Theratyping cystic fibrosis patients to guide elexacaftor/tezacaftor/ivacaftor out-of-label prescription.

BACKGROUND: Around 20% of people with cystic fibrosis (pwCF) do not have access to the triple combination elexacaftor/tezacaftor/ivacaftor (ETI) in Europe because they do not carry the F508del allele on the CF transmembrane conductance regulator (CFTR) gene. Considering that pwCF carrying rare variants may benefit from ETI, including variants already validated by the US Food and Drug Administration (FDA), a compassionate use programme was launched in France. PwCF were invited to undergo a nasal brushing to investigate whether the pharmacological rescue of CFTR activity by ETI in human nasal epithelial cell (HNEC) cultures was predictive of the clinical response. METHODS: CFTR activity correction was studied by short-circuit current in HNEC cultures at basal state (dimethyl sulfoxide (DMSO)) and after ETI incubation and expressed as percentage of normal (wild-type (WT)) CFTR activity after sequential addition of forskolin and Inh-172 (ΔI ETI/DMSO%WT). RESULTS: 11 pwCF carried variants eligible for ETI according to the FDA label and 28 carried variants not listed by the FDA. ETI significantly increased CFTR activity of FDA-approved CFTR variants (I601F, G85E, S492F, M1101K, R347P, R74W;V201M;D1270N and H1085R). We point out ETI correction of non-FDA-approved variants, including N1303K, R334W, R1066C, Q552P and terminal splicing variants (4374+1G>A and 4096-3C>G). ΔI ETI/DMSO%WT was significantly correlated to change in percentage predicted forced expiratory volume in 1 s and sweat chloride concentration (p<0.0001 for both). G85E, R74W;V201M;D1270N, Q552P and M1101K were rescued more efficiently by other CFTR modulator combinations than ETI. CONCLUSIONS: Primary nasal epithelial cells hold promise for expanding the prescription of CFTR modulators in pwCF carrying rare mutants. Additional variants should be discussed for ETI indication.

Lymphoid Gene Upregulation on Circulating Progenitors Participates in Their T-Lineage Commitment.

Extrathymic T cell precursors can be detected in many tissues and represent an immediately competent population for rapid T cell reconstitution in the event of immunodeficiencies. Blood T cell progenitors have been detected, but their source in the bone marrow (BM) remains unclear. Prospective purification of BM-resident and circulating progenitors, together with RT-PCR single-cell analysis, was used to evaluate and compare multipotent progenitors (MPPs) and common lymphoid progenitors (CLPs). Molecular analysis of circulating progenitors in comparison with BM-resident progenitors revealed that CCR9(+) progenitors are more abundant in the blood than CCR7(+) progenitors. Second, although Flt3(-) CLPs are less common in the BM, they are abundant in the blood and have reduced Cd25(+)-expressing cells and downregulated c-Kit and IL-7Rα intensities. Third, in contrast, stage 3 MPP (MPP3) cells, the unique circulating MPP subset, have upregulated Il7r, Gata3, and Notch1 in comparison with BM-resident counterparts. Evaluation of the populations' respective abilities to generate splenic T cell precursors (Lin(-)Thy1.2(+)CD25(+)IL7Rα(+)) after grafting recipient nude mice revealed that MPP3 cells were the most effective subset (relative to CLPs). Although several lymphoid genes are expressed by MPP3 cells and Flt3(-) CLPs, the latter only give rise to B cells in the spleen, and Notch1 expression level is not modulated in the blood, as for MPP3 cells. We conclude that CLPs have reached the point where they cannot be a Notch1 target, a limiting condition on the path to T cell engagement.

Innate pro-B-cell progenitors protect against type 1 diabetes by regulating autoimmune effector T cells.

Diverse hematopoietic progenitors, including myeloid populations arising in inflammatory and tumoral conditions and multipotent cells, mobilized by hematopoietic growth factors or emerging during parasitic infections, display tolerogenic properties. Innate immune stimuli confer regulatory functions to various mature B-cell subsets but immature B-cell progenitors endowed with suppressive properties per se or after differentiating into more mature regulatory B cells remain to be characterized. Herein we provide evidence for innate pro-B cells (CpG-proBs) that emerged within the bone marrow both in vitro and in vivo upon Toll-like receptor-9 activation and whose adoptive transfer protected nonobese diabetic mice against type 1 diabetes (T1D). These cells responded to IFN-γ released by activated effector T cells (Teffs), by up-regulating their Fas ligand (FasL) expression, which enabled them to kill Teffs through apoptosis. In turn, IFN-γ derived from CpG-proBs enhanced IFN-γ while dramatically reducing IL-21 production by Teffs. In keeping with the crucial pathogenic role played by IL-21 in T1D, adoptively transferred IFN-γ-deficient CpG-proBs did not prevent T1D development. Additionally, CpG-proBs matured in vivo into diverse pancreatic and splenic suppressive FasL(high) B-cell subsets. CpG-proBs may become instrumental in cell therapy of autoimmune diseases either on their own or as graft complement in autologous stem cell transplantation.

CCL22-producing CD8α- myeloid dendritic cells mediate regulatory T cell recruitment in response to G-CSF treatment.

G-CSF prevents type 1 diabetes in the NOD mouse by promoting the local recruitment of T regulatory cells (Tregs). This is an indirect effect because adoptive transfer of G-CSF-induced tolerogenic dendritic cells (DCs) promotes Treg accumulation. However, the identity of the particular DC subset and the molecule(s) mediating this effect remain unknown. We demonstrate in this study that the adoptive transfer of CD11c(high)CD8α(-) DCs isolated from pegylated G-CSF (pegG-CSF) recipients, but not that of other DC subtypes, enhanced the pancreatic recruitment of CD4(+)CD25(+)Foxp3(+) Tregs, which generated increased amounts of TGF-ß. Likewise, only CD11c(high)CD8α(-) DCs from pegG-CSF recipients secreted the chemokine CCL22 at levels that effectively attracted Tregs. PegG-CSF was more efficient at enhancing the synthesis of CCL22 by CD11c(high)CD8α(-) DCs from the pancreatic lymph nodes compared with those from the spleen. Accordingly, CD11c(high)CD8α(-) DCs from the pancreatic lymph nodes of pegG-CSF recipients were more efficient than their splenic counterparts in the recruitment of Tregs upon adoptive transfer. Predictably, CD11c(high)CD8α(-) DCs failed to recruit these Tregs both in vivo and in vitro following intracellular neutralization of CCL22. These data assign a key role to CD8α(-) DCs and CCL22 in Treg recruitment in the protection of NOD mice against type 1 diabetes following the treatment with G-CSF.

CpG-Activated Regulatory B-Cell Progenitors Alleviate Murine Graft-Versus-Host-Disease.

Development of Graft Versus Host Disease (GVHD) represents a major impediment in allogeneic hematopoietic stem cell transplantation (HSCT). The observation that the presence of bone marrow and circulating hematogones correlated with reduced GVHD risks prompted us to evaluate whether B-cell progenitors, which provide protection in various autoimmune disease models following activation with the TLR-9 agonist CpG (CpG-proBs), could likewise reduce this allogeneic disorder. In a murine model of GVHD that recapitulates an initial phase of acute GVHD followed by a phase of chronic sclerodermatous GVHD, we found that CpG-proBs, adoptively transferred during the initial phase of disease, reduced the diarrhea score and mostly prevented cutaneous fibrosis. Progenitors migrated to the draining lymph nodes and to the skin where they mainly differentiated into follicular B cells. CpG activation and IFN-γ expression were required for the protective effect, which resulted in reduced CD4+ T-cell-derived production of critical cytokines such as TGF-ß, IL-13 and IL-21. Adoptive transfer of CpG-proBs increased the T follicular regulatory to T follicular helper (Tfr/Tfh) ratio. Moreover, CpG-proBs privileged the accumulation of IL-10-positive CD8+ T cells, B cells and dendritic cells in the skin. However, CpG-proBs did not improve survival. Altogether, our findings support the notion that adoptively transferred CpG-proBs exert immunomodulating effect that alleviates symptoms of GVHD but require additional anti-inflammatory strategy to improve survival.

Toll-like receptor-9 stimulated plasmacytoid dendritic cell precursors suppress autoimmune neuroinflammation in a murine model of multiple sclerosis.

Early innate education of hematopoietic progenitors within the bone marrow (BM) stably primes them for either trained immunity or instead immunoregulatory functions. We herein demonstrate that in vivo or in vitro activation within the BM via Toll-like receptor-9 generates a population of plasmacytoid dendritic cell (pDC) precursors (CpG-pre-pDCs) that, unlike pDC precursors isolated from PBS-incubated BM (PBS-pre-pDCs), are endowed with the capacity to halt progression of ongoing experimental autoimmune encephalomyelitis. CpG activation enhances the selective migration of pDC precursors to the inflamed spinal cord, induces their immediate production of TGF-ß, and after migration, of enhanced levels of IL-27. CpG-pre-pDC derived TGF-ß and IL-27 ensure protection at early and late phases of the disease, respectively. Spinal cords of CpG-pre-pDC-protected recipient mice display enhanced percentages of host-derived pDCs expressing TGF-ß as well as an accumulation of IL-10 producing B cells and of CD11c+ CD11b+ dendritic cells. These results reveal that pDC precursors are conferred stable therapeutic properties by early innate activation within the BM. They further extend to the pDC lineage promising perspectives for cell therapy of autoimmune diseases with innate activated hematopoietic precursor cells.

Role of GM-CSF in tolerance induction by mobilized hematopoietic progenitors.

Mechanisms of protection against autoimmune diseases by transplantation of autologous hematopoietic progenitors remain poorly defined. We recently demonstrated that, unlike medullary hematopoietic stem cells (HSCs), mobilized hematopoietic progenitors (HPCs) stimulate peripheral Foxp3(+) regulatory T cell (Treg)-expansion through cell-contact activation of Notch signaling and through as yet undetermined soluble factor(s), distinct from TGF-beta1. Herein we identified one such soluble factor as granulocyte macrophage-colony stimulating factor (GM-CSF), which is produced at higher levels by HPCs than HSCs and whose neutralization significantly reduces the growth-promoting effect of HPCs on Treg. Treg express a functional GM-CSF receptor alpha-chain CD116 and proliferate in response to this cytokine independently from IL2. GM-CSF-expanded Treg-like HPC-expanded Treg-display enhanced suppressive capacity relative to control Treg. Hence, mobilized progenitors stimulate Treg expansion both by cell-contact dependent mechanisms and by their production of GM-CSF.

Role of uncoupling protein UCP2 in cell-mediated immunity: how macrophage-mediated insulitis is accelerated in a model of autoimmune diabetes.

Infiltration of inflammatory cells into pancreatic islets of Langerhans and selective destruction of insulin-secreting beta-cells are characteristics of type 1 diabetes. Uncoupling protein 2 (UCP2) is a mitochondrial protein expressed in immune cells. UCP2 controls macrophage activation by modulating the production of mitochondrial reactive oxygen species (ROS) and MAPK signaling. We investigated the role of UCP2 on immune cell activity in type 1 diabetes in Ucp2-deficient mice. Using the model of multiple low-dose streptozotocin (STZ)-induced diabetes, we found that autoimmune diabetes was strongly accelerated in Ucp2-KO mice, compared with Ucp2-WT mice with increased intraislet lymphocytic infiltration. Macrophages from STZ-treated Ucp2-KO mice had increased IL-1beta and nitric oxide (NO) production, compared with WT macrophages. Moreover, more macrophages were recruited in islets of STZ-treated Ucp2-KO mice, compared with Ucp2-WT mice. This finding also was accompanied by increased NO/ROS-induced damage. Altogether, our data show that inflammation is stronger in Ucp2-KO mice and islets, leading to the exacerbated disease in these mice. Our results highlight the mitochondrial protein UCP2 as a new player in autoimmune diabetes.

Mobilized Multipotent Hematopoietic Progenitors Promote Expansion and Survival of Allogeneic Tregs and Protect Against Graft Versus Host Disease.

Allogeneic Hematopoietic Stem Cell Transplantation (Allo-HSCT) is routinely performed with peripheral blood stem cells (PBSCs) mobilized by injection of G-CSF, a growth factor which not only modulates normal hematopoiesis but also induces diverse immature regulatory cells. Based on our previous evidence that G-CSF-mobilized multipotent hematopoietic progenitors (MPP) can increase survival and proliferation of natural regulatory T cells (Tregs) in autoimmune disorders, we addressed the question how these cells come into play in mice and humans in an alloimmune setting. Using a C57BL/6 mouse model, we demonstrate that mobilized MPP enhance the immunosuppressant effect exerted by Tregs, against alloreactive T lymphocytes, both in vitro and in vivo. They do so by migrating to sites of allopriming, interacting with donor Tregs and increasing their numbers, thus reducing the lethality of graft-versus-host disease (GVHD). Protection correlates likewise with increased allospecific Treg counts. Furthermore, we provide evidence for a phenotypically similar MPP population in humans, where it shares the capacity to promote selective Treg expansion in vitro. We postulate that G-CSF-mobilized MPPs might become a valuable cellular therapy to expand donor Tregs in vivo and prevent GVHD, thereby making allo-HSCT safer for the treatment of leukemia patients.

Mobilized Multipotent Hematopoietic Progenitors Stabilize and Expand Regulatory T Cells to Protect Against Autoimmune Encephalomyelitis.

Achieving immunoregulation via in vivo expansion of Foxp3+ regulatory CD4+ T cells (Treg) remains challenging. We have shown that mobilization confers to multipotent hematopoietic progenitors (MPPs) the capacity to enhance Treg proliferation. Transcriptomic analysis of Tregs co-cultured with MPPs revealed enhanced expression of genes stabilizing the suppressive function of Tregs as well as the activation of IL-1ß-driven pathways. Adoptive transfer of only 25,000 MPPs effectively reduced the development of experimental autoimmune encephalomyelitis (EAE), a pre-clinical model for multiple sclerosis (MS). Production of the pathogenic cytokines IL-17 and GM-CSF by spinal cord-derived CD4+ T-cells in MPP-protected recipients was reduced while Treg expansion was enhanced. Treg depletion once protection by MPPs was established, triggered disease relapse to the same level as in EAE mice without MPP injection. The key role of IL-1ß was further confirmed in vivo by the lack of protection against EAE in recipients of IL-1ß-deficient MPPs. Mobilized MPPs may thus be worth considering for cell therapy of MS either per se or for enrichment of HSC grafts in autologous bone marrow transplantation already implemented in patients with severe refractory multiple sclerosis.

Treatment with granulocyte colony-stimulating factor prevents diabetes in NOD mice by recruiting plasmacytoid dendritic cells and functional CD4(+)CD25(+) regulatory T-cells.

Accumulating evidence that granulocyte colony-stimulating factor (G-CSF), the key hematopoietic growth factor of the myeloid lineage, not only represents a major component of the endogenous response to infections, but also affects adaptive immune responses, prompted us to investigate the therapeutic potential of G-CSF in autoimmune type 1 diabetes. Treatment with G-CSF protected NOD mice from developing spontaneous diabetes. G-CSF triggered marked recruitment of dendritic cells (DCs), particularly immature CD11c(lo)B220(+) plasmacytoid DCs, with reduced costimulatory signal expression and higher interferon-alpha but lower interleukin-12p70 release capacity than DCs in excipient-treated mice. G-CSF recipients further displayed accumulation of functional CD4(+)CD25(+) regulatory T-cells that produce transforming growth factor-beta1 (TGF-beta1) and actively suppressed diabetes transfer by diabetogenic effector cells in secondary NOD-SCID recipients. G-CSF's ability to promote key tolerogenic interactions between DCs and regulatory T-cells was demonstrated by enhanced recruitment of TGF-beta1-expressing CD4(+)CD25(+) cells after adoptive transfer of DCs isolated from G-CSF- relative to vehicle-treated mice into naive NOD recipients. The present results suggest that G-CSF, a promoter of tolerogenic DCs, may be evaluated for the treatment of human type 1 diabetes, possibly in association with direct inhibitors of T-cell activation. They also provide a rationale for a protective role of the endogenous G-CSF produced during infections in early diabetes.

Fas receptor signaling is requisite for B cell differentiation.

The Fas/Fas ligand (FasL) pathway has been largely implicated in the homeostasis of mature cells. However, it is still unclear whether it plays a role at the progenitor level. To address this issue, we created chimeric mice by transferring C57BL/6 bone marrow (BM) cells of the lpr (Fas-FasL+) or gld (Fas+FasL-) genotype into Rag-2-/- hosts of the same genetic background. In this model, the consequences of a deficient Fas/FasL pathway on lymphoid differentiation could be evaluated without endogenous competition. Analysis of the chimerism revealed a differential sensitivity of hematopoietic lineages to the lack of Fas receptor signaling. While donor-derived myelo-monocytic cells were similarly distributed in all chimeric mice, mature B cells were deleted in the BM and the spleen of lpr chimera, leading to the absence of the marginal zone (MZ) as detected by immunohistology. In contrast, B cell hematopoiesis was complete in gld chimera but MZ macrophages undetectable. These defects suggest a direct and determinant dual role of FasL regulation in negative selection of B cells and in maintenance of the MZ.

Characterization and Immunoregulatory Properties of Innate Pro-B-Cell Progenitors.

Control of T-cell responses can be achieved by several subsets of B cells with immunoregulatory functions, mostly acting by provision of the anti-inflammatory cytokine IL-10 or exhibiting killing properties through Fas ligand (Fas-L) or granzyme B-induced cell death. We herein describe the characterization as well as the cellular and molecular mechanisms mediating the suppressive properties of bone marrow immature innate pro-B cell progenitors that emerge upon transient activation of Toll-like receptor 9. They are licensed by activated T-cell-derived IFN-γ to become suppressive by up-regulating their Fas-L expression and inducing effector CD4(+) T-cell apoptosis. They also up-regulate their own IFN-γ production which dramatically reduces T-cell production of a major pathogenic cytokine, IL-21. A single adoptive transfer of as little as 60,000 of them efficiently prevents the onset of spontaneous type 1 diabetes in recipient nonobese diabetes (NOD) mice, highlighting the remarkable regulatory potency of these so-called CpG-proB cell progenitors compared to regulatory cells of diverse lineages so far described. The CpG-proB cell activity is prolonged in vivo by their differentiation after migration in the pancreas and the spleen into B-cell progeny with high Fas-L expression that can keep up inducing apoptosis of effector T cells in the long term.

Treatment of ongoing autoimmune encephalomyelitis with activated B-cell progenitors maturing into regulatory B cells.

The influence of signals perceived by immature B cells during their development in bone marrow on their subsequent functions as mature cells are poorly defined. Here, we show that bone marrow cells transiently stimulated in vivo or in vitro through the Toll-like receptor 9 generate proB cells (CpG-proBs) that interrupt experimental autoimmune encephalomyelitis (EAE) when transferred at the onset of clinical symptoms. Protection requires differentiation of CpG-proBs into mature B cells that home to reactive lymph nodes, where they trap T cells by releasing the CCR7 ligand, CCL19, and to inflamed central nervous system, where they locally limit immunopathogenesis through interleukin-10 production, thereby cooperatively inhibiting ongoing EAE. These data demonstrate that a transient inflammation at the environment, where proB cells develop, is sufficient to confer regulatory functions onto their mature B-cell progeny. In addition, these properties of CpG-proBs open interesting perspectives for cell therapy of autoimmune diseases.

G-CSF mobilizes CD34+ regulatory monocytes that inhibit graft-versus-host disease.

Granulocyte colony-stimulating factor (G-CSF) is routinely used to collect peripheral blood stem cells (PBSCs) from healthy donors for allogeneic hematopoietic stem cell transplantation (allo-HSCT). We show that, in both humans and mice, G-CSF mobilizes a subset of CD34(+) cells with mature monocyte features. These cells, which are phenotypically and functionally conserved in mice and humans, are transcriptionally distinct from myeloid and monocytic precursors but similar to mature monocytes and endowed with immunosuppressive properties. In response to interferon-γ released by activated T cells, these cells produce nitric oxide, which induces allogeneic T cell death both in vitro and in vivo. These apoptotic T cells are engulfed by macrophages that release transforming growth factor-ß and promote regulatory T cell expansion. Indeed, the fraction of CD34(+) monocytes in peripheral blood CD34(+) cells inversely correlates with the incidence of acute graft-versus-host disease (GVHD) in humans. Therefore, G-CSF-mobilized cells are an attractive candidate population to be expanded ex vivo for cellular therapy against GVHD.

Jagged2-expressing hematopoietic progenitors promote regulatory T cell expansion in the periphery through notch signaling.

Cellular interactions promoting the in vivo expansion of CD4(+)CD25(+)Foxp3(+) regulatory T (Treg) cells for maintenance of immune tolerance remain poorly defined. Here we report that mobilized Lin(-)Sca-1(+)c-kit(+) (LSK) hematopoietic progenitor cells (HPCs), unlike medullary hematopoietic stem cells (HSCs), selectively drove the direct, immediate expansion of functional host-derived Treg cells, thereby preventing the progression to overt spontaneous autoimmune diabetes in nonobese diabetic mice. Treg cell expansion required cell-to-cell contact and Notch3 signaling, which was mediated selectively through the Notch ligand Jagged2 expressed by the multipotent HPC subset, as assessed by small interfering RNA (siRNA) silencing. Conversely, notwithstanding their similar multilineage microchimerism, neither sorted Jagged2(-) HPCs nor Jagged2(lo) medullary HSCs were able to expand Treg cells. These data provide evidence for a productive Notch-mediated interaction between a unique subset of mobilized hematopoietic progenitors and Treg cells. They open therapeutic perspectives for autologous transplantation of Jagged2(+) LSK progenitors to promote Treg cell expansion in T cell-mediated diseases.

Granulocyte colony-stimulating factor: a novel mediator of T cell tolerance.

In recent years, several investigators have unraveled a previously unrecognized role for G-CSF in the regulation of T cell and dendritic cell functions. The experimental evidence in favor of G-CSF-mediated immune regulation includes the ability to switch T cell cytokine secretion profile to Th2 responses and the promotion of regulatory T cell and tolerogenic dendritic cell differentiation. Interestingly, G-CSF is beneficial in animals for the prevention and/or treatment of immune-mediated diseases, e.g., graft-vs-host disease, multiple sclerosis, systemic lupus erythematosus, inflammatory bowel disease, and diabetes, suggesting a potential role in human autoimmune diseases. This review summarizes the growing body of evidence that supports a critical role for G-CSF as a novel mediator of T cell tolerance.

G-CSF treatment prevents cyclophosphamide acceleration of autoimmune diabetes in the NOD mouse.

Cyclophosphamide (CY) accelerates autoimmune diabetes in the NOD mouse at different levels, including critical targeting of a regulatory T cell subset, exacerbation of pro-Th1 IFN-gamma production and promotion of inflammation in pancreatic islets. Here we evaluated the ability of G-CSF to antagonize the acceleration of the disease induced by CY. Human recombinant G-CSF, administered daily at 200 microg/kg by s.c. injection, protected NOD mice from CY-accelerated onset of glycosuria and insulitis. G-CSF accelerated the recovery of the T cell compartment after the depletion of the lymphoid compartment triggered by CY injection. It selectively prevented the loss of the immunoregulatory T cells expressing the CD4(+)CD25+ phenotype that also stained CD62L+ in peripancreatic lymph nodes and promoted their expansion in the spleen. In addition to this, it abrogated the robust cytokine--particularly IFN-gamma- and chemokine burst triggered in immune cells by CY. G-CSF promoted only slight changes in the inflammatory effects of CY at the target tissue site, assessed by chemokine induction within the pancreas. Thus the immunoregulatory properties of G-CSF were critical in the early control of the accelerating effects of CY on autoimmune diabetes in the NOD mouse.