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Belatacept and Tregs: friends or foes? “...several lines of evidence suggest that belatacept’s targets intersect with pathways relevant to Treg generation and function. It is unclear if and how this interferes with belatacept’s effects on effector T cells...” KEYWORDS: belatacept n costimulation blockade n CTLA4Ig n organ transplantation n tolerance n Tregs
Tregs are not only a critical mechanism of self-tolerance but also an indispensable factor regulating the alloimmune responses in organ transplantation. Although Foxp3neg Tregs also exhibit potent immunoregulatory properties, we will mainly focus on the well-characterized CD4 + CD25 +FoxP3 + subset of natural Tregs and its relationship with belatacept in this article. The effects of conventional immunosuppressants on Tregs have been fairly well characterized. Calcineurin inhibitors (CNIs; cyclosporin A and tacrolimus) negatively influence Tregs by blocking IL‑2 production, which is critical for Treg development and survival [1] . Anti-IL2R
mAbs (e.g., basiliximab and daclizumab) also lead to a decrease in Treg numbers [2] ; however, recent studies suggest that basiliximab has no major effect on Treg function [3] . Alemtuzumab (anti-CD52 mAb) and rATG (rabbit antithymocyte globulin) were suggested to favor Treg conversion and expansion [4] , although the positive effect of rATG on Tregs is controversial [5] . Mycophenolate mofetil seems to have little or no direct effect on Tregs [6] . mTOR inhibitors (sirolimus and everolimus) inhibit effector T cell responses by blocking IL2 receptor signaling. Interestingly, they suppress effector T cell proliferation but spare Treg expansion [7] . Studies investigating the underlying mechanisms of this selective effect suggest that mTOR inhibition stabilizes FoxP3 expression and favors Treg conversion by inhibition of the PI3K–Akt pathway [8] . Belatacept was approved for use in renal transplantation as the first of a new class of rationally designed immunosuppressive agents known as costimulation blockers [9] . Belatacept is derived from its parent compound abatacept, which consists of the extracellular portion of human CTLA‑4 fused to the mutated Fc portion of human IgG1 (CTLA4Ig, marketed as abatacept). CTLA‑4 (whose physiological function was unknown at the time) shares the same ligands with CD28 but binds them with substantially higher affinity and avidity. The ligand-binding site of abatacept was subsequently mutated to achieve further increased binding avidity (belatacept, formerly known as LEA29Y) [10] . Phase II and III studies demonstrate that belatacept-based immunosuppression (also including mycophenolate mofetil, steroids and basiliximab) is an effective alternative to CNIs [9,11–13] . Graft function was significantly better maintained with belatacept than with
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Pharmacologic immunosuppression was originally thought of as the global inhibition of effector T-cell responses. With the emerging understanding of the role of Tregs in the acceptance of allografts, this concept had to be revisited and the potential effects of immunosuppressants on regulatory mechanisms to be considered. The costimulation blocker belatacept (Nulojix®, Bristol-Myers Squibb; an antiB7 compound designed to block CD28) has recently been approved and provides an alternative to calcineurin inhibitors in renal transplant recipients. The now-recognized complexities of the CD28–CTLA‑4–B7 costimulation pathway urge the question as to whether belatacept affects not only effector T cells but also Tregs. Here, we provide a personal perspective on the current insight of possible interactions of belatacept and Tregs and their potential clinical relevance.
“The now-recognized complexities of the CD28–CTLA‑4–B7 costimulation pathway urge the question as to whether belatacept affects not only effector T cells but also Tregs.”
Nina Pilat Division of Transplantation, Department of Surgery, Vienna General Hospital, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna, Austria
Thomas Wekerle Author for correspondence: Division of Transplantation, Department of Surgery, Vienna General Hospital, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna, Austria Tel.: +43 1 40400 5621 Fax: +43 1 40400 6872
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CNIs, while the frequency of cellular rejection was increased – surprisingly more so with higher doses of belatacept (different dosing regimens were tested in these trials). Since the initial development of CTLA4Ig two decades ago, the CD28/B7 costimulation pathway has been recognized to be considerably more complex than initially envisioned. CTLA4Ig/ belatacept was designed to block CD28 – a costimulation receptor critically required for full activation of naive T cells – through saturation of the CD28 ligands B7‑1 and B7‑2. The bulk of evidence still suggests that CD28 inhibition is the main mode of action of belatacept. However, CTLA4Ig/belatacept are likely to have additional mechanisms of action, some of which might impact Treg generation and function.
“...the CD28/B7 costimulation pathway has been recognized to be considerably more complex than initially envisioned.”
Five studies have so far looked at Tregs in transplant recipients treated with belatacept [2,14–17] . In general, interpretation of the results derived from these investigations is complicated by the concomitant use of either basiliximab or thymoglobulin induction therapy and the low number of patients investigated. Flow cytometric analysis of FoxP3 expression in peripheral blood indicates that belatacept did not induce Treg expansion in vivo [14,17] . Interestingly, while some studies report no negative long-term effect on circulating Tregs [2,14] , others show a trend towards lower percentages of CD25 bright cells among CD4 + subsets [16] under belatacept therapy. Detailed analysis revealed that due to induction therapy with basiliximab (anti-IL2R mAb) the short-term effects of belatacept could not be directly evaluated, as basiliximab treatment appeared to cause transient depletion of circulating CD4+CD25+FoxP3+ Tregs (and CD4+CD25+ effector T cells) during the first 3 months [2] . However, CD25 – Tregs were not affected by treatment, resulting in the selective increase in Tregs compared with CD4 effector cells. Preliminary analysis of graft biopsies shows that the intragraft numbers of FoxP3 + Tregs among infiltrating CD3 + cells were significantly increased during acute rejection episodes in patients treated with belatacept compared with CNI-treated patients [2,17] . Although small patient numbers are insufficient for evaluating the impact of increased intragraft FoxP3 cells on overall outcome, other studies suggest that increased FoxP3 expression favors outcome and 352
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recovery from rejection episodes [18] . These data suggest that belatacept treatment might enhance Treg migration into the allograft, leading to better resolution of rejection episodes. Interestingly, intragraft levels of FoxP3 mRNA were significantly lower under belatacept in protocol biopsies mostly devoid of rejection [16] . Remarkably, the combination of belatacept with sirolimus and induction with thymoglobulin was shown to result in a significantly higher frequency of Tregs among the memory T-cell compartment, suggesting the promotion of immunoregulatory mechanisms among these patients [15] . Although the results need to be considered preliminary due to the small number of patients evaluated, they clearly show that various belatacept regimens differ in their impact on Tregs and suggest that a belatacept-based combination of immunosuppressive drugs might be identified for renal transplantation that is beneficial to Tregs. Together these studies – albeit valuable – show a mixed and incomplete picture of the relationship of belatacept with Tregs. They also emphasize that looking at different compartments – intragraft versus peripheral blood – might yield different results. Likewise, different time points (time of rejection and protocol biopsies vs immediately post-transplant), markers (e.g., CD25 vs FoxP3) and methods (quantitative-PCR vs immunohistological analysis) might influence results considerably and inhibit interpretation. Nevertheless, the bulk of data argue that Tregs can be influenced by belatacept even though the details remain unclear.
“...the bulk of data argue that Tregs can be influenced by belatacept even though the details remain unclear.”
Beyond this limited direct clinical evidence, several theoretical considerations argue for an effect of belatacept on Treg function [19] . Importantly, CTLA‑4 (CD152) – the receptor sharing the same ligands with CD28 – has since been recognized to provide a major negative co-signal, critically regulating self-tolerance [20] and attenuating alloresponses in the experimental setting [21] . Notably, CTLA4 is constitutively expressed on Tregs and is required for their suppressive function [22] . It remains controversial, however, whether the inhibitory function of CTLA‑4 is due to cell-intrinsic (i.e., an inhibitory signal provided through the receptor) or cell-extrinsic effects (i.e., an inhibitory effect induced in a ligand-bearing target cell), or both [23] . Belatacept, by binding with increased future science group
Belatacept & Tregs: friends or foes?
affinity to B7.1 and B7.2, would be expected to prevent CTLA4 ligation and thus to block an inhibitory cell-intrinsic CTLA4 signal. Cellextrinsic mechanisms, such as the removal of B7 molecules from APC through trans-endocytosis by CTLA4-expressing Tregs [24] , might probably be affected to a lesser extent. Even though transendocytosis of B7 molecules would be inhibited through belatacept binding, these B7 molecules would not bind CD28 anyway (they are prevented from CD28 ligation as they have bound belatacept). Thus, no inhibitory mechanism would be prevented by belatacept. It thus remains currently unknown whether belatacept blocks the physiological inhibitory effects of CTLA4. CD28 signals participate in intrathymic Treg development with genetic ablation of CD28 leading to a decline of natural Tregs leaving the thymus [25,26] . By contrast, there is no evidence on the direct impairment of Treg generation by CTLA4 deficiency [22] . CD28 also contributes to the homeostasis of Tregs, potentially through CD28-dependent IL2 production [27] . This mechanism might explain the transient decline in Treg frequency observed in mice treated with CTLA4Ig [28,29] . At present, it is unclear whether belatacept monotherapy would have a similar effect in humans. Moreover, PD‑L1 is involved in the generation of adaptive Tregs [30,31] , probably through interaction with PD‑1 [32] . As PD‑L1 has recently been revealed also to bind to B7‑1 (in addition to PD‑1) [33,34] , B7‑1 saturation by belatacept might conceivably indirectly interfere with this pathway of adaptive Treg generation. However, the presence of CTLA4Ig during in vitro generation of murine adaptive Tregs by TGF‑b had no detectable effect on Treg numbers or suppressor function [Wekerle T, Pilat N, Unpublished Data] . Indeed, one paper reported that CTLA4Ig induced regulatory cells in swine mixed lymphocyte cultures [35] .
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In vivo, CTLA4Ig extends experimental allograft survival, with anergy, clonal deletion and regulation having all been proposed as mechanisms [36] . Interestingly, delayed CTLA4Ig treatment seems to be more effective in such models, perhaps as it allows upregulation and engagement of CTLA4 before both CD28 and CTLA4 signaling are blocked. In murine heart transplants, CTLA4Ig acts synergistically with donor-specific transfusion, leading to Tregdependent graft prolongation [37] . Likewise, regulation is a critical mechanism in CTLA4Ig-based mixed chimerism models [29] and Treg therapy is effective in CTLA4Ig-treated bone marrow transplant recipients [38,39] . Thus, Tregs are an important mechanism in several experimental models employing CTLA4Ig. However, the clinical relevance of these specific rodent models with regard to clinical renal transplantation remains to be determined. In summary, several lines of evidence suggest that belatacept’s targets intersect with pathways relevant to Treg generation and function. It is unclear if and how this interferes with belatacept’s effects on effector T cells in the clinical setting. The clarification of this relationship deserves further investigation as it would facilitate the definition of the most effective timing and dosing regimen of belatacept, an indispensable step for optimizing its therapeutic efficacy. Financial & competing interests disclosure T Wekerle received research support, travel grants and consultant honoraria from Bristol-Myers Squibb. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript. Feng X, Kajigaya S, Solomou EE et al. Rabbit ATG but not horse ATG promotes expansion of functional CD4 + CD25 high FOXP3 + regulatory T cells in vitro. Blood 111(7), 3675–3683 (2008). Broady R, Yu J, Levings MK. ATG-induced expression of FOXP3 in human CD4(+) T cells in vitro is associated with T‑cell activation and not the induction of FOXP3(+) T regulatory cells. Blood 114(24), 5003–5006 (2009). Velthuis JH, Mol WM, Weimar W, Baan CC. CD4 + CD25 bright + regulatory T cells can mediate donor nonreactivity in long-term
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