Memory T Cells Mediate Cardiac Allograft Vasculopathy and are Inactivated by Anti-OX40L Monoclonal Antibody

Abstract

Purpose: Cardiac allograft vasculopathy (CAV) is a major complication limiting the long-term survival of cardiac transplants. The role of memory T cells (Tmem) in the pathogenesis of CAV remains elusive. This study investigated the role of Tmem cells in the development of CAV and the therapeutic potential of targeting the OX40/OX40L pathway for heart transplant survival.

Methods: Tmem cells were generated in Rag-1(-/-) C57BL/6 (B6) mice by homeostatic proliferation (HP) of CD40L null CD3(+) T cells from B6 mice. Rag-1(-/-) B6 mice (H-2(b)) harboring Tmem cells received cardiac allografts from BALB/c mice (H-2(d)), and were either untreated or treated with anti-OX40L monoclonal antibody (mAb) (0.5 mg/mouse/day) for 10 days.

Results: Six weeks after HP, the majority of transferred CD40L(-/-) T cells in Rag-1(-/-) B6 mice were differentiated to CD44(high) and CD62L(low) Tmem cells. BALB/c heart allografts in Rag-1(-/-) B6 recipient mice in the presence of these Tmem cells developed a typical pathological feature of CAV; intimal thickening, 100 days after transplantation. However, functionally blocking the OX40/OX40L pathway with anti-OX40L mAb significantly prevented CAV development and reduced the Tmem cell population in recipient mice. Anti-OX40L mAb therapy also significantly decreased cellular infiltration and cytokine (IFN-γ, TNF-α and TGF-β) expression in heart allografts.

Conclusions: Tmem cells mediate CAV in heart transplants. Functionally blocking the OX40/OX40L pathway using anti-OX40L mAb therapy prevents Tmem cell-mediated CAV, suggesting therapeutic potential for disrupting OX40-OX40L signaling in order to prevent CAV in heart transplant patients.

Fig. 1

Phenotypic analysis of CD40L^−/− B6 mouse naive and T_mem cells. Naive CD3⁺ T cells from CD40L^−/− B6 mice were adoptively transferred into Rag-1 deficient B6 mice, and allowed to undergo homeostatic proliferation for 6 weeks. The phenotypes of both cell types were determined using a flow cytometry with flurochrome-conjugate antibody staining with anti-CD44-FITC and anti-CD62L-PE. Data are a representative of three mice

Fig. 2

CAV development in cardiac allografts in Rag-1 deficient recipients. BALB/c mouse heart allografts in Rag-1^−/− B6 mouse recipients were harvested on POD 100. CAV was evaluated in H&E stained paraffin sections. Data are presented as a typical image of vascular lesion in the arteries. a Heart grafts from naïve Rag-1^−/− recipients—lack of T_mem cells (Group 1, n =3). b Heart grafts from Rag-1^−/− recipients harboring T_mem cells (Group 2, n =8), showing severe CAV. c Heart grafts from Rag-1^−/− recipients harboring T_mem cells and receiving anti-OX40L mAb treatment (Group 3, n =8)

Fig. 3

Regulation of CD4⁺OX40⁺CD44^hi and CD8⁺OX40⁺CD44^hi T cells by anti-OX40L mAb treatment in allograft recipients. The splenocytes were harvested from the recipient mice on POD 100. a Representative dot plots of OX40 and CD44 staining of CD4⁺ cells. b Frequency of CD4⁺OX40⁺CD44^hi and CD8⁺OX40⁺CD44 ^hi T cells were analyzed by flow cytometric analysis, and expressed graphically in terms of mean percentages (±SEM) that were statistically compared using Student's t-test test (*P <0.001, vs. untreated, n =8)

Fig. 4

Regulation of cellular infiltration (CD4 and CD8 Tcells, and macrophages) in heart allogarfts by anti-OX40L mAb treatment. Cardiac allografts in Rag-1^−/− B6 recipients were harvested on POD 100. The CD4, CD8 and Mac-1 expressing cells in the sections of the grafts were localized by immunohistochemistry. Data are presented as a representative immunoperoxidase staining for each of CD4⁺, CD8⁺, and Mac-1⁺ cells. a, c and e A typical microscopic view of CD4⁺ T cells, CD8⁺ T cells, or Mac-1⁺/macrophages in the sections of the grafts from Rag-1^−/− recipients harboring T_mem cells (Group 2, untreated, n =8). The red arrows indicated positively stained (brown) cells. b, d and f A typical microscopic view of CD4, CD8, or Mac-1 staining in the sections of the grafts from Rag-1^−/− recipients harboring T_mem cells and receiving anti-OX40L mAb treatment (Group 3, n =8). Cytoplasm: blue. Nucleus: dark blue

Fig. 5

Attenuation of cytokine expression in cardiac allografts by anti-OX40L mAb treatment. Cardiac allografts in Rag-1^−/− B6 recipients were harvested on POD 100. Five tissues from each group were randomly selected for total RNA extraction. The intragraft expression of TNF-α, IFN-γ, TGF-β in the grafts in untreated versus anti-OX40L mAb-treated groups was analyzed by real-time RT-PCR. Data are presented as mean percentages (±SEM) that were statistically compared using Student's t-test test (*P <0.05 vs. untreated controls, n =5)