ABSTRACT
Following acute stress such as trauma or sepsis, most of critically ill elderly patients become immunosuppressed and susceptible to secondary infections and enhanced mortality. We have developed a virus-based immunotherapy encoding human interleukin-7 (hIL-7) aiming at restoring both innate an adaptative immune homeostasis in these patients. We assessed the impact of this encoded hIL-7 on the ex vivo immune functions of T cells from PBMC of immunosenescent patients with or without hip fracture. T-cell ex vivo phenotyping was characterized in terms of senescence (CD57), IL-7 receptor (CD127) expression, and T cell differentiation profile. Then, post stimulation, activation status, and functionality (STAT5/STAT1 phosphorylation and T cell proliferation assays) were evaluated by flow cytometry. Our data show that T cells from both groups display immunosenescence features, express CD127 and are activated after stimulation by virotherapy-produced hIL-7-Fc. Interestingly, hip fracture patients exhibit a unique functional ability: An important T cell proliferation occurred compared to controls following stimulation with hIL-7-Fc. In addition, stimulation led to an increased naïve T cell as well as a decreased effector memory T cell proportions compared to controls. This preliminary study indicates that the produced hIL-7-Fc is well recognized by T cells and initiates IL-7 signaling through STAT5 and STAT1 phosphorylation. This signaling efficiently leads to T cell proliferation and activation and enables a T cell “rejuvenation.” These results are in favor of the clinical development of the hIL-7-Fc expressing virotherapy to restore or induce immune T cell responses in immunosenescent hip fracture patients.
KEYWORDS: Immunosenescence, hip fracture, immunotherapy, virotherapy, interleukin-7, T-cell functions, trauma, immunosuppression, cytokine
Introduction
The world population is aging due to the decrease in mortality and birth rates, especially in industrialized countries. For example, more than 10% of the European population will be over 80 in 2050. This will require fundamental changes in the care provided to this population to maximize disability-free aging and improve quality of life but also to reduce economic burden of health systems.1 Aging is accompanied by complex changes associating physiological alterations and chronic comorbidities. These two elements lead to a decrease in the individual’s reserve capacities, which results in a loss of autonomy and life-quality and which sometimes leads to patients’ death.2 Among the organs and systems affected by aging, the immune system undergoes major modifications.3 The two main immune system changes during aging are immunosenescence (affecting both the innate and adaptive systems), and a systemic production of pro-inflammatory cytokines, called ‘inflammaging’.4–6
While knowledge in the field of immunosenescence has grown over the years, the impact of stress on the elderly immune system is still poorly understood. Hip Fracture (HF) represents a frequent traumatic stress in elderly patients with important consequences. For example, a delayed mortality is observed in HF patients reaching up to 20–30% mortality observed at 1-year post-trauma.3 In addition, this traumatic stress induces a reduction in the quality of autonomy and an increase of patient medical care costs.7,8 The trauma induced by HF is also associated with additional immunological changes such as a transient pro-inflammatory phenotype of the innate compartment and immunosuppressive profile of the adaptative compartment.9 These changes may participate in the poor medical outcome post-HF in some of these patients. A comprehensive investigation of the induced immune-alterations post-stress would open avenues for the development of immunotherapies enabling the restoration of immune homeostasis, especially in the situation of trauma such as HF. One of the drivers of immunosenescence and its consequences in elderly trauma patients is the thymus involution leading to lymphoid compartment changes toward a decrease of naïve T cells essential for an effective adaptative immune response against infection.10
Interleukin 7 (IL-7) is known to be a central factor for homeostatic T-cell proliferation, differentiation, and survival.11 Naïve and minimally differentiated T cells express the IL-7 receptor subunit α (IL-7 Rα or CD127) which, in combination with the common γ-chain, induces intracellular activation of STAT pathways (mainly STAT5) and PI3K/Akt/mTOR via Jak1 and Jak3.12–14 This signaling drives anti-apoptotic signals and metabolic activation.15,16 IL-7 has also been regularly described as a potential rescue therapy for immunosuppressed and/or immune senescent patients.17 Ex vivo experiments demonstrated that IL-7 could expand naïve and memory T cells18 and restored T cell functionalities of septic19 and SARS-CoV-2-infected patients.20 In addition, soluble IL-7 has been evaluated in many clinical trials in oncology,21 HIV,22 and more recently in sepsis23 and COVID-19 infection.24 In immunosuppressed septic patients, soluble IL-7 was shown to be well-tolerated and increases absolute lymphocyte counts, in particular circulating CD4 and CD8 T cells.23,25
To address the immunosuppression observed in sepsis patients in Intensive Care Unit, we have developed a new immunotherapy expressing human IL-7 (hIL-7). The therapy is based on the non-propagative recombinant Modified Vaccinia virus Ankara (MVA) encoding hIL-7 gene fused to human IgG2 Fc sequence, namely MVA-hIL7-Fc. MVA is an attractive vector platform displaying a very high safety profile demonstrated in many clinical trials.26 In addition, it can stimulate the host innate immunity,27,28 a feature of interest in settings of global immunosuppression. In preclinical experiments performed in naive mice and non-human primates administered with the MVA-hIL7-Fc, pharmacokinetics studies showed that hIL7-Fc is well expressed and detected in blood.29,30 In addition, in cecal ligation puncture (CLP) sepsis mice model, the MVA-hIL7-Fc was shown to boost innate and adaptive immunity of immunosuppressed CLP mice as well as to confer a survival advantage.29 The MVA-hIL7-Fc induced an expected increase of T cell populations (CD4 and CD8) but also B lymphocytes, NK cells, and myeloid subpopulations (monocytes/macrophages and neutrophils). Interestingly, the MVA-hIL-7-Fc induced CD8 and CD4 T cell activation and functionalities such as cytokine production in particular interferon-γ.29 In a recent study, we demonstrated that the hIL-7-Fc produced by the recombinant MVA-hIL-7-Fc is able to activate ex-vivo T-cell functionalities in immunosuppressed sepsis and COVID-19 patients.31 Thus, one could imagine to administer the novel MVA-IL-7-Fc virotherapy directly to the immunosuppressed sepsis patients (specifically by the IV route) in order to help sepsis recovery.
The encouraging results obtained with the MVA-hIL-7-Fc in the above-mentioned sepsis models supported us to explore the potential of this novel virotherapy in other immunosuppressive conditions. Interestingly, a recent review highlights similarities between aged-induced immunosenescence and sepsis induced immunosuppression and their impact on induced injuries.32 Hence, impact of the MVA-hIL-7-Fc and more specifically of the expressed hIL-7-Fc on T cells of elderly immunosenescent patients suffering from HF trauma was investigated. Peripheral Blood Mononuclear Cells (PBMCs) from the HIPAGE cohort,9 which includes HF patients and age-matched controls, were ex vivo stimulated using the MVA produced hIL-7-Fc and impacts on T cell phenotypes and functions were analyzed.
Materials and methods
HIPAGE cohort
A prospective observational clinical study, HIPAGE, including HF patients aged over 75 y admitted in emergency department of Pitié Salpêtrière hospital, was approved by the French ethical committee “Comité de Protection des personnes (CPP)” Pitié Salpêtrière, Paris, France (N° CPP-PSL25042012).
Age-matched control patients were also recruited (Table 1). All participants included were informed and gave their consents. In this study, analyses were performed on cryopreserved PBMCs from 7 old controls (CTRLo) and 10 old HF patients (HF) obtained 48–72 h after hip fracture surgery. This cohort was described previously.9 For immunosenescence status comparison, 5 young donors (25< age <40) from blood transfusion center (Etablissement Français du Sang) were also included (CTRLy).
Table 1.
Cohort’s description.
Control individuals | HF patients | t-test (*p ≤ .05) | |
---|---|---|---|
Sample size | 7 | 10 | |
Age | 87.9 | 88.9 | NS |
Gender (M/F) | 3/4 | 5/5 | NS |
CD3 count (cells/µl) | 1052 | 740.2 | * |
CD4 count (cells/µl) | 814.7 | 495.2 | * |
CD8 count (cells/µl) | 258.5 | 134.7 | * |
MVA-hIL-7-Fc engineering and hIL7-Fc supernatant production
MVA-hIL7-Fc is a recombinant Modified Virus Ankara expressing the human IL-7 fused to human IgG2 Fc and was previously described.29 MVATGN33.1 is the empty viral vector used as negative control. Both vectors were produced on primary chicken embryo fibroblasts (CEFs). Primary human hepatocytes (Biopredic International) were cultivated as recommended by the provider and plated on collagen. Cells were infected with the MVA-hIL7-Fc or MVATGN33.1 at a multiplicity of infection of 5, and supernatants were collected 24 h post-infection, filtered through 0,1 µm, aliquoted, and stored at −20°C. The concentration of hIL-7-Fc in the collected supernatants was assessed by ELISA (DuoSet, R&D system) modified to use a standard curve based on a recombinant hIL-7-Fc soluble protein produced in mammalian ExpiCHOTM cells from a coding sequence identical to that cloned in the MVA (GeneArt, ThermoFisher). The hIL-7-Fc was dosed at 1768 ng/mL in the MVA-IL-7-Fc supernatant and was undetectable in the empty MVA supernatant. The hIL-7-Fc produced by the MVA from infected human hepatocytes had previously been shown to be dimeric, glycosylated and biologically active.31
PBMC preparation and stimulation with MVA-hIL-7-Fc and MVA-N33.1 supernatants
Cryopreserved PBMCs were thawed and cultivated in R10 medium composed of RPMI 1640 medium supplemented with 1% glutamine, 1% pyruvate, 1% MEM non-essential amino-acids solution, 1% penicillin-streptomycin, and 10% fetal bovine serum. For T cell stimulation, MVA-hIL7-Fc supernatant was used at a concentration of 12.5 ng/mL of hIL7-Fc. The same dilution factor was used for MVA-N33.1 supernatant. In addition, through the whole study, the commercial hIL-7 (without Fc domain, R&D) previously used in ex vivo studies in elderly and thymectomized patients14 was included as a positive control (5 ng/mL).
Immunostaining and flow cytometry analysis
In all immunostaining experiments, T-cells were characterized by CD3 and CD8 staining, CD3+CD8+ cells represent CD8 T cells and CD3+CD8- are considered as CD4 T cells (although this definition is approximate because it can include other subsets such as regulatory T cells, NKT, or γδ T cells). Differentiation profiles based on CD27/CD45RA expression, as well as CD127 expression, receptor of IL-7, were evaluated on unstimulated cells. In addition, CD57 expression which reflects immunosenescence was also evaluated on the day of PBMC thawing. Dead cells were excluded with eFluor 780 Fixable Viability Dye (eBioscience,) first staining (10 min). Washed cells were then stained with differentiation antibodies panel (PE-Cy7 anti-CD3 (BD Pharmingen), Brilliant Violet 650 anti-CD8, Alexa Fluor 700 anti-CD27 (Biolegend), ECD anti-CD45RA (Beckman Coulter), FITC anti-CD57 (Becton Dickinson) for 15 min) or with CD127 panel (Pacific Blue anti-CD8 (Becton Dickinson) and APC-Vio770 anti-CD127 (Miltenyi) for 5 min). CD57 also known as HNK-1, is a glycoprotein expressed on the surface of natural killer cells and a subset of T cells is a marker of cell senescence33,34 which has been associated with several clinical pathologies.35,36
To evaluate the IL-7 signaling, STAT5 and STAT1 phosphorylation was assessed after stimulation with supernatants. Cells were left unstimulated or were stimulated with either hIL-7 or supernatants of MVA-hIL7-Fc or MVAGN33.1. Fixation and permeabilization were carried out with warm BD Fixation Buffer (Becton Dickinson Cytofix) and cold BD Perm Buffer III (Becton Dickinson Phosflow) (15 min each) before intracellular staining with Pacific Orange anti-CD3 (Invitrogen), Brilliant Violet 650 anti-CD8 (Biolegend), Alexa Fluor 647 anti-pSTAT5 (Becton Dickinson Phosflow), and Alexa Fluor 488 anti-pSTAT1 (Becton Dickinson Phosflow) for 30 min.
T cell proliferation, activation, and differentiation were assessed within unstimulated cells and within cells stimulated for 5 d with either MVA-hIL7-Fc or MVAGN33.1 supernatants or hIL-7. Thawed PBMCs were first stained with eFluor 450 Cell Proliferation Dye (eBioscience) before being washed and resuspended in R10 medium. Cells were left unstimulated or were stimulated with either hIL-7, MVA-hIL7-Fc, or MVAGN33.1 supernatants. Cells were incubated at 37°C for 5 d and stained with eFluor 780 Fixable Viability Dye (eBioscience) for 10 min, washed and stained with PE-Cy7 anti-CD3 (Becton Dickinson Pharmingen), Brilliant Violet 650 anti-CD8 (Biolegend), Alexa Fluor 700 anti-CD27 (Biolegend), ECD anti-CD45RA (Beckman Coulter), PE anti-CD25 (Becton Dickinson), APC anti-CD38 (Becton Dickinson) for 15 min.
All sample acquisitions were performed on a BD LSR Fortessa flow cytometer. Data were analyzed with the FlowJo v10 software. Gating strategies are represented in Figure S1.
Statistical analysis
Univariate analysis was performed to compare populations for each treatment groups by markers. For T cell characterization, groups control versus HF were compared, t-test was used to test differences between groups. Variable equality was first tested to define which test to be used (Folded Fisher or Satterthwaite).
For pSTAT and phenotyping assays, mixed models were built with Stimulation (“unstimulated,” “hIL-7,” “MVA N33.1” and “MVA-IL-7-Fc”) and Group (“CTRLo” and “HF”) and interaction of both as fixed effect and Patient ID as random effect with unstructured covariance. If group effect was found significant, post-hoc comparisons were done to compare groups and simulations with Tukey multiplicity adjustment. Analyses were conducted using SAS® 9.4. Tests were performed at the level of 5%.
For all experiments performed, no statistical difference was observed between the unstimulated group and the group treated with MVAGN33.1 supernatant. For representation convenience, statistics between hIL-7 treated group and the other groups are not represented.
Results
Patients from the HIPAGE cohort display features of immunosenescence and a slight decrease of IL-7 receptor expression
Age-gender and lymphocyte counts of the elderly populations aere summarized in Table 1. Both groups had similar gender repartition and the same age. As expected, a significant lymphopenia was observed in HF patients as compared to control counterparts. This decrease of lymphocytes counts was observed in the two sub compartments, CD8 and CD4 T cells.
In order to characterize immunosenescence from control and HF patients, CD57 expression among viable CD8 and CD4 T cells was immediately assessed after PBMC thawing. This well-known T cell senescent marker is described to be expressed on approximatively 20% of the total CD8 T cell population in healthy adults and can reach a 50–60% expression after 80 y of age.37 As expected, and shown in Figure 1a, CD8 T cells from old control subjects as well as HF patients highly express CD57 (39.0 ± 20.7% and 52.0 ± 22.2%, respectively) compared to younger individuals (9.9 ± 3.8%). Expression tended to be more pronounced in HF patients. Interestingly, CD4 T cells are not described to be the key component of immune senescence, and usually present a low CD57 expression among circulating CD4+ T cells (about 5%),38,39 as observed in our young controls (3 ± 1.7%). In our experiment, CD4 T cells also shown an increase of CD57+ expression in HF patients compared with old controls (6.4 ± 4.5% and 11.9 ± 14.1% in controls and HF patients, respectively). These results confirm that the studied elderly patients, both hip fracture and age-matched controls, display immunosenescence features. This is confirmed by a low proportion of circulating naïve cells compared to memory counterparts (Figure S2S).
Figure 1.
Ex vivo characterization of T cells in senescent patients with or without hip fracture.
PBMCs from healthy donors (young CTRLy, gray circle, n = 5; old CTRLo, white circles, n = 7) and senescent hip fracture patients (HF, black circles, n = 10) were thawed, stained with anti-CD3, anti-CD8 anti CD57 or anti CD127 and analyzed were performed by flow cytometry. The bar is indicative of mean value ± standard deviation. (a) Frequency of CD57+ cells among CD8 T cells (left) and CD4 T cells (right), representing immunosenescence. (b) Frequency of CD127+ cells among CD8 T cells (left) and CD4 T cells (right), representing the level of expression of the IL-7 receptor.
Within cell subpopulations, highest percentage of CD57+ cells were observed for CD8 and CD4 T Effector Memory re-expressing CD45RA (TEMRA) cells and for T Effector Memory (TEM) in comparison with naïve (TN) and T Central Memory (TCM) (Figure S3A). No significant difference was observed between the two elderly groups, despite a tendency for HF patients to exhibit higher CD57 expression on each subset.
Similarly, expression of the IL-7 receptor subunit alpha (IL7-Rα), also known as CD127, was evaluated on CD8 and CD4 T cell surface from both controls and HF patients (Figure 1b). All groups expressed the IL-7 receptor. A slight but unsignificant decrease of the receptor detection on CD8 T cells from HF patients as compared to age-matched controls was observed (p = .1). Within cell subpopulations, highest percentage of CD127+ cells were observed for CD8 and CD4 TN and TCM in comparison with TEM and TEMRA cells for which detection was lower in particular for TEMRA (<10%) (Figure S3B). No significant difference was observed between the two studied groups.
hIL-7-Fc produced by the MVA-IL-7-Fc induces activation of IL-7 intracellular signaling
As IL-7 activity in T-cells is driven mainly by STAT5 phosphorylation and to a lesser extent by STAT1 phosphorylation, proportions of phospho-STAT5 (pSTAT5), and phospho-STAT1 (pSTAT1) were assessed among CD8 T cells and CD4 T cells (Figure 2 and S4).
Figure 2.
Ex vivo characterization of IL-7 signaling induction by MVA-Hil-7-Fc supernatant treatment in CD8 and CD4 T cells from senescent patients with or without hip fracture.
PBMCs from old donors (CTRLo, empty circles, n = 5) and hip fracture patients (HF, filled circles, n = 10) were thawed, stained, left unstimulated (green circles) or stimulated for 30 min with IL-7 (blue circles), empty MVA supernatant (MVATGN33.1, orange circles), or with MVA-hIL7-Fc supernatant (red circles). Fixed and permeabilized cells were then stained with anti-CD3, anti-CD8 and anti pSTAT5 or anti pSTAT1 and analyzed by flow cytometry. The bar is indicative of mean value ± standard deviation. *, p < .05; **, p < .01; ****, p < .0001. (a) Frequency of pSTAT5+ cells among CD8 T cells (left) and CD4 T cells (right). (b) Frequency of pSTAT1+ cells among CD8 T cells (left) and CD4 T cells (right).
While pSTAT5 was not induced when T cells were left unstimulated or were stimulated with empty MVA supernatant, MVA-hIL7-Fc supernatant-treated CD8 and CD4 T cells displayed a highly significant pSTAT5 induction in both HF patient and old control groups (Figure 2a). HF patients displayed an unsignificant decreased proportions of pSTAT5+ CD8 T cells compared to control patients (51.9 ± 18.5% vs. 66.1 ± 7.4%, respectively).
Similarly, proportion of pSTAT1 was assessed among CD8 T cells and CD4 T cells (Figure 2b). While pSTAT1 was not induced when T cells were left unstimulated or were stimulated with empty MVA supernatant, MVA-hIL7-Fc supernatant treated T cells displayed a significant pSTAT1 induction in patient group only, but not in the control group.
Overall, similar to the control soluble hIL-7, the hIL7-Fc expressed by the MVA-hIL7-Fc is capable to initiate IL-7 signaling through pSTAT5 and, albeit to a lower level, pSTAT1.
pSTAT5 and pSTAT1 were further analyzed among CD8 and CD4 TN, TCM, TEM, and TEMRA cells (Figure S4). Whatever the cell population analyzed and as expected, pSTAT5 was not induced when cells were left unstimulated or were stimulated with empty MVA supernatant, while MVA-hIL7-Fc supernatant-treated cells displayed pSTAT5 induction in both patient and control groups (Figure S4A). The intensity of pSTAT5 induction decreased with the level of maturation CD8 and CD4 T cells, corresponding with CD127 expression levels. Induction was stronger and comparable for TN and TCM cells, it decreased for TEM cells and TEMRA cells, the latter cell type being the least significant. Of note, HF patients displayed a significant decreased proportion of pSTAT5+ CD8 TN cells compared to control patients (66.1 ± 25.8% vs. 82.5 ± 7.6%, respectively). The other cell subpopulations did not differ between the two groups.
For pSTAT1 induction (Figure S4B), MVA-hIL7-Fc supernatant-treated CD8 TN, CD4 TN and CD4 TCM cells displayed pSTAT1 induction in both groups to a similar level, whereas MVA-hIL7-Fc supernatant-treated CD8 TCM and CD8 TEM cells displayed pSTAT1 induction only for HF patients. Regarding CD8 TEMRA cells, pSTAT1 could be induced solely via the MVA-hIL7-Fc supernatant compared to the empty MVA treatment and for controls alone. Overall results obtained with the MVA-hIL-7-Fc were comparable to those observed with the soluble hIL-7 control cytokine.
hIL-7-Fc produced by MVA-IL-7-Fc treatments leads to T cell activation
First, CD8 and CD4 T cell proportions in living lymphocytes were studied after 5 d of stimulation (Figure 3a). Thereafter, cell activation of both subtypes was assessed through CD25 and CD38 expression, reflecting early and late activation, respectively.40,41 Cumulative activation (i.e., the expression of either CD25 or CD38, or both) was then calculated among CD8 and CD4 T cells (Figure 3b).
Figure 3.
Ex vivo characterization of CD8 and CD4 T cells activation after MVA-Hil-7-Fc supernatant treatment in senescent patients with or without hip fracture.
PBMCs from old t donors (CTRLo, empty circles, n = 7) and hip fracture patients (HF, filled circles, n = 10) were thawed, stained, left unstimulated (green circles) or stimulated with IL-7 (blue circles), empty MVA supernatant (MVATGN33.1, orange circle) or with MVA-hIL7-Fc supernatant (red circles). Five days later, CD3 and CD8 antibodies were used to identify CD8 T cells and CD4 T cells and cells were stained with anti-CD38/CD25. T cell phenotype and activation was assessed by flow cytometry. The bar is indicative of mean value ± standard deviation. *, p < .05; **, p < .01; ****, p < .0001. (a) Frequency of CD8 (left) and CD4 (right) T cells among total alive lymphocytes. (b) Representative staining for CD38 and CD25 activation markers. Frequency of cumulative T cell activation (i.e., sum of CD25+CD38-, CD25+CD38+, and CD25-CD38+ cells) among CD8 T cells (top panel) and CD4 T cells (bottom panel).
Results show that, regardless of the stimulations used, proportions of CD8 and CD4 T cells among total lymphocytes remain unchanged (Figure 3a). Interestingly, a significant T cell activation was induced after stimulation with the MVA-hIL7-Fc supernatant in both CD8 and CD4 T cells (Figure 3b). No activation was observed within unstimulated cells or empty MVA supernatant-treated cells. Proportions of individual subpopulations of CD25+CD38-, CD25-CD38+, and CD25+CD38+ cells among CD8 and CD4 T cells from control and HF patients are presented in Figure S5. All activated T cell populations, when originating from HF patients, were increased after stimulation with MVA-hIL7-Fc supernatant as compared to no stimulation or to the empty MVA supernatant stimulation. However, when originating from control patients, only CD8 and CD4 CD25+CD38- and CD4 CD25+CD38+ T cell populations were increased when cells were stimulated with MVA-hIL7-Fc supernatant compared to no stimulation or to the empty MVA supernatant stimulation. Whether for CD8 or CD4 T cells and for both HF and control subjects, all three activated cell populations reached comparable levels except for CD8 CD25-CD38+ which increased more significantly for HF patients. As seen with previous read-outs, these levels were similar to those observed with the soluble hIL-7 control cytokine.
MVA-hIL-7-Fc supernatant treatment drives differentiation changes
The four differentiation profiles of T cells were analyzed through CD27 and CD45RA expression among CD8 and CD4 T cell populations from control and HF patients after 5 d of stimulation to assess cell distributions (Figure 4). CD8 and CD4 TCM and TEMRA cell proportions remained the same regardless of patient groups and treatments. Stimulation with MVA-hIL7-Fc supernatant lead to an increase of TN cell proportion as well as a decreased TEM cell proportion compared to no stimulation and stimulation with empty MVA in both CD8 and CD4 T cell populations (Figure 4). These changes appeared in both HF patient and immunosenescent control groups.
Figure 4.
Immunophenotyping after 5 d of stimulation with MVA-hIL7-Fc supernatant.
PBMCs from old donors (CTRLo, empty circles, n = 7) and hip fracture patients (HF, filled circles, n = 10) were thawed, stained, left unstimulated (green circles) or stimulated with soluble IL-7 (blue circles), empty MVA supernatant (MVATGN33.1, orange circles), or with MVA-hIL7-Fc supernatant (red circles). Five days later, cells were stained with anti-CD27/CD45RA to determine frequencies of CD8 and CD4 TN,TCM,TEM and TEMRA cells. Phenotypes of CD8+ T cells are shown on the left panel; CD4 T cells, on the right. The bar is indicative of mean value ± standard deviation. *, p < .05; **, p < .01; ***, p < .001.
A significant T cell proliferation occurs for hip fracture patients following stimulation with MVA-hIL-7-Fc supernatant
2A fluorescent dye was used to track cell divisions within total CD3, CD8, CD4 T cells from control and HF patients after 5 d of stimulation (Figure 5). Stimulation with MVA-hIL7-Fc supernatant clearly drove total CD3, CD8, and CD4 T cells from HF patients to proliferate compared to no stimulation and stimulation with empty MVA. Surprisingly, proportion of proliferating cells was higher within HF patients than within controls following stimulation with MVA-hIL-7 supernatant (total T cells: 17.7 ± 11.2% vs. 5.4 ± 2.1%; p = .015; CD8 T cells: 18.7 ± 11.3% vs. 9.7 ± 3.6%; p = .085; CD4 T cells: 18.2 ± 11.7% vs. 4.5 ± 2.0%; p = .009) and this difference of proliferation between HF patients and old controls was only found for CD4 T cells following stimulation with IL-7 alone (12.1 ± 8.5% vs. 5.07 ± 2.5%; p = .04). Of note, T-cell proliferation post MVA-hIL7-Fc supernatant was similar to the one observed with the soluble IL-7 positive control.
Figure 5.
T cell proliferation after 5 d of stimulation with MVA-hIL7-Fc supernatant.
PBMCs from old donors (CTRLo, empty circles, n = 7) and hip fracture patients (HF, filled circles, n = 10) were thawed, stained, left unstimulated (green circles) or stimulated with IL-7 (blue circles), empty MVA supernatant (MVATGN33.1, orange circle) or with MVA-hIL7-Fc supernatant (red circles). Five days later, cells were stained, and T cell proliferation was assessed by flow cytometry. The bar is indicative of mean value ± standard deviation. *, p < .05; **, p < .01; ***, p < .001; ****, p < .0001. (a) Frequency of proliferating cells among CD3+ T cells. (b) Representative histogram for Cell Proliferation Dye staining. Frequency of proliferating cells among CD8 (top panel) and CD4 (bottom panel) T cells.
Within cell subpopulations, all CD4 and CD8 TN, TCM, TEM, and TEMRA cells were able to proliferate in response to IL-7 or to MVA-hIL-7 supernatant (Figure S6). No significant difference was observed between the two elderly groups, despite a tendency for HF patients to exhibit higher proliferation capacity for each subset.
Discussion
The MVA-IL-7-Fc virotherapy has been designed to induce an immunostimulation of both arms of the immune system with the aim to improve or restore immune-homeostasis in immunosuppressed sepsis patients.28,31 Our earlier studies, in agreement with the literature, have shown that innate immunity is directly activated by the MVA viral backbone known in particular to induce chemokines, pro-inflammatory cytokines as well as NK cells27,29,42 while the MVA-expressed hIL-7-Fc triggers adaptive immunity in particular.20,24,27,31 Thus, given the expansion of T-cell subsets in the presence of IL-7, it could be of interest to study the impact of the viral vector on TCR repertoires.
Here, we assessed the immune activity of the MVA-expressed hIL-7-Fc in a novel context of immunodepression linked to immunosenescent elderly subjects with or without trauma (hip fracture). Collectively, our data show that the secreted hIL7-Fc is recognized by T cells from both groups of subjects, which get activated by initiating IL-7 signaling through STAT5 and STAT1 phosphorylation. The MVA-expressed cytokine triggered T cell proliferation, especially within HF patients, which consequently lead to an increase of naïve T cell and a decrease of effector memory T cell proportions, hence globally triggered “T cell rejuvenation.” Immune rejuvenation refers to the process of enhancing and revitalizing the immune system, which is responsible for defending the body against pathogens and maintaining overall health. While there is no single method or guaranteed approach to achieve immune rejuvenation, one may suggest that improving naïve T cell counts with diverse repertoire may participate to the improvement of the overall immune fitness in elderly. Thus, enhancing proliferative capacity of naïve cells through IL-7 virotherapy could be one strategic step to improve care of HF patients. The HIPAGE cohort previously described9 is unique as it allows to address in a single study two types of geriatric populations: elderly (75 and older) known to be in an immunosenescent condition4,10 and elderly (same age group) with HF i.e. with an additional stress trauma.
Here, we first confirmed the immunosenescence status of our study groups. As expected, the expression level of CD57, an established marker of immunosenescence37 on CD8 T cells is high as compared to normal level usually find in younger population (Figure 1). In addition, we observe here a trend to an increase of CD8+CD57+ in HF patient as compared to controls. This observation is concordant with Pietschmann et al., who described an increase of senescent CD57+CD8+ T cell in osteoporosis fracture elderly patients.43 Interestingly, recent review highlights the impact of immunological stress conditions, such as viral infection or cancer, on the induction of these CD57+CD8+ senescent T cells.44 These CD8+CD57 T cells, also observed in other pathologies, demonstrated an immunosenescent phenotyping, even in younger people, as HIV/CMV or SARS-CoV-2 infected patients and in auto-immune diseases, with a worsening evolution of theses pathologies.44 Detection of CD57 was also observed although to a much lower level in CD4 T cells with a slight increase in HF patients. This increase of CD4+CD57+ immunosenescent cells was already described in an elderly cohort with acute heart failure and was associated with proinflammation phenotype and worse clinical outcome.39 This marker of senescence on T cells observed in our cohort highlights the effect of aging on immune functions. Immunosenescence impacts all immune cells from immune progenitor cells45 to other compartments, with an urge impact on adaptative immunity. The main impact of aging on adaptative immunity is driven by thymic involution which induced a decline of naïve T cell compartment and an increase of memory T cell compartment, as also observed here (Figure S2).4,10,14 In addition, immunosenescent lymphocytes have reduced renewal capacity by the reduction of their telomer length and an impairment of telomerase activity.46,47 Despite these defects, the lymphocytes of old individuals maintain functional IL-7 response pathways.14 In our cohort, we observed in the control group a low level of CD3 cells as compared to normal range of healthy people (Table 1) (normally between 1100 to 4400 cell/µL) in accordance with a slight lymphopenia usually observed in elderly. But more interestingly, the HF patients present a high lymphopenia, involving both CD8 and CD4 T cells. This lymphopenia was transitory observed in HF patients signing the impact of trauma on adaptative immunity and was accompanied by induction of innate induced inflammation.9
Beyond HF patients, the impact of trauma on immune system was intensively described with a biphasic phase. First, an inflammatory response to the systemic release of cellular components and then the induction of an immunosuppressive phase in part characterized by a transient lymphopenia observed after stroke,48 surgery,49,50 or polytraumatic events.50–53 This immunosuppression, observed in diverse traumatic events in sterile conditions, is also present after infectious diseases as observed in sepsis patients54 or after COVID-19 infection.55 Interestingly, despite diverse source of trauma (both sterile or infectious), the profile of immunosuppression is quite similar between these different populations and impacts not only adaptative but also innate immunity, as demonstrated in a recent cohort.56 As in our HIPAGE cohort, an important part of patients with trauma or supporting a heavy surgery in Intensive Care Unit (ICU) are of advanced age. These patients combine both immunosenescence and trauma effect on immune system with complex interactions worsening the clinical outcome.2,3 This is the same for sepsis patients in which immunosenescence is a critical factor associated with worse prognostic.32,57,58 In HF, elderly patient mortality can reached 20–30%, mostly due to poor comorbidities decompensation and secondary infections.59 Prevention of these infections by restoring immune-homeostasis in immunocompromised patients or improving global immunity has become an important therapeutic objective.
To this aim, IL-7 is an attractive therapeutic product, known for a long time to its capacity to induce adaptive T cell proliferation and survival.11,60,61 Animal models confirmed this capability in the context of sepsis62,63 and other pathologies as influenza64 and its potential interest in vaccine adjuvant.65–67 In human, IL-7 was shown to increase lymphocyte count in healthy donors68 and in difficult to treat fungal wound sepsis patient with a weak lymphopenia.69 IL-7 was used in clinical trials for restoration of lymphocyte count in HIV infected patients.70,71 Currently, IL-7 is tested in many clinical trials as cancer direct therapy and/or adjuvant therapy with CAR-T cells or immune check point inhibitors (For review, see ref.21
In the context of immunodepression observed in sepsis, IL-7 was intensively tested in animal-like different CLP models62,63 and used in clinical trial.23 Even without a clear benefit in terms of survival in human, clinical used of IL-7 confirmed its safety already observed in other pathologies, and improvement of clinical parameters as reduction of secondary infections.23 More recently, IL-7 was tested in the treatment of COVID-19, as a viral sepsis, with success in restoring immunity function20,72 and was proposed as vaccine adjuvant.61 Additional studies using human-surrogate assays would be of interest to further dissect the effect of the hIL-7-Fc produced by our recombinant MVA in comparison with recombinant IL-7 currently used in the clinic which either do not harbor a Fc domain (CYT107) or harbor a different one than the once we used (NT-17).
We previously demonstrated the capacity of the new IL-7 form, the MVA-expressed hIL-7-Fc to boost and/or restore T-cell functions in sepsis patients whom majority are also elderly.31 It was hence particularly attractive to evaluate the MVA vectorized hIL-7-Fc in other elderly populations including those with trauma as in the HIPAGE cohort that represent a growing medical concern.
In these patients, we confirm the capacity of our vectorized hIL7-Fc to induced lymphocyte immune function restauration via activation of intracellular STAT pathways (Figure 2) inducing activation markers (Figure 3) and finally proliferation of T cell compartments (Figure 5). Activation of both STAT5 and STAT1 phosphorylation pathways results in lymphopenia-induced proliferation of naïve T cell at the expense of differentiated cells (Figure 4).73
This activation is performed via the IL-7 receptor (CD127), which is present on CD8 and CD4 T cells on both studied groups although expression appears lower in HF patients (Figure 1). This detection was reduced on both CD8 and CD4 T cell subtypes for which a gradient of detection was observed: a greater proportion of T cells from naïve phenotype (TN and TCM) express the receptor relative to a low detection within the more differentiated (TEM and TEMRA) cells. A stronger trend decreases of CD127 expression in HF patients especially in CD8 TN and TCM subpopulations, was observed. The expression of CD127 is known to decreased with the differentiation process of T-cells.74,75 Different T cell subsets with variable level of CD127 expression were observed in TEM with function of reservoir and effector cells. Loss of CD127 expression in these cells is associated with poor outcome in some infection like HIV76–78 or HBV79,80 with a senescent phenotype profile.77
These different levels of CD127 expression on T cell subsets could in part explain the impact of hIL-7 MVA produced on the repartition of the T cell population after stimulation. In both CD8 and CD4 T cells, significant increase of TN and decrease of TEM percentages were observed (Figure 4) in control and HF patient groups. These T cell differentiation changes could be explained by extensive TN cell proliferation compared to TEM cell proliferation or specific TEM cell death. Indeed, alteration in CD127 expression, signaling, and survival responses have been reported in effector memory cells from elderly.81 However, we did not bring to light such differences in terms of TN vs. TEM cell proliferation, or in terms of T cell viability (Figure S6 & data not shown). This observation is concordant with preclinical observation in mice model of T cell depletion and recovery with IL-7 which favor naïve compartment restoration.82 In addition, this predominant naïve T cell expansion was previously observed in a first in man clinical experiment with recombinant hIL-7 in healthy donors. This expansion of naïve T cell as compared to effector memory cells was accompanied with an increase of immune repertoire diversity83 and increase lymphoid organs metabolism.84 In addition to proliferative expansion differences, recently, Frumento et al. described an epigenetic reprogramming of activated T cells into a “naïve-memory” phenotype following stimulation with homeostatic cytokines such as IL-7.85 In this study, this IL-7-driven reversion was preferentially observed in cord blood-derived T cells but was also possible for adult peripheral blood-derived T cells. This results in “naïve-revertant” T cells sharing a naïve-like phenotype with the already well-known and described stem cell memory T cells or human memory T cells with a naïve phenotype. Furthermore, chromatin regions containing binding motifs for several transcription factors, including STAT5, became more accessible during reversion in this study. and could thus be more responsive and proliferative. These hypothesis leads to the main finding of our study: MVA-produced-hIL7-Fc can boost the T cell compartment from immunosenescent patients, including and in a most significant manner for immunosenescent HF patients, increasing total and CD4 T cells and in a lesser extend CD8 T cells (Figure 5). This difference in proliferative capacity in T cell compartment could be related to the variable metabolism profile and energy needs of these different cells especially in aged senescent population.86 For example, low CD127 expressing TEM cell was described as defective for glycolysis and proliferation.87 Alteration in T cell metabolism was also described in sepsis immunosuppressed patients, with a defect of glycolysis induction after stimulation impairing proliferation. This effect was mediated by mTOR dysfunction. In this study, authors demonstrate that hIL-7 was able to restore mTOR pathway and induced proliferation.19 Therefore, the impact of MVA-IL-7 on immune metabolism of T cell compartments T could be an important factor since functional impairment of T cells in elderly has been associated with altered metabolism.88,89
Conclusion
In conclusion, we have demonstrated in this ex vivo study that hIL-7 produced by a MVA-vectorized novel immunotherapy, MVA-hIL7-Fc, is functional and capable to stimulate adaptative immune responses in the context immunosenescent elderly subjects in particular following a trauma. Phosphorylation of STAT5 and STAT1, changes in T cell differentiation status, induction of T cell activation and proliferation were demonstrated. Interestingly, T cell proliferative capacity was more significant for HF patients than non-traumatic controls. In addition, we observed that hIL-7-Fc produced is able to rescue T cell population in favor of naïve T cell, which could be of interest to restore immune function against potential secondary infections, frequently observed in elderly’s trauma patients. These results support the clinical development of the MVA-hIL7-Fc to boost immune T cell responses in immunosenescent patients, especially in HF patients who represent a medical challenge to treat.
Supplementary Material
Acknowledgments
We are very grateful to the patients and staff of the department of geriatrics of the Pitié-Salpétrière Hospital. We thank Perrine Martin for her interest and support in the realization of the study.
Funding Statement
The other authors have no financial conflicts of interest.
Disclosure statement
C Marton, CA Coupet, N Kehrer, B Bastien, L Barraud and G Inchauspé were employees of Transgene when the work was performed. Transgene SA is a publicly traded French biopharmaceutical company with Institut Merieux as major shareholder.
Supplementary data
Supplemental data for this article can be accessed on the publisher’s website at https://doi.org/10.1080/21645515.2023.2232247.
Author contributions
C. Marton designed the study, performed cytometry experiment and analysis, and participated in the redaction of the manuscript. A. Minaud, participated to the cytometry experiments. C.A. Coupet performed the production of hIL7-Fc and participated to the redaction of the manuscript. M. Chauvin and J. Dhiab performed flow cytometry analysis. N. Kehrer realized quantification of h-IL-7-Fc in supernatants. H. Vallet, recruited patients, collected clinical data. B. Bastien realized the statistical analysis. J. Boddaert, designed the clinical study and recruited the patients. L. Barraud, G. Inchauspé and D. Sauce conceived the project, designed the experiments, participated to the interpretation of data and to the redaction of the manuscript.
References
- 1.World population ageing 1950-2050 – UNESCO Bibliothèque Numérique [Internet]. [cited 2023 May 15]. https://unesdoc.unesco.org/ark:/48223/pf0000125754.
- 2.Joseph B, Scalea T.. The consequences of aging on the response to injury and critical illness. Shock. 2020;54(2):144–14. doi: 10.1097/SHK.0000000000001491. [DOI] [PubMed] [Google Scholar]
- 3.Fali T, Vallet H, Sauce D. Impact of stress on aged immune system compartments: overview from fundamental to clinical data. Exp Gerontol. 2018;105:19–26. doi: 10.1016/j.exger.2018.02.007. [DOI] [PubMed] [Google Scholar]
- 4.Aiello A, Farzaneh F, Candore G, Caruso C, Davinelli S, Gambino CM, Ligotti ME, Zareian N, Accardi G. Immunosenescence and its hallmarks: how to oppose aging strategically? A review of potential options for therapeutic intervention. Front Immunol. 2019;10:2247. doi: 10.3389/fimmu.2019.02247. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Fulop T, Larbi A, Hirokawa K, Cohen AA, Witkowski JM. Immunosenescence is both functional/adaptive and dysfunctional/maladaptive. Semin Immunopathol. 2020;42(5):521–36. doi: 10.1007/s00281-020-00818-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Santoro A, Bientinesi E, Monti D. Immunosenescence and inflammaging in the aging process: age-related diseases or longevity? Ageing Res Rev. 2021;71:101422. doi: 10.1016/j.arr.2021.101422. [DOI] [PubMed] [Google Scholar]
- 7.Boddaert J, Cohen-Bittan J, Khiami F, Le Manach Y, Raux M, Beinis J-Y, Verny M, Riou B, Ang D. Postoperative admission to a dedicated geriatric unit decreases mortality in elderly patients with hip fracture. PLoS One. 2014;9(1):e83795. doi: 10.1371/journal.pone.0083795. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Moyet J, Deschasse G, Marquant B, Mertl P, Bloch F. Which is the optimal orthogeriatric care model to prevent mortality of elderly subjects post hip fractures? A systematic review and meta-analysis based on current clinical practice. Int Orthop. 2019;43(6):1449–54. doi: 10.1007/s00264-018-3928-5. [DOI] [PubMed] [Google Scholar]
- 9.Vallet H, Bayard C, Lepetitcorps H, O’Hana J, Fastenackels S, Fali T, Cohen-Bittan J, Khiami F, Boddaert J, Sauce D. Hip fracture leads to transitory immune imprint in older patients. Front Immunol. 2020;11:571759. doi: 10.3389/fimmu.2020.571759. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Elyahu Y, Monsonego A. Thymus involution sets the clock of the aging T-cell landscape: implications for declined immunity and tissue repair. Ageing Res Rev. 2021;65:101231. doi: 10.1016/j.arr.2020.101231. [DOI] [PubMed] [Google Scholar]
- 11.Chen D, Tang T-X, Deng H, Yang X-P, Tang Z-H. Interleukin-7 biology and its effects on immune cells: mediator of generation, differentiation, survival, and homeostasis. Front Immunol. 2021;12. doi: 10.3389/fimmu.2021.747324. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Gao J, Zhao L, Wan YY, Zhu B. Mechanism of action of IL-7 and its potential applications and limitations in cancer immunotherapy. Int J Mol Sci. 2015;16(12):10267–80. doi: 10.3390/ijms160510267. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Jiang Q, Li WQ, Aiello FB, Mazzucchelli R, Asefa B, Khaled AR, Durum SK. Cell biology of IL-7, a key lymphotrophin. Cytokine Growth Factor Rev. 2005;16(4–5):513–33. doi: 10.1016/j.cytogfr.2005.05.004. [DOI] [PubMed] [Google Scholar]
- 14.Sauce D, Larsen M, Fastenackels S, Roux A, Gorochov G, Katlama C, Sidi D, Sibony-Prat J, Appay V. Lymphopenia-driven homeostatic regulation of naive T cells in elderly and thymectomized young adults. J Immunol. 2012;189(12):5541–8. doi: 10.4049/jimmunol.1201235. [DOI] [PubMed] [Google Scholar]
- 15.Drake A, Kaur M, Iliopoulou BP, Phennicie R, Hanson A, Chen J, Crispin JC. Interleukins 7 and 15 maintain human t cell proliferative capacity through STAT5 signaling. PLoS One. 2016;11(11):e0166280. doi: 10.1371/journal.pone.0166280. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Hand TW, Cui W, Jung YW, Sefik E, Joshi NS, Chandele A, Liu Y, Kaech SM. Differential effects of STAT5 and PI3K/AKT signaling on effector and memory CD8 T-cell survival. Proc Natl Acad Sci U S A. 2010;107(38):16601–6. doi: 10.1073/pnas.1003457107. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Nguyen V, Mendelsohn A, Larrick JW. Interleukin-7 and Immunosenescence. J Immunol Res. 2017;2017:4807853. doi: 10.1155/2017/4807853. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Hopkins B, Fisher J, Chang M, Tang X, Du Z, Kelly WJ, Huang Z. An in-vitro study of the expansion and transcriptomics of CD4+ and CD8+ naïve and memory T cells stimulated by IL-2, IL-7 and IL-15. Cells. 2022;11(10):1701. doi: 10.3390/cells11101701. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Venet F, Demaret J, Blaise BJ, Rouget C, Girardot T, Idealisoa E, Rimmelé T, Mallet F, Lepape A, Textoris J, et al. IL-7 restores t lymphocyte immunometabolic failure in septic shock patients through mTOR activation. J Immunol. 2017;199(5):1606–15. doi: 10.4049/jimmunol.1700127. [DOI] [PubMed] [Google Scholar]
- 20.Bidar F, Hamada S, Gossez M, Coudereau R, Lopez J, Cazalis M-A, Tardiveau C, Brengel-Pesce K, Mommert M, Buisson M, et al. Recombinant human interleukin-7 reverses T cell exhaustion ex vivo in critically ill COVID-19 patients. Ann Intensive Care. 2022;12(1):21. doi: 10.1186/s13613-022-00982-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Wang C, Kong L, Kim S, Lee S, Oh S, Jo S, Jang I, Kim T-D. The role of IL-7 and IL-7R in cancer pathophysiology and immunotherapy. Int J Mol Sci. 2022;23(18):10412. doi: 10.3390/ijms231810412. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Katlama C, Lambert-Niclot S, Assoumou L, Papagno L, Lecardonnel F, Zoorob R, Tambussi G, Clotet B, Youle M, Achenbach CJ, et al. Treatment intensification followed by interleukin-7 reactivates HIV without reducing total HIV DNA: a randomized trial. AIDS. 2016;30(2):221–30. doi: 10.1097/QAD.0000000000000894. [DOI] [PubMed] [Google Scholar]
- 23.Francois B, Jeannet R, Daix T, Walton AH, Shotwell MS, Unsinger J, Monneret G, Rimmelé T, Blood T, Morre M, et al. Interleukin-7 restores lymphocytes in septic shock: the IRIS-7 randomized clinical trial. JCI Insight. 2018;3(5):e98960, 98960. doi: 10.1172/jci.insight.98960. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Laterre PF, François B, Collienne C, Hantson P, Jeannet R, Remy KE, Hotchkiss RS. Association of interleukin 7 immunotherapy with lymphocyte counts among patients with severe coronavirus disease 2019 (COVID-19). JAMA Netw Open. 2020;3(7):e2016485. doi: 10.1001/jamanetworkopen.2020.16485. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Gasnault J, de Goër de Herve M-G, Michot J-M, Hendel-Chavez H, Seta V, Mazet A-A, Croughs T, Stankoff B, Bourhis J-H, Lambotte O, et al. Efficacy of recombinant human interleukin 7 in a patient with severe lymphopenia-related progressive multifocal leukoencephalopathy. Open Forum Infect Dis. 2014;1(2):ofu074. doi: 10.1093/ofid/ofu074. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Volz A, Sutter G. Modified vaccinia virus Ankara: history, value in basic research, and current perspectives for vaccine development. Adv Virus Res. 2017;97:187–243. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Price PJR, Torres-Domínguez LE, Brandmüller C, Sutter G, Lehmann MH. Modified vaccinia virus Ankara: innate immune activation and induction of cellular signalling. Vaccine. 2013;31(39):4231–4. doi: 10.1016/j.vaccine.2013.03.017. [DOI] [PubMed] [Google Scholar]
- 28.Waibler Z, Anzaghe M, Ludwig H, Akira S, Weiss S, Sutter G, Kalinke U. Modified vaccinia virus Ankara induces toll-like receptor-independent type I interferon responses. J Virol. 2007;81(22):12102–10. doi: 10.1128/JVI.01190-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Lélu K, Dubois C, Evlachev A, Crausaz M, Baldazza M, Kehrer N, Brandely R, Schlesinger Y, Silvestre N, Marchand J-B, et al. Viral delivery of IL-7 is a potent immunotherapy stimulating innate and adaptive immunity and confers survival in sepsis models. J Immunol. 2022;209(1):99–117. doi: 10.4049/jimmunol.2101145. [DOI] [PubMed] [Google Scholar]
- 30.Coupet C-A, Dubois C, Evlachev A, Kehrer N, Baldazza M, Hofman S, Vierboom M, Martin P, Inchauspe G. Intravenous injection of a novel viral immunotherapy encoding human interleukin-7 in nonhuman primates is safe and increases absolute lymphocyte count. Hum Vaccin Immunother. 2022;18(6):2133914. doi: 10.1080/21645515.2022.2133914. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 31.Crausaz M, Monneret G, Conti F, Lukaszewicz A-C, Marchand J-B, Martin P, Inchauspé G, Venet F. A novel virotherapy encoding human interleukin-7 improves ex vivo T lymphocyte functions in immunosuppressed patients with septic shock and critically ill COVID-19. Front Immunol. 2022;13:939899. doi: 10.3389/fimmu.2022.939899. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 32.Lu X, Yang Y-M, Lu Y-Q. Immunosenescence: a critical factor associated with organ injury after sepsis. Front Immunol. 2022;13:917293. doi: 10.3389/fimmu.2022.917293. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 33.Brenchley JM, Karandikar NJ, Betts MR, Ambrozak DR, Hill BJ, Crotty LE, Casazza JP, Kuruppu J, Migueles SA, Connors M, et al. Expression of CD57 defines replicative senescence and antigen-induced apoptotic death of CD8+ T cells. Blood. 2003;101(7):2711–20. doi: 10.1182/blood-2002-07-2103. [DOI] [PubMed] [Google Scholar]
- 34.Xu W, Larbi A. Markers of T cell senescence in humans. Int J Mol Sci. 2017;18(8):1742. doi: 10.3390/ijms18081742. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 35.Be P, B N, Ap F, Cc W. Functional and phenotypic characterization of CD57+CD4+ T cells and their association with HIV-1-induced T cell dysfunction. J Immunol (Baltimore Md : 1950) [Internet]. 2005;175(12):8415–23. doi: 10.4049/jimmunol.175.12.8415. [DOI] [PubMed] [Google Scholar]
- 36.Bayard C, Lepetitcorps H, Roux A, Larsen M, Fastenackels S, Salle V, Vieillard V, Marchant A, Stern M, Boddaert J, et al. Coordinated expansion of both memory T cells and NK cells in response to CMV infection in humans. Eur J Immunol. 2016;46(5):1168–79. doi: 10.1002/eji.201546179. [DOI] [PubMed] [Google Scholar]
- 37.Kared H, Martelli S, Ng TP, Pender SLF, Larbi A. CD57 in human natural killer cells and T-lymphocytes. Cancer Immunol Immunother. 2016;65(4):441–52. doi: 10.1007/s00262-016-1803-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 38.Alshekaili J, Chand R, Lee CE, Corley S, Kwong K, Papa I, Fulcher DA, Randall KL, Leiding JW, Ma CS, et al. STAT3 regulates cytotoxicity of human CD57+ CD4+ T cells in blood and lymphoid follicles. Sci Rep. 2018;8(1):3529. doi: 10.1038/s41598-018-21389-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 39.Youn J-C, Jung MK, Yu HT, Kwon J-S, Kwak J-E, Park S-H, Kim I-C, Park M-S, Lee SK, Choi S-W, et al. Increased frequency of CD4+CD57+ senescent T cells in patients with newly diagnosed acute heart failure: exploring new pathogenic mechanisms with clinical relevance. Sci Rep. 2019;9(1):12887. doi: 10.1038/s41598-019-49332-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 40.Morandi F, Airoldi I, Marimpietri D, Bracci C, Faini AC, Gramignoli R. CD38, a receptor with multifunctional activities: from modulatory functions on regulatory cell subsets and extracellular vesicles, to a target for therapeutic strategies. Cells. 2019;8(12):1527. doi: 10.3390/cells8121527. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 41.Kalia V, Sarkar S, Subramaniam S, Haining WN, Smith KA, Ahmed R. Prolonged Interleukin-2Rα expression on virus-specific CD8+ T cells favors terminal-effector differentiation in vivo. Immunity. 2010;32(1):91–103. doi: 10.1016/j.immuni.2009.11.010. [DOI] [PubMed] [Google Scholar]
- 42.Dai P, Wang W, Cao H, Avogadri F, Dai L, Drexler I, Joyce JA, Li X-D, Chen Z, Merghoub T, et al. Modified vaccinia virus Ankara triggers type I IFN production in murine conventional dendritic cells via a Cgas/STING/STING-mediated cytosolic DNA-sensing pathway. PLoS Pathog. 2014;10(4):e1003989. doi: 10.1371/journal.ppat.1003989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 43.Pietschmann P, Grisar J, Thien R, Willheim M, Kerschan-Schindl K, Preisinger E, Peterlik M. Immune phenotype and intracellular cytokine production of peripheral blood mononuclear cells from postmenopausal patients with osteoporotic fractures. Exp Gerontol. 2001;36(10):1749–59. doi: 10.1016/S0531-5565(01)00125-5. [DOI] [PubMed] [Google Scholar]
- 44.Tedeschi V, Paldino G, Kunkl M, Paroli M, Sorrentino R, Tuosto L, Fiorillo MT. CD8+ T cell senescence: lights and shadows in viral infections, autoimmune disorders and cancer. Int J Mol Sci. 2022;23(6):3374. doi: 10.3390/ijms23063374. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 45.Fali T, Fabre-Mersseman V, Yamamoto T, Bayard C, Papagno L, Fastenackels S, Zoorab R, Koup RA, Boddaert J, Sauce D, et al. Elderly human hematopoietic progenitor cells express cellular senescence markers and are more susceptible to pyroptosis. JCI Insight. 2018;3(13). doi: 10.1172/jci.insight.95319. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 46.Fali T, Papagno L, Bayard C, Mouloud Y, Boddaert J, Sauce D, Appay V. New insights into lymphocyte differentiation and aging from telomere length and telomerase activity measurements. J Immunol. 2019;202(7):1962–9. doi: 10.4049/jimmunol.1801475. [DOI] [PubMed] [Google Scholar]
- 47.Fali T, K’Ros C, Appay V, Sauce D. Assessing T lymphocyte aging using telomere length and telomerase activity measurements in low cell numbers. Methods Mol Biol. 2019;2048:231–43. [DOI] [PubMed] [Google Scholar]
- 48.Faura J, Bustamante A, Miró-Mur F, Montaner J. Stroke-induced immunosuppression: implications for the prevention and prediction of post-stroke infections. J Neuroinflammation. 2021;18(1):127. doi: 10.1186/s12974-021-02177-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 49.Angele MK, Chaudry IH. Surgical trauma and immunosuppression: pathophysiology and potential immunomodulatory approaches. Langenbecks Arch Surg. 2005;390(4):333–41. doi: 10.1007/s00423-005-0557-4. [DOI] [PubMed] [Google Scholar]
- 50.Kimura F, Shimizu H, Yoshidome H, Ohtsuka M, Miyazaki M. Immunosuppression following surgical and traumatic injury. Surg Today. 2010;40(9):793–808. doi: 10.1007/s00595-010-4323-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 51.Islam MN, Bradley BA, Ceredig R. Sterile post-traumatic immunosuppression. Clin Trans Immunol. 2016;5(4):e77. doi: 10.1038/cti.2016.13. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 52.Thompson KB, Krispinsky LT, Stark RJ. Late immune consequences of combat trauma: a review of trauma-related immune dysfunction and potential therapies. Mil Med Res. 2019;6(1):11. doi: 10.1186/s40779-019-0202-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 53.Hesselink L, Hoepelman RJ, Spijkerman R, de Groot MCH, van Wessem KJP, Koenderman L, Leenen LPH, Hietbrink F. Persistent Inflammation, Immunosuppression and Catabolism Syndrome (PICS) after polytrauma: a rare syndrome with major consequences. J Clin Med. 2020;9(1):191. doi: 10.3390/jcm9010191. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 54.Hotchkiss RS, Monneret G, Payen D. Sepsis-induced immunosuppression: from cellular dysfunctions to immunotherapy. Nat Rev Immunol. 2013;13(12):862–74. doi: 10.1038/nri3552. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 55.Remy KE, Mazer M, Striker DA, Ellebedy AH, Walton AH, Unsinger J, Blood TM, Mudd PA, Yi DJ, Mannion DA, et al. Severe immunosuppression and not a cytokine storm characterizes COVID-19 infections. JCI Insight. 2020;5(17):e140329, 140329. doi: 10.1172/jci.insight.140329. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 56.Immune profiling demonstrates a common immune signature of delayed acquired immunodeficiency in patients with various etiologies of severe injury: erratum. Crit Care Med. 2022;50(6):e618. doi: 10.1097/CCM.0000000000005565. [DOI] [PubMed] [Google Scholar]
- 57.Pei F, Zhang G-R, Zhou L-X, Liu J-Y, Ma G, Kou Q-Y, He Z-J, Chen M-Y, Nie Y, Wu J-F, et al. Early immunoparalysis was associated with poor prognosis in elderly patients with sepsis: secondary analysis of the ETASS study. Infect Drug Resist 2020; 13: 2053–61. 10.2147/IDR.S246513. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 58.Monneret G, Gossez M, Venet F. Sepsis and immunosenescence: closely associated in a vicious circle. Aging Clin Exp Res. 2021;33(3):729–32. doi: 10.1007/s40520-019-01350-z. [DOI] [PubMed] [Google Scholar]
- 59.Rosencher N, Vielpeau C, Emmerich J, Fagnani F, Samama CM, ESCORTE group . Venous thromboembolism and mortality after hip fracture surgery: the ESCORTE study. J Thromb Haemost. 2005;3:2006–14. doi 10.1111/j.1538-7836.2005.01545.x. [DOI] [PubMed] [Google Scholar]
- 60.de Roquetaillade C, Monneret G, Gossez M, Venet F. IL-7 and its beneficial role in sepsis-induced T lymphocyte dysfunction. Crit Rev Immunol. 2018;38(6):433–51. doi: 10.1615/CritRevImmunol.2018027460. [DOI] [PubMed] [Google Scholar]
- 61.Bekele Y, Sui Y, Berzofsky JA. IL-7 in SARS-CoV-2 infection and as a potential vaccine adjuvant. Front Immunol. 2021;12:737406. doi: 10.3389/fimmu.2021.737406. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 62.Unsinger J, McGlynn M, Kasten KR, Hoekzema AS, Watanabe E, Muenzer JT, McDonough JS, Tschoep J, Ferguson TA, McDunn JE, et al. IL-7 promotes T cell viability, trafficking, and functionality and improves survival in sepsis. J Immunol. 2010;184(7):3768–79. doi: 10.4049/jimmunol.0903151. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 63.Shindo Y, Fuchs AG, Davis CG, Eitas T, Unsinger J, Burnham C-A, Green JM, Morre M, Bochicchio GV, Hotchkiss RS. Interleukin 7 immunotherapy improves host immunity and survival in a two-hit model of Pseudomonas aeruginosa pneumonia. J Leukoc Biol. 2017;101(2):543–54. doi: 10.1189/jlb.4A1215-581R. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 64.Sheikh A, Jackson J, Shim HB, Yau C, Seo JH, Abraham N. Selective dependence on IL-7 for antigen-specific CD8 T cell responses during airway influenza infection. Sci Rep. 2022;12(1):135. doi: 10.1038/s41598-021-03936-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 65.Han K-H, Jang MS, Han H-Y, Im W-J, Jung KJ, Park KS, Choi D, Jeong HG, Kim SK, Moon K-S. Preclinical safety assessment of a therapeutic human papillomavirus DNA vaccine combined with intravaginal interleukin-7 fused with hybrid Fc in female rats. Toxicol Appl Pharmacol. 2021;413:115406. doi: 10.1016/j.taap.2021.115406. [DOI] [PubMed] [Google Scholar]
- 66.Huang J, Long Z, Jia R, Wang M, Zhu D, Liu M, Chen S, Zhao X, Yang Q, Wu Y, et al. The broad immunomodulatory effects of IL-7 and its application in vaccines. Front Immunol. 2021;12:680442. doi: 10.3389/fimmu.2021.680442. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 67.Choi YW, Kang MC, Seo YB, Namkoong H, Park Y, Choi D-H, Suh YS, Lee S-W, Sung YC, Jin H-T. Intravaginal administration of Fc-Fused IL7 suppresses the cervicovaginal tumor by recruiting HPV DNA vaccine-induced CD8 T cells. Clin Cancer Res. 2016;22(23):5898–908. doi: 10.1158/1078-0432.CCR-16-0423. [DOI] [PubMed] [Google Scholar]
- 68.Lee SW, Choi D, Heo M, Shin E-C, Park S-H, Kim SJ, Oh Y-K, Lee BH, Yang SH, Sung YC, et al. hIL-7-hyFc, a long-acting IL-7, increased absolute lymphocyte count in healthy subjects. Clin Transl Sci. 2020;13(6):1161–9. doi: 10.1111/cts.12800. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 69.Turnbull IR, Mazer MB, Hoofnagle MH, Kirby JP, Leonard JM, Mejia-Chew C, Spec A, Blood J, Miles SM, Ransom EM, et al. IL-7 immunotherapy in a nonimmunocompromised patient with intractable fungal wound sepsis. Open Forum Infect Dis. 2021;8(6):ofab256. doi: 10.1093/ofid/ofab256. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 70.Lévy Y, Sereti I, Tambussi G, Routy JP, Lelièvre JD, Delfraissy JF, Molina JM, Fischl M, Goujard C, Rodriguez B, et al. Effects of recombinant human interleukin 7 on T-cell recovery and thymic output in HIV-infected patients receiving antiretroviral therapy: results of a phase I/IIa randomized, placebo-controlled, multicenter study. Clin Infect Dis. 2012;55(2):291–300. doi: 10.1093/cid/cis383. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 71.Thiébaut R, Jarne A, Routy J-P, Sereti I, Fischl M, Ive P, Speck RF, D’Offizi G, Casari S, Commenges D, et al. Repeated cycles of recombinant human interleukin 7 in HIV-Infected patients with low CD4 T-Cell reconstitution on antiretroviral therapy: results of 2 phase II multicenter studies. Clin Infect Dis. 2016;62(9):1178–85. doi: 10.1093/cid/ciw065. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 72.Monneret G, de Marignan D, Coudereau R, Bernet C, Ader F, Frobert E, Gossez M, Viel S, Venet F, Wallet F. Immune monitoring of interleukin-7 compassionate use in a critically ill COVID-19 patient. Cell Mol Immunol. 2020;17(9):1001–3. doi: 10.1038/s41423-020-0516-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 73.Le Saout C, Luckey MA, Villarino AV, Smith M, Hasley RB, Myers TG, Imamichi H, Park J-H, O’Shea JJ, Lane HC, et al. IL-7–dependent STAT1 activation limits homeostatic CD4+ T cell expansion. JCI Insight. 2017;2(22):e96228, 96228. doi: 10.1172/jci.insight.96228. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 74.Mackall CL, Fry TJ, Gress RE. Harnessing the biology of IL-7 for therapeutic application. Nat Rev Immunol. 2011;11(5):330–42. doi: 10.1038/nri2970. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 75.Fry TJ, Mackall CL. The many faces of IL-7: from lymphopoiesis to peripheral T cell maintenance. J Immunol. 2005;174(11):6571–6. doi: 10.4049/jimmunol.174.11.6571. [DOI] [PubMed] [Google Scholar]
- 76.Crawley AM, Angel JB. The influence of HIV on CD127 expression and its potential implications for IL-7 therapy. Semin Immunol. 2012;24(3):231–40. doi: 10.1016/j.smim.2012.02.006. [DOI] [PubMed] [Google Scholar]
- 77.Mojumdar K, Vajpayee M, Chauhan NK, Singh A, Singh R, Kurapati S. Loss of CD127 & increased immunosenescence of T cell subsets in HIV infected individuals. Indian J Med Res. 2011;134(6):972–81. doi: 10.4103/0971-5916.92645. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 78.Xu W, Li J, Wu Y, Zhou J, Zhong J, Lv Q, Shao H, Rao H. CD127 expression in naive and memory t cells in hiv patients who have undergone long-term HAART. Lab Med. 2017;48(1):57–64. doi: 10.1093/labmed/lmw053. [DOI] [PubMed] [Google Scholar]
- 79.Lv G, Ying L, Ma W-J, Jin X, Zheng L, Li L, Yang Y. Dynamic analysis of CD127 expression on memory CD8 T cells from patients with chronic hepatitis B during telbivudine treatment. Virol J. 2010;7(1):207. doi: 10.1186/1743-422X-7-207. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 80.Boettler T, Panther E, Bengsch B, Nazarova N, Spangenberg HC, Blum HE, Thimme R. Expression of the interleukin-7 receptor alpha chain (CD127) on virus-specific CD8+ T cells identifies functionally and phenotypically defined memory T cells during acute resolving hepatitis B virus infection. J Virol. 2006;80(7):3532–40. doi: 10.1128/JVI.80.7.3532-3540.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 81.Kim H-R, Hong MS, Dan JM, Kang I. Altered IL-7Rα expression with aging and the potential implications of IL-7 therapy on CD8+ T-cell immune responses. Blood. 2006;107(7):2855–62. doi: 10.1182/blood-2005-09-3560. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 82.Broers AEC, Posthumus-van Sluijs SJ, Spits H, van der Holt B, Löwenberg B, Braakman E, Cornelissen JJ. Interleukin-7 improves T-cell recovery after experimental T-cell–depleted bone marrow transplantation in T-cell–deficient mice by strong expansion of recent thymic emigrants. Blood. 2003;102(4):1534–40. doi: 10.1182/blood-2002-11-3349. [DOI] [PubMed] [Google Scholar]
- 83.Kim S, Lee SW, Koh J-Y, Choi D, Heo M, Chung J-Y, Lee BH, Yang SH, Sung YC, Lee H, et al. A single administration of Hil-7-hyFc induces long-lasting T-cell expansion with maintained effector functions. Blood Adv. 2022;6(23):6093–107. doi: 10.1182/bloodadvances.2021006591. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 84.Sportès C, Hakim FT, Memon SA, Zhang H, Chua KS, Brown MR, Fleisher TA, Krumlauf MC, Babb RR, Chow CK, et al. Administration of rhIL-7 in humans increases in vivo TCR repertoire diversity by preferential expansion of naive T cell subsets. J Exp Med. 2008;205(7):1701–14. doi: 10.1084/jem.20071681. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 85.Frumento G, Verma K, Croft W, White A, Zuo J, Nagy Z, Kissane S, Anderson G, Moss P, Chen FE. Homeostatic cytokines drive epigenetic reprogramming of activated t cells into a “naive-memory” phenotype. iScience. 2020;23(4):100989. doi: 10.1016/j.isci.2020.100989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 86.Singh B, Kumar Rai A. Loss of immune regulation in aged T-cells: a metabolic review to show lack of ability to control responses within the self. Hum Immunol. 2022;83(12):808–17. doi: 10.1016/j.humimm.2022.10.002. [DOI] [PubMed] [Google Scholar]
- 87.Sim JH, Kim J-H, Park AK, Lee J, Kim K-M, Shin HM, Kim M, Choi K, Choi EY, Kang I, et al. IL-7Rαlow CD8+ T cells from healthy individuals are anergic with defective glycolysis. J Immunol. 2020;205(11):2968–78. doi: 10.4049/jimmunol.1901470. [DOI] [PubMed] [Google Scholar]
- 88.Nicoli F, Cabral-Piccin MP, Papagno L, Gallerani E, Fusaro M, Folcher V, Dubois M, Clave E, Vallet H, Frere JJ, et al. Altered basal lipid metabolism underlies the functional impairment of naive CD8+ T cells in elderly humans. J Immunol. 2022;208(3):562–70. doi: 10.4049/jimmunol.2100194. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 89.Nicoli F, Papagno L, Frere JJ, Cabral-Piccin MP, Clave E, Gostick E, Toubert A, Price DA, Caputo A, Appay V. Naïve CD8+ T-Cells engage a versatile metabolic program upon activation in humans and differ energetically from memory CD8+ T-Cells. Front Immunol. 2018;9:2736. doi: 10.3389/fimmu.2018.02736. [DOI] [PMC free article] [PubMed] [Google Scholar]
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