the total number of CD3(+) T cells ((FOXP3(+)/CD3(+)) ×100), were not significantly different (4.6%±0.9 and 2.7%±0.6). GrB(+) cells were rare in NA, but significantly increased numbers of GrB(+) cells were found in AV lesions (85±24 and 0.2±0.1 GrB(+)/mm(2), p<0.05). Perivascular tissues in the allografts showed a higher relative frequency of FOXP3(+) cells than adjacent intimal lesions (14.0%±2.7 and 4.6%±0.9, respectively, p<0.05), but a lower frequency of GrB(+) cytotoxic T cells (16.1%±2.7 and 22.6%±3.6, p<0.05).

Conclusions: Similar to NA, AV is characterized by a low frequency of intimal FOXP3(+) regulatory T cells. Moreover, significant spatial differences exist in the distribution of functional T cell subsets between the intra- and extravascular micro-environments of the graft.

Figure 1. Histological examples of regulatory- and cytotoxic T cells in a nephrectomy specimen with allograft vasculopathy.

All pictures are from the same specimen. A: H&E, overview. Bar = 100 µm. B: Same area as in A, doublestained with FOXP3 (red) and CD3 (blue). Bar = 100 µm. C: Detail from B, extra vascular area. Bar = 25 µm. Note the nuclear staining of FOXP3. D. Detail of B, intravascular area (intima). Bar = 25 µm. E: extra-vascular area (same region as in C), CD8 (blue)/Granzyme B(red) double staining. Bar = 25 µm. F: intima (same region as D), CD8 (blue)/Granzyme B(red) double staining,. Bar = 25 µm. Inset shows high power magnification, showing intracellular GranzymeB⁺ granules (red).

Figure 2. Absolute and relative numbers of CD3⁺ T cells (A), CD8⁺ T cells (B), FOXP3⁺ T cells (C and E) and Granzyme B⁺ T cells (D and F) in the intima of native atherosclerotic plaques (NA) and the intima of vessels with allograft vasculopathy (AV).

*: P<0.05, ** P<0.005.

Figure 3. A: Immunohistochemical doublestaining of a renal allograft specimen with Granzyme B (red) and CD57 (blue), illustrating a Granzyme B positive NK cell.

Bar = 10 µm. B: Doublestaining of Granzyme B (red) with CD68 (blue). Note that macrophages do not express Granzyme B. Bar = 25 µm.

Figure 4. Differences between intra- and extravascular functional T cells subsets in renal allograft vasculopathy.

A: numbers of FOXP3⁺ T cells, expressed as a percentage of the total number of CD3⁺ T cells. B: numbers of GrB⁺ T cells, expressed as a percentage of the total number of CD8⁺ cells. *: p<0.05, paired T test.