Biology of Blood and Marrow Transplantation
Volume 12, Issue 1, Supplement 1 , Pages 42-46, January 2006

Immune Reconstitution: From Stem Cells to Lymphocytes

  • Gay M. Crooks

      Affiliations

    • Divisions of Research Immunology and Bone Marrow Transplant, Children’s Hospital Los Angeles, Los Angeles, California
    • ,
  • Kenneth Weinberg

      Affiliations

    • Divisions of Research Immunology and Bone Marrow Transplant, Children’s Hospital Los Angeles, Los Angeles, California
    • Corresponding Author InformationCorrespondence and reprint requests: Kenneth Weinberg, MD, Division of Pediatric Stem Cell Transplantation, Lucile Packard Children’s Hospital, 1000 Welch Rd., Stanford, CA 94305
    • ,
  • Crystal Mackall

      Affiliations

    • Pediatric Oncology Branch, National Cancer Institute, Bethesda, Maryland

Article Outline

 

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Introduction 

Over the last decade, immune reconstitution has emerged as a general concern in hematopoietic stem cell transplantation (HSCT). Interest in immune development after transplantation is due to the high rate of susceptibility to opportunistic infection and the relationship of immune recovery to relapse of malignant diseases. Infection rates for HSCT recipients after resolution of peritransplantation neutropenia have ranged from 40% to nearly 80%, depending on the donor source and whether graft-versus-host disease (GVHD) is present [1]. Conventionally, herpes virus family infections—notably, cytomegalovirus (CMV) and Epstein-Barr virus—have been the major opportunistic viral infections that have been relevant to transplantation. However, community respiratory virus infections, including adenovirus infections, are being increasingly described. Fungal infections are another major cause of posttransplantation morbidity and mortality, as well as significant expense and morbidity from the prophylactic, preemptive, and therapeutic use of antifungal agents. Increased susceptibility to fungal infections is likely to represent defects of both T cell–mediated and innate immune functions in HSCT recipients. Besides the viral and fungal infections that are associated with defective cell-mediated immunity, late bacterial infections occur as a result of defective antibody production, including low titers and defective class switching from immunoglobulin M to immunoglobulin G. Besides infectious complications, the other evidence for the importance of post-HSCT immune reconstitution has been the decreased efficacy of hematopoietic stem cells (HSCs) for malignant disease when the graft has been manipulated by T-cell depletion (TCD) to prevent GVHD. To date, the problem of delayed or defective immune reconstitution has been addressed mainly by aggressive supportive care, including the prophylactic or presumptive use of antibiotics, antifungals, and antivirals and the administration of replacement intravenous immunoglobulin. In addition, innovative cellular therapies using adoptively transferred T-cell clones or polyclonal T-cell populations have been used experimentally.

The risk or severity of delayed immune reconstitution may be increasing because of changes in HSCT practices. Use of unrelated donor or haploidentical stem cell products increases the severity of post-HSCT immune deficiency as a result of the increased risk of GVHD, as well as the use of cell-processing methods, eg, TCD, that remove functional T cells [2]. As will be discussed, the removal of mature T cells from HSC products means that one pathway for posttransplantation T-cell generation, the expansion of adoptively transferred T cells, is severely limited. As a result, generation of T lymphocytes must occur from prethymic progenitors via a thymic-dependent pathway. Although difficult to quantify in clinical studies, increased dose intensity of both pretransplantation conditioning and prior chemotherapy may damage the thymic microenvironment and result in a reduced capacity to support de novo T-cell development. Another factor that may increase the incidence or severity of posttransplantation immune deficiency is the use of HSCT in older patients who have poorer capacity for generation of T lymphocytes than younger patients [3, 4].

Functional immunity depends on cells that mediate both the innate and adaptive immune response. After HSCT, recovery of polymorphonuclear and dendritic cells is relatively rapid, occurring within weeks. In contrast, the development of the lymphocytes that mediate the adaptive immune response is substantially delayed and in some cases may never occur. The reasons for the delayed development of functional T and B lymphocytes, compared with myeloid cells, are unclear. One reason for the delay may be differences in the ontogeny of lymphoid and nonlymphoid populations. For example, the development of immunocompetence in the fetus occurs significantly later than the development of nonlymphoid cells. The thymic anlage is colonized with hematopoietic cells at 7 to 8 weeks’ gestation, but antigen-specific T-cell function does not develop until the second trimester, and a full repertoire and normal numbers of T lymphocytes are not present until postnatal life. Thus, if T-lymphocyte development were to occur after HSCT at the same rate as normal fetal ontogeny, it might be predicted that functional numbers of T cells might appear 3 to 4 months after transplantation and that full recovery might take 6 to 12 months. Recapitulation of ontogeny can explain some of the delays in immune reconstitution and can lead to therapeutic strategies based on manipulation of lymphoid progenitors.

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Thymic-dependent and -independent pathways for t-cell generation 

Several concepts that are critical for understanding posttransplantation immune deficiency will be discussed. It is postulated that all posttransplantation T-lymphocyte populations are derived either from (1) prethymic cells that must differentiate in the host via thymic-dependent pathways or (2) postthymic cells that can undergo expansion in the host without a requirement for host thymic function (thymic-independent pathway) [5]. The thymic-dependent pathway recapitulates ontogeny in that it is slow, but it can generate a diverse repertoire of T lymphocytes that are host tolerant. Critically, the thymic-dependent pathway is predicated on the generation of lymphoid progenitors that can traffic, engraft, and develop in the thymus. Generation of lymphocytes proceeds through stages marked by different levels of commitment, differentiation, proliferation, and survival. Murine experiments have demonstrated that the number of transplanted HSCs is a critical determinant of thymic recovery. Therefore, manipulations to increase lymphoid commitment and progenitor expansion or to transplant large numbers of committed progenitors may be therapeutically useful. The thymic-dependent pathway is also predicated on the thymus providing an adequate microenvironment for the development of T lymphocytes. The function of the thymus in a transplant patient is probably never the same as that in a normal fetus. The thymic microenvironment is damaged in HSCT recipients by age-related thymic involution, exposure to cytotoxic drugs or radiation, and GVHD. Therefore, reversal of thymic involution, protection of the thymus from injury, and replacement of thymic functions are likely to be important in ensuring that the thymic-dependent pathway is functional. Because of the delays inherent in the thymic-dependent pathway, much of the T-cell function seen early after HSCT is occurring via the thymic-independent pathway. Homeostatic peripheral expansion of T lymphocytes occurs in lymphopenic hosts and is a mechanism by which adoptively transferred T cells in the graft can rapidly expand. Cells generated after HSCT by thymic-independent pathways have a narrower and skewed repertoire than T lymphocytes generated de novo in the thymus. However, the ability to undergo rapid expansion in a thymic-independent manner offers the potential for clinically significant expansion of cells with defined specificities, eg, against pathogens (CMV, Epstein-Barr virus, and adenovirus) or tumor cells.

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HSC and progenitor cell biology 

The ability to directly manipulate the earliest lymphoid progenitors in hematopoietic transplant grafts could theoretically lead to novel therapies aimed at improving immune reconstitution after transplantation. However, our understanding of how the early stages of human lymphopoiesis are regulated has been limited until recently by the lack of suitable assays for the human system. The classic model of hematopoiesis assumes that HSCs differentiate initially down 2 mutually exclusive pathways, the first restricted to the myeloerythroid lineage and the second restricted to the lymphoid lineage. Studies using adult murine bone marrow that support this concept have identified common lymphoid progenitors (CLPs), ie, progenitors derived from HSCs that have lost myeloid potential but retain the ability to differentiate into all lymphoid cell types [6]. Murine CLPs are immunophenotypically distinct from murine HSCs; the 2 populations are distinguished most importantly by differential expression of interleukin (IL)–7 receptor α (IL-7Rα). However, more recent studies in murine bone marrow and thymus have identified other types of progenitor pathways that can generate lymphoid cells. For example, the early thymocyte progenitor has been identified in mouse thymus and lacks IL-7Rα, thus leading to controversy over whether CLPs or other progenitors are the direct precursors of the earliest thymocytes.

With the development of in vitro and in vivo assay systems able to measure human lymphopoiesis, human lymphoid progenitor stages and how they are regulated can now be studied. The Crooks laboratory has isolated multilymphoid progenitors capable of generating at least B, natural killer, and dendritic cells from cord blood, bone marrow, and mobilized peripheral blood on the basis of expression of the surface antigen CD7 [7]. These progenitors express IL-7Rα and lymphoid-associated genes such as Pax5 and Tdt. However, another progenitor population can be found in cord blood that lacks IL-7Rα expression. These CD34+CD38−CD7+ cells represent an earlier step in lymphoid commitment that precedes the onset of Pax5 or Tdt expression. The existence of this population suggests that lymphoid commitment in the human is not dependent on IL-7Rα expression. The existence of analogous IL-7Rα–negative lymphoid progenitors in the human bone marrow has not yet been reported. However, CD34+CD38−CD7+ cells are found at high frequency in the bone marrow from patients with severe combined immune deficiency caused by a mutation in the signaling component of the IL-7R (ie, common γ or γc gene deficiency). This supports the notion that IL-7R signaling is not required for the earliest stages of human lymphoid differentiation.

By using the in vitro assays and immunophenotypes developed for cord blood and bone marrow, the Crooks laboratory has begun to characterize distinct progenitor subpopulations in the human thymus. CD7+ cells make up the bulk of the CD34+ progenitor population in the thymus and possess both T-cell and natural killer cell potential. More primitive progenitors with lymphomyeloid lineage potential can also be identified in the human thymus at very low frequency.

Cotransplantation of CLPs with purified HSCs has been shown to be effective at protection form infection in a model of murine CMV infection [8]. It is important to note that CLPs, which have not yet undergone selection in the thymus, do not cause GVHD in allogeneic models of bone marrow transplantation. One of the major obstacles to the clinical use of CLPs to enhance lymphopoiesis after transplantation is their rarity and relatively poor engraftment and proliferation potential. Novel strategies to overcome these biological limitations are under exploration.

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Thymic microenvironmental function and HSCT 

Besides the model of recapitulation of ontogeny, a second explanation for the delayed lymphocyte recovery in HSCT recipients is damage to the thymic microenvironment. The thymus consists of HSC-derived thymocytes that are developing into T lymphocytes, as well as diverse populations of resident microenvironmental cells, most of which are not hematopoietically derived. The thymocytes, although transient, are the vast majority (>99%) of the cells in a normal thymus. Classic models of thymopoiesis have emphasized the positive and negative signals provided by the microenvironmental cells for the developing thymocytes. The clonotypic T-cell receptor (TCR) for antigen is generated by the process of V(D)J recombination occurring in the most immature CD3−CD4−CD8− (triple-negative) thymocytes. Thymocytes with different TCR specificities are positively and negatively selected at subsequent CD3+CD4+CD8+ (double-positive) and CD3+CD4+ or CD3+CD8+ (single-positive) stages of differentiation. Because of the large amount of culling of cells that occurs via negative selection, the maintenance of a pool of thymocytes throughout life requires a continuous stream of newly produced triple-negative cells. The expansion of triple-negative cells is mediated in the mouse by IL-7 and Kit ligand signals. The role of IL-7 in human thymocyte development has been established in severe combined immune deficiency syndromes caused by IL-7Rα or γc deficiencies, but the role of Kit signaling is not well established for humans. IL-7 and Kit ligand are both produced by subsets of thymic epithelial cells (TECs), which can be classified on the basis of anatomic location (cortical versus medullary), gene expression, and function. In a revision of classic unidirectional models of interactions between the microenvironment and thymocytes, there is evidence that TEC development and maintenance are reciprocally dependent on signals derived from developing thymocytes. An example of these reciprocal interactions is the production of fibroblast growth factors (FGFs) by thymocytes that interact with an epithelial-specific FGF receptor (FGFR2-IIIb) expressed by the TECs.

Multiple mechanisms for damage to the thymic microenvironment may be operative in HSCT recipients. In the 1970s, it was noted that in animals with experimental GVHD, the alloreactive cells infiltrated the thymus and caused inflammation. Subsequent studies have shown that TECs are a target of this allogeneic response. Although an effect of posttransplantation immune suppressive drugs, particularly glucocorticoids, might intuitively be expected to suppress thymopoiesis by killing thymocytes, such effects have been shown only in animal models, but not in clinical studies of patients receiving prophylactic immunosuppression who did not develop GVHD [3, 9]. Thus, GVHD itself is likely to be more inhibitory of thymopoiesis than the drugs used to prevent GVHD. Other studies have shown that TECs are decreased in number by aging and are killed by cytotoxic agents, such as radiation therapy and alkylating agents, that are used in pretransplantation conditioning. The loss of TECs likely results in decreased intrathymic IL-7 production, thus decreasing the ability of the thymus to sustain the de novo generation of T cells.

The recognition of TEC damage as a common pathway for thymic microenvironmental dysfunction opens up many avenues for research aimed at improving immune reconstitution. Among the strategies that are in need of translational study are the replacement of intrathymic IL-7 production by either systemic administration of IL-7 or cellular therapy and the protection and regeneration of TECs by administration of ligands for FGFR2-IIIb: eg, FGF-7 (keratinocyte growth factor) [10, 11].

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Homeostatic proliferation in immune reconstitution 

Until the 1990s, it was generally believed that most T lymphocytes were not in the cell cycle but could be made competent for proliferation by recognition of antigenic peptides by the TCR and engagement of costimulatory receptors. IL-2–dependent activation and proliferation of the stimulated T cells, activated induced apoptosis, or induction of anergy of the stimulated T cells would then result. However, it is now clear that low-level proliferation of all T-lymphocyte subsets occurs under normal conditions, even when there is no nominal antigenic stimulation. During fetal and early postnatal life, homeostatic cycling expands the number of mature T lymphocytes so that T-cell numbers are not totally constrained by the capacity of the thymus to produce new cells. Later in postnatal life, homeostatic cycling allows the continued maintenance of a broad repertoire of T-cell specificities and functions, even as the thymus involutes. Under conditions of lymphopenia, the homeostatic proliferation of mature T lymphocytes increases, thus resulting in homeostatic peripheral expansion. Homeostatic peripheral expansion has been demonstrated under experimental conditions, as well as clinically in lymphopenic HSCT and in human immunodeficiency virus–infected patients, and is largely responsible for the thymic-independent reconstitution of immunity that is observed early after HSCT.

The regulation of homeostatic cycling and peripheral expansion has been elucidated for different lymphocyte subsets [12]. Such homeostatic cycling or proliferation of naive CD4+ and CD8+ T lymphocytes is induced by interactions of the TCR with self-peptides and IL-7 in the periphery. In the absence of either IL-7 or major histocompatibility molecules to present self-peptide antigens, adoptively transferred naive T cells will fail to survive. IL-7 levels have been shown to be inversely related to lymphocyte number. The increased levels of IL-7 observed in lymphopenic conditions may explain the homeostatic peripheral expansion of naive T cells that is observed in lymphopenic hosts and, indeed, can be replicated in nonlymphopenic hosts by the administration of pharmacologic doses of IL-7. Stimulation of naive T cells via TCR and IL-7 signals may result in expansion of the naive T-cell pool or of conversion to memory T lymphocytes. Memory T lymphocytes differ from naive cells in their requirements for homeostatic cycling and peripheral expansion. CD4+ memory T cells are mainly dependent on IL-7 alone, but optimal proliferation occurs in the presence of both IL-7 and TCR signals. In contrast, CD8+ memory T-cell homeostatic cycling or proliferation requires another γc cytokine, IL-15—not IL-7—and does not require TCR signals.

The regulation of homeostasis may be more complex than simply combinations of cytokine and TCR signals. For example, activation of human dendritic cells by thymic stroma-derived lymphopoietin results in their ability to support proliferation of mature T lymphocytes that acquire a central memory phenotype [13]. The mechanism of expansion is dependent on TCR and B7 costimulation signals but, like homeostatic proliferation, is not dependent on foreign antigens.

The application of our knowledge of T-cell homeostatic cycling or proliferation is highly relevant to clinical HSCT. The skewing of the repertoire toward tumor antigens is likely to be desirable for effective immunotherapy of cancer. In addition, proliferation of cells with weak affinities for antigen are magnified during homeostatic peripheral expansion, a property that may be particularly useful for antitumor responses. Evidence of the ability of IL-7 treatment to duplicate some of the conditions of lymphopenia-associated homeostatic peripheral expansion suggests that pharmacologic IL-7 therapy may be useful in augmenting immune responses to tumor vaccines. The ability of adoptively transferred antigen-specific T lymphocytes to survive and expand to clinically relevant numbers may be augmented by IL-7 and IL-15 therapy.

In addition to the opportunities for therapy offered by the understanding of the processes of homeostatic cycling and peripheral expansion, there are several pitfalls and unresolved questions that need to be addressed. Although IL-7 therapy is an attractive way to address the problem of thymic microenvironmental damage, the dual effects of IL-7 in promoting the expansion and differentiation of both thymocytes and homeostatic cycling and expansion of mature T lymphocytes mean that studies of IL-7–mediated thymic stimulation should be viewed with caution. IL-7 is necessary for the survival of alloreactive T lymphocytes that mediate GVHD and may exacerbate GVHD in an allogeneic setting. Clinical trials of IL-7 to stimulate thymopoiesis may require either TCD of the stem cell product in allogeneic HSCT or initial study in an autologous setting. Furthermore, because the effects of IL-7 on mature T-cell subsets seem to be dose dependent, the optimal dose for thymic stimulation may differ from that needed for stimulation of homeostatic peripheral expansion.

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Summary 

Although the problem of immune reconstitution after HSCT has been addressed indirectly by supportive care measures, new knowledge of the biology of transplantation should allow us to move forward in directly attacking the problem. Strategies to improve the development of T lymphocytes via thymic-dependent pathways involve the generation, isolation, and manipulation of prethymic cells, including CLPs. Understanding of the reciprocal relationships between lymphoid progenitors and the thymic microenvironment have allowed small-animal model testing of either IL-7 replacement or FGF-mediated protection and regeneration of TECs. The elucidation of the TCR and cytokine requirements for the thymus-independent homeostatic cycling and peripheral expansion of mature T cells is leading to studies of either enhancement of immune responses to antigens or to improved survival and efficacy of adoptively transferred T lymphocytes. Although all of these approaches have solid preclinical supporting data, a major challenge to the HSCT community will be the implementation of clinical trials that will allow proper evaluation of their safety, efficacy, and best use.

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Acknowledgments 

Supported by National Institutes of Health grant nos. HL77912, HL73104 (G.M.C.), AI50765, HL54729, HL70005, and HL73104 (K.W.).

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PII: S1083-8791(05)00720-2

doi:10.1016/j.bbmt.2005.10.015

Biology of Blood and Marrow Transplantation
Volume 12, Issue 1, Supplement 1 , Pages 42-46, January 2006

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