Greater than the sum of their parts: Combination strategies for immune regeneration following allogeneic hematopoietic stem cell transplantation

Abstract

Cytoreductive conditioning regimes designed to allow for successful allogeneic hematopoietic stem cell transplantation (allo-HSCT) paradoxically are also detrimental to recovery of the immune system in general but lymphopoiesis in particular. Post-transplant immune depletion is particularly striking within the T cell compartment which is exquisitely sensitive to negative regulation, evidenced by the profound decline in thymic function with age. As a consequence, regeneration of the immune system remains a significant unmet clinical need. Over the past decade studies have revealed several promising therapeutic strategies to address ineffective lymphopoiesis and post-transplant immune deficiency. These include the use of cytokines such as IL-7, IL-12 and IL-15; growth factors and hormones like keratinocyte growth factor (KGF), insulin-like growth factor (IGF)-1 and growth hormone (GH); adoptive transfer of ex vivo-generated precursor T cells (preT) and sex steroid ablation (SSA). Moreover, recently several novel approaches have been proposed to generate whole thymii ex vivo using stem cell technologies and bioscaffolds. Increasingly, however, when transferred to the clinic, these strategies alone are not sufficient to restore thymopoiesis in all patients leading to the potential of combination strategies as a way to reign in non-responders. Synergistic enhancement in combination may be due to differential targets may therefore be effective in improving clinical outcomes in the transplant settings as well as in other lymphopenic states induced by high dose chemotherapy/radiation therapy or HIV, and may also be useful in improving responses to vaccination and augmenting anti-tumor immunotherapy.

The importance for immune regeneration

Allogeneic hematopoietic stem cell transplant (allo-HSCT) is a potentially curative therapy for leukemia patients and others with hematological malignancies. Its use, however, is restricted by several major complications, including graft versus host disease (GVHD), malignant relapse and post-transplant immune deficiency. Following transplantation, recipients can suffer a period of prolonged immune deficiency, especially devastating to the T cell compartment, which leads to an increase in opportunistic infections and higher treatment-associated morbidity and mortality due to the extended period of time that it takes to recover from such insult (1). Furthermore, one of the most significant physiological problems underlying a broad spectrum of diseases is the well-recognised progressive decline in immune competence with age. This is manifest at many levels including increased opportunistic infections, incidence and burden of cancer and, somewhat paradoxically, autoimmune disease. There is also a markedly reduced capacity to respond to overt vaccines but of paramount importance is the very poor recovery from immune insults such as common cancer cytoreductive treatments of chemotherapy and radiotherapy, particularly when in combination with allo-HSCT. This delay in immune recovery can precipitate high morbidity, and often mortality, from opportunistic infections and may even facilitate cancer relapse. In fact, the risk of opportunistic infection in the post-transplant period is directly correlated to the recovery of T cells. The severity and duration of this immune deficiency is influenced by several factors such as previous chemo- or radiation- therapy, GVHD and donor/host incompatibility, however, there is a clear inverse relationship between transplant recipient age and the capacity to generate T lymphocytes. Therefore, strategies to enhance T cell recovery could significantly improve the outcome of allo-HSCT.

Optimal T cell development requires a functional thymus and many patients who could benefit from enhancing immune regeneration after allo-HSCT have poor thymic function due to their age or their exposure to chemo- and radiation therapy. Thymopoiesis is a complex process involving the cross-talk between developing thymocytes and the non-hematopoietic supporting stromal microenvironment (2). Moreover, thymic involution caused by age or exposure to chemo- and radiation therapy impacts on both developing thymocytes and the thymic stromal compartment (3). Consequently, regeneration of intrathymic hematopoietic and stromal compartments is of critical importance for sustained and T cell production following immunodepletion.

Immune Degeneration: aetiology and implications

Treatment with chemotherapy or radiation therapy results in the severe depletion of all hematopoietic cells of the immune system. Both alkylating chemotherapeutics and irradiation target highly proliferative cells (4–6), including developing and naïve lymphocytes making them particularly depleted following treatment (7). Delayed recovery following immunodepletion is associated with a high degree of morbidity and mortality (7–9). Lymphoid recovery is critically dependent on functioning primary immune organs. In particular CD4⁺ T cells are dependent on a functioning thymus for recovery (10). Compared to children, adults whose thymii have already involuted, are significantly impaired in their ability to recover following chemotherapy (11–13). Interestingly, while CD8⁺ T cell recovery in both young and aged patients is quite rapid (14), these are predominantly extrathymically derived by clonal expansion (15, 16). While delayed, full immune recovery is possible up until middle age, however, in older patients the peripheral naïve T cell receptor repertoire is never fully restored (17). Similarly, recovery of B cells and NK cells are also severely impaired in aged compared to young patients (17). Furthermore, much like the TCR repertoire, the B cell repertoire is severely diminished after chemotherapy and suffers a prolonged recovery (18).

Interestingly while quiescent HSCs are largely numerically spared from many chemotherapeutic and low-dose radiation regimes (19), their hematopoietic function appears to be significantly impaired (20, 21). Importantly this was also shown with human CD34⁺ HSCs (22) suggesting that the standard clinical practice of collecting autologous HSCs in remission, oftentimes after prolonged chemotherapy treatment, may lead to poorer transplant outcomes. Furthermore, immunodepletion from radiation or chemotherapy was shown to cause enhanced senescence in HSCs that was coupled with an upregulation in the cyclin-dependent kinase inhibitors p19^Arf and p16^ink4A (23, 24) mimicking some of the effects seen with age.

The profound involution of the thymus with age is one of the most widely studied effects of age on the immune system (25–27). While the proportion of thymocyte subsets remains unaffected with age, there is a profound decline in the supply of BM and intrathymic lymphoid progenitors (28, 29) and a significant disruption to the thymic architecture (30–34). This results in a decline in the emigration of naïve T cells into the periphery (35) and a consequent homeostatic expansion of pre-existing peripheral memory T cells (36, 37). Taken together, this results in a decline in the peripheral T cell receptor repertoire (38) and subsequently a reduced responsiveness to both new and previously encountered antigens (39, 40). Age-related thymic involution has profound implications for designing regenerative strategies after immunodepleting therapies as a functioning thymus is crucial to the recovery of T cells following immunodepletion (41).

Toolkit for Immune Regeneration

Previous studies have demonstrated several novel treatments with beneficial regenerative effects on the thymus, including the administration of growth factors such as keratinocyte growth factor (KGF), IL-7 and fms-like tyrosine kinase 3 ligand (Flt3L), withdrawal of sex steroids and adoptive transfer of in vitro generated precursor T (pre-T) cells (31, 34, 42–50). Administration of growth factors to aid in thymic regeneration specifically targets either intrathymic hematopoietic cells (IL-7 and Flt3L) or the stromal microenvironment (KGF). Rejuvenation of either compartment leads to a commensurate regeneration within the other, suggesting that absolute thymic renewal can be achieved by promoting either lymphoid or stromal expansion.

Cytokines and Hormones

Several exogenously administered cytokines and hormones have been nominated for their potential to regenerate lymphopoiesis. Keratinocyte Growth Factor (KGF), IL-7, Flt-3 Ligand (Flt3L) and Growth Hormone (GH) have all shown promise in their regenerative abilities (1, 51–53).

Exogenous administration of KGF has been found to increase thymic cellularity up to four fold in the aged and following radiation or chemotherapy (42, 54, 55) and significantly enhances response to plasmid tumor vaccines (56). Furthermore, KGF can actually protect TECs from GVHD mediated thymic damage (55) and KGF-induced thymopoiesis is mediated by proliferation and expansion of TECs (57). The pro-lymphopoietic cytokine IL-7, which has long been recognised for its role in steady-state lymphopoiesis (58, 59), has also been studied for its potential in enhancing immune regeneration. Several studies have demonstrated the beneficial effects of exogenous administration of IL7 which enhances thymopoiesis and recent thymic emigrants as well as aiding peripheral T cell function in aged mice or following allogeneic BMT (43, 60–63). The mechanism behind IL7 induced thymic regeneration lies in its ability to reverse age-related increases in apoptosis (64), while simultaneously enhancing the proliferation (43) of lymphocytes and lymphoid precursors. Administration of Flt3L enhances both thymic dependent and independent T cell reconstitution (48, 65). The effects of Flt3L are predominantly due to an expansion in Flt3⁺ progenitors in the BM (66). However, increases in T cell reconstitution can be at the expense of B-lymphopoiesis which is significantly declined with exogenous Flt3L administration and, in particular, its effects on early progenitors with both lymphoid and myeloid potential (67, 68). Use of growth hormone (GH) has also been proposed as a possible regenerative therapy. Treatment with exogenous GH regenerates the aged thymus (27, 69) and enhances HPC function in the BM (70). GH has also been shown to reverse irradiation-associated loss of BM function determined by colony formation (70).

Several other cytokines and growth factors have been evaluated for a beneficial role in regenerating the immune system. These include IGF-1, which promotes TEC expansion and enhances reconstitution following HSCT (71–73); IL-15, which predominantly promotes proliferation of circulating NK and T cells (74, 75); and IL-12, which stimulates thymic expression of IL-7 and enhances hematopoietic engraftment after transplant (76–78), although IL-12 and IL-15 have also been recently found to also act on regulatory lymphoid-tissue inducer cells and NK cells (79, 80).

Sex steroid withdrawal

As sex steroids have been implicated in the degeneration of BM and thymic lymphopoiesis (81, 82), sex steroid ablation (SSA), which can be achieved in a reversible chemical fashion (83, 84), has been investigated for its potential in enhancing the immune system. Studies have found that removal of sex steroids leads to reorganised thymic architecture, an enhanced ability to import circulating progenitors (85) and subsequently enhances thymopoiesis in aged mice and humans (31, 34, 45, 50, 85–89). The effects of SSA, however, are not restricted to the thymus with enhanced B-lymphopoiesis and lymphoid progenitors (29, 90–94)) also observed, as well as enhancing overall immune recovery following autologous (95) and allogeneic (44) BMT as well as cytoablative therapy (29, 50, 89). Taken together these studies indicate the wide-ranging implications on immune recovery following SSA and provide evidence for clinical application of SSA in treatments where immune depletion is an unavoidable side effect.

Precursor Cell Therapies and Artificial Organs

The length of time to recovery following HSCT is at least in part due to the length of time required to develop and mature from HSC to functional T cell. While early strategies seeking to fill the void in T cell development after transplant employed isolation and co-transplant of BM-derived lymphoid precursor cells (96), the advent of the robust OP9-DL1 in vitro T cell development system, in which large numbers of highly purified T cell precursors can be generated by incubating hematopoietic stem cells with the bone marrow stromal cell line OP9, transduced with the Notch ligand Delta-like 1 (OP9-DL1), has meant that even more mature T cell precursors can be used to offer a ready supply of T cell precursors well before they develop from HSCs. Adoptive transfer of OP9-DL1-derived T cell precursors into lethally irradiated allo-HSCT recipients caused significant increases in thymic cellularity and chimerism, as well as enhanced peripheral T and NK cell reconstitution compared with recipients of allogeneic hematopoietic stem cells (HSCs) only (46, 97). In addition to their significant benefit for immune regeneration following transplant, in vitro generated pre-T cells can also be genetically engineered for tumor-specificity and subsequently used for targeted tumor immunotherapy (47). However, the impact of pre-T cell treatment on the supporting stromal microenvironment will be of critical importance in promoting long-term thymic regeneration.

Consequently, in addition to providing a supply of T cell precursors, several groups are attempting to identify and isolate populations of thymic epithelial progenitor cells (TEPC) that could be used to directly enhance the function of the thymus by providing regenerative benefit to the supporting stromal microenvironment. While using different strategies TEPC have been successfully isolated from fetal thymii and coaxed into generating a new thymus in FoxN1^−/− recipients (98–101), the identity and existence of a similar population in the adult thymus has thus far remained elusive (102, 103). Recently, however, in a glimpse of the possibilities and potential of iPS technologies, a recent study has directed the reprogramming of thymic epithelial cells into functional multipotent skin stem cells (104). A reverse of this approach, by reprogramming skin epithelium into TEPC, would offer an excellent opportunity to either regenerate the thymus by grafting TEPC directly or even ex vivo generation of a transplantable thymus.

There are currently several approaches being considered to rejuvenate immunity that do not rely on the endogenous thymus at all but rather concentrate on forming whole organs ex vivo that can be transplanted into patients as required (52, 105). These include the decellularization of an existing organ and using biomatrices in addition to vascularising chambers. One such strategy gaining momentum is to decellularize an existing organ, which removes all cellular compartments of the organ, leaving only the extracellular matrix components. Critically, this approach removes the significant immune barriers preventing xenogenic transplantation. This technique has so far been achieved only in heart, liver and lung (106–109). Another promising alternate approach for generating an ectopic thymus is the use of biomatrices that can be used to seed TEPC and mesenchymal elements to form an ex vivo generated thymus. One study, which used this approach in combination with implantable chambers that promote vascularization, found that this approach could be used to generate a functional thymus in vivo (110). However, at this stage these approaches still require TEPC to initiate thymic organogenesis highlighting the dependence of fetal tissue and the importance of discovering an adult TEPC.

Immune crosstalk: Rationale for combination strategies

The considerable crosstalk that occurs between developing thymocytes and the supporting microenvironment has been known for some time. However, while most of these interactions remain poorly understood, thymic crosstalk has been implicated in regulating Notch signalling (111), IL-7 expression (112, 113) and thymic organogenesis. Deficiency in specific lymphoid or stromal subsets has led to findings of commensurate deficiency in the corresponding compartment, for instance, presence of CD44⁻CD25⁺ TN2 thymocytes is critically required for cTEC organisation while single positive (CD4⁺ or CD8⁺) thymocytes are crucial for the maintenance of mTECs (114, 115). Moreover, while differentiation from TEC progenitors into mTEC or cTEC is not dependent on crosstalk with lymphoid progenitors (116, 117), development of the thymus itself is critically dependent on interactions with the earliest lymphoid progenitors in a highly specific window (118) and conditional deletion of TECs leads to profound loss in T cell production (119).

These studies suggest the significant contribution of each compartment to the development and maintenance of the other. Despite, or perhaps even because of it, this crosstalk between the thymic stroma and the hematopoietic compartment suggests that a combination of strategies targeting different compartments contributing towards thymopoiesis will lead to a greater regenerative boost than could be achieved with either strategies alone. In fact KGF has been proposed as an effective agent when used in conjunction with other regenerative therapies, successfully enhancing the sole regenerative benefits of preT cells (46), SSA (120) and temporary inhibition of p53 (121). Contrary to this, it is unlikely that IL-7 could be used effectively in combination with KGF or SSA as both of these strategies promote intrathymic production of IL-7 and studies in knockout mice demonstrate both of these regenerative strategies are dependent on IL-7 (42, 44).

The crosstalk between developing T cells and the supporting thymic stromal microenvironment is critical for normal thymus function and has been exploited to great effect for thymic regeneration. However, the fundamental relationship between the BM and the thymus, which is primarily predicated on the supply of lymphoid precursors, has been far less exploited as a means for promoting immune regeneration. While the precise identity of the circulating progenitor released from the BM to seed the thymus is unclear (122–126) their potential for improving immune reconstitution is not (96). Moreover, fundamental defects in HSC function, particularly in their ability to differentiate down the lymphoid lineage (127), may contribute towards some of the age-related changes observed in the thymus (94). Moreover, several strategies have been studied for their impact on BM recovery. These include parathyroid hormone (PTH), which can enhance HSC numbers (128) and retinoic acid (129), which accelerates B lymphopoiesis. However, at least in the aged setting, improving HSC function alone is not sufficient to restore thymopoiesis (130) and the reduced importation of progenitors is not enough to cause thymic involution (131) suggesting that these defects in HSC function merely contribute rather than cause age-related declines in lymphopoiesis. Nevertheless, strategies combining different aspects of immune renewal, from thymus-restricted KGF and preT cells to BM-restricted PTH and retinoic acids as well as more systemic therapies like IL7, Flt3L and SSA – aid the overall regeneration of the immune system. Taken together these findings lay the groundwork for the effective use of combination strategies for systemic immune renewal.

Concluding Remarks

These novel approaches to restore immune capacity through the translation of pre-clinical research will likely result in the development of new strategies to improve the outcome for a variety of patients who incur considerable morbidity and mortality from infections and relapse after transplant. Finally these strategies could also be used in a variety of clinical settings to overcome lymphocytopenia or to stimulate lymphocyte regeneration, including autologous HSCT, high dose chemotherapy, AIDS, vaccination, tolerance-induction or directed tumour therapies. Tying these strategies together, it stands to reason that using KGF to act directly on the thymic stroma, SSA to give an overall regenerative boost to the thymus and the supply of endogenous BM-derived precursors, IL7 to enhance precursor T cells and circulating mature T cells and finally, administering a ready-supply of ex vivo generated precursor T cells, will give the greatest clinical outcomes in modalities of immune depletion.

Figure 1. Strategies to enhance T cell immunity following allo-HSCT.

Several therapeutic strategies have been developed for boosting T cell reconstitution following immunodepletion. Exogenous cytokines and hormones such as IL-7, IL-12, IL-15, KGF, Flt3L, GH and IGF-1 have all been used to directly or indirectly boost T cell development in the thymus. T cell development prior to thymic involvement can also be targeted using factors such as PTH, administration of BM-derived lymphoid precursors or sex steroid inhibition. Several of these strategies, such as IL-7, IL-15, GH and IGF-1 also target peripheral lymphocytes mediating the expansion of pre-existing clones and leading to rapid, albeit limited, immune reconstitution. However, all of these therapies use the pre-existing thymic architecture, which can be critically damaged due to age or the conditioning regimes required for successful engraftment. One alternative solution would be to generate thymic tissue ex vivo using embryonic stem cell (or iPS)-derived TEPCs or even developing entire artificial organs using biomatrices or decellularization protocols.

Table 1.

Mechanisms underlying the most promising strategies for immune regeneration

Therapy	Impact on immunity	References
IL-7	Directly promotes expansion of lymphoid progenitors and peripheral T cells	(43, 49, 62, 64, 75, 132–135)
IL-12	Enhances thymopoiesis by inducing expression of IL-7 and IL-2	(77, 78)
IL-15	Proliferation and expansion of circulating NK and T cells	(74, 75)
KGF	Promotes proliferation and expansion of thymic epithelial cells	(42, 54, 55, 57, 136)
Flt3L	Directly promotes expansion and differentiation of lymphoid progenitors	(48, 65, 66, 68, 137)
IGF-1	Promotes expansion of thymic epithelial cells	(71–73)
GH/Ghrelin	Enhances thymopoiesis and BM hematopoietic function	(69, 70)
SSA	Impacts on proliferation of BM and intrathymic lymphoid progenitors. Also enhances TECs.	(29, 31, 34, 44, 45, 50, 89, 95, 138)
preT	Provides a ready-supply of T cell progenitors for thymopoiesis	(46, 47, 97, 139)