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Molecular basis oftransplantation 403

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indications for allogeneic HCT, and thus promoted the use of DLI in several diseases. Similarly, the use of T- cell depleting conditioning regimens to reduce the risk of developing early, severe acute GvHD may be tempered by using later postHCT pre- emptive or prophylactic DLI to provide T- cell addback that may protect against relapse. The rate of response after reduced intensity allograft in patients with high- risk acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) has been reported to be comparable with the results obtained using standard intensity conditioning regimens. Durable responses, that exceed the response durations seen both after conventional transplantation and following previous chemotherapy cycles, were reported in a small cohort of patients with Hodgkin lymphoma and in lowgrade non- Hodgkin lymphomas. Initial enthusiasm with multiple myeloma has been subsequently disappointing, since it has become apparent that responses can be achieved only in association with severe GvHD. It is possible that higher doses of donor lymphocyte are necessary, but then they would be incompatible with the side effects.

The reason for the different sensitivity to DLI among various malignancies remains unclear. A possible explanation for low susceptibility to DLI of acute lymphoblastic leukemia has been attributed to the limited duration and magnitude of leukemia- specific T- cell responses generated in vivo, which has been attributed to clonal exhaustion. As widely documented by studies on solid cancers, several mechanisms implicated in tumor evasion from immune surveillance can explain the resistance to graft- versus- tumor responses.

Furthermore, prevention is likely to be more efficacious than therapy when considering how best to apply DLI, at least in the context of active disease, in the case of AML and MDS, conditions which may rapidly overwhelm and/or evade the alloreactive response. Prophylactic or pre- emptive DLI are the most routinely used treatments post- HCT aiming to boost GvL activity. Prophylactic DLI is given regardless of recipient chimerism and has been reported in small studies to be effective at preventing leukemic relapse, although there is a significant risk of provoking GvHD. Selective use of DLI as a pre-

emptive strategy in patients deemed at higher risk of relapse due to falling recipient donor chimerism may minimize the chances of inducing significant GvHD. These two strategies have been compared in the PRO- DLI clinical trial, the results of which are eagerly awaited.

Strategies to limit the induction of GvHD in the context of DLI have included using lower starting doses for unrelated versus sibling pairs, fractionating rather than administering DLI as a bulk dose and not initiating treatment with DLI until at least six months post- HCT. As such, the likelihood of inducing GvHD following DLI has been reported to range between 20% and 60% depending on the strategy used. Delaying initiation of DLI beyond six months post- HCT may lower the likelihood of inducing GvHD, through

avoidance of infusing T- cells into the early post- HCT inflammatory microenvironment. However, this also risks the rapid kinetics of the underlying disease, such as AML (more likely to relapse within 6–12 months of transplantation), overwhelming the patient before DLI can exert its protective effects. Alternative approaches have included the use of CD8+ T- cell depleted DLI or selectively depleting in vitro recipient- reactive donor T- cells before their infusion. Limited success has been seen so far with these approaches and current practice continues to employ the use of unmanipulated DLI. Similarly, naïve T- cell depleted peripheral blood stem cell grafts have been studied in early phase trials enrolling patients with acute leukemia/MDS, demonstrating very low rates of cGvHD, and although rates of aGvHD were still substantial, the vast majority of these were grade 2in severity with a low incidence of Grade 3 or higher aGvHD.

An important component for the induction of GvL reactions is fully functional antigen presentation. There is emerging evidence that host dendritic cells (DCs) of leukemic origins may be harnessed in this process by inducing the generation of antileukemic cytotoxic T- cells in invitro and in vivo pre- clinical models. Early efforts to translate this approach in the clinical setting seem encouraging. The principle has been adopted by exploiting the ability of cytarabine and azacytidine to induce the maturation of leukemic blasts into DCs and the consequent provision of antigenic and accessory stimulation of donor naïve T- cells. There are reports of complete hematological remissions in AML patients relapsed after HCT by using a combination of low- dose cytarabine, G- CSF- mobilized donor blood cells, and GM- CSF post- transplant or combination of azacytidine and DLI. The efficacy of such a strategy has been associated with the differentiation of central memory T- cells and the induction of a selective, GvHD- sparing, anti- leukemia effect. An additional beneficial effect of azacytidine is the ability of this hypomethylating agent to increase expression of tumor- associated antigens (TAAS) (specifically cancer testis antigens) on leukemic blasts, rendering the tumor more immunogenic. This effect may then synergize with the co-

administration of DLI to

boost induction of GvL.

Another cell subset involved in the elimination of residual leukemic cells is NK cells. NK are fundamental players of the innate immunity. Their killing activity is tightly tuned by the balance of activating and inhibitory signals transmitted by the interaction of different receptors on the surface of NKcells such as killer- cell immunoglobulin- like receptors (KIRs), NK Group2member D (NKG2D) or NKG2A/CD94 and the corresponding ligands on the target cell membrane. NK- cells lyse target cells that lack MHC class I molecules specific to the inhibitory receptors KIRs (“missing- self hypothesis”). Therefore, NK cytotoxicity can be particularly important in HLA- mismatched or haploidentical donorrecipient pairs. Importantly, since NK- cells are the first immune population to recover after HCT, they are also the

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first cell subset to recognize normal and malignant allogeneic targets. The involvement of “alloreactive” NK- cells in GvL, suggested in pre- clinical models, has been corroborated in clinical studies whereby the transplantation of haplo- identical NK- “alloreactive” donors in high- risk AML patients has been shown to prevent disease relapse and improve survival. Accordingly, a slow NK recovery after HCT has been correlated with a higher risk of relapse. Furthermore, it has been suggested by some groups that cytomegalovirus reactivation following allogeneic HCT may trigger expansion of particular NK- cell subpopulations that may also be protective against relapse of selected hematological malignancies, such as AML, even in fully MHCmatched donor- recipient pairs.

Are tumor- specific T- cells generated after allografting?

There is evidence that the immune system can mount a response against a tumor by recognizing antigens that are either specific or associated with the tumor. Tumor- specific antigens (TSAs) include antigenic peptides generated by genetic mutations specific to an individual cancer. TAAs come from normal non- polymorphic proteins overexpressed or aberrantly expressed in the tumor.

Almost all patients with CML harbor a specific molecular abnormality, the reciprocal chromosome translocation t(9;22)(q34;q11) which juxtaposes BCR and ABL genes. The fusion gene encodes for a chimeric protein, namely p210

BCR- ABL

. Peptides derived from p210

BCR- ABL

can be presented by both MHC class I and class II molecules on CML cells in vitro and p210

BCR- ABL

specific CD8+ T- cells have been detected in the peripheral blood of CML patients. However, it is unclear whether these T- cells have any purpose in the control of leukemia. Several years ago, earlyphase clinical trials using p210

BCR- ABL

vaccination have shown some encouraging results. In patients with CML receiving a peptide-

based vaccination, the development of bcr- abl peptide- specific immune responses was associated with clinical responses and/or further reduction of residual disease. However, the limitation of HLA- class I restriction for the immunogenic peptides and the lower incidence of disease in these haplotypes have questioned the feasibility of such an approach.

Other non- polymorphic proteins, such as proteinase- 3 (Pr3) and Wilm’s tumor 1 (WT1) have also been investigated as targets for T- cells in CML and other myeloid malignancies, including AML and MDS, because of their high expression levels in these diseases. Very low frequency of Pr3- or WT1- specific T- cells have been identified in patients with CML in remission after allogeneic HCT, but the findings have never been confirmed and their significance not supported by proper clinical trials.

Several other tumor-

associated and TSAs, such as preferentially expressed antigen in melanoma (PRAME), melanoma family antigen, receptor for hyaluron- mediated motility, or NewYork esophageal squamous cell carcinoma- 1 cancer- testis antigen (NY3ESO) are often expressed in leukemic blasts. Cytotoxic T- cells specific for the cognate antigen have been isolated from the blood and bone marrow of healthy donors as well as from non- transplanted AML, ALL, and CML patients, indicating that the alloreactivity posttransplant is more prominent than leukemia- reactive immune responses. The presence of higher titers of PRAME- specific CD8+ T- cells after HCT has been correlated with a higher probability of longer remission. However, most immune reactivities against any of these antigens traced after DLI are weak. This is likely due to the fact that these antigens comprise naturally expressed “self” peptides, therefore potent T- cells recognizing them would usually have been deleted during thymic selection. The results so far obtained with the use of TAA- specific T- cells or TAA- directed cancer vaccines as immunotherapy have yielded some interesting observations with respect to evidence of invitro anti- leukemic responses, although these have not clearly translated into demonstrable clinical benefit. However, NPM1- mutated AML represents a potentially interesting immunotherapeutic target. This mutation generates a novel “neoantigen” within the leukemic cells that differs from the natural protein. NPM1- specific T- cells have been observed to emerge following DLI in recipients and can be generated following invitro culture of either healthy donor or patient peripheral blood mononuclear cells. These observations pave the way for future studies that could combine immunotherapeutic modalities, e.g. administration of DLI alongside NPM1 peptide vaccination, to prevent relapse in patients at high risk of recurrence post- transplant.

Graft- versus- host disease

GvHD is the main drawback of allogeneic SCT and is largely responsible for non- relapse mortality. In relation to the time of onset, GvHD can be classified as acute or chronic, depending on whether it develops within 100

days from the transplant. However, although there are clearly two types of diseases in terms of symptoms, organ involvement, and pathological changes, there is not necessarily a temporal correlation, with “overlap” cases increasingly recognized, presenting with distinctive clinical features attributable to both disorders. The uncertainty in the clinical classification is the result of the poor understanding of the pathogenesis of the disease and lack of standardized biomarkers.

Acute GvHD (aGvHD)

Acute GvHD is a potentially lethal disease that usually targets skin, liver, and the gastrointestinal tract. The typical pathological finding in the skin is the epithelial apoptotic

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damage with characteristic nuclear alterations and a small inflammatory infiltrate. In the liver, the first injury involves vascular endothelial cells and later the biliary epithelium is destroyed as a result of portal lymphocytic infiltration. In the gastrointestinal tract, there is destruction of intestinal crypts with acute or chronic inflammatory infiltrates and flattening of the villous architecture. The clinical score is graded at 4levels, with grade 4 being very often lethal.

The cellular effectors of alloreactivity after HCT are cytotoxic T- cells and NK- cells, depending on the histoincompatibility of the donor–recipient pair. Cytotoxic T- cells are activated CD8+ T- cells that recognize their specific antigen bound to MHC class I molecules on the target cells. They exert their functions through two main contact- dependent cytolytic pathways: Fas/Fas- ligand (Fas–FasL, CD95- CD178) mediated apoptosis and the release of cytotoxic granules containing perforin/granzyme. Studies performed in mice genetically deficient for these molecules showed a prominent but not exclusive role of the perforin/granzyme pathway in GvHD induced organ damage. In contrast, the same pathway appears to be critical for the GvL effect, thus suggesting the possibility to selectively impair the alternative pathways to inhibit GvHD without compromising GvL. In fact, during experimental and clinical GvHD, FasL expression on donor T- cells is increased, elevated serum levels of soluble FasL and Fas are correlated with the severity of GvHD and the inhibition of Fas–FasL pathway markedly reduces GvHD of the liver in animal models.

NK- cells mediate direct cytotoxicity with the same pathways of cytotoxic lymphocytes albeit recognizing different targets. Experimental studies have shown that NK- cells are neither necessary nor sufficient to induce GvHD, but in the setting wherein GvHD is already underway, NK- cells contribute to the overall outcome. The effect of donor NKmediated cytotoxicity invivo, in the absence of alloreactive T- cells is shown to be confined to recipient lymphohematopoietic cells. Although the mechanism responsible for such a selectivity is unclear, it has been hypothesized that donor “alloreactive” NK-

cells could prevent GvHD manifestations by killing of host DCs thus avoiding alloantigen presentation to donor T- cells.

Chronic GvHD (cGvHD)

Chronic GvHD is a syndrome characterized by multi- organ involvement, with clinical similarity to an autoimmune disorder. It may involve skin, liver, gastrointestinal, joints, and respiratory mucosa among other targets, but several immunological functions are impaired. The incidence of cGvHD ranges from 6% to 80% mainly depending on the degree of disparity in the major histocompatibility antigen and the previous appearance of acute GvHD. Pathological findings are not typical and characterized by epithelial atrophy,

increased hyaline deposits, fibrosis, and chronic inflammatory infiltrates. An internationally agreed scoring system is used to grade the severity of cGvHD based on clinical parameters. Doing so allows determination of mortality risk from cGvHD, guides therapeutic approach, allows monitoring of response to treatment and standardizes reporting to clinical trials.

The pathogenesis of GvHD

Acute GvHD and the “cytokine storm”

Donor T- cells recognizing major and/or mHag disparities on recipient tissues are certainly critical in the induction of aGvHD. However, other factors need to be considered. In particular, the incidence of aGvHD has been reported to be much higher if allogeneic donor lymphocytes are administered in concomitance with the conditioning regimen than if they are infused a few months later in the form of DLI. The reason for this is that the injuries produced by cytotoxic agents and infections at the time of transplant activate the innate immune system and induce pro- inflammatory changes in endothelial and epithelial cells. This process, described as the “cytokine storm,” consists of three main phases: (i) the destruction of natural barriers by the conditioning regimens (first phase), (ii) the activation of DC and presentation of alloantigens (second phase), and (iii) the recruitment and proliferation of activated effector cells (third phase).

In the first phase, the destruction of natural barriers by the conditioning regimens and the release of inflammatory cytokines, like TNF- α and IL- 1, allows bacterial products to permeate in the tissues. The T- cells exposed to bacterial products like lipopolysaccharide exhibit enhanced migration and survival. In accord with this notion, the use of lowintensity preparatory regimens substantially reduces the incidence of acute GvHD. There is increasing evidence that lower diversity of the microbiota in the gut at the time of the HCT is associated with higher incidence of GvHD rates and mortality. Studies in human and mice indicate that active GvHD is associated with an imbalance in favor of specific bacterium phyla with a predominance of Enterococci. Furthermore, the use of specific antibiotic associations active against anaerobic bacteria, such as imipenempiperacillin- tazobactam seem to correlate with higher GvHD grades and mortality. Conversely, the use of antibiotics with less perturbative effects on the gut microbiota (such as rifaximin) was associated with a lower incidence of gut GvHD and GvHD- related mortality.

Studies of microbiota composition highlight that its role in GvHD development is not merely related to the release of bacterial particles. In fact, microbiota seem to actively participate in the generation of a specific environment, which can be

cilastatin and

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pro- or anti- GvHD through the production of metabolites. Short- chain fatty acids, for example, stimulate the extrathymic maturation of T

and one of these acids (butyric

regs

acid) can reduce the production of pro- inflammatory cytokines while inducing the production of indoleamine 2,3- dioxygenase (IDO), which can ameliorate GvHD. More recently, it has been suggested that the composition and metabolism of the gut microbiome prior to GvHD onset may even affect specific organ involvement with GvHD as well as being predictive of GvHD- related patient outcome. Based on these findings, several approaches have been tested in clinical trials with the aim of restoring the diversity of the microbiota by the use of probiotics or specific combinations of antibiotics for a decontamination of the gut at the HCT. Increasingly, the use of fecal microbiota transplantation is being investigated in early- phase clinical trials for the prevention and management of gut GvHD. The rationale of these approaches is very intriguing, but further large- scale clinical trials are needed before this approach enters routine clinical practice.

The second step in the cytokine storm involves the activation of DCs and the presentation of the alloantigens. The inflammatory changes promoted in recipient tissues by conditioning regimens can initiate DC maturation and license them to potently induce T- cell responses. While host APCs are necessary and sufficient for GvHD development, the role of donor APC appears confined to the intensification of ongoing disease. The differential role of donor and recipient APC can be accounted by the fact that endogenously synthesized mHag- derived peptides are presented to CD8+T- cells on host MHC class I (induction phase). Subsequently, the same antigens can be processed by donor APC through the exogenous route of antigen processing and presented to the T- cells, thus accounting for augmenting the GvHD initiated by host APC (maintenance phase). This two- step process is similar to that described for the rejection of solid organ transplants (direct and indirect alloresponses).

For its full development, GvHD finally requires cellular effectors to be recruited. Although activated donor T-

and NK- cells affect the killing of host cells by contact- dependent cytotoxicity, the release of inflammatory cytokines also plays an important role. In accordance, target organ injury can be partially prevented by the neutralization of TNF- α and IL- 1in animal models and the blockade of TNF- α is of some efficacy in pilot clinical studies. The importance of multiple inflammatory effectors in GvHD suggests that more than one pro- inflammatory cytokine should be simultaneously inhibited to effectively antagonize systemic GvHD. The case of IL- 6 blockade can well explain the complexity of the matter. IL- 6 is a cytokine usually associated with proinflammatory function, but it also plays an important role in controlling local and systemic inflammation. This dual activity depends on the fact that IL- 6 can transmit signals through alternative pathways that can target different subsets of

CD4+ cells. While the small proportion of CD4+ cells expressing IL6R is subject to a suppressive signal, the vast majority respond to IL-

6 linked to a soluble form of IL- 6R present in the serum and that drives an inflammatory response. Blockade of IL- 6 signaling has been successfully used in murine models of GvHD, but its efficacy in patients seems to be confined to its prophylactic use.

Acute GvHD and T- cell trafficking

Although essentially all tissues express transplantation antigens, clinical manifestations of acute GvHD display remarkable tissue tropism involving primarily gut, liver, and skin. Recent studies have suggested that the skewed organ involvement is related to the homing properties of activated allogeneic T- cells and the presence of local tissue inflammation.

Naïve T- cells injected in lethally irradiated allogeneic murine recipients are initially retained within secondary lymphoid tissues where, activated by recipient- derived APC, they undergo a rapid burst of proliferation, enter the peripheral circulation, and subsequently accumulate in the gut, liver, and skin. However, local inflammation also appears crucial in permitting the entry of activated T- cells to target tissues, because the infusion of alloreactive T- cells produces GvHD in irradiated recipients but not in recipients in whom mixed hemopoietic chimerism has already been established, despite similar levels of alloreactive T- cells in the periphery. To confirm the role of local inflammation, GvHD can also be induced in mixed chimeric mice if inflammatory stimuli are administered together with the donor T- cell transfer.

Chronic GvHD

The pathogenesis of chronic GvHD largely remains to be elucidated. Mature donor alloreactive T- cells infused with the graft appear to play a key role also in chronic GvHD. Similarly, APCs have been shown to be important although, in contrast to acute GvHD, the development of the chronic form is dependent on the APC of donor rather than recipient origin, thus justifying the possibility of a broader repertoire of antigens and more widespread organ involvement.

However, one of the most remarkable features of chronic GvHD is the clinical and pathological similarities with systemic autoimmune disease. Elevated levels of serum autoantibodies (i.e. antinuclear, anti- dsDNA, anti- smooth muscle antibodies) in up to 70% of chronic GvHD patients support the hypothesis that functionally relevant autoreactive T- and B- cells can be generated during chronic GvHD. It has been suggested that antibodies to ubiquitously expressed mHags may be analogous to pathogenic autoantibodies in systemic autoimmune diseases, thus justifying the similarities of tissue pathology. The importance of B- cell responses in chronic GvHD was documented by clinical improvement following B- cell depletion with anti- CD20monoclonal antibody.

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DLI cells

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Studies in animal models have been carried out with the aim of investigating the autoimmune manifestations of cGvHD. Using an HCT model in which donor and recipient differed for multiple mHags, it was possible to isolate from mice developing chronic GvHD, autoreactive CD4+ T- cells with specificity for a MHC class II encoded determinant common to donor and recipient. Moreover, donor CD4+ T- cells isolated from recipients with chronic GvHD cause autoimmune disease if transferred to new syngeneic recipients. The development of donor T- cells that recognize antigens shared by donor and host has also been attributed to impaired mechanisms of deletion or regulation during chronic GvHD. It has been hypothesized that autoreactive T- cells may be the consequence of a damaged recipient thymus, unable to select the new T- cell repertoire following the pre- transplant therapy or acute GvHD. However, it seems that also thymectomized animals can develop GvHD.

1. Reduce the dose

2. Deplete CD8*/ Enrich CD4*

Not dissimilarly from the acute disease, cytokine dysregulation has a causative role in chronic GvHD. High levels of IL- 1β, IL- 6, IFN- γ, TNF- α, and low levels of the inhibitory cytokine IL- 10 are associated with more severe forms. Multiple cytokines produced by activated T- cells, such as INF- γ and TGF- β were shown to promote the increased collagen deposition in preclinical models. Other soluble mediators, such as the CCL2 and CCL3 chemokines seem to modulate collagen turnover and deposition by sending signals to fibroblast via macrophages or indirectly through stimulation of TGF- β.

Can GvHD and GvL be dissected?

The “holy grail” of allogeneic HCT is to separate the beneficial GvL effect from unwanted GvHD (summarized in Figure26.3). As yet, how to do so remains elusive, despite the best efforts of the transplant community.

Figure26.3 Strategies to separate GvHD from GvL.

5. Th1 Th2 shift

7. Block cytokines

TNFα, IFNγ IL-2, IL-6 JAK Inhibitors

+ IL-15/IL-2 + BiKEs

8. NK cells infusion

9. DLI in association with differentiating factors

3. Remove alloreactive T cells (suicideinducible gene)

4. Induce anergy to alloantigens (co-stimulation, T

6. Selection of leukaemia-specic clones

Leukaemic blasts

)

reg

Haemopoieticrestricted

minor Ags

Cytotoxic pathways

(FAS vs perforin)

+ Cytarabine + Azacytidine

Ags

Leukaemic APCs

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408 Molecular Hematology

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Prevention and treatment of GvHD

Current standard global practice involves the use of at least two treatment modalities within transplant preparative regimen to prevent induction of GvHD. These would comprise a combination of either a calcineurin inhibitor, post- transplant methotrexate, mycophenolate mofetil, or a lymphodepleting agent such as alemtuzumab (anti- CD52) or anti- thymocyte globulin (ATG). While some data have suggested increased relapse risk following the use of lymphodepletion, this may be countered by the judicious application of pre- emptive/ prophylactic DLI (see section The role of Donor Lymphocytes in induction of GvL).

The use of post- transplant cyclophosphamide (PTCy) to diminish the activity of rapidly proliferating alloreactive T- cells early following infusion of haploidentical stem cells has effectively reduced the incidence of acute and chronic GvHD in patients lacking conventional donors. As such, this GvHD prophylaxis platform has seen a rise in popularity and is being compared to standard GvHD prevention strategies even in fully MHC- matched transplants. The rationale for such an approach includes avoidance of the prolonged immunosuppression and delayed immune recovery observed with agents such as alemtuzumab or anti- thymocyte globulin used for GvHD prophylaxis. Studies of immune reconstitution have highlighted differential kinetics of immune subset recovery following PTCy compared to conventional GvHD prophylaxis, including higher numbers of T

cells and myeloid- derived suppressor cells. What remains

reg

to be understood are the implications of this differential immune signature following PTCy on GvL.

Alternative approaches to diminish GvHD have been based on the use of donor T- cells genetically engineered with suicide genes that can be activated in case of GvHD. Such a strategy allows donor T- cells to be safely exploited for immune reconstitution and the GvL effect, with the option of switching them off in case of GvHD. The positive experience with the gene encoding herpes simplex virus thymidine kinase

(HSV-

TK) is being confirmed by the use of caspase 9 transgene. While the former approach relies on the administration of the antiviral drug ganciclovir to induce necrosis in transduced donor T- cells, the second is based on the druginduced activation of the intrinsic apoptotic pathway. The results of new clinical trials are keenly awaited.

Based on the observation that some of the cytokines involved in GvHD development act through the STA3/JAK2, JAK1, or JAK3 pathways, it is not surprising that the availability of JAK inhibitors has paved the way to testing these reagents in the management of GvHD. The JAK2 inhibitor ruxolitinib has demonstrated significant clinical efficacy in the treatment of steroid- refractory acute and chronic GvHD and is routinely used now for this indication with newer generation JAK/STAT pathway inhibitors entering clinical trials.

Anti- IL6 antibodies have also been tested both as a prophylactic agent and for the treatment of acute GvHD with interesting results. The addition of anti-

IL6 antibodies to the standard GvHD prophylaxis produced a significant reduction of both grade 2–4 and more severe grade 3–4in a phase 1/2 trial. More recently, the same drug has been tested to treat steroid- resistant GvHD with prompt and durable responses in about one- third of the patients.

As technological advances in T- cell engineering spiral, demonstrated by the explosion of chimeric antigen TCR therapy (CAR T) for treatment of lymphoma and myeloma, application of CAR T to prevention of GvHD and induction of GvL has appeared on the horizon. Preclinical studies for example have demonstrated the feasibility of an OX40directed T- cell co- expressing a CD19 CAR to boost GvL while limiting GvHD induction through OX40 blockade.

Active immune regulation of donor/recipient reactive cells is emerging as a key mechanism for inducing and maintaining tolerance to alloantigens (summarized in Figure26.4). T

are probably the main player in this process and we have

regs

previously elaborated on this concept.

Treg cells MSCs MDSCs

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↓ Tissue damage ↓ GvHD severity ↓ GvHD mortality

No effect on GvL

Figure26.4 Cell- based immunosuppression in HSCT.

Molecular basis oftransplantation 409

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Initial studies have suggested that T

isolated from

regs

umbilical cord blood and expanded invitro or freshly isolated from adult peripheral blood may have a positive impact on GvHD prophylaxis. In both cases, there was a reduction in the development of GvHD in comparison with historical controls and the treatment was well tolerated. However, T

reg

treated patients exhibited a higher frequency of opportunistic infections than controls.

Mesenchymal stromal cells (MSCs) have been explored as therapy for GvHD given their ability to modulate immune responses and virtually absent. These cells exhibit a potent immunosuppressive activity invitro and invivo, which targets virtually any cells of the immune system. A number of Phase I–II clinical trials have been conducted to evaluate the use of MSCs to prevent or treat usually steroid- refractory GvHD with variability in reported outcomes, although some success has been experienced. The heterogeneous clinical results are likely to result from the poor knowledge of the mechanisms underlying immunosuppression and the lack of criteria to stratify patients for treatment with MSC. Recent studies have shown that MSC must undergo apoptosis to be effective and that this apoptosis is induced by the recipient cytotoxic cells responsible for GvHD. Apoptotic MSCs are efferocytozed by phagocytes that are then reprogrammed to become immunosuppressive after upregulating indoleamine- 2,3 dioxygenase. This observation, confirmed by subsequent studies, can provide a new perspective to stratify patients for treatment and refine the use of this therapeutic tool. Preliminary evidence shows that only patients harboring activated cytotoxic cells, responsible for inducing MSC apoptosis, are the ones who respond to treatment.

Survivin, and NY-

ESO) derived from the original HCT donor showed invitro evidence of induced responses to these TSAs post- infusion alongside clinical responses albeit at early follow- up.

Further selectivity could be achieved by concentrating on lineage- specific polymorphisms as demonstrated for B cell malignancies (Figure26.5). A promising approach can be the production of effector CD4+ T- cells specific for mHags presented in the context of HLA- class II molecules, which are mainly expressed on cells of the hematopoietic system. However, limitations to this approach are represented by the observation that non- hematopoietic precursors are induced to express HLA- class II peptides in the presence of inflammation. Furthermore, quiescent leukemic stem cells do not express HLA- class II epitopes.

The selective infusion of “CD4- enriched DLI” would also induce the reversal of host anti- leukemic CD8+ cells with exhausted phenotype by “awakening” dormant endogenous GvL effector cells. The use of CD4+ selected DLI has been associated with a high incidence of durable responses and a low rate of GvHD. Responding patients exhibited a significant down- regulation of exhaustion- related genes in their bone marrow CD8+ T- cells, with intriguing data suggesting that the exhaustion profiles of these cells prior to DLI may predict for responsiveness to therapeutic DLI. These approaches could also benefit with the association with drugs able to block the immunological checkpoints, thus further acting against the immunosuppressive environment of the tumor, although induction of GvHD remains a concern in this setting.

Induction of selective GvL

Selective induction of GvL using unmanipulated DLI remains problematic, not least since the numbers and avidity of T- cells within the infused product that selectively promote GvL through recognition of mHags, TAAs, and/or TSAs expressed on tumor cells is small. It has been shown that patients with evidence of GvL but not GvHD following DLI show more restricted diversity of mHag-

directed CD8+ T- cell responses compared with those who developed GvHD. Furthermore, these CD8+ T- cells were less reactive against non- hematopoietic cells even in the presence of inflammatory stimuli. It is conceivable that modulating the inflammatory microenvironment in the recipient at the time of DLI could skew more toward a selective GvL response, although this is yet to be evaluated prospectively. When considering TAA/TSAs as targets, induction of a polyclonal response may be considered beneficial to avoid immune escape through downregulation of the cognate antigen. Some progress toward separation of GvL from GvH has been made by using selected DLI that target multiple TAAs. A Phase I study of multispecific DLI (targeting PRAME, WT1,

GVHDGVHD

Normal

tissues

Polymorphisms expressed on all normal and malignant tissues

Haemopoietic-restricted polymorphisms

Figure26.5 Minor histocompatibility antigens in GvHD and GvL.

failure

Normal

haemopoietic tissues

Alloreactive donor T cellAlloreactive donor T cell

GVLGVL

LeukaemiaLeukaemia

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410 Molecular Hematology

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Long- term responses can be achieved with more aggressive strategies based on the use of pre- emptive or prophylactic DLI in association with differentiating factors able to induce the maturation of DCs from leukemic blasts and the stimulation of naïve donor lymphocytes. Promising is also the combination of DLI with other therapeutic agents like lenalidomide and sorafenib, although the mechanistic bases of their interaction are still poorly understood. Clinical trial data demonstrate that combining DLI and azacytidine can be an effective treatment strategy for AML/MDS recurrence post- HCT. The potential for demethylation to increase the expression of TAAs that render leukemic blasts more immunogenic to infused donor lymphocytes provides a supportive scientific rationale to the observed clinical responses.

Much research is still needed to improve the therapeutic efficacy of these procedures and to further extend their use in the clinical practice. It is conceivable that in the near future, a combination of strategies will become the most successful approach to maximize the GvL effect after HCT.

New directions

The evidence that NK- cells can produce a potent antileukemic effect with minimal risk of GvHD makes such an approach ideal. However, significant limitations are represented by the relatively small number of NK- cells obtained with leukapheresis and limited understanding of how to maintain NK therapeutic activity after their exvivo expansion. The administration of recombinant IL- 2 and IL- 15has been used in patients after NK infusions with a view of augmenting their number and activation invivo.

The use of bispecific killer engagers (BiKEs) has been recently tested in the clinic. BiKEs are small molecules made by 2 antibody regions of different specificity, the first specific for a TAA/TSA and the second CD16a- spcific, thus crosslinking NK- cells to a specific target cells. Such approaches have been tested for the treatment of MDS and AML using a combination of CD16a and CD33.

More recently, the use of chimeric antigen receptor Thas gained increasing use within routine clinical practice with a number of commercially licensed products in use across Europe and the United States. While toxicity may now be considered manageable, antigen escape and lack of persistence hinder long- term disease control. In malignancies where there is a lack of TSAs, identifying suitable antigens without offtumor, on- target toxicity may be difficult. Efforts to improve the efficacy of these products continue with advances in CAR T- cell technology and manufacturing. These advances include but are not limited to multi- targeting CAR T- cells, gating strategies and “armoring” CAR T- cells to overcome the immunosuppressive tumor microenvironment.

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Conclusions

HCT is a potent clinical methodology which, by leveraging on both recipient and donor immune systems, generates long- term immunological tolerance and anti- tumor immunity. Despite the limitations and toxicities of the procedure, the many successes of HCT in malignant and non- malignant conditions have provided a unique platform for the development of new cellular therapies that are currently being tested in other ailments.