An overview of IL-7 biology and its use in immunotherapy

Interleukin (IL)-7 is required for T-cell development as well as for the survival and homeostasis of mature T-cells. In the thymus, the double negative (DN) CD4^? CD8^? thymocyte progenitor transition into double positive CD4⁺ CD8⁺ cells requires Notch and IL-7 signaling. Importantly, IL-7 seems to have a dose effect on T-cell development and, at high doses, DN progression is blocked. Naïve T-cells in the thymus, and after their exit to the periphery, are dependent on IL-7 and TCR signaling for survival. Upon antigen exposure, they proliferate and differentiate into memory T-cells. Because IL-7 intervenes at all stages of T-cell development and maintenance, it has been introduced recently into clinical trials as an immunotherapeutic agent for cancer patients (of particular note, those who had undergone T-cell depleting therapy) in an attempt to increase their population sizes of CD4⁺ and CD8⁺ cells overall, and specifically of CD8⁺ (CD45RA⁺CCR7⁺ and/or CD27⁺), CD4⁺ (CD45RA⁺CD31⁺), and CD4⁺ central memory T-cells (CD45RA^?CCR7⁺). Interestingly, IL-7 in humans induced a preferential expansion of naïve T-cells, resulting in a broader T-cell repertoire than before the treatment; this effect was independent of age. This suggests that IL-7 therapy could enhance immune responses in patients with limited naïve T-cell numbers as in aged patients or after disease-induced or iatrogenic T-cell depletion. This overview highlights the role of IL-7 on T-cells in mice and humans.     

Characterization of CXCR5+ CD8+ T-cells in humanized NSG mice

Humanized NOD/SCID/IL-2 receptor γ-chain^null (huNSG) mice recapitulate some features of human T-cell populations that can be exploited in basic and pre-clinical research. CXCR5⁺ T CD8⁺ T-cells play an important role in the control of viral infections and tumors. Indeed, they have been associated with low-level HIV replication, making them a possible novel correlate of protection, and potentially useful in the eradication of HIV reservoirs. Here, by flow cytometry, we evaluated the reconstitution of CXCR5⁺ CD8⁺ T-cells in huNSG mice engrafted with CD34⁺ hematopoietic stem cells. This population was readily generated in huNSG mice, and where particularly confined to spleen and lymph nodes. These cells exhibited a follicular-like phenotype, with expression of Programmed Death (PD)-1, Inducible T-cell costimulatory (ICOS), and absence of CCR7. Moreover, CXCR5⁺ CD8⁺ T-cells had a higher expression of interleukin (IL)-21 and a higher cytotoxic potential compared with CXCR5⁻ cells. HIV infection did not affect the frequencies of CXCR5⁺ CD8⁺ T-cells in secondary lymphoid organs. Finally, taking advantage of the high proportion of naïve T-cells in huNSG mice, we evaluated the in vitro response of splenic T-cells to the follicular profile-polarizing cytokines Transforming Growth Factor (TGF)-β1 and IL-23. After in vitro treatment, there was an increase in CXCR5⁺ CD8⁺ T-cells, which exhibited high levels of PD-1, CD40 L and low expression of CCR7. Thus, there is a reconstitution of CXCR5⁺ CD8⁺ T-cells in huNSG mice, supporting the use of this model for exploring the biology and role of this cell population in healthy and diseased conditions.     

Adenosine signalling in T-cell activation favours development of IL-17 positive cells with suppressive properties

Evidence suggests that the anti-inflammatory nucleoside adenosine can shape immune responses by shifting the regulatory (T_reg)/helper (Th17) T-cell balance in favour of T_reg. Since this observation is based on in vivo and in vitro studies mostly confined to murine models, we comprehensively analysed effects of adenosine on human T-cells. Proliferation, phenotype and cytokine production of stimulated T-cells were assessed by flow cytometry, multiplex assay and ELISA, gene expression profiling was determined by microarray. We found that the pan-adenosine agonist 5′-N-ethylcarboxamidoadenosine (NECA) skews human CD3⁺ T-cell responses towards non-inflammatory Th17 cells. Addition of NECA during T-cell activation increased the development of IL-17⁺ cells with a CD4⁺ RORγt⁺ phenotype and enhanced CD161 and CD196 surface expression. Remarkably, these Th17 cells displayed non-inflammatory cytokine and gene expression profiles including reduced Th1/Th17 transdifferentiation, a stem cell-like molecular signature and induced surface expression of the adenosine-producing ectoenzymes CD39 and CD73. Thus, T-cells cultured under Th17-inducing conditions together with NECA were capable of suppressing responder T-cells. Finally, genome-wide gene expression profiling revealed metabolic quiescence previously associated with non-pathogenic Th17 cells in response to adenosine signalling. Our data suggest that adenosine induces non-inflammatory Th17 cells in human T-cell differentiation, potentially through regulation of metabolic pathways.     

Interleukin-7 (IL-7) Knockout Mice. Implications for Lymphopoiesis and Organ-Specific Immunity

Interleukin-7 (IL-7) is produced by both immune and non-immune cells including stromal cell lines, B-cells, monocytes/macrophages, follicular dendritic cells, keratinocytes, and gut epithelial cells. The development of IL-7 knockout mice aided to elucidate the role of this multifaceted cytokine in lymphopoiesis. Additionally, IL-7 genedeleted mice may represent an excellent model in order to define the functional role of locally secreted IL-7 in organ-specific immunity and in anti-microbial responses as well. For instance, analysis of IL-7 gene-deleted mice revealed reduced numbers of total T-lymphocytes with preservation of the CD4/CD8 ratio and increased ratio of αβ⁺ T-cells compared to γδ⁺ T-cells. Transition of pro-T-cells to pre-T-cells was impaired. Cell marker analysis of thymocytes in IL-7-/-mice suggested that IL-7 may induce expression of as yet unidentified cytokine receptors, and that IL-7 may also be critically involved in T-cell differentiation. However, there are clear differences in the requirements of αβ or γβ T-cells for IL-7. In general, IL-7 appears to serve as the major growth and differentiation factor for γβ T-cells. IL-7-/-mice are characterized by a block of maturation of Vγ3^low, CD24⁺ T-cells to Vγ3^high, CD24^low T-cells. Thus, IL-7 does not only represent a ‘maintenance factor’, but rather a cytokine required for sucessful thymic and extrathymic development and maturation of γδ T-cells. γδ⁺ intestinal intraepithelial lymphocytes (iIEL) are absent in IL-7-/-animals. In contrast, αβ⁺ iIEL can be detected in IL-7 gene-deleted animals, but not in γc, or in JAK-3 deficient mice suggesting that alternative cytokines may be involved in development of iIEL αβ⁺ T-cells, but not necessarily for γδ T-cells. To this end, IL-7 has predominantly been studied in the context of B- and T-cell development. With the availability of IL-7 gene-deleted mice, the paracrine effects of IL-7, which may be secreted in vivo by non-immune cells including keratinocytes or gut epithelial cells, can now be critically examined.     

Antigen-specific expansion of human regulatory T cells as a major tolerance mechanism against mucosal fungi

Foxp3⁺ regulatory T cells (Treg) have a central role for keeping the balance between pro- and anti-inflammatory immune responses against chronically encountered antigens at mucosal sites. However, their antigen specificity especially in humans is largely unknown. Here we used a sensitive enrichment technology for antigen-reactive T cells to directly compare the conventional vs. regulatory CD4⁺ T-cell response directed against two ubiquitous mucosal fungi, Aspergillus fumigatus and Candida albicans. In healthy humans, fungus-specific CD4⁺CD25⁺CD127⁻Foxp3⁺ Treg are strongly expanded in peripheral blood and possess phenotypic, epigenetic and functional features of thymus-derived Treg. Intriguingly, for A. fumigatus, the strong Treg response contrasts with minimal conventional T-cell memory, indicating selective Treg expansion as an effective mechanism to prevent inappropriate immune activation in healthy individuals. By contrast, in subjects with A. fumigatus allergies, specific Th2 cells were strongly expanded despite the presence of specific Treg. Taken together, we demonstrate a largely expanded Treg population specific for mucosal fungi as part of the physiological human T-cell repertoire and identify a unique capacity of A. fumigatus to selectively generate Treg responses as a potentially important mechanism for the prevention of allergic reactions.     

Identification of CD4+ T-cell epitopes on iron-regulated surface determinant B of Staphylococcus aureus

Iron-regulated surface determinant B (IsdB) of Staphylococcus aureus (S. aureus) is a highly conserved surface protein that can induce protective CD4⁺ T-cell immune response. A pivotal role of CD4⁺ T-cells in effective immunity against S. aureus infection has been proved, but CD4⁺ T-cell epitopes on the S. aureus IsdB have not been well identified. In this study, MHC binding assay was firstly used to predict CD4⁺ T-cell epitopes on S. aureus IsdB protein, and six peptides were synthesized to validate the probable epitopes. Two novel IsdB CD4⁺ T-cell epitopes, P1 (residues 159–178) and P4 (residues 287–306), were for the first time identified using CD4⁺ T-cells obtained from IsdB-immunized C57BL/6 (H-2^b) and BALB/c (H-2^d) mice spleen based on cell proliferation and cytokines response. The results showed that P1 and P4 emulsified in Freund's adjuvant (FA) induced much higher cell proliferation compared with PBS emulsified in FA. CD4⁺ T-cells stimulated with peptides P1 and P4 secreted significantly higher levels of IFN-γ and IL-17A. However, the level of the cytokine IL-4 almost remained unchanged, suggesting that P1 and P4 preferentially elicited polarized Th1-type responses. In addition, BALB/c mice just respond to P4 not P1, while C57BL/6 mice respond to P1 not P4, implying that epitope P1 and P4 were determined as H-2^b and H-2^d restricted epitope, respectively. Taken together, our data may provide an explanation of the IsdB-induced protection against S. aureus and highlight the possibility of developing the epitope-based vaccine against the S. aureus.     

Evolution of the Immune Response to Chronic Airway Colonization with Aspergillus fumigatus Hyphae

Airway colonization by the mold Aspergillus fumigatus is common in patients with underlying lung disease and is associated with chronic airway inflammation. Studies probing the inflammatory response to colonization with A. fumigatus hyphae have been hampered by the lack of a model of chronic colonization in immunocompetent mice. By infecting mice intratracheally with conidia embedded in agar beads (Af beads), we have established an in vivo model to study the natural history of airway colonization with live A. fumigatus hyphae. Histopathological examination and galactomannan assay of lung homogenates demonstrated that hyphae exited beads and persisted in the lungs of mice up to 28 days postinfection without invasive disease. Fungal lesions within the airways were surrounded by a robust neutrophilic inflammatory reaction and peribronchial infiltration of lymphocytes. Whole-lung cytokine analysis from Af bead-infected mice revealed an increase in proinflammatory cytokines and chemokines early in infection. Evidence of a Th2 type response was observed only early in the course of colonization, including increased levels of interleukin-4 (IL-4), elevated IgE levels in serum, and a mild increase in airway responsiveness. Pulmonary T cell subset analysis during infection mirrored these results with an initial transient increase in IL-4-producing CD4⁺ T cells, followed by a rise in IL-17 and Foxp3⁺ cells by day 14. These results provide the first report of the evolution of the immune response to A. fumigatus hyphal colonization.     

Human cytomegalovirus-specific CD4 and CD8 T cell responses in primary infection of the immunocompetent and the immunocompromised host

The kinetics of primary human cytomegalovirus (HCMV) infection and specific T-cell responses were investigated in 16 immunocompetent pregnant women and 8 solid-organ transplant recipients (SOTR). T-cell responses to whole HCMV and to pp65 and IE-1 peptides were determined by flow cytometry evaluation of IFNγ production. HCMV-specific CD4⁺ and CD8⁺ T-cells appeared earlier and simultaneously in immunocompetent subjects, whereas specific CD8⁺ T-cells preceded CD4⁺ T-cells in half of the SOTR examined. The magnitude of the HCMV-specific T-cell pool was comparable. HCMV load reached peak levels 100–1000 times higher in SOTR than in immunocompetent women, while the virus persisted for months in blood of both groups. T-cells directed to pp65 and IE-1 were only detected in a portion of subjects developing a full T-cell response to the whole virus. Thus, the development of cell-mediated immune response in primary HCMV infection may be missed when looking at pp65 and IE-1 peptide-stimulated T-cells only.     

Regulatory T-cell-derived interleukin-15 shapes cytotoxic T cell memory

It is well known that regulatory T-cells (Tregs) are required to prevent autoimmunity, but they may also have some less-well understood immune-stimulatory effects. In particular, in CD8⁺ T-cell responses Tregs select high-affinity clones upon priming and promote memory by inhibiting inflammation-dependent generation of short-lived effector cells. In the current issue of the European Journal of Immunology [Eur. J. Immunol. 2023. 53: 2149400 ], Madi et al. report the surprising finding that human and murine FOXP3⁺ Tregs are a physiologically relevant source of IL-15, a homeostatic cytokine that promotes antigen-independent maintenance of CD8⁺ memory T-cells. In mice that lack IL-15 selectively in FOXP3⁺Tregs the authors show that the composition of the CD8⁺ T-cell memory pool is altered in the absence of Treg-derived IL-15, since a subset of terminally effector memory cells is drastically reduced. Otherwise Treg-derived IL-15 is dispensable for antiviral immune responses and the generation of anti-viral CD8⁺ memory T-cells. These findings add to our understanding of the multifaceted role of Tregs in immune responses, and how IL-15 derived from different cellular sources maintains anti-viral T-cell memory.     

Triggering of T Cell-Mediated Immune Responses by Allogenic Tumor Cell Vaccine in Patients with Oral Cancer

We investigated the immunomodulatory activity of allogenic whole tumor cell vaccine in oral cancer patients in vitro by two-color flow cytometry. Vaccine treatment significantly increased the expression of CD69 and HLA-DR in CD3⁺ T-cell subsets. The frequency of Interferon-gamma and Interleukin (IL)-2 expressing CD4⁺/CD8⁺ T-cell subsets was significantly higher with a concomitant reduction in IL-4 and IL-10 expressing T-cells in the vaccine treated group as compared with the untreated controls. Vaccine treatment significantly increased T-cell receptor (TCR), Vβ3, Vβ5 and Vβ8 usage. The results indicate that the allogenic whole tumor cell vaccine is able to trigger T-cell mediated immunity in patients with intraoral squamous cell carcinoma.     

The Increase of IFN-γ Production through Aging Correlates with the Expanded CD8CD28CD57 Subpopulation

The use of flow cytometry to detect intracellular cytokines at the single cell level has the potential to quantify cytokine production together with the possibility of phenotypic identification of the cell population concerned. The unbalanced presence of intracellular cytokines produced by T cells has been recognized in some pathological conditions. To better address this issue, we studied the production of IFN-γ and IL-4 in CD4⁺ and CD8^+high T cells in healthy donors of a broad range of age (17–62 years). Given that an increase of IFN-γ and IL-4 with aging had been reported by some authors in healthy controls, we have performed a multivariate analysis to assess the intrinsic role of aging or of other external factors, such as chronic antigenic exposures (i.e., viruses), over the cytokine production of phenotypically characterized T cells. In this respect we show that, mainly in CD8^+high T cells, the production of IFN-γ is directly correlated with age. Besides, the cytokine production correlates with the CD8^+highCD28⁻CD57⁺ T-cell population, which we have recently reported elevated in aged individuals. Perhaps this T-cell subpopulation plays a regulatory role as a Tc1 response in aging individuals.     

In vitro effect of 4-pentylphenol and 3-methyl-4-nitrophenol on murine splenic lymphocyte populations and cytokine/granzyme production

Gasoline exhaust particles (GEP) and diesel exhaust particles (DEP) are considered to be some of the most important air pollutants. Among the many constituents in these pollutant particles, 4-pentylphenol (PP) and 3-methyl-4-nitrophenol (PNMC) are considered important phenolics in GEP and DEP, respectively. The aim of this study was to investigate the effect of in vitro exposure to commercially-supplied PP and PNMC on populations of, and production of interleukin (IL)-2, IL-4 and granzyme-B by, mouse splenic lymphocytes. After in vitro exposure to PP or PNMC for 48 h, splenocyte viability was measured, cell phenotypes, e.g. B-cell (CD19), T-cells (CD3), T-cell subsets (CD4 and CD8), were quantified by flow cytometry and production of IL-2, IL-4 and granzyme-B was assessed via ELISA. The oxidative toxicity of PP and PNMC toward the splenocytes was also evaluated using measures of hydroxyl radical and malondiadehyde production and changes in glutathione peroxidase and superoxide dismutase activities. Results showed that in vitro exposure to PP and PNMC inhibited splenic cell parameters in a dose-related manner. Exposure to PP and PNMC decreased splenic T-lymphocyte populations and splenocyte production of cytokines and granzyme B, as well as induced oxidative stress in the splenocytes. The results also showed that the percentages of CD3⁺ T-cells overall and of CD4⁺ and CD8⁺ T-cells therein, among exposed splenocytes, were reduced; neither compound appeared to affect levels of CD19⁺ B-cells. Overall, the suppressive effects of PP were stronger than PNMC. The data here provide support for the proposal that PP-/PNMC-induced toxicity in splenocytes may be due at least in part to oxidative damage and that PP and PNMC – as components of GEP and DEP – might significantly impact on splenic T-cell formation/release of cytokines/granzymes in situ.     

Comparative Analysis of Mycobacterial Heat Shock Proteins-Induced Apoptosis of Peripheral Blood Mononuclear Cells in Sarcoidosis and Tuberculosis

Sarcoidosis (SA) is a granulomatous disorder of an unknown etiology. Mycobacterium tuberculosis heat shock proteins (Mtb-hsp), considered as causative agents, play an important role in apoptosis. A role for apoptosis has been proposed in pathogenesis of SA and tuberculosis (TB) granuloma formation but results remain controversial. Differences in Mtb-hsp-induced apoptosis between SA, TB, and healthy subjects found in this study might put some light on the etiology of SA. Early apoptotic peripheral blood mononuclear cells (PBMC) were determined in 22 SA patients, 20 TB patients, and 20 healthy volunteers by flow cytometry (Annexin-V-FITC). Our results revealed that spontaneous apoptosis of monocytes and CD8⁺ T-cells was comparable between tested groups. Apoptosis of unstimulated CD4⁺ T-cells was significantly lower in TB versus controls and insignificantly lower versus SA. Mtb-hsp- and PHA (Phytohemagglutinin)-induced monocytes apoptosis was significantly lower in TB versus controls and SA. Mtb-hsp-induced CD4⁺ T-cell apoptosis was significantly lower in TB versus controls and SA. There were no differences of PHA-induced CD4⁺ T-cell and CD8⁺ T-cell apoptosis between tested groups. Apoptosis of Mtb-hsp-induced CD8⁺ T-cells was significantly lower in TB and SA versus controls. Analysis of PBMC apoptosis before and after stimulation in each tested group revealed that, in contrast to TB, sarcoid monocytes were resistant to Mtb-hsp- and PHA-induced apoptosis and CD4⁺ T-cells were resistant to PHA- but not Mtb-hsp-induced apoptosis. CD8⁺ T-cell apoptosis, before and after Mtb-hsp or PHA stimulation, was significantly increased in all tested groups. It seems likely that dysregulated apoptosis of CD4⁺ T-cells and resistant apoptosis monocytes may be involved in pathogenesis of SA.     

Properties of HTLV-I transformed CD8 T-cells in response to HIV-1 infection

HIV-1 infection studies of primary CD8⁺ T-cells are hampered by difficulty in obtaining a significant number of targets for infection and low levels of productive infection. Further, there exists a paucity of CD8-expressing T-cell lines to address questions pertaining to the study of CD8⁺ T-cells in the context of HIV-1 infection. In this study, a set of CD8⁺ T-cell clones were originated through HTLV-I transformation in vitro, and the properties of these cells were examined. The clones were susceptible to T-cell tropic strains of the virus and exhibited HIV-1 production 20-fold greater than primary CD4⁺ T-cells. Productive infection resulted in a decrease in expression of CD8 and CXCR4 molecules on the surface of the CD8⁺ T-cell clones and antibodies to these molecules abrogated viral binding and replication. These transformed cells provide an important tool in the study of CD8⁺ T-cells and may provide important insights into the mechanism(s) behind HIV-1 induced CD8⁺ T-cell dysfunction.     

Ex vivo detection of adenovirus specific CD4 T-cell responses to HLA-DR-epitopes of the Hexon protein show a contracted specificity of THELPER cells following stem cell transplantation

Human adenovirus (HAdV) is a cause of significant morbidity and mortality in immunocompromised patients, especially after stem cell transplantation (SCT). Viral clearance has been attributed to CD4⁺ T-cell responses against the Hexon-protein, but the frequency of specific T_HELPER cells is extremely low or not detectable ex vivo and preference for different CD4⁺ T-cell epitopes is variable among individuals. We therefore analyzed 44 healthy donors and 6 SCT-recipients for Hexon-specific CD4⁺-responses ex vivo, to identify epitopes which would be broadly applicable. We selected 19 candidate epitopes with predicted restriction to HLA-DR1/DR3/DR4/DR7; 16 were located within the highly conserved regions, indicating cross-reactivity of T cells among HAdV-subspecies. Ten epitopes induced CD4⁺-proliferation in >50% of individuals, confirmed by intracellular IFN-γ detection. Three SCT recipients who recovered from an infection with HAdV displayed reactivity towards only a single hexon epitope, whereas healthy individuals were responsive to two to eight epitopes (median 3). The ex vivo detection of Hexon-specific CD4⁺ T-cells, without any long-term culture in vitro, enables the detection and generation of HAdV-specific CD4⁺ T cells for adoptive T-cell transfer against HAdV-infection post SCT.     

Ex Vivo Triggering of T-Cell-Mediated Immune Responses by Autologous Tumor Cell Vaccine in Oral Cancer Patients

We investigated immunomodulatory activity of autologous tumor cell vaccine from oral cancer patients ex vivo by lymphoproliferation assay and two color flow cytometry. Vaccine treatment lead to 10-fold higher proliferation of lymphocytes compared with the untreated controls. A significant increase in CD69⁺ and HLA-DR⁺ T-cells was observed in vaccine pulsed cultures compared with untreated (p<0.0001) controls. The frequency of IFN-γ and IL-2 expressing CD4⁺/CD8⁺T-cell subsets was significantly higher with a concomitant reduction in IL-4 and IL-10 expression in the vaccine-treated group (p<0.0001) compared with the untreated controls. Vaccine treatment further increased T-cell receptor Vβ3, Vβ5, and Vβ8 usage. It seems that the autologous tumor cell vaccine triggers T‐cell responses ex vivo, hence it may have a protective role in oral cancer patients.     

The influence of HIV on CD127 expression and its potential implications for IL-7 therapy

Interleukin-7 (IL-7) is critical for early T-cell development and plays an important role in T-cell homeostasis, differentiation and function. Signalling via the IL-7 receptor is dependent on the expression of its components, IL-7Rα (CD127) and IL-2Rγ (CD132) and is mediated in part by alterations in CD127 expression levels in different cell subsets. Naïve and memory T-cells express high levels of CD127, while effector cells are CD127^lo and retention of the receptor is thought to influence the development of memory cells. Reduced expression of CD127 has been associated with markers of disease severity in HIV infection and other chronic viral infections as well as in various cancers. In HIV infection, decreased CD127 expression on T-cells is correlated with reduced CD4⁺ T-cell counts, increased viral replication and immune activation. The loss of IL-7 activity, due to decreased CD127 expression, may contribute to the observed loss of CD8⁺ cytotoxic T lymphocyte (CTL) activity in HIV infection. The downregulation of CD127 expression in HIV infection may be due to host (e.g. IL-7, IL-4, immune activation) and/or viral (e.g. HIV-tat) factors and mechanisms of receptor regulation may differ by cell type. In addition, the expression of a soluble form of CD127 (sCD127) has been shown to be increased in HIV infection. This protein may affect IL-7 activity in vivo and therefore may have implications for IL-7-based therapies which are currently being tested in clinical trials. Understanding how CD127 is regulated during HIV infection will provide insight for the development of novel therapeutics to improve immune function and anti-viral T-cell activity.