Strategies for reconstituting and boosting T cell-based immunity following haematopoietic stem cell transplantation: pre-clinical and clinical approaches

Poor immune recovery is characteristic of bone marrow transplantation and leads to high levels of morbidity and mortality. The primary underlying cause is a compromised thymic function, resulting from age-induced atrophy and further compounded by the damaging effects of cytoablative conditioning regimes on thymic epithelial cells (TEC). Several strategies have been proposed to enhance T cell reconstitution. Some, such as the use of single biological agents, are currently being tested in clinical trials. However, a more rational approach to immune restoration will be to leverage the evolving repertoire of new technologies. Specifically, the combined targeting of TEC, thymocytes and peripheral T cells, together with the bone marrow niches, promises a more strategic clinical therapeutic platform.     

Multiparameter evaluation of human thymic function: interpretations and caveats

After the provision of highly active antiretroviral therapy (HAART), the level of circulating CD4+ T cells increases in many adults infected with the human immunodeficiency virus, type 1 (HIV). To study factors involved in immune reconstitution, we have measured thymic abundance by CT scans, circulating naive-phenotype CD4+ T cells by flow cytometry, and T cell receptor (TCR) rearrangement excision circles (TRECs) by quantitative PCR in 40 virologically suppressed, HIV-infected adults and 33 age-matched, HIV-uninfected controls. In HIV-uninfected subjects, naive T cell numbers, thymic abundance, and the frequency of circulating naive CD4+ T cells bearing TRECs decreased with age, as expected. When corrected for this relationship with age, naive T cell numbers correlated significantly with naive T cell TREC frequencies. Virologically suppressed HIV-infected subjects had higher TREC frequencies, and subjects over the age of 39 were more likely to have abundant thymus compared to age-matched, HIV-uninfected adults. Nevertheless, all HIV-infected subjects had reduced absolute numbers of naive T cells, irrespective of thymic size, age, or TREC frequencies. These data illustrate the complex relationship between these measures of thymic size and function and underscore the need to develop more definitive measures of thymic function in the future.     

Reconstitution of the lymphocyte compartment after lymphocyte depletion: a key issue in clinical immunology

Loss of existing T cell populations is a significant clinical problem following cytoreductive therapies or in disease states such as HIV. Characterization of the pathways of T cell regeneration, their limitations and regulation, is central to the development of new approaches for the correction of T cell lymphopenia. Recently, there has been an increasing appreciation that subsets of T cells differ not only in requirements for homeostasis, but in the mechanisms of initial generation and later reconstitution of lost populations. In this issue, Cox et al. determine that variations in cytokine/chemokine levels may affect the biology of T cell recovery which impacts on the development of treatment strategies for lymphopenia.     

Understanding the mechanism of the age-change of thymic function to promote T cell differentiation

Immunological functions peak at around puberty and gradually decline thereafter with advancing age. The immunological decline mainly occurs in the T cell-dependent immune system and is generally associated with an increase in not only susceptibility to infections but also incidence of autoimmune phenomena. The age-related changes in T-cell dependent immune functions can be mainly ascribed to the physiological thymic involution which starts in the early phase of life. The age-related thymic involution can be ascribed to either extrinsic or intrinsic factors. Bone marrow stem cells can be one of the extrinsic factors for the thymic involution, but their role is estimated to be marginal as compared with alteration of the thymic microenvironment. With advancing age, the thymic capacity to promote T-cell differentiation declines together with a change in the composition of T-cell subsets produced. Such an alteration of the thymic environment is responsible for the age-related change in peripheral T cells in number and in composition. Age change is observed in several intrinsic factors in the thymic environment which influence proliferation of thymocytes. These thymic intrinsic factors can either promote or inhibit proliferation of thymocytes, and promoting factors generally decrease with age with a concomitant increase in inhibitory factors. Various endocrine hormones are important extrinsic factors influencing the thymic function. In fact, physiological thymic involution can be intervened by manipulation of the endocrine system, sometimes resulting in rejuvenation of immune functions to a certain extent. A specific strain of Buffalo rats has an unusual thymus, which does not involute with age, and the functions of the T-cell dependent immune system do not decline with age. Such animals having hyperplastic thymus are relatively resistant to various kinds of stress. Thus, restoration of immune functions in the aged animals appears to be beneficial for them in coping with various diseases associated with age.     

Medullary thymic epithelial stem cells: role in thymic epithelial cell maintenance and thymic involution

The thymus consists of two distinct anatomical regions, the cortex and the medulla; medullary thymic epithelial cells (mTECs) play a crucial role in establishing central T-cell tolerance for self-antigens. Although the understanding of mTEC development in thymic organogenesis as well as the regulation of their differentiation and maturation has improved, the mechanisms of postnatal maintenance remain poorly understood. This issue has a central importance in immune homeostasis and physiological thymic involution as well as autoimmune disorders in various clinicopathological settings. Recently, several reports have demonstrated the existence of TEC stem or progenitor cells in the postnatal thymus, which are either bipotent or unipotent. We identified stem cells specified for mTEC-lineage that are generated in the thymic ontogeny and may sustain mTEC regeneration and lifelong central T-cell self-tolerance. This finding suggested that the thymic medulla is maintained autonomously by its own stem cells. Although several issues, including the relationship with other putative TEC stem/progenitors, remain unclear, further examination of mTEC stem cells (mTECSCs) and their regulatory mechanisms may contribute to the understanding of postnatal immune homeostasis. Possible relationships between decline of mTECSC activity and early thymic involution as well as various autoimmune disorders are discussed.     

Characterization of T cell maturity in thymic epithelial cell tumors from BUF/Mna spontaneous thymoma rats and BUF/Mna-Rnu/+ rats showing delayed thymomagenesis

BUF/Mna rats develop thymomas spontaneously, which histologically mimic human thymomas. Although neoplasms in this rat strain contain a large number of immature lymphocytes, the phenotype has not been sufficiently assessed. We characterized T cell phenotypes in tumors from BUF/Mna rats in the present study. We also analyzed BUF/Mna-Rnu/+ rats, a heterozygous strain with suppressive thymomagenesis, and compared the histology and T cell maturation with those from the BUF/Mna rats. A total of 11 BUF/Mna and 10 BUF/Mna-Rnu/+ rats were used. Three-color flow cytometry was performed with anti-CD3, CD4, and CD8 antibodies to identify infiltrated lymphocytes, while tumor histology was evaluated with hematoxylin-eosin staining. The weight ratios of the entire thymic tissue including thymoma as compared to the BUF/Mna and BUF/Mna-Rnu/+ rat bodies were 0.8±0.8% and 1.2±1.8%, respectively. Histological findings for both rat congenic strains showed abundant lymphocytes surrounding large polygonal neoplastic thymic epithelia, which was compatible with the type B1 classification of the World Health Organization for human thymoma. CD4+CD8+ T cells accounted for 73.7±8.0% of the cells in tumors from BUF/Mna and 67.2±9.4% in those from BUF/Mna-Rnu/+ rats. Further, CD3-CD4-CD8+ T cells, intermediate between CD4-CD8-and CD4+CD8+ cells, accounted for 47.7±17.5% and 38.0±14.0% of the cells in tumors from the BUF/Mna and BUF/Mna-Rnu/+ strains, respectively. Thus, the proportion of developing thymic lymphocytes in and histology of thymomas from BUF/Mna and BUF/Mna-Rnu/+ rats were similar. These results suggest that both BUF/Mna and BUF/ Mna-Rnu/+ strains are suitable animal models for human thymoma to understand the development of immature thymic lymphocytes.     

Positive and negative thymic selection in T cell receptor-transgenic mice correlate with Nur77 mRNA expression

The orphan nuclear receptor Nur77 has been implicated in thymic negative selection. We studied the effect of two T cell receptor (TCR) transgenes on positive selction and Nur77 mRNA expression in thymus. DO11.10 mice, expressing a transgenic TCR specific for an ovalbumin (OVA) 323–339 peptide presented by I-A^d, were found to have an enlarged thymus with a reduced apoptotic activity, measured by flow cytometry, reduced mitochondrial membrane potential and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) techniques. In contrast, in F5 mice expressing a transgenic TCR recognizing the influenza virus nucleoprotein (NP) 366–374 peptide restricted by D^b, this positive selection effect was much less pronounced. Positive thymic selection in DO11.10 TCR⁺ mice correlated with a reduced level of Nur77 mRNA expression shown by Northern blot. F5 mice expressed levels close to those expressed by the wild type. Both transgenic mouse strains responded with extensive cortical apoptosis, and with up-regulation of Nur77 mRNA, to injection of cognate peptides. As 9-cis-Retinoic acid (9-cis-RA) inhibits Nur77-dependent apoptosis in T cell hybridomas in vitro, mice were pretreated with the drug to investigate a similar effect in vivo. However, the drug itself, at saturating concentrations, caused extensive apoptosis in immature CD4⁺/CD8⁺ thymocytes. The result demonstrates a correlation between Nur77 expression and thymic apoptotic activity, both during positive and negative selection events.     

Thymic heterotypic cellular complexes in gene-targeted mice with defined blocks in T cell development and adhesion molecule expression

Thymocytes form unique multicellular complexes with epithelial cells (thymic nurse cells, TNC) and rosettes (ROS) with macrophages, epithelial cells and dendritic cells. To investigate the role of differentiation checkpoints in the formation of the thymic heterotypic complexes in vivo, we used mutant mice which have genetically defined blocks at early and late stages of T cell development. We show that RAG-1^−/−, TCRβ^−/−, and p56^lck−/− mice lack thymocyte ROS formation with epithelial cells, macrophages, or dendritic cells. TNC formation was not affected by TCRβ and p56^lck gene mutations but partially decreased in RAG-1^−/− mice, indicating that TNC are the earliest thymocyte-stromal cell complexes formed in development, whereas ROS only appear after thymocytes have rearranged and expressed a functional TCRβ chain. Genetic blocks in CD8 lineage commitment (CD8^−/− and IFN regulatory factor-1^−/− mice) and positive and negative T cell selection (CD45^−/−, TCRα^−/−, and CD30^−/− mice) did not affect thymocyte-stromal cell complexes. Surprisingly, CD4^−/− mice, but not MHC class II^−/− mice, had significantly reduced numbers of TNC and ROS, in particular, a severe defect in ROS formation with thymic dendritic cells. The CD4^−/− block in ROS and TNC formation was rescued by the introduction of a human CD4 transgene. Moreover, we show that the adhesion receptors CD44 and LFA-1 cooperate in the formation of the thymic microenvironment. These results provide genetic evidence on the role of defined stages in T cell development and adhesion molecules on thymocyte/stromal cell interactions in vitro.     

Disparate peptide-dependent thymic selection outcomes in beta2M-deficient mice versus TAP-1-deficient mice: implications for repertoire formation

Fetal thymic organ cultures of N15-transgenic RAG-2-/- H-2b mice on normal, beta-2 microglobulin (beta2M)-/- or transporter associated with antigen processing (rAP-1)-/- MHCl-deficient backgrounds were used to examine differentiation of thymocytes bearing a TCR specific for a viral peptide bound to H-2Kb. Strong agonists mediate negative selection in all mice whereas weak agonists are positively selecting in beta2MW-/- mice but negatively selecting on TAP-1-/- or normal backgrounds. Very weak agonists and very weak antagonists are generally without effect in beta2M-/- mice yet foster differentiation in TAP-1-/- animals. The 20-40-fold reduction in beta2M4-/- thymic H-2Kb surface expression suggests that the avidity of the TCR for peptide-MHCI accounts for these differences, consistent with effects of TCR density and individual thymic-peptide abundance in peptide-MHC complexes. TCR-self-MHC interaction dominates Kb-based selection, subtly modulated by peptides as revealed by X-ray crystallography.     

T cell lymphoblastic leukaemia/lymphoma associated with a microenvironment of thymic asteroid B cells in the bone marrow

Naresh K N, May P C, Reid A G, Marks A J, Macdonald D & Kanfer E
(2010) Histopathology 57, 549–554
T cell lymphoblastic leukaemia/lymphoma associated with a microenvironment of thymic asteroid B cells in the bone marrow Aims: Asteroid B cells are a component of normal thymus. It is currently unclear whether these cells are identifiable in T cell lymphoblastic leukaemia/lymphoma (T‐ALL/LBL) of the thymus. The aim of this study was to identify asteroid B cells both in thymic and extrathymic tissue involved by T‐ALL/LBL. Methods and results: Thymic, lymph node (LN) and bone marrow trephine biopsy (BMTB) samples from eight patients with T‐ALL/LBL were reviewed. All had been investigated by immunohistochemistry and one by fluorescent in situ hybridization (FISH). The BMTB samples of two of eight T‐ALL/LBLs and LN sample in one of them showed the presence of asteroid‐shaped B cells with dendritic cytoplasmic processes. These B cells also expressed CD23 and the features were akin to the unique thymic asteroid B cells. Both patients had aggressive/resistant disease. Cytogenetic analysis in one showed a complex translocation involving the T cell receptor beta (TCRB) gene at 7q35 and a distal region of 9q known to harbour the NOTCH1 gene. Conclusion: This is the first report of T‐ALL/LBL documenting the presence of an asteroid B cell‐rich microenvironment at bone marrow and LN sites. In this small subset, T‐ALL/LBL cells are possibly dependent upon asteroid B cells, and whether targeting of asteroid B cells with anti‐CD20 monoclonal antibody in such cases will result in clinical benefit remains to be determined.     

Inhibition of I-Ad-, but not Db-restricted peptide-induced thymic apoptosis by glucocorticoid receptor antagonist RU486 in T cell receptor transgenic mice

Thymocytes differentiate by positive and negative selection of immature CD4⁺ CD8⁺ T cells. Negative selection occurs by default or by high-affinity recognition of peptides bound to proteins encoded by the major histocompatibility complex (MHC). MHC class I molecules are expressed on many different cell types, although at different levels, whereas MHC class II molecules are selectively expressed on thymic epithelial cells (TEC) and dendritic cells (DC). We investigated the role of the glucocorticoid receptor (GR) in thymic negative selection using the receptor antagonist RU486. Glucocorticoids (GC) are known to be potent inducers of apoptosis in CD4⁺ CD8⁺ thymocytes, and we have earlier shown that anti-CD3-induced thymic apoptosis can be blocked by RU486 in vivo. We now show that anti-CD3 induces thymic apoptosis in mice that have been adrenalectomized (ADX), and that RU486 inhibits anti-CD3 antibody-mediated thymocyte killing in newborn thymic organ cultures. Thymocyte apoptosis induced by ovalbumin peptide OVA323–339 treatment of mice transgenic for the DO11.10T cell receptor (TCR), which recognizes this peptide in the context of I-A^d, was found to be inhibited by RU486. These mice responded to peptide treatment by an extensive activation of the peripheral immune system, which became lethal in 60% of the mice when accompanied by simultaneous RU486 treatment. In contrast, RU486 had no effect on thymic apoptosis induced by the influenza A nucleoprotein NP366–374 peptide, recognized in context of D^b, in F5 TCR transgenic mice. We interpret the results to demonstrate that different deletion systems operate in the thymus. We propose that endogenous GC may be important for negative selection by default and by high-affinity recognition of endogenous MHC-presented peptides on TEC.     

The emerging role of ECM crosslinking in T cell mobility as a hallmark of immunosenescence in humans

Immunosenescence is thought to result from cellular aging and to reflect exposure to environmental stressors and antigens, including cytomegalovirus (CMV). However, not all of the features of immunosenescence are consistent with this view, and this has led to the emergence of the sister theory of “inflammaging”. The recently discovered diffuse tissue distribution of resident memory T cells (T_RM) which don't recirculate, calls these theories into question. These cells account for most T cells residing in barrier epithelia which sit in and travel through the extracellular matrix (ECM). With almost all studies to date carried out on peripheral blood, the age-related changes of the ECM and their consequences for T cell mobility, which is crucial for the function of these cells, have been largely ignored. We propose an update of the theoretical framework of immunosenescence, based on a novel hypothesis: the increasing stiffness and cross-linking of the senescent ECM lead to a progressive immunodeficiency due to an age-related decrease in T cell mobility and eventually the death of these cells. A key element of this mechanism is the mechanical stress to which the cell cytoplasm and nucleus are subjected during passage through the ECM. This hypothesis is based on an “evo-devo” perspective bringing together some major characteristics of aging, to create a single interpretive framework for immunosenescence.     

Analyses of T cell phenotype and function reveal an altered T cell homeostasis in systemic sclerosis: Correlations with disease severity and phenotypes

We investigated in systemic sclerosis (SSc) patients the T cell homeostasis and its relationship with the clinical course of the disease. Distribution of peripheral T cell subsets, thymic output, lymphocyte proliferation and apoptosis were analyzed by flow cytometry or ELISA. Age inappropriate levels of naive CD4⁺ T cells and thymic output were observed. Proliferation of CD4⁺ T cells, lymphocyte apoptosis and CD4⁺ regulatory T (Treg) cell frequency were significantly higher than those observed in controls and significantly correlated with clinical phenotypes and clinical progression parameters i.e., diffusing capacity of the lung for carbon monoxide (DLCO) and disease activity. These data indicate that the evaluation of the T cell homeostasis can represent a valuable prognostic tool for SSc patients and it is useful to distinguish between limited and diffuse phenotypes. A therapeutic intervention targeted at reversing T cell homeostasis abnormalities would therefore potentially be helpful in counteracting disease progression.     

T cell function in the aged: Lessons learned from animal models

The deterioration of immune function with advancing age contributes to the increased incidence of morbidity and mortality among the elderly from infectious disease and possibly cancer. The innate and adaptive arms of immunity are affected by the deleterious effects of aging, but it is adaptive immunity, and in particular T lymphocytes, that is most susceptible to these effects. The aging of the immune system, referred to as immunosenescence, is associated with a dramatic decline in responsiveness as well as functional dysregulation. Age-associated alterations in T cells are evident at all stages of its development, and it is these changes that contribute to the overall increased susceptibility to infection and possibly cancer. Although there is an enormous effort worldwide to develop vaccines, much of the research is performed with young animals. Because the immune system of the aged is different from that of the young, many of the findings cannot be extrapolated. In this review, we will discuss those differences in T cell immunity of the aged, relate how these differences influence the immune response of the aged to pathogens as well as to tumors, and describe the current approaches to rejuvenate the aged immune response. Although the majority of the conclusions on T cell immunity in the aged are based on studies in the murine model, many of these findings can be extended to the human model. Throughout this review, we have noted those findings that are specific to humans.     

Effect of Antihuman T Lymphocyte Globulin on Immune Recovery after Myeloablative Allogeneic Stem Cell Transplantation with Matched Unrelated Donors: Analysis of Immune Reconstitution in a Double-Blind Randomized Controlled Trial

We recently conducted a randomized double-blind study in which we demonstrated that moderate/severe chronic graft-versus-host disease (cGVHD) but not cGVHD-free survival was reduced in patients receiving anti-T lymphocyte globulin (ATLG) versus placebo. In a companion study we performed immunophenotypic analysis to determine the impact of ATLG on immune reconstitution (IR) and to correlate IR with clinical outcomes. The randomized study (n?=?254) included patients (aged 18 to 65 years) who underwent myeloablative transplants for acute myeloid leukemia, myelodysplastic syndrome, or acute lymphoblastic leukemia from HLA-matched unrelated donors. Ninety-one patients consented for the companion IR study (ATLG?=?44, placebo?=?47). Blood samples were collected on days 30, 100, 180, and 360 after hematopoietic cell transplantation (HCT), and multiparameter flow cytometry was performed in a blinded fashion. Reconstitution of CD3⁺ and CD4⁺ T cells was delayed up to 6 months post-HCT in the ATLG arm, whereas absolute regulatory T cell (Treg) (CD4⁺25⁺127-) numbers were lower only in the first 100 days. Analysis of the CD4⁺ Treg and conventional T cells (Tconv) (CD4⁺25^–127⁺) compartments showed a profound absence of naive Tregs and Tconv in the first 100 days post-HCT, with very slow recovery for 1 year. B cell and natural killer cell recovery were similar in each arm. Higher absolute counts of CD3⁺, CD4⁺, CD8⁺ T, Tregs, and Tconv were associated with improved overall survival, progression-free survival, and nonrelapse mortality but not moderate/severe cGVHD. Although ATLG delays CD3⁺ and CD4⁺ T cell recovery post-transplant, it has a relative Treg sparing effect after the early post-HCT period, with possible implications for protection from cGVHD. ATLG severely compromises the generation of naive CD4⁺ cells (Treg and Tconv), potentially affecting the diversity of the TCR repertoire and T cell responses against malignancy and infection.     

Diagnostic potential of tetramer-based monitoring of cytomegalovirus-specific CD8+ T lymphocytes in allogeneic stem cell transplantation

Cytomegalovirus (CMV) infection remains a significant problem in allogeneic stem cell transplant (SCT) recipients despite the availability of effective antiviral drugs. This problem concerns patients which are unable to mount an effective T-lymphocyte response against CMV. Therefore, the development and use of tetramer technology to enumerate CMV-specific T cells will be valuable to identify these patients as early as possible. Here, we review clinical studies in which CMV-specific CD8(+) T cells have been monitored in allogeneic SCT recipients using tetramers in the context of similar studies that are based on functional assays of CMV-specific T cells. The results thus far warrant the further development of tetramer technology as a diagnostic tool to monitor CMV-specific T cells in SCT recipients and other groups of immunocompromised patients threatened by CMV.