Veronica Basso

Prolonged TCR/CD28 Engagement Drives IL-2-Independent T Cell Clonal Expansion through Signaling Mediated by the Mammalian Target of Rapamycin

Proliferation of Ag-specific T cells is central to the development of protective immunity. The concomitant stimulation of the TCR and CD28 programs resting T cells to IL-2-driven clonal expansion. We report that a prolonged occupancy of the TCR and CD28 bypasses the need for autocrine IL-2 secretion and sustains IL-2-independent lymphocyte proliferation. In contrast, a short engagement of the TCR and CD28 only drives the expansion of cells capable of IL-2 production. TCR/CD28- and IL-2-driven proliferation revealed a different requirement for PI3K and for the mammalian target of rapamycin (mTOR). Thus, both PI3K and mTOR activities were needed for T cells to proliferate to TCR/CD28-initiated stimuli and for optimal cyclin E expression. In contrast, either PI3K or mTOR were sufficient for IL-2-driven cell proliferation as they independently mediated cyclin E induction. Interestingly, rapamycin delayed cell cycle entry of IL-2-sufficient T cells, but did not prevent their expansion. Together, our findings indicate that the TCR, CD28, and IL-2 independently control T cell proliferation via distinct signaling pathways involving PI3K and mTOR. These data suggest that Ag persistence and the availability of costimulatory signals and of autocrine and paracrine growth factors individually shape T lymphocyte expansion in vivo.

The soluble D2D388-274 fragment of the urokinase receptor inhibits monocyte chemotaxis and integrin-dependent cell adhesion

We have previously shown that chymotrypsin-cleaved soluble uPAR (D2D388-274) elicits migration of monocytic cells through interaction with FPRL-1, a G protein-coupled receptor that is homologous to the fMLP receptor. Here, we report that D2D388-274 also modulates the ability of monocytes to migrate in response to other chemokines. Pretreatment of monocytes with increasing amounts of D2D388-274 prevents cell migration in response to MCP-1, RANTES and fMLP. We demonstrate that D2D388-274 does not inhibit MCP-1 receptor binding, elicit CCR2 internalization and prevent MCP-1-induced intracellular Ca2+ increase. Thus, CCR2 receptor desensitization cannot account for D2D388-274-mediated inhibition of MCP-1-induced cell migration. Rather, we show that pretreatment of monocytes with D2D388-274 dramatically decreases chemokine-induced integrin-dependent rapid cell adhesion by interacting with FPRL-1. Together, our results indicate that chemokine-dependent cell migration can be regulated not only by homologous and heterologous receptor desensitization, but also by inhibition of integrin-dependent cell adhesion, an important step in cell transmigration.

Targeted inactivation of the COP9 signalosome impairs multiple stagesof T cell development

Genetic programs promoting cell cycle progression, DNA repair, and survival are coordinately induced in developing T cells and require rapid turnover of effector molecules. As the COP9 signalosome (CSN) has been placed at the crossroads of these programs in lower organisms, we addressed its role by conditionally deleting CSN5/JAB1, its catalytic subunit, in developing thymocytes. CSN5/JAB1del/del thymocytes show defective S phase progression and massive apoptosis at the double-negative (DN) 4–double-positive (DP) transition stage, which is paralleled by altered turnover of selected CSN-controlled substrates, including p53, IκB-α, and β-catenin. Combined dysregulation of the p53 and NF-κB pathways affects thymocyte survival by altering the mRNA and protein levels of selected Bcl-2 family members. Genetic complementation analysis performed on p53−/−, Bcl-xL/Bcl-2A1, or T cell receptor transgenic backgrounds indicates that CSN5/JAB1 acts at distinct developmental stages to coordinate proliferation, survival, and positive selection of thymocytes by controlling the induction of defined genetic programs acting downstream of CSN-regulated transcription factors.

Autonomic Denervation of Lymphoid Organs Leads to Epigenetic Immune Atrophy in a Mouse Model of Krabbe Disease

Lysosomal β-galactosylceramidase deficiency results in demyelination and inflammation in the nervous system causing the neurological Krabbe disease. In the Twitcher mouse model of this disease, we found that neurological symptoms parallel progressive and severe lymphopenia. Although lymphopoiesis is normal before disease onset, primary and secondary lymphoid organs progressively degenerate afterward. This occurs despite preserved erythropoiesis and leads to severe peripheral lymphopenia caused by reduced numbers of T cell precursors and mature lymphocytes. Hematopoietic cell replacement experiments support the existence of an epigenetic factor in mutant mice reconcilable with a progressive loss of autonomic axons that hampers thymic functionality. We propose that degeneration of autonomic nerves leads to the irreversible thymic atrophy and loss of immune-competence. Our study describes a new aspect of Krabbe disease, placing patients at risk of immune-related pathologies, and identifies a novel target for therapeutic interventions.

Clonal Anergy Is Maintained Independently of T Cell Proliferation

Ag encounter in the absence of proliferation results in the establishment of T cell unresponsiveness, also known as T cell clonal anergy. Anergic T cells fail to proliferate upon restimulation because of the inability to produce IL-2 and to properly regulate the G1 cell cycle checkpoint. Because optimal TCR and CD28 engagement can elicit IL-2-independent cell cycle progression, we investigated whether CD3/CD28-mediated activation of anergic T cells could overcome G1 cell cycle block, drive T cell proliferation, and thus reverse clonal anergy. We show here that although antigenic stimulation fails to elicit G1-to-S transition, anti-CD3/CD28 mAbs allow proper cell cycle progression and proliferation of anergic T cells. However, CD3/CD28-mediated cell division does not restore Ag responsiveness. Our data instead indicate that reversal of clonal anergy specifically requires an IL-2-dependent, rapamycin-sensitive signal, which is delivered independently of cell proliferation. Thus, by tracing proliferation and Ag responsiveness of individual cells, we show that whereas both TCR/CD28 and IL-2-generated signals can drive T cell proliferation, only IL-2/IL-2R interaction regulates Ag responsiveness, indicating that proliferation and clonal anergy can be independently regulated.

FOXP3 + T Cells Recruited to Sites of Sterile Skeletal Muscle Injury Regulate the Fate of Satellite Cells and Guide Effective Tissue Regeneration

Muscle injury induces a classical inflammatory response in which cells of the innate immune system rapidly invade the tissue. Macrophages are prominently involved in this response and required for proper healing, as they are known to be important for clearing cellular debris and supporting satellite cell differentiation. Here, we sought to assess the role of the adaptive immune system in muscle regeneration after acute damage. We show that T lymphocytes are transiently recruited into the muscle after damage and appear to exert a pro-myogenic effect on muscle repair. We observed a decrease in the cross-sectional area of regenerating myofibers after injury in Rag2-/- γ-chain-/- mice, as compared to WT controls, suggesting that T cell recruitment promotes muscle regeneration. Skeletal muscle infiltrating T lymphocytes were enriched in CD4+CD25+FOXP3+ cells. Direct exposure of muscle satellite cells to in vitro induced Treg cells effectively enhanced their expansion, and concurrently inhibited their myogenic differentiation. In vivo, the recruitment of Tregs to acutely injured muscle was limited to the time period of satellite expansion, with possibly important implications for situations in which inflammatory conditions persist, such as muscular dystrophies and inflammatory myopathies. We conclude that the adaptive immune system, in particular T regulatory cells, is critically involved in effective skeletal muscle regeneration. Thus, in addition to their well-established role as regulators of the immune/inflammatory response, T regulatory cells also regulate the activity of skeletal muscle precursor cells, and are instrumental for the proper regeneration of this tissue.

Involvement of Prep1 in the αβ T-Cell Receptor T-Lymphocytic Potential of Hematopoietic Precursors

ABSTRACT Prep1 is a homeodomain transcription factor that acts by dimerizing with Pbx. Since Prep1 null embryos die at gastrulation, we studied Prep1 i/i hypomorphic mice to study the physiological role of Prep1. A low percentage of homozygous Prep1 i/i mice survived at birth, and their postnatal functions could be investigated. Reduced Prep1 expression caused an abnormal thymic T-cell development: increased CD4− CD8− double-negative thymocytes, decrease in αβTCRhigh cells (cells with high levels of the αβΤ-cell receptor [αβTCR]) and CD4+ and CD8+ single-positive (SP) thymocytes, and increase in γδTCR cells. Peripheral lymphoid organs of Prep1 i/i mice contained fewer αβTCR mature T cells and more γδTCR T cells than wild-type littermates. Moreover, Prep1 i/i CD4+ CD8+ double-positive thymocytes underwent more apoptosis, and SP thymocytes proliferated less than control littermates. Mice that were lethally irradiated and then had Prep1 i/i fetal liver cells transplanted showed the same defects as the Prep1 i/i mice did. Among PBC family members, Pbx2 and very low levels of Pbx3 were observed in the thymi of wild-type mice. In Prep1 i/i mice, the level of Pbx2 protein was profoundly decreased, while for Pbx3 no definitive conclusion could be reached. Therefore, the deficient postnatal T-lymphocytic potential of the Prep1 hematopoietic progenitors depends on the combined, not compensated, absence of Prep1 and at least Pbx2.

Phenotype and Homing of CD4 Tumor-Specific T Cells Is Modulated by Tumor Bulk

Technical difficulties in tracking endogenous CD4 T lymphocytes have limited the characterization of tumor-specific CD4 T cell responses. Using fluorescent MHC class II/peptide multimers, we defined the fate of endogenous Leishmania receptor for activated C kinase (LACK)-specific CD4 T cells in mice bearing LACK-expressing TS/A tumors. LACK-specific CD44highCD62Llow CD4 T cells accumulated in the draining lymph nodes and had characteristics of effector cells, secreting IL-2 and IFN-γ upon Ag restimulation. Increased frequencies of CD44highCD62Llow LACK-experienced cells were also detected in the spleen, lung, liver, and tumor itself, but not in nondraining lymph nodes, where the cells maintained a naive phenotype. The absence of systemic redistribution of LACK-specific memory T cells correlated with the presence of tumor. Indeed, LACK-specific CD4 T cells with central memory features (IL-2+IFN-γ−CD44highCD62Lhigh cells) accumulated in all peripheral lymph nodes of mice immunized with LACK-pulsed dendritic cells and after tumor resection. Together, our data demonstrate that although tumor-specific CD4 effector T cells producing IFN-γ are continuously generated in the presence of tumor, central memory CD4 T cells accumulate only after tumor resection. Thus, the continuous stimulation of tumor-specific CD4 T cells in tumor-bearing mice appears to hinder the systemic accumulation of central memory CD4 T lymphocytes.

IL-7 is superior to IL-2 for ex vivo expansion of tumour-specific CD4+ T cells

It is well established that tumours hinder both natural and vaccine‐induced tumour‐specific CD4+ T‐cell responses. Adoptive T‐cell therapy has the potential to circumvent functional tolerance and enhance anti‐tumour protective responses. While protocols suitable for the expansion of cytotoxic CD8+ T cells are currently available, data on tumour‐specific CD4+ T cells remain scarce. We report here that CD4+ T cells sensitized to tumour‐associated Ag in vivo, proliferate in vitro in response to IL‐7 without the need for exogenous Ag stimulation and accumulate several folds while preserving a memory‐like phenotype. Both cell proliferation and survival accounts for the outgrowth of tumour‐sensitized T cells among other memory and naive lymphocytes following exposure to IL‐7. Also IL‐2, previously used to expand anti‐tumour CTL, promotes tumour‐specific CD4+ T‐cell accumulation. However, IL‐7 is superior to IL‐2 at preserving lymphocyte viability, in vitro and in vivo, maintaining those properties, that are required by helper CD4+ T cells to confer therapeutic efficacy upon transplantation in tumour‐bearing hosts. Together our data support a unique role for IL‐7 in retrieving memory‐like CD4+ T cells suitable for adoptive T‐cell therapy.

Concomitant Tumor and Minor Histocompatibility Antigen–Specific Immunity Initiate Rejection and Maintain Remission from Established Spontaneous Solid Tumors

Nonmyeloablative hematopoietic cell transplantation can cure patients with hematologic malignancies but has reported limited success against solid tumors. This is possibly because of profound peripheral tolerance mechanisms and/or suboptimal tumor recognition by effector T lymphocytes. We report that in mice developing spontaneous prostate cancer, nonmyeloablative minor histocompatibility mismatched hematopoietic stem cell transplantation, and donor lymphocyte infusion of unmanipulated lymphocytes combined with posttransplant tumor-specific vaccination circumvents tumor-specific tolerance, allowing acute tumor rejection and the establishment of protective immunosurveillance. Although donor-derived tumor-specific T cells readily differentiated into effector cells and infiltrated the tumor soon after infusion, they were alone insufficient for tumor eradication, which instead required the concomitance of minor histocompatibiltiy antigen-specific CD8(+) T-cell responses. The establishment of protective immunosurveillance was best induced by posttransplant tumor-specific vaccination. Hence, these results provide the proof of principle that tumor-specific T-cell responses have to be harnessed together with minor histocompatibility responses and sustained by posttransplant tumor-specific vaccination to improve the efficacy of allotransplantion for the cure of solid tumors.