Allogeneic haematopoietic cell transplantation (HCT) establishes a new lymphohaematopoietic system in patients who suffer from severe abnormalities of normal haematopoiesis or immune dysfunction. In the case of malignant disorders of haematopoiesis such as acute lymphoblastic leukaemia (ALL) or acute myeloid leukaemia (AML), the success of HCT critically depends on a graft-vs.-leukaemia (GvL) effect, an immunological reaction in which donor T cells track down and eliminate minimal residual leukaemic cells. HCT creates one of the deepest immunosuppressive states in medicine, sharing many features with naturally occurring states like congenital immune deficiency or human immunodeficiency (HIV) infection. For long it has been known that immune reconstitution (IR) after HCT has to recapitulate immune ontogeny but follows different pathways than nature (1–3). In normal ontogeny, lymphopoiesis begins under protected circumstances in utero, is equipped with a perfectly broad repertoire of naïve T cells at delivery, and continues to mature in early childhood when thymic tissue is most active. In contrast, lymphopoiesis post HCT is happening in an aberrant environment, where the thymus is only partially active, organs are damaged from chemotherapy and inflammation, and the body is strongly exposed to internal and external antigens. Furthermore, immune function has to be suppressed around HCT by serotherapy or immunosuppressive drugs to prevent or treat graft-vs.-host disease (GvHD)—an immune-mediated iatrogenic disorder that is caused by the artificial encounter of two immune systems in one organism. Still, the capacity to reconstitute the immune system through the generation and proliferation of immune effector cells is immense (3), and, if guided and supported by targeted interventions, immunity can be restored within months.

IR is a multidimensional process that is unique and variable among different patients (4). It may depend on the graft source, cell dose, human leukocyte antigen (HLA) barriers, conditioning of the patient prior to HCT and post-transplantation interventions, including those to prevent or treat HCT complications. The multitude of variables that influence IR post HCT have been reviewed before (5, 6) and levels of innate and adaptive immune cell reconstitution in transplanted children over time have been reported (7). The transfused graft, in addition to being a source of haematopoietic stem cells for restoration of haematopoiesis, acts as reservoir of immune cells and initiates the complex process of IR. This process is achieved by two different but complementary waves of immune cell regeneration which are closely interlocked and hard to segregate (illustrated in Figure 1). The first wave is mediated by donor lymphocytes present in the graft. Upon transfusion into a lymphodepleted host, these mature lymphocytes have the capability to expand and proliferate in response to antigenic or cytokine-mediated stimulation in a process termed homeostatic peripheral expansion (HPE), providing an early but incomplete immune defence against invading pathogens. More complete IR relies on de novo lymphopoiesis from donor-derived stem cells in the bone marrow and/or thymus, a process which can take up to several months.

Allogeneic HCT grafts include naïve and memory T-cell subsets of which the ratio may differ tremendously between cord blood (mostly naïve cells) and bone marrow or peripheral blood (which have more memory subsets). T memory stem cells (T_SCM) are of special interest as they show superior reconstitution capacity in preclinical models and contribute to peripheral reconstitution by differentiating into effectors in the early days following haploidentical HCT with post-transplant cyclophosphamide (8, 9). The abundance of naïve T cells in the graft may influence the outcome of patients after allogeneic HCT as long as thymic function has not been restored. Although total numbers of CD4⁺ T cells have been shown to directly correlate with survival post GvHD (10, 11), levels of naïve T cells have been identified as the most potent drivers of alloreactivity (12). In agreement, high levels of CD4⁺ naïve T cells (but not of CD8⁺ T cells) in allografts have been observed to correlate with an increased incidence of acute GvHD (aGvHD) post transplantation (13). These findings led to the initiation of clinical trials using peripheral blood stem cell (PBSC) grafts depleted of naïve T cells, which showed lower rates of aGvHD and chronic GvHD (cGvHD) in HLA-matched HCT, with no apparent increase in relapse rates (14). On the other hand, cord blood grafts (in which almost all T cells are naïve) show great anti-leukaemic potential with reduced relapse risk but a similar likelihood of developing GvHD when compared to bone marrow grafts (15), indicating that T-cell intrinsic factors are contributing to the risk of GvHD development and anti-leukemic efficacy as well. Most significant associations between IR and clinical events were described for CD4⁺ rather than for CD8⁺ T cells, maybe because CD8⁺ T cell numbers fluctuate more swiftly in response to infections (e.g., CMV) or other events post-transplant (16). Still, CD8⁺ T cells numbers have been positively associated with the likelihood to develop GvHD (17, 18), lower relapse rates as well as better overall survival (19). Furthermore, IR of CD8⁺ T cells is highly dependent on the graft type used for transplantation (20): Unmanipulated BM- or PBSC-grafts generally show a more rapid CD8⁺ than CD4⁺ T-cell reconstitution, due to faster homeostatic or antigen-driven expansion of memory-type CD8⁺ T cells. In contrast, after T-cell replete CBT frequently a rapid reappearance of thymus-derived CD4⁺ T cells can be observed (21, 22). However, as IR is influenced by many patient-specific and transplant-related factors, the impact of these patterns on individual outcome is hardly predictable.

Naïve CD4⁺ T cells in particular have been found to undergo HPE and rapidly shift toward a central memory phenotype (22). Although no side-by-side comparisons were made with other graft sources, some authors hypothesised that this CD4⁺ phenotype shift may be a particular characteristic of cord blood T cells (21). In a follow-up study, they showed that the transcription profile of the naïve CD4⁺ T cells from the cord blood grafts overlapped with the profile of foetal CD4⁺ T cells. Likewise, reconstituting cells that were induced in the lymphopenic environment shortly after transplant maintained these overlapping features with foetal CD4⁺ T cells. Interestingly, it was suggested that enhanced T-cell receptor (TCR) signalling via the transcription factor AP-1 after ligation of the TCR with self-major histocompatibility complex molecules was responsible for the rapid T-cell reconstitution (23). As expansion of T cells in lymphopenic situations is affected by the strength of TCR activation (24, 25), a skewing toward cells expressing high-affinity TCRs against host and microbiome-associated antigens during HPE may be observed. Thus, beyond monitoring numbers and phenotypes of T cells after HCT, the epigenetic programming and functional status of reconstituting naïve cells should be studies in more detail.

After an age-dependent recovery period in which HPE prevails, the thymus starts to replenish the naïve T-cell pool with new thymic emigrants. Up to this point, the diversity of the TCR repertoire is limited as new TCR recombination events do not take place in donor T cells undergoing HPE. During the first year after T-cell depleted CD34⁺ haploidentical HCT in children, early reconstituting T cells display a predominantly primed, activated phenotype with a severely skewed TCR repertoire (26). Nevertheless, rapidly expanding cells can differentiate into virus-specific T cells that are able to clear an infection within 2 months, as was shown in patients receiving umbilical cord blood (21). Thymopoiesis includes TCR recombination events and positive and negative selection thereby increasing TCR diversity tremendously (27). Ex vivo evaluation of thymic function is generally performed by molecular analyses of signal joint TCR excision circles (sjTRECs), which strongly correlate with flow cytometric measurements of recent thymic emigrants (CD45RA⁺CD27⁺CD31⁺ T cells) (28). T-cell diversity analysis can be evaluated by spectratyping the size of the β-chain complementarity determining region 3 (CDR3) (29). Nowadays, next-generation sequencing methods allow high-resolution clonotyping providing quantitative TCR assessments that can be applied to better understand clonotype dynamics during viral infections or GvHD (30) and to identify pathogenic or protective T-cell clones following HCT. In addition, screening the TCR repertoire for absence of sequences with annotated specificity for cytomegalovirus (CMV) (the public CMV repertoire) may also help to identify patients at risk for CMV reactivation and disease who may benefit from prophylactic antiviral strategies (31).

An increase in TCR diversity has been related to a better clinical outcome in multiple studies (30, 32–35). Talvensaari et al. studied TCRs in patients who underwent cord blood transplantation (harbouring an intrinsic, broad, polyclonal TCR repertoire) or bone marrow transplantation (36); they showed abnormal TCR repertoires and low TREC values during the first year after transplantation in both groups. After 2 years, TCR diversity was higher in recipients of cord blood vs. bone marrow HCT (34), suggesting a more efficient thymic regeneration pathway from cord blood lymphoid progenitors despite the lower numbers of CD34⁺ cells in the graft. In turn, recipients of unmanipulated bone marrow from matched sibling donors showed increased TCR diversity and faster T-cell reconstitution compared with children receiving selected CD34⁺ PBSCs from unrelated donors (37). In patients receiving T-cell depleted PBSCs from a matched donor or T-cell depleted haploidentical PBSCs in combination with an independent cord blood product, both GvHD and relapse were independently correlated with lower TCR repertoire diversity (35). In addition, within 6 months, adult cord blood recipients had approximately the same TCR diversity as healthy individuals, whereas recipients of T-cell-depleted PBSC grafts had much lower diversities of CD4⁺ and CD8⁺ T cells. Interestingly, these deficiencies improved 12 months post-transplant for the CD4⁺ but not for CD8⁺ T cell compartment (38). Both TCR repertoire diversity and sjTREC levels can decline during GvHD or infections as a reflection of decreased thymic output under these conditions (39, 40).

Analyses of the diversity of the TCR repertoire were mostly based on the TCR Vβ repertoire in TCRαβ⁺ T cells so far. However, it has been demonstrated that reconstitution of the TCRγδ repertoire is an important marker post HCT as well (41, 42). γ/δ T cells constitute up to ~10% of all T cells in blood; they are effective against virus reactivation and their presence is associated with lower relapse rates after HCT (43, 44). In line with this, Vδ2^neg γδ cells isolated from CMV-reactivating patients specifically reacted with both CMV-infected cells as well as leukemic cell lines and primary myeloid leukemic and myeloma cells (45). The interplay between CMV and γδ cell subsets and the result on clinical outcome measures has not been fully elucidated yet (46). In future studies, it would be interesting to assess the predictive value of TCR diversity in specific T-cell subsets with regard to clinical outcomes in more detail, in particular regulatory T (Treg) cells. The latter subset is of special interest as in a murine model adoptively transferred Tregs at the time of HCT accelerated broadening of the TCR Vβ repertoire diversity by preventing GvHD-induced damage in the thymus and secondary lymphoid microenvironment (47).

Given the decisive impact of T-cell IR on survival chances, this issues has to be considered in the design of conditioning regimens. For instance, serotherapy (e.g., with anti-thymocyte globulin; ATG) may reduce the risk of developing GvHD and graft rejection, but dosing should be individualised (based on graft source, absolute lymphocyte count and weight) to prevent dramatically reduced T-cell IR in patients after high ATG exposure (48–51), in particular when given in combination with filgrastim (52).

Factors affecting IR have been actively investigated for almost 30 years now. Besides other contributing factors such as stem cell dose (53), donor age (54, 55), and mixed chimerism (56), patient age at transplantation has been recognised as a prime determinant of the speed and quality of IR from the start of this research (57). The T-cell compartment (both CD4⁺ and CD8⁺) reconstitutes slower in adults than in children, which translates into a higher rate of life-threatening opportunistic infections in older patients. Storek et al. already reported in 1995 that T-cell phenotypes in adult HCT recipients were strikingly different from neonatal T cells and that these changes were more pronounced in the CD4⁺ compartment (58). Numerous later studies confirmed this finding and supported the notion that the second, thymus-dependent wave of T-cell reconstitution is enhanced in children (59). Prediction models of thymic output based on TREC measurements revealed that thymic reconstitution can start as early as 83 days post-transplant in infants and that each additional year of patient age adds 2 weeks to that starting point (60). Interestingly, this advantage of children with regard to improved naïve T-cell regeneration seems to confer protection against viral infections (57), non-relapse mortality and cGvHD (61) but not aGvHD and leukemic relapse, because relapse incidences in paediatric and adult ALL patients after allogeneic HCT are not strikingly different (62, 63). Whether the increased thymic output contributes to a better GvL effect is unknown. However, regeneration of functional Tregs, probably derived from thymic Treg precursors, is a prerequisite for resolution of cGvHD in children (64). Therefore, it is conceivable that the addition of new, potentially leukaemia-reactive clonotypes to the TCR repertoire is counterbalanced by the regeneration of tolerizing Tregs. Mechanistic studies addressing this issue are lacking so far. Furthermore, the precise mechanisms underlying improved thymic reconstitution (increased thymic cellularity, higher susceptibility of thymic precursors to cytokines, or enhanced influx of committed lymphoid progenitors) have not been elucidated so far. Nevertheless, enhancing thymic reconstitution in adults to achieve the same level as that observed in children is a pivotal strategy to boost IR (see below).

Another, less-examined difference between children and adults may be the better preservation of the B-cell bone marrow niche in children. Children show faster reconstitution of total numbers of B cells (56), have more B-cell precursors in regenerating bone marrow (65), and exhibit more B-cell neogenesis as measured by kappa-deleting recombination excision circles than do adults (66). Moreover, cGvHD has been demonstrated to have little impact on B-cell neogenesis and bone marrow precursor composition in children (67), which is in stark contrast to observations in adults (68, 69). Therefore, the microenvironment of the thymus as well as the bone marrow seems to be more resilient to noxious influences such as conditioning regimens and alloreactivity in children compared to in adults. These complex interactions (e.g., regenerating CD4⁺ T cells providing help to transitional and naïve B cells), contribute to facilitate new humoral immune responses (56) and lower production of autoantibodies (67).

In contrast to the aberrant pathways of adaptive immunity regeneration after allogeneic HCT, natural killer (NK)-cell reconstitution after HCT seems to resemble NK-cell ontogeny in early childhood, with a preponderance of immature NK cells in the early post-transplant phase (70). Type of graft manipulation (NK-replete vs. NK-depleted grafts) seems to have a greater impact on NK reconstitution than patient age, although no comparative studies directly addressing this question are available. In a heterogenous cohort of paediatric ALL patients who underwent haploidentical HCT, T-cell depletion techniques that also depleted graft-derived NK cells (i.e., CD34⁺ selection) resulted in faster NK-cell recovery post-transplant than techniques like CD3/CD19-depletion, which keep NK cells in the graft (71), underlining the importance of cytokine sinks such as interleukin (IL)-7 and−15 for NK-cell development (72). Especially in the haploidentical transplant setting, potential NK cell alloreactivity has gained a lot of attention. Differences in the killer-cell immunoglobuline-like (KIR) gene haplotype could lead to a donor NK cell activation caused by the lack of an inhibitory receptor on host leukemic cells. The clinical relevance of this scenario remains controversial. One study analysing 85 children with ALL transplanted with ex vivo T-cell depleted haploidentical PBSCs showed a benefit if the donor had a KIR B content score (5-year event-free survival of 51 vs. 30% in KIR B vs. KIR A haplotype, respectively) (73). However, this was not confirmed in a subsequent study of 80 children with acute leukaemias receiving TCRab/CD19-depleted haplo grafts. Here, KIR-KIR-L mismatching was not associated with any difference in leukaemia-free survival (74). For more details on that issue we refer to one of the excellent reviews published recently (75).

GvHD is a frequent complication of HCT. Although the incidence is lower in paediatric compared to in adult patients, GvHD significantly contributes to transplant-associated morbidity and mortality. It is broadly accepted that aGvHD and cGvHD involve different effectors and targets and have different pathologic pathways, therefore being seen as two different diseases. Nevertheless, aGVHD remains the major risk factor for development of cGvHD in the paediatric population (96, 97). This review focuses on parameters and kinetics of early IR of mainly the adaptive immune system, and therefore this chapter will cover primarily aGVHD.

In general, aGVHD is mediated by alloreactive donor T cells activated by host antigen-presenting cells followed by donor cell reactivity against a variety of target tissues of the host. aGVHD is associated with significantly impaired IR, but which is the cause and which is the effect? This question applies to a number of interacting aspects: (1) the T- and the B-cell compartment, as the antigen-presenting cells involved in aGvHD could be B cells; (2) the number and function of subpopulations of the adaptive immune system (quantity and quality); (3) HPE vs. impaired thymic production; (4) the composition of the graft and the microenvironment of the host; and (5) the effects of aGvHD itself and the administration of immunosuppressive agents for GvHD prophylaxis and treatment.

Immune cell function does not equate to cell number: it is important to distinguish between quantitative immune cell reconstitution and qualitative IR. For instance, T cells often remain dysfunctional after HCT, with a skewed TCR repertoire even after recovery to normal number (98). Hence, the normalisation of B- and T-cell numbers does not necessarily indicate reconstitution of their function and it has been suggested to differentiate between “immune reconstitution” and “immune recovery” rather than using IR alone (99).

Data regarding the influence of aGVHD on IR profiles and vice versa lack detailed information on reconstituting cell subsets and on effector functionality. Moreover, as IR is age dependent, this and other reviews are hampered by the lack of data from a primarily paediatric setting (7). Table 1 provides published data on immune cell subsets in adaptive IR and their relation to aGvHD after HCT in paediatric and adult patients (10, 11, 17–19, 49, 98, 100–110).

Immune attack of donor T cells against residual host leukaemic cells is a major pathway by which allogeneic HCT combats haematological malignancies. In general, a higher number of T cells in the graft is associated with lower relapse rates but at the cost of a higher incidence of GvHD (136). Patients with early recovery of antiviral T-cell responses have a higher probability of relapse-free survival (137), and high numbers of interferon gamma (IFNg)-reactive T cells during early IR have been shown to be associated with improved overall survival (138). However, certainly not all donor T cells contribute to the supposed GvL effect and the involved specific T-cell subpopulations are not known so far.

Since the first reports of the contribution of an immunological GvL effect on the success of HCT in the 1980s (139), the segregation of the GvL effect from GvHD has been considered the “holy grail” of HCT. In the quest to enhance the GvL effect without increasing the risk for GvHD, two general approaches have been studied. The first approach aims to discriminate subpopulations of T cells that can mediate GvL from those that mediate GvHD, thereby enabling a safer and more effective T-cell composition by graft engineering. The second approach tries to define minor histocompatibility antigens that are restricted to the haematopoietic lineage and to elicit specific T-cell responses against these antigens post HCT. Though both approaches have not yet been translated into clinical routine, progress has been achieved and some modalities are currently tested in clinical trials.

Zheng et al. have shown in a murine model of chronic myeloid leukaemia that donor memory CD4⁺ T cells (CD4⁺CD62L⁻CD44⁺CD25⁻) can kill leukaemic cells without causing GvHD, as opposed to the action of naïve cells that cause GvHD (140). The authors speculated that the reason for this difference is that memory T cells can generate only a limited immune response that is sufficient for GvL but not sufficient to cause GvHD (which requires a high-magnitude response and high systemic levels of cytokines to cause tissue invasion and systemic inflammation). The same group has also shown that adoptive transfer of CD8⁺ memory T cells from donors vaccinated against the recipient minor histocompatibility antigen H60 augmented the GvL effect without increasing GvHD (141).

Given these pre-clinical data about the central role of memory T cells in GvL, Triplett et al., conducted a clinical trial (142) in 17 paediatric patients with relapsed/refractory acute leukaemia, performing reduced intensity HCT from haploidentical donors after naïve (CD45RA⁺) T-cell depletion of the graft. At a median follow-up of 223 days, aGvHD rate was acceptable, and there were only two cases of relapse: both of these were in patients with advanced AML in whom primary induction had failed. Interestingly, nine patients had detectable disease at time of transplant, yet relapse rates were still low, highlighting the potential of memory T cells to mediate GvL effect. A second trial using transfer of CD45RA-depleted T cells in 35 patients with high-risk acute leukaemia confirmed low rates of cGvHD (9% with a follow-up of 932 days). aGvHD rates were similar to T-replete HCTs (66%; 95% CI 41–74%) but all cases of aGvHD were steroid responsive and no patient required second line treatment. Overall survival was 78% at 2 years, which is encouraging in this high-risk population (14). These data suggest that CD45RA⁻ memory T cells are not devoid of any GvHD potential; however, GvHD seems more controllable for this type of HCT. The combination of CD45RA with other surface antigens such as CD276 as a depletion marker can confer superior protection against GvHD initiation (143).

Potential targets for donor T cells in the HCT setting are any polymorphic proteins of the host against which donor T cells have not been tolerized during their education in the host thymus. Recent molecular analyses have revealed that 12% of the human exome is polymorphic but only 0.5% of all single nucleotide variants (SNVs) are finally presented as HLA class I peptides (144). From this huge number of possible antigens, about 50 candidates have been biologically validated as bona fide minor histocompatibility antigens with relevance for HCT (145). Early mechanistic studies revealed that T-cell responses against minor histocompatibility antigens are oligoclonal in nature and CD4⁺ dominated (146, 147); one or a few minor histocompatibility antigen mismatches can be sufficient to cause GvHD and massive thymic infiltration (148). Disappointingly, a closer look at donor–host minor histocompatibility antigen disparities has not allowed clear separation between GvL and GvHD so far. The UGT2B17 truncating gene deletion has been shown to lead to increased incidence of aGvHD and reduced survival in HCT recipients (149), while HA-8 and ACC-1 SNVs in the recipient have been associated with an increased incidence of cGvHD (150). In a large retrospective analysis, Spierings et al. (151) investigated in 849 HLA-matched HCTs the impact of 10 autosomal and 10 HY-encoded minor histocompatibility antigens on GvHD and relapse incidence. Their most striking observation was a lower relapse rate and higher overall survival in patients mismatched for haematopoiesis-restricted minor histocompatibility antigens compared to patients who were matched in these antigens. Notably, this association was only given in the context of GvHD (not without).

The introduction of immune checkpoint inhibitors as an efficient method of immune-based anti-cancer therapy made its use in the context of allo HCT an intriguing way to augment the GvL effect. Pilot reports about patients with Hodgkin disease who relapsed post allo HCT have shown that this modality can be effective, though carrying the risk of occurrence of de novo GVHD (152). Davids et al. have prospectively treated 28 adult patients with relapsed haematological malignancies post HCT with aCTLA4 blockade and other 28 adult patients with aPD-1 blockade (153, 154). While some responses were noted (more in lymphoid diseases and some complete responses in extramedullary myeloid leukaemia), severe GvHD and other serious immune-mediated adverse events occurred in a significant proportion of patients. Of note only a single ALL patient was included in these studies. Further and more homogenous studies are required to better characterise patients in whom the potential benefit of immune checkpoint blockade overrides its risks. Noteworthy, in a study of 85 patients after allo HCT for various haematologic malignancies, LAG-3 and TIM-3 rather than PD-1 were overexpressed on T-cells of relapsing patients, indicating that other exhaustion markers beyond the PD-1-PD-L1 axis might be interesting and druggable targets to enhance GvL after allo HCT (127).

In summary, these data indicate that natural IR will most likely not distinguish between GvHD and GvL effects. However, adoptive transfer of minor-antigen-directed T cells, the generation of which is challenging but feasible (155, 156), in a T-cell depleted setting should be the subject of further research. Another approach to skew IR toward preferred regeneration of minor-antigen-specific T cells is the vaccination of the recipient with minor-peptide-loaded dendritic cells in combination with donor lymphocyte infusions (DLIs) (157). Given the very tight association between GvL and GvHD, a clearer separation of these two effects will only be possible by controlling IR through tailored grafts and targeted add-back of TCR specificities, e.g., antiviral T cells in the first 2–3 months to avoid or control viral reactivations followed by adoptive transfer of donor T cells reactive against leukaemic epitopes.

Studies about the reconstitution kinetics of different cellular subsets after HCT have revealed important insights about the basic principles of this treatment. They helped us to understand the artificial immune ontology after HCT as well as the pathophysiology of GvHD, viral reactivation and other transplant-related complications. By continuous efforts to dissect the phenomenon of alloreactivity, IR studies have opened the door to understand the GvL effect, at least in part. In recent years, research on IR has evolved from merely descriptive studies into a highly dynamic and innovative field which actively shapes the future design of HCT. Novel insights have fostered the continuous evolution of T-cell-depletion techniques to a level by which HCTs employing this method now yield comparable results to T-replete HCTs. Clinical trials over the coming years will show whether adoptive transfer of memory DLI, veto T_CM cells or selective allodepletion approaches will give superior results. Strategies of restoring thymic cellularity by soluble factors, targeted influx of committed lymphoid progenitors or tissue engineering not only intend to lift IR kinetics of adult patients to that of an infant but will beyond that impact on ageing research since thymic involution is considered a major contributor of immune senescence.

Future studies on IR will aim to develop more precise prediction models for complications such as GvHD, viral disease or relapse. To this end, multifactorial models of IR will have to take the complex interactions around HCT into account and include not only lymphocyte subset numbers but also other factors such as graft type, graft manipulation, HLA disparity and minor histocompatibility differences. The first examples of such multidimensional IR analyses have already been published (71, 82, 132). Control over IR with targeted interventions in a timely orchestrated fashion will help to reduce transplant-related morbidity and mortality and improve GvHD-free, relapse-free survival.

AY, AS, AL, SN, and ME contributed specific chapters to this review article. All authors contributed to the article and approved the submitted version.

This study received funding from the St. Anna Children's Cancer Research Institute, Vienna, Austria. The funders were not involved in the study design, collection, analysis, interpretation of data, the writing of this article, or the decision to submit it for publication.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.