The Evolving Role of Mtor Inhibition in Transplantation Tolerance

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The Evolving Role of mTOR Inhibition in Transplantation Tolerance

Gearoid McMahon,* Matthew R. Weir,† Xian C. Li,‡ and Didier A. Mandelbrot§

*Division of Nephrology, Children’s Hospital Boston, Massachusetts; †Division of Nephrology, University of Maryland School of Medicine, Baltimore, Maryland; ‡Department of Medicine, Beth Israel Deaconess Medical Center/Harvard Medical School, Boston, Massachusetts; and §Division of Nephrology, Beth Israel Deaconess Medical Center, Boston, Massachusetts

ABSTRACT The mammalian target of rapamycin (mTOR) plays a key role in the immune mTORC2.8,9 mTORC1 is sensitive to response. mTOR inhibitors suppress T cell activation and proliferation and are rapamycin and is involved in the regu- effective immunosuppressants. Today there is growing interest in their potential lation of cell growth by controlling role in inducing tolerance after transplantation. mTOR inhibitors induce anergy in translation, transcription, and nutrient naïve T cells, promote the expansion of regulatory T cells, and inhibit the matura- transport.10 mTORC2 is classically tion of dendritic cells, thus promoting immunologic tolerance. Here we review the thought to be rapamycin insensitive, mechanisms by which mTOR inhibitors promote tolerance. We discuss the clinical but has recently been shown to be dis- relevance of these mechanisms and suggest how they might be used in the design rupted after long-term treatment.11 of future protocols to induce tolerance. mTORC2 regulates the spatial aspects of cell growth through its effects on the ac- J Am Soc Nephrol 22: 408–415, 2011. doi: 10.1681/ASN.2010040351 tin cytoskeleton and also phosphorylates and activates Akt.12

The ultimate goal in transplantation is covered in the soil of Easter Island (Rapa long-term graft acceptance by achieving Nui), from which it derives its name. It is RAPAMYCIN AND T CELL graft tolerance in the recipient with mini- produced by the bacterium Streptomyces ANERGY mal or no immunosuppression. The mam- hygroscopicus, and, although it was ini- malian target of rapamycin (mTOR) path- tially proposed as an anti-fungal agent,2 Naïve T cells encountering antigen re- way plays a central role in the activation it was subsequently developed as an im- quire co-stimulation for full activation. and proliferation of T cells. The mTOR in- munosuppressive drug.3,4 Recognition When the TCR is stimulated in the ab- hibitor, rapamycin, has been used clinically of antigen by the T cell receptor (TCR) in sence of co-stimulation, T cells do not to prevent graft rejection since being ap- the presence of CD28 co-stimulation proliferate or produce IL-2, and they re- proved for use in transplantation in 1999.1 leads to downstream activation of main unresponsive on re-challenge with Increasing experimental data also suggest mTOR, which is a 289-kD serine/threo- the same antigen.13 This state is called T that blocking the mTOR pathway pro- nine protein kinase.5 This ultimately leads cell clonal anergy, and it plays an impor- motes immunological tolerance. This re- to increased translation of multiple pro- tant role in tolerance to self-antigens and view summarizes the molecular mecha- teins important for cell division. mTOR in- hence the prevention of autoimmunity. nisms by which mTOR inhibitors might hibitors prevent progression of the cell cy- An immunosuppressive protocol that in- promote tolerance and suggests how these cle from the G1 to the S phase and thus duces anergy in donor-reactive T cells mechanisms may be considered in design- block proliferation of T cells.5 Rapamycin ing future therapeutic regimens to prevent binds and forms a complex with the FK506 rejection. binding protein, which inhibits mTOR.6 In Published online ahead of print. Publication date response to growth factors, phosphatidyl- available at www.jasn.org. inositol 3-kinase (PI3K) phosphorylates Correspondence: Dr. Gearoid McMahon, Children’s Hospital Boston, Enders 826, 300 Longwood Avenue, THE mTOR PATHWAY Akt, which in turn inactivates tuberous Boston, MA 02115. Phone: 617-999-7876; Fax: 617-730- sclerosis complex 1, an inhibitor of 0130; E-mail: [email protected] 7 Rapamycin, also known as sirolimus, is a mTOR (Figure 1). mTOR forms two Copyright © 2011 by the American Society of macrocyclic antibiotic that was first dis- distinct complexes: mTORC1 and Nephrology

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CD28 TCR IL-2R regulatory T cells (Tregs). Tregs were first identified in the 1990s when it was discovered that depletion of a subset of CD4ϩ T cells leads to autoimmune disease in mice. These cells highly express PI3K the IL-2 receptor ␣-chain (CD25).20 ϩ mTORC1 Nude mice inoculated with CD4 T

mTOR Akt cells depleted of CD25 develop autoim- Rictor mune disease, which is prevented by the inoculation of CD4ϩ, CD25ϩ T TSC2 ? cells. Tregs are anergic and do not produce proinflammatory cytokines when mTORC1 stimulated in vitro. Tregs play a role in Rapamycin mTOR Raptor the prevention of autoimmunity by suppressing T cell responses to self-antigens and by limiting the response to p70S6K 4EBP1 foreign antigens. Tregs are also characterized by the ex- Protein translation pression of the forkhead family tran- + cell division scription regulator, Foxp3. Foxp3 is vital for the development and function of Figure 1. Recognition of antigen by the TCR in the presence of CD28 co-stimulation leads Tregs, and the lack of Foxp3 leads to ab- to phosphorylation and activation of PI3K. PI3K activates Akt, which inhibits TSC2, en- abling formation of the mTOR/Raptor complex (mTORC1). mTORC1 phosphorylates p70 sence of Tregs in mice. Defects in the S6 kinase and 4EBP1, leading to an increase in new protein translation in preparation for Foxp3 gene associate with autoimmune entry into the cell cycle. The binding of IL-2 to its receptor also activates the PI3K/mTOR disease in mice, and humans with muta- pathway. mTOR forms another complex with Rictor (mTORC2), which phosphorylates and tions in Foxp3 develop a similar autoim- further activates Akt. Rapamycin acts by preventing formation of mTORC1 and thus mune disease called IPEX.21,22 The role of inhibiting T cell proliferation. It may also inhibit the action of mTORC2. Foxp3 in Treg development was shown when naïve CD4ϩ, CD25Ϫ T cells devel- could allow for long-term graft accep- ine inhibit the induction of clonal anergy oped regulatory activity after retroviral tance without maintenance immuno- in vitro,18 even when T cells are simulta- gene transfer of Foxp3.23 Foxp3 acts in T suppression. Inhibition of mTOR with neously treated with rapamycin.14 cells in part by blocking NFAT-mediated rapamycin induces anergy in naïve T Similar effects have been noted in vivo. transcription of IL-2.24 IL-2 is necessary cells. T cells treated with rapamycin in Rapamycin treatment during antigen for the survival and function of Tregs. vitro, even in the presence of normal stimulation induces clonal anergy in Mice lacking the IL-2 receptor develop TCR/CD28/IL-2R co-stimulation, be- mouse T cells.15,16 This is seen even in the autoimmune disease,25 and the number come anergic and fail to produce IL-2 on presence of CTLA-4 blockade, which pre- of mature, peripheral Tregs is reduced in repeated stimulation.14,15 The key role vents anergy induction.15 In a mouse IL-2–deficient mice.26 IL-2 also induces played by mTOR activation, as measured model, rapamycin, combined with co- proliferation of Tregs when accompa- by the phosphorylation of S6 kinase, is stimulation blockade in the form of CTLA- nied by TCR stimulation.27 This has im- significantly reduced in anergic T cells, 4Ig plus anti-CD40L, leads to long-term plications for the choice of immunosup- whereas T cells that express a mutant ra- survival of skin allografts. These mice sub- pressive therapy, because calcineurin pamycin-resistant mTOR do not be- sequently accept second skin allografts inhibitors reduce the production of IL-2 come anergic in the presence of rapamy- from the same donor, whereas third party by activated T cells, whereas rapamycin, cin.16 This anergic state is reversed if the grafts are rejected, suggesting this combi- in contrast, inhibits cellular responses to cells are subsequently treated with exog- nation induces tolerance. Treatment with IL-2.28 Distinguishing Tregs from con- enous IL-2.16 IL-2–mediated reversal of rapamycin, co-stimulation blockade, or ventional T cells (Tconv) is complicated anergy is also mTOR dependent. Anergic cyclosporine alone leads to rapid rejection by the fact that the markers for Tregs T cells stimulated in vitro after treatment of the grafts.19 have limited specificity. For example, with IL-2 respond similarly to control stimulated CD4ϩ, CD25Ϫ Tconv tran- 29 cells. However, when these cells are RAPAMYCIN AND REGULATORY T siently express Foxp3. Also, Tregs that treated with IL-2 in the presence of rapa- CELLS are expanded in vitro lose Foxp3 expres- mycin, they fail to respond to stimula- sion and hence their regulatory pheno- tion and remain anergic.17 In contrast, Rapamycin may also be useful in pro- type on repeated stimulation.30 calcineurin inhibitors such as cyclospor- moting tolerance through its effects on Rapamycin has beneficial effects on

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the survival and proliferation of Tregs in Tregs. Tregs from normal humans prolif- pared with cyclosporine.46 Mycopheno- vivo and in vitro. Mice treated with a erate in response to rapamycin, whereas late mofetil also preserves Treg function combination of an IL-2 agonist, an IL-15 the proliferation of Tconv does not. These in a mouse model of allogeneic bone antagonist, and rapamycin increase the expanded Tregs express high levels of marrow transplantation,47 particularly apoptosis of Tconv and increase the per- CD25 and Foxp3, are resistant to apopto- compared with cyclosporine. After con- ϩ ϩ centage of CD4 , CD25 cells.31 This sis, and have suppressive activity in vitro.38 version from cyclosporine to mycophe- regimen promotes long-term engraft- Rapamycin also expands a highly suppres- nolate mofetil, liver transplant recipients ment of skin, heart, and islet cell allo- sive subset of human CD4ϩ, CD25ϩ T cells were found to have higher numbers of ϩ grafts. These mice are tolerant to subse- that are CD27 . These cells do not expand circulating Tregs.48 quent skin transplants from the same in the presence of cyclosporine. Interest- donors even after cessation of immuno- ingly, human Tregs cultured in the pres- suppression, whereas grafts from new ence of cyclosporine lose their suppressive donors are promptly rejected.31 Subse- capabilities within 48 hours, whereas rapa- INTRACELLULAR MECHANISMS quent studies showed that rapamycin ex- mycin-treated Tregs retain this function- UNDERLYING THE EFFECTS OF pand Tregs in vitro and in vivo, that these ality.39 Tregs expanded in vitro may have RAPAMYCIN ON TREGS Tregs are anergic on TCR stimulation,32 a role in the future treatment of autoim- and that they retain their suppressive mune disease, because the suppressive Several mechanisms may explain rapa- ability.33 Although rapamycin preserves activity of Tregs expanded in vitro from mycin’s preferential expansion of Tregs these T cell subsets,34 when mice are patients with autoimmune disease re- compared with Tconv. One is the differ- treated with cyclosporine, the number of mains. In one study of patients with mul- ential effect of IL-2 receptor signaling. A peripheral Tregs and the maturation of tiple sclerosis, rapamycin expanded number of pathways are activated in re- thymic Foxp3ϩ cells are significantly re- Tregs from both normal donors and sponse to IL-2R signaling, including the duced; cyclosporine treatment in the first those with the disease, and the suppres- JAK/STAT, MAPK, and the PI3K/Akt/ week of life also leads to autoimmune sive phenotype was retained in each.40 mTOR pathways. In Tregs, JAK/STAT disease similar to that seen in IL-2–defi- Similarly, Tconv isolated from patients signaling is induced preferentially, cient mice.35 Rapamycin in addition pro- with type I diabetes are highly sensitive to whereas PI3K/Akt/mTOR signaling is motes the differentiation of naïve T cells the anti-proliferative effects of rapamy- reduced relative to Tconv (Figure 2).49 into Tregs in the periphery. When cin, whereas Tregs expand to be suppres- STAT5 binds to the Foxp3 promoter in Foxp3Ϫ T cells are activated in vitro,2to sive. In contrast to other studies, rapa- Tregs, thus regulating Foxp3 expres- 3% expressed Foxp3 and developed a mycin treatment did not promote sion.50,51 Phosphatase and tensin ho- regulatory phenotype. However, when anergy.41 molog (PTEN) is an endogenous inhibi- these cells are treated with IL-2 and rapa- Unlike cyclosporine, corticosteroids tor of PI3K. Tregs constitutively express mycin at the time of activation, the per- do not inhibit Tregs and may promote PTEN and, therefore, when the IL-2 re- centage of cells expressing Foxp3 in- their expansion. The combination of dexa- ceptor is activated, there is minimal creases to 7%.36 Similarly, Foxp3 methasone and IL-2 treatment expands downstream activation of mTOR. In Ϫ expression induces in vitro when Foxp3 Tregs in mice.42 Similarly, an increased contrast, PTEN activity is low in Tconv, T cells activate in the presence of TGF␤ percentage of Tregs is seen in the thymus so the PI3K/Akt/mTOR pathway acti- or rapamycin, whereas cyclosporine fails and spleen of mice receiving dexametha- vates in response to IL-2 receptor signal- to induce Foxp3. In the same study, sone relative to controls. However, this ing.52 Thus, rapamycin treatment inhib- when naïve T cells are adoptively trans- may be the result of increased resistance its the PI3K/Akt/mTOR pathway in ferred into mice, 2% became Foxp3ϩ. to dexamethasone-induced apoptosis in Tconv, whereas Tregs, because this path- After treatment with rapamycin, 6% ex- Tregs relative to Tconv, because their ab- way is not activated, are relatively insen- pressed Foxp3, and the combination of solute numbers were reduced.43 Addi- sitive to the anti-proliferative effects of rapamycin and anti-CD154 (CD40L) in- tion of dexamethasone and IL-7 to hu- rapamycin.47,53 There also is evidence duced Foxp3 expression in 25% of T man CD4ϩ, CD25Ϫ cells results in an that activation of Akt may itself be det- cells. In contrast, when the mice were increased percentage of CD4ϩ, CD25ϩ rimental to Tregs. When naïve T cells treated with cyclosporine, none of the T cells in the culture,44 whereas increased are treated in vitro with IL-2 and TGF␤, cells became Foxp3ϩ. Rapamycin and Foxp3 expression is noted in CD4ϩ T Foxp3 is induced. However, when Akt anti-CD154 treatment for 14 days pro- cells of asthmatic patients after systemic is overexpressed in these cells, Foxp3 motes tolerance of skin allografts (sur- treatment with glucocorticoids.45 The expression is inhibited. This effect is vival Ͼ100 days), whereas cyclosporine- few studies assessing the effect of myco- partially reversed by treatment with treated mice eventually reject their grafts phenolate mofetil on Tregs show little or rapamycin.54 This latter finding sug- after cessation of immunosuppression, no effect on their function. One study gests the decreased activation of the with a mean survival of 45 days.37 showed reduced apoptosis of Tregs in the PI3K/Akt/mTOR pathway in Tregs Rapamycin also expands human presence of mycophenolate mofetil com- helps ensure expression of sufficient

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Conventional T cells Regulatory T cells they were on immunosuppression. In contrast, there was no increase in the number IL-2R IL-2R of Tregs in those who had an episode of acute rejection after withdrawal, suggesting that increased Tregs associate with tol- JAK3 JAK3 erance to the graft.61 In a study of 37 patients with subclin- PI3K STAT5 PTEN PI3K STAT5 ical rejection diagnosed on protocol renal biopsies 3 years after transplant, a higher percentage of Tregs in the T cell Akt FOXP3 Akt FOXP3 infiltrates correlated with better renal function. Most of the patients with increased Tregs were on rapamycin, al- RAPA mTOR PIM-2 RAPA mTOR PIM-2 though even in the group taking calcineurin inhibitors, Treg numbers correlated with renal function.62 Increased expression 4EBP1 4EBP1 4EBP1 4EBP1 of Foxp3 was noted in T cells of renal allograft recipients with long-term (8 years) Figure 2. In Tconv, stimulation of the IL-2 receptor leads preferentially to activation of stable renal function relative to those who the PI3K/Akt/mTOR pathway. Because of their dependence on this pathway for pro- had chronic rejection.63 Increased Foxp3 liferation, Tconv are sensitive to the effects of rapamycin. In contrast, because of their expression was also noted in renal trans- high baseline expression of PTEN, in Tregs, IL-2R stimulation leads to activation of plant recipients with donor-specific hypo- JAK/STAT signaling, which regulates Foxp3. The Pim-2 pathway is not sensitive to responsiveness, and all of the hyporespon- rapamycin and is constitutively expressed in the presence of Foxp3. This allows sive patients were taking rapamycin.64 expansion of Tregs in the presence of rapamycin while suppressing the proliferation of Tconv. When T cells are depleted with the anti- CD52 mAb, Campath-1H, T cell numbers slowly recover over the first year. The use of levels of Foxp3 to allow their prolifera- and it is constitutively expressed.57 Thus, a rapamycin-containing regimen associ- tion and survival. in Tregs, Pim-2 confers resistance to the ates with higher numbers of circulating Rapamycin also influences the ex- anti-proliferative effects of rapamycin by Tregs relative to those patients treated with pression of pro- and anti-apoptotic pro- offering an alternative pathway indepen- cyclosporine. These Tregs are capable of teins on T cells. Tregs cultured in the dent of PI3K/Akt/mTOR. suppressing T cell alloreactivity to donor presence of rapamycin express high lev- antigens in vitro, and this hyporespon- els of the anti-apoptotic proteins Bcl-2 siveness is reversed when CD4ϩ, CD25ϩ and Bcl-xL. In contrast, when Tconv are REGULATORY T CELLS AND T cells are depleted.65 However, the in- cultured with rapamycin, they down- TRANSPLANTATION creased numbers of Tregs is not associ- regulate the expression of the anti-apop- ated with better outcomes in a small totic proteins and upregulate the expres- Higher levels of circulating Tregs associ- number of patients 3 years after trans- sion of pro-apoptotic Bax. As a result, ate with better outcomes after solid-or- plant. In fact, higher tubular CD4 stain- after treatment with rapamycin, Tregs gan transplantation. Decreased numbers ing and increased clinical proteinuria are relatively resistant to apoptosis com- of Tregs are found in the blood of lung were noted in the patients treated with pared with Tconv.53 transplant recipients with bronchiolitis rapamycin.66 A third mechanism for the relative re- obliterans compared with those with Rapamycin is effective both in treat- sistance of Tregs to rapamycin is through normal grafts 3 years after transplant. ing established graft versus host disease the Pim-2 pathway. Pim-2 is a serine- None of the recipients were treated with (GVHD)67 and for the prophylaxis of threonine kinase that shares downstream rapamycin.58,59 Similarly, decreased num- GVHD68,69 in allogeneic bone marrow targets of mTOR and Akt, including Bad bers of Tregs are seen in liver transplant transplantation in humans. The benefits and 4-EBP1.55 In Tconv, Pim-2 is acti- recipients who have an episode of acute re- may be partially mediated through the vated in response to cytokine stimulation jection in the first year after transplant.60 effect of rapamycin on Tregs. For exam- through the JAK/STAT pathway and ex- Another study of liver transplant recipients ple, interstitial Tregs are increased in the pression is quickly downregulated in the reported the numbers of Tregs before and skin of mice treated with rapamycin after absence of these cytokines. Absence of after withdrawal of cyclosporine. In those bone marrow transplantation.70 Human Pim-2 causes Tconv to be highly sensi- patients who tolerated withdrawal of im- studies showed in patients with acute tive to rapamycin.56 In Tregs, however, munosuppression, there was a threefold GVHD that there are reduced numbers Pim-2 expression is controlled by Foxp3, increase in Tregs compared with when of Tregs in the periphery and in the af-

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fected organs.71,72 The infusion of donor matory cytokine IL-18 by mature DCs in like to acknowledge Dr. David Briscoe for Tregs is an effective treatment for GVHD response to lipopolysaccharide. Cyclo- thoughtful comments during the preparation in mice and prevents GVHD induced by sporine treatment has no effect on IL-18 of this manuscript. donor Tconv when they are transfused in production.85 Rapa-DCs also prolong graft high numbers relative to the numbers of survival in mice. Multiple infusions of al- Tconv. Importantly, this does not affect loantigen-pulsed, rapa-DCs lead to long- DISCLOSURES the anti-tumor activity of the trans- term cardiac allograft survival.80 In the M.R.W. has served as a scientific advisor to 73,74 plant. same model, a single infusion of rapa- Wyeth. DCs in combination with a short postop- erative course of rapamycin promotes RAPAMYCIN AND DENDRITIC indefinite graft survival. 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