Nuno L. Alves

IL-15 trans-presentation promotes human NK cell development and differentiation in vivo

The in vivo requirements for human natural killer (NK) cell development and differentiation into cytotoxic effectors expressing inhibitory receptors for self–major histocompatability complex class I (MHC-I; killer Ig-like receptors [KIRs]) remain undefined. Here, we dissect the role of interleukin (IL)-15 in human NK cell development using Rag2−/−γc−/− mice transplanted with human hematopoietic stem cells. Human NK cell reconstitution was intrinsically low in this model because of the poor reactivity to mouse IL-15. Although exogenous human IL-15 (hIL-15) alone made little improvement, IL-15 coupled to IL-15 receptor α (IL-15Rα) significantly augmented human NK cells. IL-15–IL-15Rα complexes induced extensive NK cell proliferation and differentiation, resulting in accumulation of CD16+KIR+ NK cells, which was not uniquely dependent on enhanced survival or preferential responsiveness of this subset to IL-15. Human NK cell differentiation in vivo required hIL-15 and progressed in a linear fashion from CD56hiCD16−KIR− to CD56loCD16+KIR−, and finally to CD56loCD16+KIR+. These data provide the first evidence that IL-15 trans-presentation regulates human NK cell homeostasis. Use of hIL-15 receptor agonists generates a robust humanized immune system model to study human NK cells in vivo. IL-15 receptor agonists may provide therapeutic tools to improve NK cell reconstitution after bone marrow transplants, enhance graft versus leukemia effects, and increase the pool of IL-15–responsive cells during immunotherapy strategies.

Characterization of the thymic IL-7 niche in vivo

The thymus represents the “cradle” for T cell development, with thymic stroma providing multiple soluble and membrane cues to developing thymocytes. Although IL-7 is recognized as an essential factor for thymopoiesis, the “environmental niche” of thymic IL-7 activity remains poorly characterized in vivo. Using bacterial artificial chromosome transgenic mice in which YFP is under control of IL-7 promoter, we identify a subset of thymic epithelial cells (TECs) that co-express YFP and high levels of Il7 transcripts (IL-7hi cells). IL-7hi TECs arise during early fetal development, persist throughout life, and co-express homeostatic chemokines (Ccl19, Ccl25, Cxcl12) and cytokines (Il15) that are critical for normal thymopoiesis. In the adult thymus, IL-7hi cells localize to the cortico-medullary junction and display traits of both cortical and medullary TECs. Interestingly, the frequency of IL-7hi cells decreases with age, suggesting a mechanism for the age-related thymic involution that is associated with declining IL-7 levels. Our temporal-spatial analysis of IL-7-producing cells in the thymus in vivo suggests that thymic IL-7 levels are dynamically regulated under distinct physiological conditions. This IL-7 reporter mouse provides a valuable tool to further dissect the mechanisms that govern thymic IL-7 expression in vivo.

IL-21 Sustains CD28 Expression on IL-15-Activated Human Naive CD8+ T Cells

Human naive CD8+ T cells are able to respond in an Ag-independent manner to IL-7 and IL-15. Whereas IL-7 largely maintains CD8+ T cells in a naive phenotype, IL-15 drives these cells to an effector phenotype characterized, among other features, by down-regulation of the costimulatory molecule CD28. We evaluated the influence of the CD4+ Th cell-derived common γ-chain cytokine IL-21 on cytokine-induced naive CD8+ T cell activation. Stimulation with IL-21 did not induce division and only slightly increased IL-15-induced proliferation of naive CD8+ T cells. Strikingly, however, IL-15-induced down-modulation of CD28 was completely prevented by IL-21 at the protein and transcriptional level. Subsequent stimulation via combined TCR/CD3 and CD28 triggering led to a markedly higher production of IL-2 and IFN-γ in IL-15/IL-21-stimulated cells compared with IL-15-stimulated T cells. Our data show that IL-21 modulates the phenotype of naive CD8+ T cells that have undergone IL-15 induced homeostatic proliferation and preserves their responsiveness to CD28 ligands.

Differential regulation of human IL-7 receptor alpha expression by IL-7 and TCR signaling.

IL-7Rα is essential for the development and homeostatic maintenance of mature T cells. Studies in humans and mice have shown that IL-7Rα expression is reduced by its cognate cytokine, IL-7, and Ag, suggesting that active regulation of IL-7 responsiveness is necessary to balance T cell numbers. We show that IL-7- or TCR/CD28-mediated signaling induced a rapid down-regulation of IL-7Rα expression on naive T cells on the mRNA and protein level, with a mild (10-fold) or strong (50-fold) gene suppression, respectively. In both situations, the down-regulation of IL-7Rα was blocked by cyclohexamide and actinomycin D, indicating the involvement of an active mechanism dependent on new transcription and protein synthesis. Upon IL-7 withdrawal, IL-7Rα mRNA and surface protein reappeared in a transcription-dependent manner within 7 h. Yet, IL-7Rα was hardly re-expressed during the same period after TCR/CD28-activation. Likewise, T cells that were activated through CMV in vivo did not re-express IL-7Rα after in vitro culture. Functionally, IL-7-induced down-regulation of IL-7Rα did not hinder the responsiveness of naive T cells to IL-7. Conversely, down-regulation of IL-7Rα on TCR/CD28-activated cells limited IL-7 responsiveness. Strikingly, ectopic expression of IL-7Rα cells on TCR/CD28-activated cells conferred a selective advantage in the response to IL-7. In conclusion, our data show that IL-7- and TCR/CD28-mediated signaling differentially regulate IL-7Rα expression on human T cells with a transient and chronic effect, respectively. The stringent and active regulation of IL-7Rα may constitute a homeostatic mechanism to curtail unwarranted T cell expansion.

IL-15 transpresentation promotes both human T-cell reconstitution and T-cell–dependent antibody responses in vivo

Cytokine immunotherapies targeting T lymphocytes are attractive clinical interventions against viruses and tumors. In the mouse, the homeostasis of memory α/β CD8+ T cells and natural killer (NK) cells is significantly improved with increased IL-15 bioavailability. In contrast, the role of “transpresented” IL-15 on human T-cell development and homeostasis in vivo is unknown. We found that both CD8 and CD4 T cells in human immune system (HIS) mice are highly sensitive to transpresented IL-15 in vivo, with both naïve (CD62L+CD45RA+) and memory phenotype (CD62L−CD45RO+) subsets being significantly increased following IL-15 “boosting.” The unexpected global improvement in human T-cell homeostasis involved enhanced proliferation and survival of both naïve and memory phenotype peripheral T cells, which potentiated B-cell responses by increasing the frequency of antigen-specific responses following immunization. Transpresented IL-15 did not modify T-cell activation patterns or alter the global T-cell receptor (TCR) repertoire diversity. Our results indicate an unexpected effect of IL-15 on human T cells in vivo, in particular on CD4+ T cells. As IL-15 promotes human peripheral T-cell homeostasis and increases the frequency of neutralizing antibody responses in HIS mice, IL-15 immunotherapy could be envisaged as a unique approach to improve vaccine responses in the clinical setting.

Serial progression of cortical and medullary thymic epithelial microenvironments.

Thymic epithelial cells (TECs) provide key instructive signals for T‐cell differentiation. Thymic cortical (cTECs) and medullary (mTECs) epithelial cells constitute two functionally distinct microenvironments for T‐cell development, which derive from a common bipotent TEC progenitor. While seminal studies have partially elucidated events downstream of bipotent TECs in relation to the emergence of mTECs and their progenitors, the control and timing of the emergence of the cTEC lineage, particularly in relation to that of mTEC progenitors, has remained elusive. In this review, we describe distinct models that explain cTEC/mTEC lineage divergence from common bipotent progenitors. In particular, we summarize recent studies in mice providing evidence that mTECs, including the auto‐immune regulator+ subset, derive from progenitors initially endowed with phenotypic properties typically associated with the cTEC lineage. These observations support a novel “serial progression” model of TEC development, in which progenitors serially acquire cTEC lineage markers, prior to their commitment to the mTEC differentiation pathway. Gaining a better understanding of the phenotypic properties of early stages in TEC progenitor development should help in determining the mechanisms regulating cTEC/mTEC lineage development, and in strategies aimed at thymus reconstitution involving TEC therapy.

Crosstalk among Bcl-2 family members in B-CLL: seliciclib acts via the Mcl-1/Noxa axis and gradual exhaustion of Bcl-2 protection.

Seliciclib (R-roscovitine) is a cyclin-dependent kinase inhibitor in clinical development. It triggers apoptosis by inhibiting de novo transcription of the short-lived Mcl-1 protein, but it is unknown how this leads to Bax/Bak activation that is required for most forms of cell death. Here, we studied the effects of seliciclib in B-cell chronic lymphocytic leukemia (B-CLL), a malignancy with aberrant expression of apoptosis regulators. Although seliciclib-induced Mcl-1 degradation within 4 h, Bax/Bak activation occurred between 16 and 20 h. During this period, no transcriptional changes in apoptosis-related genes occurred. In untreated cells, prosurvival Mcl-1 was engaged by the proapoptotic proteins Noxa and Bim. Upon drug treatment, Bim was quickly released. The contribution of Noxa and Bim as a specific mediator of seliciclib-induced apoptosis was demonstrated via RNAi. Significantly, 16 h after seliciclib treatment, there was accumulation of Bcl-2, Bim and Bax in the ‘mitochondria-rich’ insoluble fraction of the cell. This suggests that after Mcl-1 degradation, the remaining apoptosis neutralizing capacity of Bcl-2 is gradually overwhelmed, until Bax forms large multimeric pores in the mitochondria. These data demonstrate in primary leukemic cells hierarchical binding and crosstalk among Bcl-2 members, and suggest that their functional interdependence can be exploited therapeutically.

Thymocyte Selection Regulates the Homeostasis of IL-7–Expressing Thymic Cortical Epithelial Cells In Vivo

Thymic epithelial cells (TECs) help orchestrate thymopoiesis, and TEC differentiation relies on bidirectional interactions with thymocytes. Although the molecular mediators that stimulate medullary thymic epithelial cell (mTEC) maturation are partially elucidated, the signals that regulate cortical thymic epithelial cell (cTEC) homeostasis remain elusive. Using IL-7 reporter mice, we show that TECs coexpressing high levels of IL-7 (Il7YFP+ TECs) reside within a subset of CD205+Ly51+CD40low cTECs that coexpresses Dll4, Ccl25, Ccrl1, Ctsl, Psmb11, and Prss16 and segregates from CD80+CD40high mTECs expressing Tnfrsf11a, Ctss, and Aire. As the frequency of Il7YFP+ TECs gradually declines as mTEC development unfolds, we explored the relationship between Il7YFP+ TECs and mTECs. In thymic organotypic cultures, the thymocyte-induced reduction in Il7YFP+ TECs dissociates from the receptor activator of NF-κB–mediated differentiation of CD80+ mTECs. Still, Il7YFP+ TECs can generate some CD80+ mTECs in a stepwise differentiation process via YFP−Ly51lowCD80low intermediates. Il7YFP+ TECs are sustained in Rag2−/− mice, even following in vivo anti-CD3ε treatment that mimics the process of pre-TCR β-selection of thymocytes to the double positive (DP) stage. Using Marilyn-Rag2−/− TCR transgenic, we find that positive selection into the CD4 lineage moderately reduces the frequency of Il7YFP+ TECs, whereas negative selection provokes a striking loss of Il7YFP+ TECs. These results imply that the strength of MHC/peptide–TCR interactions between TECs and thymocytes during selection constitutes a novel rheostat that controls the maintenance of IL-7–expressing cTECs.

A New Subset of Human Naive CD8+ T Cells Defined by Low Expression of IL-7Rα

Concomitant with an increased number of memory-type cells, the amount of naive T cells steadily declines with age. Although the regulatory mechanisms behind this conversion are not fully understood, the suggestion is that both alterations in thymic output and homeostatic signals mold the naive T cell pool. In this study, we identify a new subset of circulating CD27highCD45RAhigh CD8+ T cells characterized by low IL-7Rα message and protein expression. Analysis of TCR repertoire and TCR excision circle content together with ex vivo recovery of IL-7Rα expression indicated that these cells should be placed into the naive T cell pool. Compared with conventional IL-7Rαhigh naive T cells, this subset displayed significantly lower levels of CD28 and higher levels of HLA-DR. Proliferative responses to anti-CD3/CD28 mAbs were indistinguishable from conventional naive T cells, but the responsiveness to IL-7 was limited. Strikingly, IL-7Rαlow naive T cells were particularly increased in circumstances of naive CD8+ T cells shortage, as in the elderly, in patients early after hemopoietic stem cell transplantation, and in HIV-infected individuals. As common γ chain cytokines induce rapid down-regulation of IL-7Rα, we propose that this new subset of naive T cells may encompass cells that have recently received homeostatic signals.

Intermediate expression of CCRL1 reveals novel subpopulations of medullary thymic epithelial cells that emerge in the postnatal thymus

Cortical and medullary thymic epithelial cells (cTECs and mTECs, respectively) provide inductive microenvironments for T‐cell development and selection. The differentiation pathway of cTEC/mTEC lineages downstream of common bipotent progenitors at discrete stages of development remains unresolved. Using IL‐7/CCRL1 dual reporter mice that identify specialized TEC subsets, we show that the stepwise acquisition of chemokine (C–C motif) receptor‐like 1 (CCRL1) is a late determinant of cTEC differentiation. Although cTECs expressing high CCRL1 levels (CCRL1hi) develop normally in immunocompetent and Rag2−/−thymi, their differentiation is partially blocked in Rag2−/−Il2rg−/− counterparts. These results unravel a novel checkpoint in cTEC maturation that is regulated by the cross‐talk between TECs and immature thymocytes. Additionally, we identify new Ulex europaeus agglutinin 1 (UEA)+ mTEC subtypes expressing intermediate CCRL1 levels (CCRL1int) that conspicuously emerge in the postnatal thymus and differentially express Tnfrsf11a, Ccl21, and Aire. While rare in fetal and in Rag2−/− thymi, CCRL1int mTECs are restored in Rag2−/−Marilyn TCR‐Tg mice, indicating that the appearance of postnatal‐restricted mTECs is closely linked with T‐cell selection. Our findings suggest that alternative temporally restricted routes of new mTEC differentiation contribute to the establishment of the medullary niche in the postnatal thymus.