THERAPEUTIC
STRATEGIES
DRUG DISCOVERY
TODAY
Allogeneic hematopoietic stem cell transplantation for malignant disease:
How to prevent graft-versus-host disease without jeopardizing the graft-versus-tumor effect?
Philippe Lewalle, Re´douane Rouas, Philippe Martiat *
Laboratory of Experimental Hematology, Institut Jules Bordet, 121, Boulevard de Waterloo, University of Brussels, 1000 Brussels, Belgium
The graft-versus leukemia (GVL) effect, closely related to allogeneic stem cell tranplantation, plays a crucial role in curing the disease but is often associated with a deleterious reaction, called graft-versus-host disease (GVHD). So far, most strategies aiming at reducing GVHD also diminish the GVL effect. We try here to give an overview of strategies that could reduce GVHD without affecting GVL, or augment GVL without increasing GVHD or achieve both at the same time.
Section Editors:
Claudine Bruck – GlaxoSmithKline, King of Prussia, USA Michel Goldman – University of Brussels, Brussels, Belgium
Introduction
The graft-versus leukemia effect (GVL), closely related to the postallogeneic stem cell transplant (allo-SCT) immune recon- stitution, plays a crucial role in the improvement of disease- free survival, as demonstrated by T-cell depletion [1] and reduced intensity conditioning (RIC) [2]. Therefore, it is important to analyze how the immune system recovers after transplant, to design how it can be manipulated to enhance this GVL effect without increasing the deleterious graft-ver- sus-host disease (GVHD), also closely related to this immune reconstitution. Different strategies have been investigated to reach this goal, but so far, many of them diminish both
GVHD and GVL effects. In this survey, we will try to give an overview of strategies aiming at reducing GVHD without affecting GVL, at augmenting the GVL effect without incre- asing the risk of GVHD and perspectives to achieve both at the same time.
Lessons from autologous vaccines
In vitro, most of the tumors express tumor-associated antigens recognizable by the immune system, [3–7] but cancer patients lack an effective immune response [8]. This could be due to a defect caused by the tumor cells, or by the microenvironment that plays an important role in affecting the balance between immune surveillance and tolerance induction [9].
The idea that a patient’s anti-cancer immune response is inhibited by tumor-induced regulatory or suppressor T-cells has recently encountered a renewed interest [10]. In vitro recognition of tumor epitopes by high-avidity T-cells does not warrant theirin vivoefficiency [11,12]. The administration of anti-tumor T lymphocytes is more effective at inducing tumor regression after immunosuppressive chemotherapy [13]. The immune response inhibition is largely mediated Editors-in-Chief
Raymond Baker– formerly University of Southampton, UK and Merck Sharp & Dohme, UK Eliot Ohlstein– GlaxoSmithKline, USA
Immunological disorders and autoimmunity
*Corresponding author:P. Martiat (pmartiat@ulb.ac.be)
by the CD4+/CD25+ regulatory T-cells (Treg) [14]. Unfortu- nately, this picture is further complicated by the interaction of immature dendritic cells (DC) with Treg and naive T-cells, which might cause T-cell depletion, anergy and the induc- tion of other subsets of Treg cells which secrete IL-10 and TGF-Beta [15–17]. The tumor can also affect myelopoiesis by inhibiting the differentiation and maturation of antigen- presenting cells. The blocked immature myeloid precursors promote tumor evasion from immune attack [18,19]. Two important conclusions from autologous situations are the following: in the right environment, the immune system is capable of eradicating malignancies and allotransplantion is a unique chance to create this environment. Second, if an anti-tumor immune reaction is demonstrated in autologous situations, then GVHD and GVL effects could be separated.
Those exciting new observations provide new opportunities in allo-SCT translational research.
Post allo-SCT immune reconstitution
Immune deficiency can persist for years after allo-SCT.
Reconstitution of the T-lymphocyte compartment follows two distinct pathways: rapid expansion of post-thymic, mainly memory cells, from the graft and later, thymus- dependent, regeneration of naive cells derived from pre- thymic donor stem cells [20,21]. Donor T-cells are clearly implicated in the pathogenesis of GVHD and GVL [22-24]. In humans, CD8+ T-cells are associated with GVHD and GVL, whereas the role of CD4+ cells is less clear and more depen- dent on the naive or memory subsets. From mice studies, it
seems that donor-memory CD4+ T-cells facilitate post-tran- plant immunological reconstitution without causing GVHD, whereas donor naive CD4+ T-cells can lead to GVHD.
Clinical application of these results must also take in account the difference in the kinetic recovery of the CD4+ and the CD8+ T-cells. Although CD4+ T-cells reconstitution is slow, CD8+ T-cells counts are close to normal values at 3 months.
After allo-SCT, the increase in TCR rearrangement excision circles (TRECs) (monitoring thymic output and naive T-cells number), occurs between 6 and 12 months, suggesting that the naive T-cell population involved in GVHD depends on the naive T-cell population from the graft [25].
Although GVHD is more related to naive T-cells than memory T-cells, there is no direct correlation between TRECs level at an early time point after transplant and development of GVHD, suggesting that only a small fraction percentage of them are alloreactive and able to provoke GVHD. Anti-tumor CTL against some tumor antigens can be detected in the central memory and effector memory subsets of donor’s lymphocytes, showing that memory cells are crucial in the GVL effect; nevertheless, some specific CTL are only detected after transplant and not in healthy donors, suggesting a GVL role for the naive subset of the T-cells from the graft [26].
Furthermore, initiation of GVL effect is also dependent on a complex series of bi-directional interactions between DC and cells of the innate immune system, including NK and NKT-cells [27].
The clinical significance of donor NK-CELLSengraftment is not well defined because T-cells from the graft affect Nk-cells recovery and their alloreactive therapeutic potential [28]. Nk- cells and T-cells with inhibitory NK receptors (KIRs) have been shown to possess a suppressive activity against GVHD while preserving the GVL effect. Early establishment of donor NK-cell chimerism in RIC transplant is associated with a lower risk of relapse without increasing acute GVHD. Because Nk-cells are negatively regulated by MHC class I-specific inhibitory receptors, in mismatched transplants, failure to recognize the appropriate HLA class-I ligand, triggers NK-cell alloreactivity. Recently, it has been shown that HLA class-I ligand incompatibility in the GVH direction is associated with a better outcome in haplo-identical allo-SCT, when following profound T-cell depletion, in AML [29]. Because KIR genotypes in Nk-cells are very diverse in the population, the donor KIR genotype can also affect the GVL effect in the related HLA-identical allo-SCT setting [30]. The presence of two activating KIRs, 2DS1 and 2DS2, in the donor is signifi- cantly associated with a decreased leukemic relapse rate suggesting that a joint effect of these two selected activating KIRs in the donor confers some protection against relapse [31]. A higher number of B-cells in the graft is also associated with a decreased risk for relapse but with a greater occurence of cGVHD [32]. In RIC transplants, where full chimerism is not immediately achieved, the mixed chimerism status after Glossary
GVHD:deleterious effect of immune cells contained in the graft, directed against either minor histocompatibility or major complexes (in case of mismatched tranpsplant) resulting in injury to host’s non leukemic cells (mainly skin, GI tract and biliary endothelium) that can lead to host’s death. According to its severity, can be treated successfully, but the usual therapeutics, beside their side-effects, wipe out the GVL effect.
GVL:anti-leukemic effect of immune cells contained in the graft, directed against either minor histocompatibility restricted to host’s hematopoiesis or true leukemia associated antigens, towards which the donor is not tolerized.
Nk-cells:part of the innate (non antigen specific) immunity. One of their major role is to destroy foreign cells that do not share self HLA class I antigens. Especially efficient against hematopoietic cells, their role in NK mismatched situation seem to enhance GVL, without increasing GVHD (partly because they destroy hosts’ APC).
Regulatory T-cells:family of T-cells (CD4+ CD25+, TR1, TH3) that can anergize cytotoxic reactions. Among them, CD4+ CD25+ are possibly antigen-specific and could therefore be used to separate GVL from GVHD.
TH1/TC1 T-cells:T-cells involved in cytotoxic reactions, responsible for both acute GVHD and GVL effect.
TH2/TC2 T-cells:T-cells, normally involved in defence against parasits, thought to be involved in chronic GVHD. These cells, if prominent are not tolerizing, but rather lead to a chronic toxicity similar to auto-immune disease.
the allo-SCT has an impact on GVH and GVL. In patients above 30, mixed chimerism is related to relapse, although this is not true in younger patients, indicating that mixed chi- merism is an important prognostic factor in older patients.
All these results indicate that the immune recovery of the differenT-cells subsets can be used and manipulated to mod- ulate GVHD and GVL in clinical settings.
Immune interventions in allogeneic stem cell transplantation
T-cell depletion (TCD)
The first attempts at reducing GVHD were based onin vitroor in vivo T-cell depletion. But, with the exception of haplo- type-mismatched donor (where Nk-cells were found to play a major role), removal of mature donor T-cells prevents GVHD, but at the expense of an increased relapse rate. In the end, there was no benefit in terms of DFS. Attempts have been made to keep the benefit of TCD on the GVHD inci- dence but without the loss of GVL effect, the first of which was to carefully add back increasing dose of T-cells after the initial procedure [33]. In TCD transplant, naive CD4+ T-cells count is low until one year after transplant although the total CD4+ T-cell count, memory CD4+ T-cell count, CD8+
T-cell count and Nk-cell count are only different until Day 45 [34]. Nevertheless, T-cell diversity tested by spectratyping showed that RIC and unmanipulated transplant patients regenerate an almost complete TCR repertoire within the first 3 months although TCD transplant patient showed a skewed repertoire even on day 100 despite donor lympho- cytes infusion (DLI) (107CD3+ T-cells/kg at D45) [25,35–39].
The results show that the repertoire diversity depends on the number, as a reflect of the repertoire diversity, of the infused cells. This shows the enormous potential of even a small T- cells inoculum to fill an empty peripheral lymphocyte com- partment. This is why, in non TCD transplant, GVHD risk is determined not by the total dose of T-cells received as add- back but by the number of T-cells given at the time of transplant (greater or less than 105/kg) and in the end TCD with DLI on days 45–60 did not show more efficacy than the infusion of unmanipulated stem cells. This led to more sophisticated methods of T-cells depletion.
Future trends
Selective or semi-selective depletion
CD8+ T-cells depletions, sometimes followed by add-backs This methods seems promising, but, so far, in most of these studies, the comparison is made with historical controls, and importantly, the results are contradictory, possibly related to the intensity of CD8+ depletion. One point that these studies have confirmed is that this procedure seems to preserve the GVL effect, but the situation is less clear in terms of GVHD [40,41]. Further studies, focusing on the level of CD8+ depletion, and, ideally randomized, are
necessary before one can have a definitive judgment on this procedure.
Allo-reactive lymphocytes specific depletion
This technique relies on co-cultivating host target cells with donor T-cells, and destroying allo-reactive donor T-cells, using mainly an anti-CD25 antibody coupled with an immu- notoxin [42,43]. This approach has proven feasible, but its results less convincing in separating GVHD from GVL. There are two possible drawbacks using this methods: first, the host cells that are usually used as targets are PBMCs. These cells are not the usual target of GVHD and, moreover, might express useful mHAgs restricted to the hematopoietic lineage. The consequence could be a lack of depletion in allo-reactive T- cells directed against skin, gut or biliary tract, and the destruc- tion of T-cells directed at hematopoietic mHAgs, useful for exerting a GVL effect. The second potential drawback is the removal of one subset of T-regulatory cells (CD4+, CD25+) that has been shown to dramatically increase GVHD in murine models.
A potential way of circumventing the first drawback could be the use of other target cells (keratinocytes for example) and to avoid the second one, to use a technique that would preserve T-reg cells. Such techniques are now emerging, such as the use of the photodynamic cell purging (PDP) process and of sorting memory T-cells for the selective elimination of alloreactivity [44]. Rhodamine purging is one method, which selectively reduces allo-reactive T-cells preventing GVHD [45].
Total depletion with add-backs of genetically engineered donor lymphocytes
The genetic engineering of donor lymphocytes with the herpes simplex virus-thymidine kinase (HSV-TK) suicide gene confers the ability to modulate GVHD byin vivoganciclovir- induced elimination of the transduced cells. The suicide-gene strategy has applications in both donor lymphocyte infusion (DLI) for disease relapse and in add-back infusions after T-cell depleted allo-SCT. TK cell DLI resulted in anti-tumor activity in a relevant proportion of treated patients [46].
Specific immunotherapy, adoptive (T-cells) or active (DCs) In postallograft immunotherapy, timing is crucial, the win- dow for efficient T-cell immunotherapy being, before appear- ance of Treg-inhibiting effective immune cytotoxic response, at a time homeostatic expansion favors the T-cells infused.
Because early DLI (earlier than day 45) also increase GVHD, specific CTL immunotherapy must take place early after transplantation.
The same leukemia-associated epitopes, recognized by class I and class II cytotoxic T-cells in autologous situations, can be targeted after allogenic-SCT. More specifically, mHAgs play a major role in graft rejection, GVHD and GVL [47,48]. Their
tissue distribution plays a significant role in the clinical out- come. Recognition by T-cells of mHAgs specifically expressed in hematopoietic cells, including the malignant cells might result in GVL reactivity without concurrent GVHD.In vitro generation of T-cell lines against these mHAgs, or leukemia associated antigens can improve efficacy and specificity of anti-leukemic allogeneic immunotherapy.
The use of cytokines, like IL-7 and IL-15 can improve survival and expansion of specific T-cells. Their usein vitro could overcome the GVHD risk associated with their usein vivo. T-cells can cure tumors, but we must leave the old fashioned concept that the immune system, once turned on, continues to fight until the antigen is gone. It will do so only until the danger signal is gone, and leukemic cells do not send enough alarm signals [49].
Expanding anti-tumor lymphocytes in vitro is a step towards effective treatment, but each T-cell will only kill a limited number of tumor cells before it needs to be reacti- vated. To eradicate a tumor, we should cause it repeated damage to alert local APCs or vaccinate repeatedly with a tumor vaccine that stimulates patient immunity again and again. The concept of specific anti-leukemic TCR introduc- tion into donor T-cells has also recently emerged [50].
Infusion of donor’s T-regulatory cells
It might be possible to diminish the incidence of GVHD, without affecting GVL by using the intrinsic regulatory capa- city of the transplanted immune system, as represented by T- cells subpopulations with suppressive activity, such as CD4+
CD25+REGULATORY T-CELLS[51]. In mouse models, these cells have been shown to suppress GVHD while preserving the GVL effect. In these same models, depleting the graft from Treg cells dramatically increases the incidence of GVHD.
Their application in allogeneic SCT might soon be explored in clinical trials. A possible advantage of this strategy is that it is unlikely that donor Treg contain cells tolerizing to leuke- mic antigens, which are absent in the donor.
Donor vaccination
As mentioned above, the autologous immune system of the patient can be tolerized. This should not be the case in donor cells and thus allows to contemplate vaccination of the donor against leukemic antigens expressed in patients. Several methods could be used: DC vaccination, whole or fragment of relevant proteins with adjuvant. Again, this will need further investigation with regard to inocuity for the donor and increased frequency of anti-leukemic T-cells in the graft.
Infusion of Nk-cells or refined choice of the donor based on Nk-cells characterization
Understanding the mechanisms that determine KIR expres- sion might allow novel therapeutic strategies based first on better donor selection and second on donor purified Nk-cells
infusions to exploit Nk-cells alloreactivity [52]. Considering the interaction between T-cells and Nk-cells, NK therapies must be applied in the context of deep TCD. Donor allo- reactive Nk-cells transfusion, early post-HSCT, can also reduce GVHD by destroying patient residual hematopoietic cells (APCs, myeloid cells, lymphocytes).
A recent study of NK-cell recovery in AML patients who received haplo-mismatched SCT, among whom no GVL effect was observed, despite GVH NK alloreactivity, showed that, in these patients, Nk-cells generated after SCT exhibited an immature phenotype. The impaired lysis of the leukemic blasts is correlated with CD94/NKG2A expression in Nk-cells.
Blockade of CD94/NKG2A restored lysis against the blasts.
This shows that Nk-cells, generated even after haplo-mis- matched SCT can be blocked in an immature state with impaired function [53]. In this context, immunotherapy with carefully purified and IL-2 activated, intentionally mis- matched Nk-cells, could overcome killing inhibition, and be a practical approach to induce GVL while avoiding GVHD.
The GVL effect of Nk-cells supports the use of TCD that would not use antibody killing Nk-cells (anti-CD2), but rather anti-CD3, and support strategies based on early infusion of selected (activated) Nk-cells together with the graft.
NKT-cells
A lower number of NKT-cells can be found in patients with cancer or severe GVHD.
When activated, NKT-cells release suppressive cytokines (IL-4, IL-10, and IL-13), as well as inflammatory cytokines, (INFG, TNFa). NKT-cells therefore act as a double-edged sword in their progressive or suppressive effects. Furthermore, activation of NKT-cells followed by activation of APCs and IL- 12 production can lead to activation of Nk-cells. Control and modification of NKT-cell function can play a role in regulat- ing GVHD/GVL effects [54].
Mesenchymal stem cell
Recent studies suggest that mesenchymal stem cells (MSC) play an important role in the leukemia microenvironment.
MSC are not immunogenic, have immunomodulatory capa- cities, and appear to escape the immune system [55]. In co- culture experiments, MSC failed to elicit a proliferative response from allogeneic lymphocytes and this inhibition appears to include both naive and memory T-cells [56,57].
MSC have many effects on both T-cells and APCs, indicating a complex interplay between MSC, T-cells, APC and inflamma- tory cells [58–61]. Co-transplantation of culture-expanded MSCs and allogeneic stem cells has recently been proposed to facilitate engraftment and lessen graft-versus-host disease.
Recombinant cytokines and cytokines antagonists
Cytokines can influence immune responses, and as such, characterization of their roles in the complexity of cellular-
microenvironment cross-talk provides clinicians with tools for modulating immune outcome.
G-CSF and analogs (pegylated or conjugated to FLT3-L), GM-CSF, IL-11, IL-18, TNF-a antagonists, TGF-b inhibitor have shown, mainly in mice models, properties to partially separate GVHD from GVL [27,62–67]. Although already giving some rationale for separating GVHD from GVL, the use of cytokines or cytokines antagonists in human HSCT still needs optimization. A better understanding of post- HSCT immunity dynamics should allow better or combined use of these approaches, in a perhaps more sequential and/or time dependent use.
Blocking non-specific inflammatory process
The skin, intestinal tract, and liver are the three major targets of GVHD. The damage inflicted to these organs, epithelial and endothelial cells in particular, by the conditioning regi- men, release various cytokines and lead to penetration of endotoxin into the systemic circulation. Thus, non-immuno- logic aspects of GVHD are, probably, due to non-specific inflammatory processes. Blocking them could decrease GVHD without interfering with the GVL reaction [68]. A substantial gain could be achieved by decreasing organ damage and endotoxin release. In animal models, KGF has been shown to decrease GVHD, without affecting GVL, possibly by decreas- ing GI necrosis due to the conditioning regimen [69]. Further- more, there is growing evidence that the mechanisms involved in GVHD can differ from organ to organ (for example, Fas/Fas- ligand interactions in the liver versus tumor necrosis factor alpha/receptor interactions in the intestinal tract), and from a therapeutic point of view, the time of onset of clinical GVHD can be important in choosing the appropriate therapy. Thus, combinations of interventions, appropriately timed, can be more effective in preventing GVHD than the standard across- the-board approaches that have been used so far.
Conclusion
The previous decade has brought much progress, in terms of increasing the number of patients candidate for allogeneic transplant, further specifying their indications and increas- ing our therapeutic tools to treat GVHD and infectious com- plications of immune suppression. The next decade will teach us how to better distinguish GVL from GVHD. Some of these studies are in progress at the clinical level, some are about to start and other still need further laboratory work. Neverthe- less, the amount of data accumulated so far opens a promis- ing era, in which classical allo-SCT will be regarded in ten years as a middle-aged approach.
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