Costimulation blockade and regulatory T-cells in a non- human primate model of kidney allograft transplantation Haanstra, K.G.

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human primate model of kidney allograft transplantation

Haanstra, K.G.

Citation

Haanstra, K. G. (2008, March 13). Costimulation blockade and regulatory T- cells in a non-human primate model of kidney allograft transplantation.

Retrieved from https://hdl.handle.net/1887/12636

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Summary and discussion

7.1 Costimulation blockade with anti-CD40 and anti- CD86 in the rhesus monkey kidney allograft model

An organ graft transplanted in a non-identical recipient invokes a vigorous immune response. Recipient T-cells recognise allogeneic donor MHC (direct presentation) and self MHC presenting donor peptides (indirect presentation) by means of their TCR [1, 2]. This signal 1 needs to be accompanied by a second signal, mediated by costimulation receptor-ligand interaction [3]. The two most important pathways of costimulation are CD40-CD40L and B7-CD28. TCR engagement without a second signal leads to anergy of recipient T-cells. This dogma was ﬁrst discovered in vitro [4], but has been extensively investigated in vivo. In rodents, blockade of a single costimulation pathway using CTLA4-Ig already led to indeﬁnite survival of an allograft [5, 6]. When this treatment was translated to NHP, the success could not be reproduced [7]. Not only costimulation blockade, but also various other tolerance induction protocols did not work in NHP, whereas they did in rodent models [8]. This difference could be caused by the different immune repertoires of rodents and NHP.

Mice used for tolerance induction testing are kept in an SPF environment and remain relatively immunologically naive. Naive cells cannot be activated in the absence of costimulation blockade, whereas memory cells are less dependent on costimulation [9–12]. Mice allowed to acquire immune memory against a virus were not susceptible to costimulation blockade induced tolerance [13, 14]. It is therefore important to evaluate costimulation blockade, as well as other tolerance induction strategies, in a valid preclinical model. NHP acquire immune memory, which accumulates with age [15]. They are not kept under SPF conditions and their immune status is similar as in humans.

NHP are a better model for studying tolerance induction protocols for several other reasons as well. They are outbred, which resembles the genetic variation present in humans. Furthermore, biologicals, such as monoclonal antibodies, are often cross-reactive with NHP. In rodents, mostly different agents, although with the same speciﬁcity, can be tested, whereas in NHP exactly the same reagents as used in humans can be tested.

Blockade of the CD40-CD40L pathway using the humanised anti-human CD40L mAb hu5C8 has been extensively studied in NHP. Hu5C8 monotherapy induced long-term graft survival in monkeys, provided that treatment is given for a sufﬁ- ciently prolonged period and antibody levels are high [16, 17]. However, the trans- lation of these results into the clinic was less successful. Effects on the graft were not as promising as could be expected from the NHP studies, and the studies were hampered by thromboembolic complications [18]. Experiments in NHP treated with anti-CD40L antibodies showed that microthrombi formed due to the aggregation of activated platelets that express CD40L, which was bound by the antibody, not only the hu5C8 antibody, but also by other anti-CD40L antibodies [19, 20]. Although this effect could be blocked by the use of anti-coagulation factors [21], scientists have changed their strategy to blocking the receptor of the pathway, CD40.

Antibodies targeting CD40 can have agonistic or antagonistic properties i.e. they

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stimulate or block CD40 signalling. An anti-CD40 mAb (Chi220) with agonistic properties was tested in the NHP models of kidney transplantation [22] and pancreatic islet allotransplantation [23]. Chi220 depleted peripheral B-cells and prolonged kidney and islet graft survival [22, 23]. The effect of Chi220 on graft survival is unlikely to be due solely to the depletion of B-cells, since rejection can occur in B- cell deﬁcient mice [24]. Recently, an antagonistic anti-CD40 mAb, 4D11, was tested at dosages of 1, 5, 10, and 20 μg/kg, for a total of 180 days. Due to neutralisation of the anti-CD40 antibody, only a few animals of the groups treated with the two highest dosages maintained high levels of circulating antibody and could maintain their graft beyond the treatment period [25]. When combined with Rapamycin, the 5 μg/kg treatment was considerably more effective [26].

The BPRC has a longstanding experience in testing immunosuppressive drugs and strategies in the rhesus monkey kidney allograft model. A number of protocols for prolongation of kidney allograft survival, including blood transfusions, polyclonal anti-T-cell antibodies, monoclonal anti-T-cell antibodies and costimulation blockade have been tested in the past [27–32]. This thesis investigates the use of costimulation blockade with chimeric anti-human anti-CD40 and anti-CD40 plus chimeric anti-human anti-CD86, clones ch5D12 and chFun-1, respectively, as a pos- sible way to induce tolerance (chapter 2). When anti-CD40 treatment resulted in high serum trough levels, it was found to prolong graft survival after cessation of treatment for 30 to 150 days. When serum concentrations were low during main- tenance therapy, grafts were rejected during treatment. The addition of anti-CD86 mAb (chFun-1) to the treatment, overcame the need for high anti-CD40 mAb serum levels. All animals treated with both antibodies survived the treatment period, although in some animals signs of rejection were seen already during treatment.

Addition of anti-CD86 to the treatment lowered the amount of inﬁltrating cells in the interstitium and tubules in day 21 biopsies, and even more in day 42 biopsies.

Since the animals treated with both costimulation blocking antibodies had shorter graft survival than animals treated with only anti-CD40 mAb, we hypothesised that the infiltrates seen on day 42 may have a beneficial, regulatory function. However, treatment of patients with subclinical rejection resulted in improvement of graft survival as opposed to when subclinical infiltrates were not treated. These results indicated that at least in patients treated with conventional immunosuppressive drugs there is no evidence to support the possibility of benign graft infiltrates [33]. Whether the same holds true for subclinical infiltrates seen during treatment with costimulation blockade needs to be investigated further.

A remarkable finding was the absence of significant levels of anti-donor antibodies in monkeys receiving either of the treatments. Blockade of CD40 prevented activation of B-cells and inhibited T-cell dependent immunoglobulin production in vitro [34, 35]. The ch5D12 antibody prevented the production of donor-specific an- tibodies even until after the treatment was stopped. The agonistic anti-CD40 mAb Chi220 did not have this effect and the antagonistic mAb 4D11 only prevented anti- donor antibody formation during treatment [22, 25]. The formation of rhesus anti- chimeric antibodies (RACAs) against both treatment antibodies ch5D12 and chFun-1 was not prevented. There was no correlation between formation of RACAs and anti-

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ch5D12 RACAs chFun-1 RACAs anti-donor Ab

ch5D12 30 + ND +

low 42 + ND -

ch5D12 91 + ND -

high 134 - ND +

217 - ND +

ch5D12 + 61 - +/- -

chFun-1 71 + +/- -

75 - - -

78 + + -

116 + + -

Table 7.1: Overview of the production of RACAs against ch5D12 and chFun-1 and anti-donor antibodies. ND: not done, no chFun-1 was administered to these animals.

donor antibodies (Table 7.1). The reason for this discrepancy is unknown but we have observed this previously in another study as well [36]. We like to propose the following explanation, which is based on two assumptions. First, allo-antigens are much weaker and/or less frequent antigens than chimeric Ig molecules; suppressive treatment will therefore have a stronger impact on production of anti-donor antibodies than on production of RACAs. Second, while CD40 is expressed both on B-cells and DC, CD86 is expressed on DC, but expression on B-cells is induced only upon activation. Predictably, a certain proportion of anti-CD40 mAb will also bind to CD40 on DC. Due to the mild agonistic effect of the antibody, as observed in MLR [37], IL-12 production may be induced. So, ch5D12 and chFun-1 may be presented via MHC class II to helper T-cells and at the same time ch5D12 activates DC, which leads to IL-12 production. IL-12 can act as a adjuvant for antibody production against soluble antigen that can be processed by DC [38]. The absence of RACAs against anti-CD40 seems to be correlated with prolonged graft survival. The formation of RACAs against anti-CD40 lowers anti-CD40 mAb trough levels, which might have been a critical factor leading to early rejection. Development of a humanised version of the antibody might overcome these problems, leading to prolonged graft survival, even with the same treatment schedule and dosages.

The survival obtained with anti-CD40 and anti-CD40 plus anti-CD86 treatment is comparable to other studies with costimulation blockade as single modality treatment of similar dose and duration (see Tables 1.2 and 1.3 in the Introduction). Dura- tion of treatment and level of circulating antibodies are a critical factor determining graft survival after cessation of treatment. If serum trough levels could be maintained, for instance by using humanised versions of the antibodies and prolongation of the treatment period, survival beyond the treatment period might be enhanced, similar to the effects of prolonged hu5C8 treatment. However, costimulation blockade as a monotherapy for a limited period does not induce tolerance, as evidenced by the fact that a number of animals rejected after withdrawal from hu5C8 mainte-

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nance therapy for as long as one year [17].

7.2 Costimulation blockade with anti-CD40 plus anti- CD86 in combination with CsA or ATG plus CsA

The treatment success of chimeric anti-CD40 antibody depends much on the given dosages and on the unpredictable factor whether or not animals formed antibodies against the anti-CD40 antibody, thereby lowering serum trough levels. The addition of anti-CD86 mAb to the treatment led to a more predictable graft survival, i.e. no rejection within the treatment period, and led to lower numbers of graft in- ﬁltrating cells in day 42 biopsies. Costimulation blockade as a single treatment is not a realistic option for clinical application. It will always be tested ﬁrst as add-on treatment. However, certain conventional immunosuppressive drugs may counter- act the effect of costimulation blockade on graft survival prolongation. Calcineurin inhibitors such as CsA and FK506 may have a negative impact on graft survival induced by CTLA4-Ig and/or anti-CD40L mAb in mice and NHP [16, 39–41], although this could not be demonstrated in another study [42]. Rapamycin, a conventional immunosuppressive drug that prevents signalling through the commonγ-chain of cytokine receptors, the receptor for cytokines such as IL-2 and IL-15, may be a better candidate for combination with costimulation blockade [40, 41], possibly because it allows initial activation of the cells via the TCR and allowing subsequent activation induced cell death (AICD) [40, 43]. In vitro, combination of anti-CD40 plus anti- CD86 costimulation blockade with tacrolimus or Rapamycin did not induce tolerance/anergy, while combination with CsA did [44]. However, CsA is a nephrotoxic drug. To allow for an initial conventional immunosuppressive drug-free period, we have combined anti-CD40 plus anti-CD86 costimulation blockade treatment with a delayed onset (day 42) of CsA treatment (chapter 3). On day 42, none of the anti- CD40 plus anti-CD86 treated animals had started to reject, so CsA was given as sup- plementary immunosuppression and not as anti-rejection treatment. CsA was given at tapering dosages until day 126 after which all immunosuppression was stopped.

This treatment prevented the graft rejection immediately after costimulation blockade withdrawal. However, two animals rejected after cessation of CsA treatment, one on day 140 and one in a more delayed fashion on day 231. Two animals lived more than six years after cessation of all treatment. Eventually, both animals had a slowly deteriorating kidney function, as measured by serum creatinine levels and both showed signs of CAN, also called IF/TA, in kidney biopsies. This outcome of chronic renal failure is very similar to what has been observed in NHP treated with hu5C8. A number of animals survive beyond the treatment period, but in the end, all develop CAN [45]. IF/TA is a poorly understood process. Attempts to reverse acute and chronic rejection with hu5C8 rescue therapy in monkeys initially treated with hu5C8, but drug-free for periods ranging from 8 to 395 days, were only partially successful [45]. Acute rejection after initial hu5C8 treatment could be reversed, but acute rejection after initial conventional immunosuppressive drugs and hu5C8 treatment could not be reversed. In addition, graft failure could not be reversed by

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the hu5C8 rescue therapy in monkeys with IF/TA.

We conclude that costimulation blockade with anti-CD40 and anti-CD86 is very effective in preventing graft rejection during treatment, but after cessation of treatment, alloreactive cells (re-)emerged, leading to graft destruction. Although delayed CsA treatment had an additive effect, it was insufﬁcient to prevent rejection in two of the four animals. We therefore hypothesised that induction therapy with ATG would remove alloreactive cells present in the normal repertoire, leaving a population that could be more easily tolerised by costimulation blockade [13, 46, 47].

CsA treatment was initiated on day 42. Before day 42, all animals treated with anti- CD40 plus anti-CD86 were treated equal, except for the ATG induction treatment plus methylprednisolone to reduce the side effects of first-dose intravenous ATG (chapter 4). Surprisingly, two out of five ATG treated animals experienced rejection during these first 42 days (days 21 and 38), while none of the nine non-ATG treated animals rejected their kidneys in this period. Rejections were defined as a 50% rise of the serum creatinine levels within three to four days. First rejections in the ATG treated animals were treated with steroids if they occurred after day 42, and all animals received high dose CsA from day 42 onwards. However, this did not reverse the ongoing rejection process, leading to shorter graft survival times as compared to costimulation blockade plus CsA treated animals. It appeared that our first as- sumption was not correct. CD4⁺and CD8⁺T-cells were analyzed for the naive subset CD95^low/CD11a^low. Memory T-cells were defined as the remaining non-naive CD4⁺and CD8⁺T-cells. We found a reduction in absolute numbers of naive CD4⁺ and CD8⁺T-cells in the peripheral blood of ATG-treated animals and significantly lower ratios of naive to memory T-cell populations were present in ATG-treated animals. This process of reappearance of memory T-cells in a lymphopenic environment, termed homeostatic proliferation, could explain our observations [48–51]. Ex- periments in mice demonstrated that cells undergoing homeostatic proliferation are not susceptible to tolerance induction by costimulation blockade [52]. Memory cells also have an intrinsic lower activation threshold, reducing the requirements for costimulation [9–12]. CD4⁺memory T-cells can provide help to CD8⁺cells even in the presence of costimulation blockade [53] and they can provide help to B-cells for iso- type switching, which may be responsible for the presence of donor-specific alloan- tibodies in ATG-treated animals, whereas they are absent in costimulation blockade treated animals.

In conclusion, costimulation blockade by anti-CD40 plus anti-CD86 mAb can be safely combined with delayed CsA treatment for prolongation of graft survival. ATG does not have synergistic effects on graft function and survival with costimulation blockade and delayed CsA. It could even have adverse effects and should therefore be avoided.

7.3 Characterization of graft-inﬁltrating cells

Transplantation research would beneﬁt from the identiﬁcation of a marker or set of markers that would indicate whether it is safe to withdraw immunosuppression in

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long-term stable patients. The monkey model of transplantation provides a unique opportunity to study markers inside the graft that may be indicative of tolerance because immunosuppressive treatment is always withdrawn at some point, in all studies. The survival and function of the graft after drug withdrawal can then be related to immune parameters prior to drug withdrawal. Expression proﬁles of leu- cocyte subset markers and Treg markers were studied inside the graft in a number of animals, which were grouped according to the presence or absence of rejection and the type of immunosuppression (chapter 5).

We have analysed our tissue samples according to two main categories, interstitial diffuse infiltrates and nodular infiltrates. They were analysed separately for expression profiles of subset markers. Nodular infiltrates develop over time, when the tissues are rejected, forming structures resembling lymphoid follicles with clear T-cell and B-cell areas. These structures are called tertiary lymphoid organs and can be found in a variety of chronic inflammatory conditions [54]. The lymphoid- like structures suggest that antigen presentation takes place in these structures. The composition of nodular infiltrates differs considerably from the composition of diffuse infiltrates; higher percentages of CD4⁺T-cells, CD20⁺B-cells and CD83⁺DCs, and lower percentages CD68⁺ macrophages were present in the nodular infiltrates.

In addition, the composition of the infiltrates also differs considerably in rejected versus non-rejected grafts. Rejected grafts had higher percentages of CD20⁺cells in nodular infiltrates; furthermore they had higher percentages of CD4⁺cells, CD20⁺ cells, CD68⁺cells and CD83⁺cells in diffuse infiltrates.

Rejected and non-rejected kidney speciments were each subdivided according to the type of immunosuppression, e.g. costimulation blockade, CsA, no immunosuppression. For non-rejected tissues the additional category no immunosuppression and graft survival for more than six months without immunosuppression, the long- term survivors (LTS) was defined. We observed that the type of immunosuppression was generally of little influence on the composition of the infiltrates.

These results prompted us to investigate whether the percentages of cells positive for Treg markers differed between rejected and non-rejected tissues and whether the type or absence of immunosuppression inﬂuenced these percentages. We hypothesized that non-rejected tissues and in particular in tissues from animals without immunosuppression may have higher percentages of Treg marker positive cells in the grafts. Currently, FOXP3, a nuclear transcription factor, is the most speciﬁc marker for Tregs. Suppressive natural CD4⁺T-cells express FOXP3, but in humans, non-suppressive cells may also (temporarily) express FOXP3, which makes them anergic, but not suppressive [55–57]. Other markers expressed on Tregs such as CD25, CTLA-4, CD103 and GITR were also investigated, although these markers can also be expressed on activated cells.

The percentage FOXP3⁺ cells is higher in nodular infiltrates as compared to in diffuse infiltrates. In both nodular and diffuse infiltrates percentages FOXP3⁺ cells are higher in samples from rejected than from non-rejected kidneys. Percentages of CD25⁺ and CTLA-4⁺ cells followed this same pattern. The percentages GITR⁺ cells were also higher in nodular infiltrates, but no differences were found between rejected and non-rejected tissues. High percentages of CD103⁺ cells were found

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in diffuse infiltrates of rejected tissues, but even more in tubular infiltrates. CD8⁺ T-cells were found in tubular infiltrates of rejected tissues in similar numbers as CD103⁺ cells, indicating that these were most likely CD8⁺CD103⁺ CTLs infiltrat- ing the tubules [58].

The observation that higher percentages of FOXP3⁺, CD25⁺ and CTLA-4⁺ T- cells were associated with rejection was further strengthened by the observation that only low percentages of cells positive for these markers were found in four animals in the LTS group. Percentages of cells expressing FOXP3, CD25 and CTLA-4 did not signiﬁcantly differ between the other subgroups deﬁned by type of immunosuppression.

Veronese et al. have investigated the presence of FOXP3⁺ cells in tissues with acute cellular and humoral rejection [59]. Although they did not compare this with non-rejected tissues, they also found increased numbers of FOXP3⁺cells in patients with shorter graft survival. Their observation that the majority (96%) of FOXP3⁺ cells are CD4⁺ T-cells is also consistent with our ﬁndings. Interestingly, they found FOXP3⁺ cells in tubuli, which was not conﬁrmed in our study. This discrepancy could be due to the differences between humans and NHP, the antibodies used or the fact that they investigated tissues which had been rejected in the presence of immunosuppression, whereas most of our rejected tissue samples came from animals without immunosuppression.

We conclude that high percentages of the Treg markers FOXP3, CD25 and CTLA- 4 are not indicative for acceptance of the graft, as percentages of cells positive for these markers were signiﬁcantly higher in nodular inﬁltrates of rejected tissues. Two not mutually-exclusive explanations may account for the presence of regulatory markers inside rejected grafts. Either, these markers are not representative for a regulatory function of these cells, as was shown in vitro [55–57], or regulatory cells expressing CD25, FOXP3 and CTLA-4 represent a failed counter-active mechanism, limiting immune activation. When during rejection the number of effector T-cells increases, the number of regulatory T-cells, already present in healthy and diseased individuals, is also increased (Fig. 7.1). This process occurs in the absence of immunosuppression, when no long-term drug free survival is achieved, but may also occur in the presence of immunosuppression, when the immunosuppressive treatment is unable to prevent an anti-donor response in the graft.

7.4 Long-term drug-free surviving kidney allograft re- cipients

True tolerance is not a complete utopia, as proven by a monkey in our cohort of long-term drug-free survivors. This monkey, YM, was transplanted in 1981, after three random pre-transplant blood transfusions and a six-month period of CsA treatment [60, 61]. After almost 26 years rejection free survival, the general health of the monkey deteriorated due to old age, but with good kidney function. The tolerance mechanism is not fully understood. The protocol of random blood transfusions and CsA led to a number of animals surviving several years after cessation of treatment,

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Figure 7.1:Hypothetical representation of the immune response in the graft. In case of stable transplant function, there is little need for the presence of active immune regulation in the graft. Upon anti-donor T-cells entrance in the graft, regulatory T- cells might also enter the graft, explaining the presence of high numbers of cells with regulatory T-cell markers in rejected tissues.

although only YM never rejected its graft [60, 61]. Important for the success may have been the transplantation over a negative (SI<3) MLR with an almost complete MHC-II match on one haplotype. The other haplotype was not matched. Further in vitro experiments with PBMC have not yielded deﬁnite answers, although the cy- tokines TGF-β and IL-10 may play a role.

Soulillou et al. have demonstrated that tolerant human recipients have a skewed TCR Vβ repertoire [62, 63]. The Vβ repertoire of monkey YM is also reduced, which may be due to clonal deletion of donor-reactive cells (J.P. Soulillou, personal com- munication).

Possibly, the suppressive cytokine TGF-β is involved in transplantation tolerance.

Latent TGF-β was found in biopsies of tolerant animals, but not in biopsies of rejected tissues [64]. The latter showed cells staining positive for the active form of TGF-β, which was conversely not found in tolerant animals. In a trans vivo delayed type hypersensitivity model (DTH) it was demonstrated that donor-speciﬁc linked suppression by PBMC from these tolerant monkeys could be inhibited by the addition of antibodies against TGF-β [64].

TGF-β was reported to be responsible for the induction of a regulatory phenotype in CD25⁻cells that were stimulated with anti-CD3. TGF-β is also implicated in the signalling cascade leading to FOXP3 expression. We were interested whether TGF-β was expressed in our collection of tissue samples taken from monkeys transplanted

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with a kidney allograft. TGF-β can be found either in the latent form, membrane- bound and noncovalently bound to latency-associated peptide (LAP), or in the active form, after LAP has been cleaved off [65]. Thrombospondin can be detected on LAP⁺cells, which can convert latent TGF-β into the active form. We have used two different antibodies for the detection of TGF-β. One antibody detects latent TGF-β1, and a second antibody detects active TGF-β. We found a weak inverse correlation between FOXP3 and latent TGF-β in our tissue samples. Chapter 3 describes the longitudinal analysis of the presence of latent TGF-β in the biopsies and at rejection.

Animals that were off immunosuppression but continued to live with a functioning graft had high numbers of latent TGF-β⁺ cells per tubulus, while rejected kidneys were negative for latent TGF-β staining, as also reported by Torrealba et al. [64]. La- tent TGF-β staining seemed to be decreased in CsA treated animals as compared to in biopsies taken at the same time point from animals in which costimulation blockade was withdrawn without subsequent CsA treatment. However two of the animals treated with CsA from day 42 onward survive long-term, with continued presence of TGF-β in the graft. We have stained tissues from all four LTS animals that did not reject and have had a drug-free survival of at least six months, including the two described in chapter 3, and monkeys YM and R142 (Fig. 7.2). The latter animal survived long-term after a treatment with anti-CD3 toxin, donor blood transfusions and CsA and sirolimus [36]. Biopsies of these monkeys show increased amounts of latent TGF-β, as compared to non-rejectors (no rejection, not drug-free, or drug-free less than six months), or as compared to rejectors, which have virtually no latent TGF-β staining. Latent TGF-β staining was found in biopsies even in the absence of T-cell inﬁltrates. The diffuse type of staining suggests that the cytokine is not membrane bound, but secreted by ﬁbroblast-like cells.

Thus, LTS monkeys seem to be able to control the invasion of immune cells into the graft, preventing the need for active regulation of the immune reactive cells by FOXP3⁺Tregs. Longitudinal analysis of percentages of FOXP3, CTLA-4 and CD25 in nodular inﬁltrates of the animals treated with costimulation blockade and CsA (Fig 5.3) demonstrates that in the two animals surviving long-term, percentages FOXP3⁺cells remain low.

The numbers of latent TGF-β positive areas per tubulus were counted. In tissues from animals without rejection, TGF-β is expressed in the graft, especially early (less than six months) after withdrawal of immunosuppression (Table 7.2, no rejection, no immunosuppression), but it continues to be present in LTS animals (Table 7.2, LTS)).

By comparison, the number of positive areas per tubulus in rejected tissues is much lower (Table 7.2, rejected tissues). Figure 7.3 shows the longitudinal follow-up on latent TGF-β expression in the kidney of monkeys YM and the two LTS monkeys described in chapter 3 which were treated with anti-CD40, anti-CD86 and CsA.

A schematic representation of this proposed mechanism is depicted in Figure 7.4.

The mechanism responsible for the appearance of latent TGF-β is unknown and whether TGF-β has a direct immunosuppressive effect is not known, it may also be a feature of another unknown underlying mechanism that is responsible for the quiescent state in the graft. The role and mechanism of action of TGF-β should be further studied using immunohistochemistry double staining for TGF-β and other

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Figure 7.2:Latent TGF-βstaining. Latent TGF-βstaining is absent in normal kidney (A). TGF-βstaining can be found in biopsies of LTS monkeys, is mainly located in the interstitium, and does not represent surface staining but typically presents as secreted cytokine staining (C, D, F). Rejected kidneys display a more diffuse interstitial staining (B). Kidney biopsies of an animal with more than 24 years of drug-free survival appear normal, without rejection and without CD3⁺ cells (E), but with large numbers of TGF-β⁺areas (F). Positive areas are localized around cells that have the morphology of ﬁbroblast-like cells.

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TGF-β No rejection, no immunosuppression 0.85±0.17 (11)

LTS 0.41±0.05 (21)

Rejected tissues 0.18±0.05 (45)

Table 7.2:Latent TGF-βpositive areas per tubulus, expressed as mean± SEM (number of tissue samples).

 

 

Figure 7.3: Longitudinal analysis of latent TGF-βpositive areas per tubulus. YM is represented by the closed diamonds. Closed triangles and open rounds represent the two LTS monkeys, identiﬁed in chapter 3 as >1320 and >1290 respectively.

markers, in vitro analysis and the trans vivo DTH model. However, attempts to set up the trans vivo DTH model were hampered by technical limitations of the assay, in our hands.

7.5 Naturally occurring Tregs in rhesus monkeys

Recent publications indicate that Tregs are present in NHP and that they can be expanded [66–69]. We have investigated whether rhesus monkey Tregs are comparable to human Tregs or whether they are more comparable to mouse Tregs (chapter 6).

Human Tregs are characterized by higher expression of CD25 and lower expression of CD4 as compared to Th cells. Up to 46% of total CD4⁺ cells may be positive for CD25, but only 1-3% are CD25^high. Mouse Tregs have a more uniform CD25 expression and all CD25⁺ cells have a regulatory function [70–73]. Chap- ter 6 describes the in-depth characterization of naturally occurring Tregs in rhesus monkeys. Rhesus monkey CD4⁺ cells have a uniform expression of CD25, as described in chapter 6 and reported by others [66, 68, 74]. This expression pattern resembles more the CD25 expression pattern as found in mice, and in human cord

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Figure 7.4:Hypothetical representation of the immune response in the graft leading to long-term drug free survival. A low anti-donor response is kept in balance by a limited natural regulatory response and possibly induced regulatory responses.

After cessation of treatment, the induced regulatory response, in the form of latent TGF-β, becomes more prominent in the graft. This state of unresponsiveness may last for many years.

blood [72]. Mouse and cord blood CD4⁺CD25⁺ cells are immunologically naive, but rhesus monkey CD4⁺CD25⁺are not immunologically naive, as indicated by the low percentage of CD45RA⁺CCR7⁺cells in this population. The difference in CD25 staining pattern between human and rhesus monkey CD25⁺cells is most likely due to the species difference, but a difference in binding kinetics of the CD25 antibody cannot be excluded. Arbitrary separation of the CD4⁺CD25⁺ population into a CD4⁺CD25^intpopulation and a CD4⁺CD25^highpopulation indicates that both populations express CTLA-4 and FOXP3 at higher levels as compared to CD4⁺CD25⁻ cells, and that highest expression can be found in the CD4⁺CD25^highpopulation. We conclude that rhesus monkey CD4⁺CD25⁺ share phenotypical characteristics with human CD4⁺CD25^highcells, although Th cells may be included in the rhesus monkey CD4⁺CD25⁺population.

Isolation of the CD4⁺CD25⁺cells selectively enriched for the CD4⁺cells expressing CD25 at high levels. Functional analysis of this population revealed that this population was anergic and suppressed the activity of CD4⁺CD25⁻ cells.

CD4⁺CD25⁺ cells did not respond to polyclonal ConA stimulation, plate-bound anti-CD3 or allogeneic stimulation, although individual variation was detected. Fur- thermore, rhesus monkey CD25⁺cells inhibited CD25⁻cells stimulated with plate- bound anti-CD3 or allogeneic cells. Inhibition was demonstrated at the level of proliferation as well as IL-2 and IFN-γ production. The mechanism of suppression de- pended on cell-cell contact and suppression was not inhibited by the addition of antibodies against the suppressive cytokines TGF-β and IL-10. We conclude that rhesus monkey CD4⁺CD25⁺have a comparable functional phenotype as human Tregs [71, 75].

These results warrant further development of the rhesus monkey model as model to study the induction of tolerance by Tregs. The low amount of naturally occurring

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Tregs in the periphery necessitates enlargement of the number of Tregs before therapeutic application of Tregs is feasible. Enlargement of the number of Tregs can be achieved either by induction of Tregs from non-regulatory CD4⁺CD25⁻cells, or by expansion of the naturally occurring Treg population. Knowledge of the phenotype and functional suppressive capacities of naturally occurring Tregs in NHP will guide the evaluation of the phenotype and function of NHP induced or expanded Tregs.

7.6 Induced Tregs in rhesus monkeys

FOXP3 is still the most speciﬁc marker for Tregs. In mice, a clear correlation is seen between the expression of Foxp3 and regulatory function of the Foxp3 expressing cells. In humans FOXP3 cannot be used as such a clear marker for regulation. Al- though human natural Tregs express FOXP3 intracellularly, as in mice, FOXP3 expression is also found in other cell types, such as CD4⁺CD25⁻cells and CD8⁺cells and the correlation with suppressive activity is less clear [57, 76–78]. It has been hypothesized that FOXP3⁺ cells are anergic, but not always suppressive [55–57].

Rhesus monkey CD4⁺CD25⁺ peripheral T-cells also express FOXP3 at high levels, whereas low level FOXP3 expression is detected in CD4⁺CD25⁻cells (chapter 6 and Gansuvd et al. [68]).

Induction of FOXP3 in CD4⁺CD25⁻ cells has been reported after activation of these cells in vitro [56, 57, 77, 79–84]. Activation-induced FOXP3 expression may re- sult in two opposite outcomes. Induced CD4⁺CD25⁺FOXP3⁺cells may be regulatory [80–83] or not [55–57]. Addition of TGF-β may favour the induction of FOXP3⁺ regulatory cells from CD4⁺CD25⁻cells [81, 84, 85].

We have investigated whether rhesus monkey CD4⁺CD25⁻cells stimulated with plate-bound anti-CD3 in the presence of TGF-β convert to FOXP3⁺regulatory cells.

First, CD25⁻ cells were stimulated with plate-bound anti-CD3 in the presence of TGF-β and a lower proliferative response as compared to CD25⁻cells stimulated in the absence of TGF-β was found (Fig. 7.5). This effect was observed at the level of proliferation as well as IFN-γ and IL-10 production.

Subsequently, FOXP3 expression in CD25⁻ cells stimulated with anti-CD3 was measured by FACS. Activated CD25⁻ cells express high levels of FOXP3 as compared to unstimulated CD25⁻ cells. The presence of 1 ng/ml TGF-β did not inﬂu- ence the level of FOXP3 staining (Fig. 7.6).

To investigate whether FOXP3 induction correlated with to the induction of a suppressive phenotype, CD25⁻ cells were again stimulated with plate-bound anti- CD3 in the presence of TGF-β. The resulting CD25⁺FOXP3⁺ cells were added to autologous CD25⁻ cells, thawed after short-term frozen storage. The cells were stimulated with plate-bound anti-CD3 and proliferation was measured. Cultured

’suppressor cells’ were, unlike natural Tregs, not anergic. Furthermore, they could not inhibit the proliferation of the freshly activated CD25⁻ cells, total proliferation was even increased (Fig. 7.7)

These results indicate that cells expressing FOXP3 after in vitro anti-CD3 stimu- lation do not have a regulatory phenotype. In conclusion, rhesus monkey CD25⁻

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Figure 7.5:CD25⁻cells were stimulated with plate-bound anti-CD3 in the presence or absence of TGF-β. Proliferation of CD25⁻ cells was measured by³H-thymidin incorporation during the last 18 hrs of a 5-day culture. IFN-γand IL-10 levels were measured in supernatant taken at day 2 by Elisa.

Figure 7.6: Intracellular FOXP3 expression was measured in unstimulated and in anti-CD3 stimulated CD4⁺CD25⁻cells. Stimulation was performed with or without 1 ng/ml TGF-β. Mean ﬂuorescence intensity (MFI) was determined in the CD25⁻ fraction of unstimulated cells and compared with the MFI of the CD25⁺fraction of stimulated cells.

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Figure 7.7:CD25⁻cells were stimulated with plate-bound anti-CD3 in the presence or absence of TGF-β. After three days of culture, cells were rested for three days in the presence of IL-2 and IL-15. These ’suppressor cells’ were harvested and added to anti-CD3 stimulated cultures of autologous CD25⁻ cells, which were thawed after short-term storage.³H-thymidin incorporation was measured during the last 18 hrs of a 4-day culture.

cells can express FOXP3 after activation, but this does not correspond with the induction of a regulatory population. This is in contrast to the reported induction of Tregs when CD25⁻ cells were stimulated in the presence of TGF-β [81, 84, 85].

Possible explanations for this discrepancy may be that it is a species-speciﬁc effect or that culture conditions may not be optimal. Since the conversion of CD4⁺CD25⁻ cells into regulatory CD4⁺CD25⁺Foxp3⁺cells under the inﬂuence of TGF-β has also been described in mice [86–89], it is unlikely that the effect is absent in NHP. Whether adjustment of culture conditions leads to the induction of Tregs from CD4⁺CD25⁻ cells in rhesus monkeys needs to be further investigated.

7.7 Conclusions and future directions

This thesis describes that costimulation blockade as a monotherapy with antibodies blocking CD40 or CD40 and CD86 prevents graft rejection during treatment, given that antibody levels are sufficiently high, but does not induce tolerance. These results and results of other NHP studies indicate that clinical application of costimulation blockade as a monotherapy is insufficient to induce life-long drug free survival. Pos- sibly, additional costimulation pathways more specific for memory cells, such as the ICOS-ICOSL pathway needs to be blocked. Alternatively, graft survival could be prolonged by targeting memory T-cells by different means.

We tested two strategies to combine costimulation blockade with other immunosuppressive drugs already used in patients for prevention of graft rejection. Addi-

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tion of ATG induction therapy to costimulation blockade by anti-CD40 plus anti- CD86 shortens the rejection free interval, possibly caused by the increased numbers of CD8 memory T-cells appearing in the circulation. Addition of a delayed course of CsA treatment has the potential to induce prolonged drug free survival, although tolerance was not induced.

Analysis of graft inﬁltrating cells reveals clear differences between non-rejected and rejected tissues. A lymphoid like structure for antigen presentation is present in rejected as well as in non-rejected tissues. We questioned whether non-rejected tissues would contain increased numbers of regulatory T-cells. However, in contrast, higher numbers of cells positive for markers of Tregs such as FOXP3, CTLA-4 and CD25 are found in rejected tissues. Furthermore, no evidence was found for increased amounts of Tregs in costimulation blockade treated animals. This is in line with the absence of evidence for costimulation blockade dependent induction of Tregs. A marker that seems to correlate better with absence of rejection after cessation of treatment is latent TGF-β. Positive latent TGF-β staining is found early after withdrawal of treatment, but continues to be expressed in kidney grafts of monkeys that are off immunosuppression over six months.

The failure to reproduce the induction of tolerance through costimulation blockade in primates has steered the search for alternative approaches such as Tregs. Be- fore Treg based treatments can be applied in patients, these treatments need to be evaluated in a relevant preclinical model. We have investigated CD25 expressing cells in the rhesus monkey. We concluded that they are phenotypically and function- ally very similar to human Tregs.

The amount of naturally occurring Tregs in the periphery is too low for direct clinical application, thus the amount of Tregs needs to be enlarged. Several reports describe the induction of Tregs from CD4⁺CD25⁻ cells. This induction of Tregs is enhanced by culturing the cells with TGF-β. Preliminary experiments with rhesus monkey CD4⁺CD25⁻ cells demonstrate that they become CD25⁺ after activation and that they express FOXP3 after activation, similar as in human activated CD25⁻ cells, but this did not correlate with the induction of a regulatory phenotype, as described in humans. The addition of TGF-β to the culture, which led to the induction of regulatory cells in humans, did not induce regulatory cells in these preliminary rhesus monkey cultures.

A second strategy to obtain larger numbers of Tregs is expansion of naturally occurring Tregs. Tregs can be expanded using either polyclonal or antigen-specific stimulation. In addition to expansion of the cells, activated (donor-specific) Tregs are also more effective in suppressing in vitro cell proliferation as compared to fresh Tregs. Highly effective Tregs can be obtained by subsequent selection for specific subsets, such as CD27⁺ or CD127^lowcells. Three groups have reported that natural Treg expansion is feasible in NHP. These findings need to be confirmed and these expanded Tregs will have to be tested in vivo for their suppressive effects, with the aim to bring therapeutic application of Tregs to the clinic.

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