Impaired Thymopoiesis Research Articles

Induction of Thymus Atrophy and Disruption of Thymocyte Development by Fipronil through Dysregulation of IL-7-Associated Genes.

The susceptibility of the immune system to immunotoxic chemicals is evident, particularly in the thymus, a vital primary immune organ prone to atrophy due to exposure to toxicants. Fipronil (FPN), a widely used insecticide, is of concern due to its potential neurotoxicity, hepatotoxicity, and immunotoxicity. Our previous study showed that FPN disturbed the antigen-specific T-cell functionality in vivo. As T-cell lineage commitment and thymopoiesis are closely interconnected with the normal function of the T-cell-mediated immune responses, this study aims to further examine the toxic effects of FPN on thymocyte development. In this study, 4-week-old BALB/c mice received seven doses of FPN (1, 5, 10 mg/kg) by gavage. Thymus size, medulla/cortex ratio, total thymocyte counts, double-positive thymocyte population, and IL-7-positive cells decreased dose-dependently. IL-7 aids the differentiation of early T-cell precursors into mature T cells, and several essential genes contribute to the maturation of T cells in the thymus. Foxn1 ensures that the thymic microenvironment is suitable for the maturation of T-cell precursors. Lyl1 is involved in specifying lymphoid cells and maintaining T-cell development in the thymus. The c-Kit/SCF collaboration fosters a supportive thymic milieu to promote the formation of functional T cells. The expression of IL-7, IL-7R, c-Kit, SCF, Foxn1, and Lyl1 genes in the thymus was significantly diminished in FPN-treated groups with the concordance with the reduction of IL-7 signaling proteins (IL-7, IL-7R, c-KIT, SCF, LYL1, FOXO3A, and GABPA), suggesting that the dysregulation of T-cell lineage-related genes may contribute to the thymic atrophy induced by FPN. In addition, FPN disturbed the functionality of thymocytes with an increase of IL-4 and IFN-γ production and a decrease of IL-2 secretion after T-cell mitogen stimulation ex vivo. Collectively, FPN significantly deregulated genes related to T-cell progenitor differentiation, survival, and expansion, potentially leading to impaired thymopoiesis.

A Novel CDC42 Variant with Impaired Thymopoiesis, IL-7R Signaling, PAK1 Binding, and TCR Repertoire Diversity

Genetic variants in cell division cycle 42 (CDC42) can manifest with dysmorphic features, autoinflammation, hemophagocytic lymphohistiocytosis, and thrombocytopenia, whereas defective thymopoiesis is a rare disease manifestation. We report a novel CDC42 missense variant (c.46A > G, p.Lys16Glu) resulting in infection and HPV-driven carcinogenesis in the mosaic mother and impaired thymopoiesis and profound T cell lymphopenia in the heterozygous daughter identified through newborn screening for SCID. We found that surface expression of IL-7Rα (CD127) was decreased, consistent with reduced IL-7-induced STAT5 phosphorylation and accelerated apoptotic T cell death. Consistent with the vital role of IL-7 in regulating thymopoiesis, both patients displayed reduced T cell receptor CDR3 repertoires. Moreover, the CDC42 variant prevented binding to the downstream effector, p21-activated kinase (PAK)1, suggesting this impaired interaction to underlie reduced IL-7Rα expression and signaling. Here, we provide the first report of severely compromised thymopoiesis and perturbed IL-7Rα signaling caused by a novel CDC42 variant and presenting with diverging clinical and immunological phenotypes in patients.

Kinetics of endogenous thymic regeneration following chemotherapy-induced involution

Abstract The thymus is a primary lymphoid organ where T cell development occurs. Besides the naturally transpiring age-dependent involution, the thymus also undergoes acute involution from cytotoxic stimuli. Establishing thymic regeneration strategies would, thus, be immensely critical for pediatric cancer and transplantation patients receiving cytoablative chemotherapies. Several signaling pathways (e.g., KGF, BMP4) promote thymic regeneration, albeit with suboptimal therapeutic potential, mainly due to poor insight of the detailed mechanisms of endogenous thymic repair. To address this issue, we developed a mouse model of cyclophosphamide-induced thymic involution and monitored thymic reconstitution for up to 5 weeks post-treatment. Macroscopic assessment via thymic weight index and MRI-based volumetric analysis, along with histopathology, revealed that endogenous thymic regeneration is a non-linear, multistep process, consisting of a: (a) Reconstitution phase, during which recuperation occurs at an exponential rate, (b) Rebound phase, in which abnormal thymic hyperplasia is observed, and (c) Recoil phase, where thymic homeostasis ensues. Quantification of peripheral blood lymphocytes, however, revealed that thymic hyperplasia at Rebound does not correlate with restored thymic functions and output. Detailed investigation via flow cytometry revealed the stalling of T cell development at the Double Positive (CD4 +CD8 +) stage, implying cortical defects during thymic repair. Currently, we employ single-cell RNA sequencing with single cell resolution imaging, to define the molecular pathways disrupting cortical Thymic Epithelial Cell (cTEC) renewal post-chemotherapy, leading to impaired thymopoiesis. Supported by AAI's Career in Immunology Fellowship PI's funding supported by Albert Einstein College of Medicine: New Investigator Start-up funds

The proprotein convertase furin regulates the development of thymic epithelial cells to ensure central immune tolerance

SummaryThe generation of mature T cells and establishment of central tolerance is predominantly orchestrated by thymic epithelial cells (TECs). Proprotein convertases are responsible for the proteolysis of proproteins into their mature bioactive counterparts. Here, we found that Furin, a member of the subtilisin/kexin-like PCs family, is highly expressed in TECs compared with other members of this family. TEC-specific deletion of Furin caused severe thymic atrophy and predominantly reduced the number of medullary TECs and thymic tuft cells, and to a less degree, cortical TECs. Furin deletion attenuated the proliferation of TECs, impaired thymopoiesis, and led to autoimmune disorders in mice. Furin promotes the development of TECs via cleavage of proIGF1 receptor and pro-Insulin receptor and the activation of downstream ERK/MAPK and Akt signaling pathways. Thus, this study uncovered the role of furin in TEC development and function and highlighted the importance of post-translational modification of immature proproteins in TEC biology.

Cytosolic Nuclear Sensor Dhx9 Controls Medullary Thymic Epithelial Cell Differentiation by p53-Mediated Pathways.

Thymic epithelial cells (TECs) critically participate in T cell maturation and selection for the establishment of immunity to foreign antigens and immune tolerance to self-antigens of T cells. It is well known that many intracellular and extracellular molecules elegantly have mastered the development of medullary TECs (mTECs) and cortical TECs (cTECs). However, the role played by NTP-dependent helicase proteins in TEC development is currently unclear. Herein, we created mice with a TEC-specific DExD/H-box helicase 9 (Dhx9) deletion (Dhx9 cKO) to study the involvement of Dhx9 in TEC differentiation and function. We found that a Dhx9 deficiency in TECs caused a significant decreased cell number of TECs, including mTECs and thymic tuft cells, accompanied by accelerated mTEC maturation but no detectable effect on cTECs. Dhx9-deleted mTECs transcriptionally expressed poor tissue-restricted antigen profiles compared with WT mTECs. Importantly, Dhx9 cKO mice displayed an impaired thymopoiesis, poor thymic T cell output, and they suffered from spontaneous autoimmune disorders. RNA-seq analysis showed that the Dhx9 deficiency caused an upregulated DNA damage response pathway and Gadd45, Cdkn1a, Cdc25, Wee1, and Myt1 expression to induce cell cycle arrest in mTECs. In contrast, the p53-dependent upregulated RANK-NF-κB pathway axis accelerated the maturation of mTECs. Our results collectively indicated that Dhx9, a cytosolic nuclear sensor recognizing viral DNA or RNA, played an important role in mTEC development and function in mice.

Impaired thymopoiesis predicts for a high risk of severe infections after reduced intensity conditioning without anti-thymocyte globulin in double umbilical cord blood transplantation

Umbilical cord blood stem cell transplantation (UCBT) is associated with retarded hematopoietic recovery and immune reconstitution and a high infection-related morbidity and mortality, especially after conditioning including anti-thymocyte globulin (ATG). However, data on immune recovery, incidence of infections, and outcome in double UCBT (dUCBT) recipients receiving an ATG-free reduced intensity conditioning (RIC) are lacking. In this study, recovery of lymphocyte subsets, thymopoiesis, and its association with severe infections and clinical outcome was assessed in a group of 55 recipients of a dUCBT ATG-free RIC regimen. T cell recovery was severely protracted in the majority of patients. However, T cell receptor excision circle TREC+ T cells were detectable in 62% of patients at 3 months post-transplantation. A total of 128 common toxicity criteria grade 3-4 infections were observed in the first year post-transplantation. Non-relapse mortality at 12 months post-transplant was 16%, of which 78% infectious mortality. One-year overall survival was 73%. Patients who failed to recover thymopoiesis at 3 months post-transplantation were at a 3.3-fold higher risk of subsequent severe grade 3-4 infections.

Loss of thymic innate lymphoid cells leads to impaired thymopoiesis in experimental graft-versus-host disease

AbstractGraft-versus-host disease (GVHD) and posttransplant immunodeficiency are frequently related complications of allogeneic hematopoietic transplantation. Alloreactive donor T cells can damage thymic epithelium, thus limiting new T-cell development. Although the thymus has a remarkable capacity to regenerate after injury, endogenous thymic regeneration is impaired in GVHD. The mechanisms leading to this regenerative failure are largely unknown. Here we demonstrate in experimental mouse models that GVHD results in depletion of intrathymic group 3 innate lymphoid cells (ILC3s) necessary for thymic regeneration. Loss of thymic ILC3s resulted in deficiency of intrathymic interleukin-22 (IL-22) compared with transplant recipients without GVHD, thereby inhibiting IL-22–mediated protection of thymic epithelial cells (TECs) and impairing recovery of thymopoiesis. Conversely, abrogating IL-21 receptor signaling in donor T cells and inhibiting the elimination of thymic ILCs improved thymopoiesis in an IL-22–dependent fashion. We found that the thymopoietic impairment in GVHD associated with loss of ILCs could be improved by restoration of IL-22 signaling. Despite uninhibited alloreactivity, exogenous IL-22 administration posttransplant resulted in increased recovery of thymopoiesis and development of new thymus-derived peripheral T cells. Our study highlights the role of innate immune function in thymic regeneration and restoration of adaptive immunity posttransplant. Manipulation of the ILC–IL-22–TEC axis may be useful for augmenting immune reconstitution after clinical hematopoietic transplantation and other settings of T-cell deficiency.

FLT3 ligand regulates thymic precursor cells and hematopoietic stem cells through interactions with CXCR4 and the marrow niche.

Impaired immune reconstitution after hematopoietic stem cell transplantation (HSCT) is attributed in part to impaired thymopoiesis. Recent data suggest that precursor input may be a point of regulation for the thymus. We hypothesized that administration of FLT3 ligand (FLT3L) would enhance thymopoiesis after adoptive transfer of aged, FLT3L-treated bone marrow (BM) to aged, Lupron-treated hosts by increasing murine HSC (Lin[minus]Sca1+c-Kit+ [LSK] cells) trafficking and survival. In murine models of aged and young hosts, we show that FLT3L enhances thymopoiesis in aged, Lupron-treated hosts through increased survival and export of LSK cells via CXCR4 regulation. In addition, we elucidate an underlying mechanism of FLT3L action on BM LSK cells-FLT3L drives LSK cells into the stromal niche using Hoescht (Ho) dye perimortem. In summary, we show that FLT3L administration leads to: (1) increased LSK cells and early thymocyte progenitor precursors that can enhance thymopoiesis after transplantation and androgen withdrawal, (2) mobilization of LSK cells through downregulation of CXCR4, (3) enhanced BM stem cell survival associated with Bcl-2 upregulation, and (4) BM stem cell enrichment through increased trafficking to the BM niche. Therefore, we show a mechanism by which FLT3L activity on hematopoeitic and thymic progenitor cells may contribute to thymic recovery. These data have potential clinical relevance to enhance thymic reconstitution after cytoreductive therapy.

Brief Report: Role of Thymic Reconstitution in the Outcome of AIDS-Related PML.

Implications of thymopoiesis in AIDS-related opportunistic infections remain unexplored. We used progressive multifocal leukoencephalopathy (PML), caused by JC virus (JCV), as an opportunistic infection model, and we simultaneously investigated thymic output and T-cell responses against JCV in 22 patients with PML treated with combined antiretroviral therapy. Thymic output was significantly associated with JCV-specific CD4⁺ and CD8⁺ T-cell responses and improved survival. Our data suggest that patients with AIDS-related PML and impaired thymopoiesis are less likely to develop a robust JCV-specific cellular immune response and consequently are at an increased risk for a poor clinical outcome.

Impact of Pre-Transplant Anti-T Cell Globulin (ATG) on Immune Recovery after Myeloablative Allogeneic Peripheral Blood Stem Cell Transplantation

BackgroundPre-transplant infusion of rabbit anti-T cell globulin (ATG) is increasingly used as prevention of graft-versus-host disease (GVHD) after allogeneic peripheral blood stem cell transplantation (PBSCT). However, the precise impact of pre-transplant ATG on immune recovery after PBSCT is still poorly documented.MethodsIn the current study, we compared immune recovery after myeloablative PBSCT in 65 patients who either received (n = 37) or did not (n = 28) pre-transplant ATG-Fresenius (ATG-F). Detailed phenotypes of circulating T, B, natural killer (NK) and invariant NKT (iNKT) cells were analyzed by multicolor flow cytometry at serial time-points from day 40 to day 365 after transplantation. Thymic function was also assessed by sjTREC quantification. Serious infectious events were collected up to 2 years post-transplantation.ResultsPre-transplant ATG-F had a prolonged (for at least up to 1-year) and selective negative impact on the T-cell pool, while it did not impair the recovery of B, NK nor iNKT cells. Among T cells, ATG-F selectively compromised the recovery of naïve CD4+, central memory CD4+ and naïve CD8+ cells, while it spared effector memory T and regulatory T cells. Levels of sjTRECs were similar in both cohorts at 1-year after PBSCT, suggesting that ATG-F unlikely impaired thymopoiesis at long-term after PBSCT. Finally, the incidence and rate of serious infections were similar in both groups, while ATG-F patients had a lower incidence of grade II-IV acute graft-versus-host disease.ConclusionsPre-transplant ATG-F induces long-lasting modulation of the circulating T-cell pool after myeloablative PBSCT, that may participate in preventing graft-versus-host disease without deeply compromising anti-pathogen defenses.

Thymus involution and regeneration: two sides of the same coin?

In vertebrates, the thymus is the main site of T cell development. The thymus reaches its maximum output during adolescence, after which it shrinks and generates fewer and fewer T cells. Physiological age-related involution of the thymus and failure to recover after injury are associated with impaired cellular immunity; hence, there is considerable interest in developing strategies to combat these deficiencies. In this Opinion article, we briefly review the phylogenetic and ontogenetic hallmarks of thymus development and function, and we discuss experimental models of impaired thymopoiesis and the molecular mechanisms of thymopoietic recovery. At each stage of the discussion we highlight the major gaps in our current knowledge.

Coactivator-Associated Arginine Methyltransferase 1 Regulates Fetal Hematopoiesis and Thymocyte Development

Coactivator-associated arginine methyltransferase 1 (CARM1) is a protein arginine methyltransferase that methylates histones and transcriptional regulators. We previously reported that the absence of CARM1 partially blocks thymocyte differentiation at embryonic day 18.5 (E18.5). In this study, we find that reduced thymopoiesis in Carm1(-/-) mice is due to a defect in the fetal hematopoietic compartment rather than in the thymic stroma. To determine the cellular basis for impaired thymopoiesis, we examined the number and function of fetal liver (FL) and bone marrow cells. Despite markedly reduced cellularity of hematopoietic progenitors in E18.5 bone marrow, the number of long-term hematopoietic stem cells and downstream subsets was not reduced in Carm1(-/-) E14.5 or E18.5 FL. Nevertheless, competitive reconstitution assays revealed a deficit in the ability of Carm1(-/-) FL cells to contribute to hematopoiesis. Furthermore, impaired differentiation of Carm1(-/-) FL cells in a CARM1-sufficient host showed that CARM1 is required cell autonomously in hematopoietic cells. Coculture of Carm1(-/-) FL cells on OP9-DL1 monolayers showed that CARM1 is required for survival of hematopoietic progenitors under conditions that promote differentiation. Taken together, this report demonstrates that CARM1 is a key epigenetic regulator of hematopoiesis that affects multiple lineages at various stages of differentiation.

GSK3β Inhibition Promotes T Cell Reconstitution and Preserves Naive T Cell Phenotype in Bone Marrow Reconstituted Mice

AbstractAbstract 1890Stem cell transplantation has become a widely used procedure in the treatment of haematological and non-haematological clinical disorders. Unfortunately, cure is often hampered by relapse of the underlying disease, graft-versus-host disease (GVHD), or severe opportunistic infections. Slow T-cell reconstitution is regarded as primarily responsible for infections, GVHD, and relapse, therefore, enhancing immune reconstitution is important. Glycogen synthase kinase-3β (GSK3β) was recently identified as an important regulator of T cell function acting through the Wingless (Wnt) pathway. The effect of in vivo administration of GSK3β inhibitor 6-Bromoindirubin 3’-oxime (BIO) was examined in a humanised mouse model. Mice transplanted with highly purified cord blood CD34+ stem cells demonstrated efficient multilineage reconstitution including myeloid, B and T cell lymphoid compartments. The presence of human CD4 and CD8 single positive human T cells was abundant in peripheral blood (PB). De novo generated T cells exhibited low CD31 expression in the naïve CD4+ T cells suggesting prolonged post-thymic proliferative history of these cells. This is not completely surprising considering that graft recipient mice are characterized by impaired thymopoiesis following irradiation. Human T cells at various stages of differentiation including late effector T cells were recorded by detecting the expression of CD62L, CD45RA and CD45RO. Late memory T cell skewing was observed in PB and spleen of graft recipient mice. Activation of human T cells expressing CD25 was registered in the spleen, however, the recipients of the graft did not exhibit any signs of GVHD suggesting normal positive and negative selection occurring in the thymus during human T cell development in this mouse model. Human T cells isolated from the spleen of transplanted mice exhibited strong proliferative responses to mitogenic and allogeneic stimulation, however, they did not demonstrated any CTL activity tested following vaccination with human leukaemia U937 cells. In vivo administration of GSK3β inhibitor promoted T cell reconstitution in mice transplanted with human CD34+haematopoietic progenitor cells. Per cell output of T cells from CD34+ and CD34+CD38- primitive bone marrow (BM) progenitor cells was higher in BIO-treated mice while CD19+ B cell output was reduced suggesting T-cell developmental skewing in expense of B cell development. In vitro analysis of CD34+ progenitor cells co-cultured with bone marrow stroma MS5 cells has demonstrated inhibited B-cell development following BIO-treatment. CD31 expression in naïve CD4+ T cells was not up-regulated by BIO suggesting that GSK3β inhibition does not act to increase thymic output of T cells. GSK3β inhibition also increased naïve/memory T cell ratio in reconstituted mice. A similar effect was observed in mice transplanted with mature cord blood (CB)-derived T cells. delayed naive to memory T cell transition is likely related to decreased T cell activation and proliferation demonstrated ex vivo. BIO reduced IFNγ and TNFα production in human T cells. BIO increased naïve T cell production in mitogenically stimulated T cells and in mixed lymphocyte cultures. GSK3β inhibition preserved naïve T cell gene expression profile and suppressed the expression of genes activated during effector T cell differentiation. BIO actrivated β-catenin sigbnaling and up-regulated IL7Rα expression. IL7 signalling prevents activated T cell death following effector differentiation suggesting that the mechanism triggered by BIO may act through the inhibition of activated T cell death. In addition, BIO down-regulated negative regulator of IL7Rα SOCS1 as well as CTLA4 and PDCD1 both up-regulated during effector differentiation. Thus clinically GSK3β inhibition acting to prevent late memory T cell skewing and preserving a subset of naïve T cells may increase T cell diversity and improve T cell responses in the recipients of CB transplant particularly in adult patients with impaired thymic function. Disclosures:No relevant conflicts of interest to declare.

Severe Chronic Graft-Versus-Host Disease Is Associated with Impaired Thymopoiesis and Peripheral Lymphocyte Expansion

Severe Chronic Graft-Versus-Host Disease Is Associated with Impaired Thymopoiesis and Peripheral Lymphocyte Expansion

Genetic Evidence for an Evolutionarily Conserved Role of IL-7 Signaling in T Cell Development of Zebrafish

In mammals, the cytokine IL-7 is a key regulator of various aspects of lymphocyte differentiation and homeostasis. Because of the difficulty of identifying cytokine homologs in lower vertebrates and the paucity of assay systems and reagents, the degree of functional conservation of cytokine signaling pathways, particularly those pertaining to lymphocyte development, is unclear. In this article, we report on the analysis and characterization of three zebrafish mutants with severely impaired thymopoiesis. The identification of affected genes by positional cloning revealed components of the IL-7 signaling pathway. A presumptive null allele of the zebrafish homolog of the IL-7Rα-chain causes substantially reduced cellularity of the thymus but spares B cell development in the kidney. Likewise, nonsense mutations in the zebrafish homologs of janus kinases JAK1 and JAK3 preferentially affect T cell development. The functional interactions of the cytokine receptor components were examined in the three groups of fish hetero- or homozygous for either il7r and jak1, il7r and jak3, or jak1 and jak3 mutations. The differential effects on T cell development arising from the different genotypes could be explained on the basis of the known structure of the mammalian IL-7R complex. Because IL-7 signaling appears to be a universal requirement for T cell development in vertebrates, the mutants described in this article represent alternative animal models of human immunodeficiency syndromes amenable to large-scale genetic and chemical screens.

CMV Infection after Transplant from Cord Blood Compared to Other Alternative Donors: The Importance of Donor-Negative CMV Serostatus

Cytomegalovirus (CMV) infection and disease are important complications after hematopoietic stem cell transplant, particularly after transplant from alternative donors. Allogeneic cord blood transplantation (CBT) is being increasingly used, but immune recovery may be delayed. The aim of this study was to compare CMV infection in CBT with transplants from unrelated or mismatched related donors, from now on defined as alternative donors. A total of 165 consecutive transplants were divided in 2 groups: (1) alternative donors transplants (n = 85) and (2) CBT recipients (n = 80). Donor and recipient (D/R) CMV serostatus were recorded. The incidence of CMV infection, its severity, timing, and outcome were compared. Median follow-up was 257 days (1-1328). CMV infection was monitored by CMV antigenemia and expressed as CMV Ag positive cell/2 × 10(5) polymorphonuclear blood cells. There was a trend toward a higher cumulative incidence of CMV infection among CBT than alternative donor transplant recipients (64% vs 51%, P = .12). The median time to CMV reactivation was 35 days, and was comparable in the 2 groups (P = .8). The maximum number of CMV-positive cells was similar in the 2 groups (11 versus 16, P = .2). The time interval between the first and the last positive CMV antigenemia was almost 4 times longer in CBT compared with alternative donor transplants (109 vs 29 days, respectively, P = .008). The incidence of late CMV infection was also higher in CBT (62% vs 24%, P < .001). The incidence of early and late CMV infection in CBT was similar to D-/R+ alternative transplants, and higher than in D+/R+ alternative transplants: early infection, 72% in CBT versus 69% in D-/R+ alternative versus 55% in D+/R+ alternative (P = .21); and late infection, 67% in CBT versus 60% in D-/R+ alternative versus 7% in D+/R+ alternative (P < .001). Transplant-related mortality and overall survival were similar between the groups: 34% versus 36% (P = .6) and 54% versus 46% (P = .3) for alternative transplant and CBT, respectively. Longer duration and higher incidence of late CMV infection was seen in CBT patients, when compared with alternative donor transplants, whereas no difference in mortality was observed. The duration and incidence of late CMV infection were similar when D-/R+ CBT were compared with D-/R+ alternative donor transplants.

Impaired Thymopoiesis With Normal T Regulatory Cell Numbers is Associated With Severe Chronic Graft-Versus-Host Disease

Chronic GVHD (cGVHD) is a significant complication of allogeneic hematopoietic stem cell transplantation (AHSCT). Although T cells have been implicated in cGVHD pathobiology, the role of the thymus in this process has yet to be defined. We characterized thymus and spleen T cell subsets in a murine model of cGVHD to elucidate the role of the thymus in this process. The murine model of minor-histocompatibility mismatch cGVHD, B10.D2- > BALB/c, was validated by pathologic review that correlated with the clinical scoring and showed cGVHD pathology in skin, stomach, small intestine, colon and liver.

Cross-talk between IL-7Ra signaling and p53 pathway during thymopoiesis and lymphomagenesis (86.8)

Abstract Thymopoiesis in IL-7Rα-/- mice is blocked at the CD4-CD8- double negative stage with failures in T cell receptor (TCR) γand β rearrangement. Early studies suggest that unsuccessful TCRβ rearrangement in mice defective in DNA damage repairs, incurring DNA double-strand breaks, results in p53 activation. However, it remains unknown whether p53 plays a role in the thymopoietic defect in IL-7Rα-/- mice. In this study, we demonstrated that p53 is highly activated in the thymocytes of IL-7Rα-/- mice. Considering the importance of p53 activity during TCR rearrangement and the observation of incomplete rescue of thymopoiesis in IL-7Rα-/- mice by modulating activities of Bcl-2 family members, we hypothesize that p53 activation may also contribute to the impaired thymopoiesis in IL-7Rα-/- mice and that temporal suppression of p53 activity by the IL-7Rα signaling facilitates thymopoiesis. With the generated IL-7Rα-/-p53-/- (DKO) mice, we found that p53 inactivation in IL-7Rα-/- background resulted in a 30-fold increase in thymic cellularity with increased TCRβ? cells. Furthermore, DKO mice developed thymic lymphoma at a higher incidence and died significantly earlier than p53-/- mice. Our study is the first to illustrate a functional interplay between IL-7Rα signaling and p53 pathways in facilitating TCRβ+ T cell development during thymopoiesis and lymphomagenesis.

Delayed immune reconstitution after cord blood transplantation is characterized by impaired thymopoiesis and late memory T-cell skewing

Advances in immune assessment, including the development of T-cell receptor excision circle (TREC) assays of thymopoiesis, cytokine-flow cytometry assays of T-cell function, and higher-order phenotyping of T-cell maturation subsets have improved our understanding of T-cell homeostasis. Limited data exist using these methods to characterize immune recovery in adult cord blood (CB) transplant recipients, in whom infection is a leading cause of mortality. We now report the results of a single-center prospective study of T-cell immune recovery after cord blood transplantation (CBT) in a predominantly adult population. Our primary findings include the following: (1) Prolonged T lymphopenia and compensatory expansion of B and natural killer (NK) cells was evident; (2) CB transplant recipients had impaired functional recovery, although we did observe posttransplantation de novo T-cell responses to cytomegalovirus (CMV) in a subset of patients; (3) Thymopoietic failure characterized post-CBT immune reconstitution, in marked contrast to results in other transplant recipients; and (4) Thymopoietic failure was associated with late memory T-cell skewing. Our data suggest that efforts to improve outcomes in adult CB transplant recipients should be aimed at optimizing T-cell immune recovery. Strategies that improve the engraftment of lymphoid precursors, protect the thymus during pretransplant conditioning, and/or augment the recovery of thymopoiesis may improve outcomes after CBT.

Comparison of Immune Reconstitution after Umbilical Cord Blood (UCB) and Allogenic Peripheral Blood Stem Cell (APBSC) Transplantation in Patients with Advanced Multiple Myeloma (MM) and Acute Myeloid Leukemia (AML).

Background. T-cell reconstitution is usually delayed after UCB transplantation, in addition to an impaired thymopoiesis and late memory T cell skewing. NK cells are usually variable, with an initial increase in % and absolute number. The role of these cells is unclear, particularly on tumor control, maybe participating to the T-cell lymphopenia. Patients and methods. F rom 01/03 to 04/07, we retrospectively analysed data concerning lymphocyte recovery for patients having MM and AML, who received allogenic transplantation (AT). We excluded patients with progressive disease before AT, with short follow-up (<120days) and without dominant donor chimerism. 43 patients (MM, n=19; AML, n=24) were included, and they received UCBT (n=13: MM=7 and AML=6; single: n=2 or double: n=11), all with reduced intensity conditioning (RIC) regimen adapted from the Minneapolis protocol, or APBSC (n=30) [RIC (n=23; MM=12 and AML=11), including ATG (n=12/23; AML=10/11) or myelo-ablative conditioning (MAC: n=7, all having AML,]. Immune reconstitution in PB was followed, including CD3+, CD4+, CD8+, CD3−/CD56+, and CD19+ cells, monthly. Prophylaxis of GVHD included cyclosporin A from d-3 to months +3 or +6, according to clinical manifestations of GVHD, and mycophenolate mofetil from d-3 to d+28 after RIC regimen and standard prophylaxis with cyclosporine and short course methotrexate after MAC regimen. Anti-infectious prophylaxis and follow-up were standard. Results. Median age was comparable between groups (MM: DUCB (57y, 42–61), PBSC (55y, 50–68); AML: DUCB (45y, 30–63), PBSC (53y, 21–64). Sex ratio (F/M) was 6/13 for MM, and 11/13 for AML. In the group MM/UCB, NK cells was above 80/mm3 from D-7 to 1 year and peaked at D260 (median=193/mm3), with no statistical difference with the MM/PBSC group (64 at D-7 to 132/mm3). NK cells were significantly higher (p=0.04) for AML/UBC group (from D90: 187/mm3) to D260: 328/mm3), in comparison to AML/PBSC (from 116 to 138/mm3). For CD4 T-lymphocytes, there was a significant difference between MM/UCB and MM/PBSC during the first 3 months [39 to 75/mm3 vs 152 to 175/mm3, p=0.01]. A more rapid recovery of CD4+ was observed in AML/UBC (from 227 to 829/mm3, D90 to 1y), 2-fold higher than in the AML/PBSC at any time. Those differences could be explained by the high-dose therapy given to patients with MM. In addition, patients who received ATG (all AML/PBSC/RIC) had a delayed CD4 recovery. CD8-cell recovery was delayed for UBC groups with a CD8 cell count >200 starting at D230 as compared to the PBSC group (D150 for MM and D90 for AML). B cell recovery was faster in the UCB group [MM/UCB and AML/UCB, >200/mm3 at D230 and D150 as compared to D300 and D230 respectively in MM/PBSC and AML/PBSC (p=NS)]. Conclusion. NK cells increased after UBC more intensively and rapidly than after PBSC AT. T-cell recovery appeared to be associated to pre-treatment intensity and use of ATG. CD4, CD8 counts were highly reduced in UCB compared to PBSC except for CD4+ in AML/UCB. B-cell recovery is not different between both groups (i.e. UCB and PBSC). NK cells from 2 patients are now collected for analysis of their functions particularly their anti-tumoral effect in vitro.