Decreased gene expression of common γ-chain receptor (CD132) in tissues of Crohn’s disease patients

doi:10.21203/rs.3.rs-3213124/v1

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Decreased gene expression of common γ-chain receptor (CD132) in tissues of Crohn’s disease patients

https://doi.org/10.21203/rs.3.rs-3213124/v1

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Background.

A deficiency of γδ T cells has been described in Crohn's disease (CD). Our goal was to analyze the gene expression of IL-7 and its receptors in tissues of patients with CD.

Methods

We studied the peripheral blood of 80 patients with Crohn's disease, comparing them with a group of 80 healthy subjects. The number and apoptosis of αβ and γδ T cells in peripheral blood and the proportion of αβ and γδ T cells in intestinal tissues of CD patients (n = 25) were studied. IL-7 gene expression, IL-7 protein, common γ-chain receptor (CD132), receptor α (CD127) and caspase 3 in tissues were analyzed by qPCR. Serum IL-7 levels were analyzed.

Results

An increase in apoptosis of CD56 + αβ and γδ T cells in peripheral blood was observed, beside IL-7 gene expression and IL-7 protein in tissues of these patients were increased. The titers of caspase 3 in tissues were low. The number of γδ T cells decreased, and was directly related to IL-7 levels in peripheral blood. The expression of common γ-chain receptor (CD132) is greatly decreased in the tissues of Crohn's disease patients.

Conclusion

There may be a cause-effect relationship between the lower gene expression of the common receptor of the γ chain (CD132) and this immunodeficiency.

Crohn&rsquo

s Disease

IL-7 gene expression

common γ-chain receptor (CD 132)

Caspase 3

γδ T cells

The pathogenesis of Crohn's disease (CD) remains understudied due to lack of knowledge about its etiology. Recent studies suggest that a defective innate immune response after disruption of the epithelial barrier is the primary defect in CD [1, 2]. In the immune system, γδ T cells, due to their frequent intraepithelial location in the mucous membranes and their functional characteristics, have qualities of innate and acquired immunity, and constitute a first line of defense against attacks from the outside world [3]. We previously described a reduction in γδ T cell subsets in peripheral blood from CD patients [4]. In addition, this reduction was repeated in all three clinical scenarios studied: new patients, active disease, and remission. Therefore, the study of γδ T cells is a valuable candidate to understand the pathophysiology of CD [5].

Interleukin 7 (IL-7) plays a very important role in the normal functioning of the immune system, exerting a signal necessary for the survival, differentiation and proliferation of B, T, NK and other immune cells. It exerts its action by binding to the IL-7 receptor (IL-7R), a heterodimer composed of two subunits, IL-7Rα (CD127) and common γ-chain receptor (CD132) [6]. Alterations in both IL-7 and its receptors are associated with immunodeficiency and other autoimmune and inflammatory diseases, including cancer. Therefore, IL-7 was proposed as a promising actor in the therapeutic strategy for many diseases [7]. IL-7 is absolutely essential for the existence of γδ T cells. In fact, IL-7R deficient mice develop few αβ T cells and B cells, but γδ T cells are absent, demonstrating that IL-7R signaling is indispensable for the development of thymic and extrathymic γδ T cells [8]. IL-7 is produced by mesenchymal stromal cells of bone marrow and epithelial cells of thymus, lymph nodes, skin, liver and intestines. Thus, IL-7 expression by enterocytes is sufficient for extrathymic development of γδ T cells [9, 10]. We observed that levels of IL-7 in peripheral blood of CD patients were decreased [11]. The role of IL-7 and its receptors in tissues of CD patient has been studied by Lyssia Belarif et al. In this paper, the authors described that failure of inflammatory bowel disease (IBD) treatments (CD and Ulcerative colitis) is strongly associated with an overexpressed IL-7R signaling pathway of these patients, although only IL-7Rα (CD127) was analyzed [12].

Apoptosis or programmed cell death of T cells is an important mechanism of peripheral immune tolerance. Observations by several laboratories showed that mucosal T cells of patients with IBD were highly resistant to apoptosis [13–18]. Due to these findings, it has been reasoned that the persistence of activated T cells in the mucosa would explain that the inflammatory reaction of IBD patients is resistant to resolution. However, lymphocyte apoptosis in peripheral blood and tissues of patients with CD has hardly been studied. Similarly, IL-7 in tissues and its relationship with apoptosis, has not been studied.

Our objective was to analyze the gene expression of IL-7 and its receptors in tissues of patients with CD, to determine the relationship of this cytokine with the deficiency of γδ T cells.

2.1. Study population

In this prospective case-control study, we studied the peripheral blood of 160 subjects, 80 patients with CD, comparing them with 80 healthy subjects (control group). In addition, we analyzed the tissues of 25 of these patients compared to a group of 25 healthy subjects. The tissues (intestinal biopsies) were obtained from patients with CD who underwent endoscopy and surgery at the Arnau de Vilanova Hospital in Valencia. The Lennard-Jones criteria were used for the diagnosis of CD [19]. Activity was measured using the CD activity index (CDAI) [20]. CD patients were divided according to three clinical scenarios: “new patients” with active CD presenting at diagnosis with no prior CD treatment, or with only treatment within the previous 24 hours, “remission” (CDAI < 150 for at least 12 months) and “active disease” (CDAI > 150 and signs and symptoms of disease). Control healthy subjects were required to have the following characteristics: they were not suffering from immunodeficiency or autoimmune or inflammatory diseases, had not been vaccinated in the last 3 months, and were not undergoing immunosuppressive therapy. Tissues from healthy subjects were biopsied by routine colonoscopies program to detect colon cancer, and diagnosed as normal. Cases and controls were matched by sex and age ± 5 years. The study was approved by the Ethics and Investigation Committee of the Arnau de Vilanova-Lliria University Hospital (Valencia, Spain).

2.2. Tissue sampling of intestinal biopsies

Three to five biopsies of ileum and colon were taken at 1–2 cm intervals in each patient. Some samples were fixed through immersion in a solution of 10% buffered formalin. Specimens were routinely processed by paraffin embedding. Tissue was stained by Hematoxylin-eosin. Some samples were frozen without fixing for cellular and molecular analyses. Small biopsy samples of tissues were homogenized and washed in separation medium (PBS, 1% fetal bovine serum, 1 mM DTT, 1 mM EDTA) for 15 min at 37°C.

After centrifugation, pellet was subsequently incubated in digestion medium consisting of 5% fetal bovine serum (FBS), 0.5 mg/ml collagenase type VIII (Sigma-Aldrich) for 30 min at 37°C by gentle shaking. Cells were passed through a mesh, centrifuged and stained with lineage markers. Human lymphocytes were isolated from intestinal tissues of healthy and CD patients.

2.3. Cell isolation for analysis of γδ and αβ T cells and apoptosis evaluation

2.3.1. Blood sample analysis

Blood cell counts were obtained in a cell counter (LH750 Beckman Coulter, Inc, Fullerton, CA). We obtained by centrifugation on density gradient from EDTA anticoagulated blood sample with Lymphoprep™ (Palex Medical SA), an enrichment of the sample in mononuclear cells. After two washes in phosphate buffered saline (PBS), the cells obtained were resuspended in 200 ml of binding buffer from the ANNEXIN V-FITC/7-AAD Kit (Beckman Coulter, Inc) with the presence of Ca²⁺.

2.3.2. Functional analysis of γδ and αβ T cells

To evaluate the functional analysis of γδ and αβ T cells —peripheral blood and intestinal tissue— we performed flow cytometry analysis with these monoclonal antibodies: anti-TCR PAN αβ- PE, anti-TCR PAN γδ- PE and FITC, CD19- PC7, CD56- PC7 and PE and CD4-PC7, CD3-PC5 and ECD, CD8-PC7 and FITC, CD5- FITC, CD45-ECD (Beckman Coulter, Inc, Miami, USA). A total of 100.000 events were acquired in a multiparameter Navios flow cytometer (Beckman Coulter, Inc) and later analyzed with Kaluza software.

2.3.3. Apoptosis evaluation

The apoptosis detection in peripheral blood was performed with ANNEXIN V-FITC/7-AAD Kit (Beckman Coulter, Inc), based on the binding properties of annexin V to phosphatidylserine and on the specificity of 7-amino-actinomycin D (7-AAD) for DNA guanine-cytosine base pair, following instructions of the manufacturer.

Gating strategy for T cell subsets in healthy controls vs patients with CD is show in Supplementary file 1.

2.3.4. IL-7 in peripheral blood.

The presence of serum IL-7 was measured by a solid phase sandwich ELISA (Quantikine® HS ELISA, R&D Systems). According to the product information, the detection limit of the human IL-7 was 0.1 pg/mL with an assay range for serum samples of 0.25-16 pg/mL.

2.3.5. Gene expression of IL-7, IL-7 receptor α (CD127) and IL-7 common γ-chain receptor (CD 132) in tissues.

2.3.5.1. RNA extraction

The samples that were homogenized with 1 mL of Trizol® Reagent (Ambion, Life Technologies,) for RNA isolation following manufacturer’s instructions. RNA concentration was measured using a GE NanoVue Spectrophotometer (GE Healthcare Life Sciences, UK).

2.3.5.2. Reverse Transcription-Polymerase Chain Reaction

A reverse transcription reaction was performed from 1 mg of extracted RNA using the Thermo Scientific RevertAid H Minus First Strand cDNA Synthesis kit (Thermo Fisher Scientific, USA). Resulting cDNA was amplified and subjected to qPCR in GeneAmp 5700 (PE Biosystems). The primer sequences are shown in Supplementary Table 1. The relative expression of each gene was normalized against GADPH expression, used as reference standards.

2.3.6. Analysis of IL-7 and caspase-3 by Western-Blot.

Western-Blot was performed with protein extracts from intestinal biopsies. Trizol/Guanidine protein purification (Trizol® Reagent (Ambion, Life Technologies) and Guanidine hydrochloride (SIGMA) following manufacturer’s instructions, preserved in TNT buffer (20 mM Tris–HCl, pH 7.5, 0.2 M NaCl, 1% Triton X-100) supplemented immediately before use with protease inhibitor cocktail tablets (10%) (Roche). Protein concentration was determined using the Pierce BCA Protein Assay Reagent (Pierce). The samples (20 µg of protein) were subjected to electrophoresis in 12% polyacrylamide gels in the presence of sodium dodecyl sulfate (SDS). The proteins were transferred to a nitrocellulose membrane (BIO-RAD), using the Biorad Mini Protean II system in accordance with the conditions recommended by the commercial company.

The membranes were incubated with the corresponding primary antibody (anti IL-7 or anti-caspase 3 (Supplementary Table 2) in TBS (20mM Tris-HCl pH 7.5 and 150 mM NaCl) with 0.1% blocking agent, for 12h at 4ºC. Membranes were to remove excess primary antibody with TBS supplemented with 0.05% tween, and then incubated with a 1/1000 dilution of the secondary antibody (SIGMA). Membranes were again washed, visualizing the positive bands by an image digitizer with the Enhanced Chemiluminescence (ECL) reagent from Amersham and were quantified by densitometry using the Image J software.

2.4. Statistical Analysis

Mann-Whitney U test was used to compare differences between means of quantitative variables. Correlation studies using Spearman’s Correlation Coefficient were performed to compare IL-7 gene expression, serum IL-7 levels and T cell subsets. Data were analyzed using the Statistical Package for Social Sciences (SPSS 19.0; SPSS Inc., Chicago, IL, USA). Figures were performed using GraphPad Prism version 8.0.0, GraphPad Software, San Diego, California USA.

Supplementary Table 3 shows the clinical characteristics of CD patients (n = 80).

3.1. Cell number and percentage of apoptotic αβ and γδ T cell subsets in peripheral blood of CD patients and healthy controls.

There were no differences between the number of αβ T cells between CD patients and healthy subjects (Fig. 1A). However, a significant decrease in γδ T cells in peripheral blood of patients with CD compared to control subjects was observed (Fig. 1B).

Apoptosis of CD3 + CD56 + αβ, CD3 + CD56 + γδ and CD3 + CD4 + γδ T cells increased in CD patient’s vs healthy subjects (Figs. 1C and 1D). According to clinical scenarios, we found no significant differences between the number or apoptosis of αβ and γδ T cell subsets, except in CD3 + CD8 + γδ T cell number in active disease (0.0057 ± 0.0056 x10⁶/mL) vs remission (0.0193 ± 0.01991 x10⁶/mL), p = 0.008 (Bonferroni test). There also were no differences between T cell subsets according to patients who were receiving (n = 44, 55.0%) or not treatment at the time of the study (Supplementary Table 4.).

3.2. Relationship between the number and apoptosis of αβ and γδ T cells.

In CD patients, there was an inverse correlation (Spearman r test) among CD3 + γδ+ (-0.373, p = 0.002), CD3 + CD4 + γδ (− 0.304, p = 0.016) and CD3 + CD8 + γδ (− 0.521, p < 0.0001) T subsets and apoptosis.

In healthy controls, there was an inverse correlation among both αβ and γδ T cell subsets and apoptosis CD3 + αβ (− 0.313, p = 0.007), CD3 + CD4 + αβ (-0.310, p = 0.019), CD3 + CD8 + αβ (− 0.220, p = 0.069) and CD3 + CD56 + αβ (− 0.456, p = 0.002), CD3 + γδ (− 0.242, p = 0.037), CD3 + CD4 + γδ (− 0.409, p < 0.0001), CD3 + CD8 + γδ (− 0.397, p < 0.0001) and CD3 + CD56 + γδ (− 0.267, p < 0.049) T cells.

3.3. IL-7 gene expression and IL-7 protein.

Figure 2A shows IL-7 gene expression and levels of IL-7 cytokine in tissues from CD patients, compared to healthy controls. Values were significantly higher in the CD group. In addition, there was a positive correlation between IL-7 gene expression and its tissue production (Fig. 2B). However, there were no significant differences in serum IL-7 levels between patients and controls. Panel C shows the IL7 gene expression and Panel D shows the IL-7 protein.

3.4. Expression of Caspase 3 in tissues and its relationship with IL-7 and γδ T cells.

There was a lower expression of caspase 3 in tissues from CD patient’s vs controls (Figs. 3A and 3C). Negative correlations were observed among expression of caspase 3 and frequencies of γδ T cells in the healthy control group (Fig. 3B). Moreover, caspase 3 was negatively correlated to both IL-7 cytokine and IL-7 gene expression in tissues from CD patients (Fig. 3D).

We did not find significant differences in serum and tissue IL-7 cytokine, IL-7 gene expression and caspase 3 according to clinical scenarios and treatments.

3.5. Percentages of αβ and γδ T cells in tissues.

Figures 4A and 4B show differences between tissue αβ and γδ T cells of CD patients vs healthy controls, respectively. CD3 + γδ and CD3 + CD8 + γδ T cells were significantly lower in tissue of CD patients compared to the control group. Serum IL-7 levels were positively correlated with CD3 + γδ and CD3 + CD8 + γδ T cells in tissue of CD patients (Fig. 4C). There were no differences in the percentages of tissue T cells between patients with and without treatment. Panels D-G show flow cytometric imaging of αβ and γδ T cells.

3.6. IL-7 receptor expression: IL-7 receptor α (CD127) and common γ-chain receptor (CD132).

Since there was no decrease in the gene expression of IL-7 and IL-7 cytokine in the tissues of patients with CD, we analyzed the expression of their receptors. As we can see in Figs. 5A and 5B, the expression of the common γ-chain receptor (CD132) was significantly lower in tissues of CD patients compared to the control group. There was a significant positive correlation between the gene expression of IL7Rα (CD127) and serum IL-7 of all subjects (Fig. 4C). In the case of CD132, the correlation, although positive, was not significant (Fig. 5D). In four patients with CD, the gene expression of IL7R was studied. Three of the four patients were in remission and one in active disease. The patient with active disease was being treated with anti-TNF-α. No significant differences were observed in the values of CD127 and CD132 between the patients.

The expression of the IL-7 gene and its receptors and the possible relationship with immunity and apoptosis of conventional and non-conventional αβ and γδ T cells, respectively, were study in intestinal tissue and peripheral blood of patients with CD. In the present study, we confirmed the previously observed deficiency of γδ T cell subsets in peripheral blood of CD patients [4]. Likewise, the lowest frequencies were observed in CD3 + CD56 + γδ T cells. In addition, the apoptosis of CD3 + CD56 + αβ, CD3 + CD4 + γδ and CD3 + CD56 + γδ T cells was higher in CD patients compared to the control group. CD3 + CD56 + γδ T cells, share receptors with NK cells, have high cytotoxic activity and it was proposed to name them γδ NKT cells [21]. We previously described the importance of this subpopulation in immunity against infections and tumors. We also showed that both a deficiency in the number of cells and an increase in their apoptosis were related to a worse prognosis of the sepsis and cancer disease [22–24]. Therefore, it is not surprising that, in the present study, CD patients had lower CD3 + CD56 + γδ (γδ NKT cells) frequencies but with a higher degree of apoptosis.

The importance of IL-7 and its relationship in activation of T cells and specifically in γδ T cells has been exposed already in the introduction. In the present work, we have shown significantly higher tissue IL-7 gene expression in CD patients compared to healthy controls. This increase was directly proportional to the production of the IL-7 cytokine. In addition, there were lower caspase 3 levels in CD patients that were inversely correlated to expression of tissue IL-7 both gene and protein. IL-7 prevented the apoptosis of γδ intestinal intraepithelial lymphocytes in a murine model, although caspase activity was only slightly inhibited [25]. This paper suggested that IL-7, stimulated by some unknown factor, could maintain a sufficient number of γδ T cells by preventing their apoptosis. Our results are in the same line, the increase in the protein levels and gene expression of IL-7 together with the reduction of Caspase 3 in the tissues of CD patients suggests an attempt to keep the number of γδ T cells stable. However, this situation is not achieved, generating an immunosuppression at mucosa-associated lymphoid tissue (MALT) level that can be used by opportunist pathogens that intend to infect through the mucous membranes.

However, discrepancies between the tissue and serum levels of IL-7 were observed. The tissue levels of IL-7 increased to try to compensate for the γδ T cell deficit in CD patients. However, serum IL-7 levels were similar in patients and healthy controls.

This lack of increase in serum could be explained by a problem in the paracrine and autocrine effect of the cytokine under study. The cytokine may be being produced in large quantities but is being consumed by binding to the receptor, hence the importance of analyzing the IL-7 receptor.

The common gamma chain (γc or CD132 or interleukin-2 receptor subunit gamma or IL-2RG) integrates the IL-7 receptor heterodimer together with IL-7 receptor α (CD127). CD132 is a cytokine receptor subunit that, in addition to IL-7, is common to the other interleukin receptors: IL-2, IL-4, IL-9, IL-15 and IL-21. This receptor, expressed on most lymphocyte subsets, contributes to the proper functioning of the immune system, regulating development, proliferation, survival, and differentiation of immune cells [26]. Mutations of the gene encoding CD132 results in X-linked severe combined immunodeficiency in humans. Mechanisms of signaling and gene regulation of the related cytokines, provide information for immunodeficiency, autoimmunity, allergic diseases, and cancer [27]. Mutations in cytokine receptor genes have been implicated in IBD, such as polymorphisms in IL-2, IL-21, IL-2RA, and IL-15RA [28].

According to the results shown in this study, the expression of the common γ-chain receptor (CD132) is greatly decreased in tissues of CD patients. Gene expression levels of the IL-7 receptor α (CD127) were similar in both healthy controls and CD patients. The integrity of the two subunits is necessary for the correct receptor functioning.

Therefore, we propose that the increase in IL-7 gene expression, which also produces an increase of IL-7 protein, and the lower expression of caspase 3, which can lead to decreased apoptosis, is insufficient to maintain adequate levels of γδ T cells in peripheral blood and tissues of CD patients. It seems that the marked decrease in the gene expression of the γ subunit (CD132) of the IL-7 receptor would prevent the proper functioning of IL-7, leading to immunodeficiency of γδ T cells that would favor superinfection or lack of elimination of some factor. Infectious etiology with consequent chronic inflammation. In a previous study, we described a relationship of an opportunistic fungus (microsporidia) with CD [29]. Based on the above evidence, we suggest that the infection by microsporidium, (either directly the pathogen or some protein thereof), could interferes with the expression of the receptor (CD132) as a mechanism of evasion of the immune response against the pathogen.

There is an immunodeficiency of γδ T cells in peripheral blood and tissues of CD patients with increased apoptosis in peripheral blood. The increase in IL-7 gene expression and IL-7 protein, and lower expression of caspase 3 in tissues, suggest an attempt to recover normal values of γδ T cells unsuccessfully. More research would be necessary to determine if there is a cause-effect relationship between the lower gene expression of the common receptor of the γ chain (CD132) and this immunodeficiency.

Ethical Approval

Each volunteer participant signed an informed consent document. This study was conducted following the recommendations of the Spanish Bioethics Committee, the Spanish legislation on Biomedical Research (Law 14/2007) and Personal Data Protection (Spanish Law 3/2018 and European Law UE676/2018). The anonymity of the subjects participating in the study has been ensured. The study was approved by the Ethics and Investigation Committee of the Arnau de Vilanova-Lliria University Hospital (Valencia, Spain).

Competing interests:

The authors declare they have no financial interests

Authors' contributions

Conception and design of the work (Andreu-Ballester JC; Hurtado C and Cuéllar C); collecting data and recruitment of patients (Gil-Borras R); Flow cytometry analysis of T cell subsets and apoptosis (García-Ballesteros C and López-Chuliá F); IL-7 analysis and preparation of tissues for cytometry analysis (Pérez-Griera J and Cuéllar C); PCR analysis of gene expression tissues (Hurtado C, Ollero D, Izquierdo, F); Tissue sampling (Jiménez A); Statistical method (Andreu-Ballester JC); interpretation of data for the work (Andreu-Ballester JC, Hurtado C and Cuéllar C); Final approval of the version to be submitted (Andreu-Ballester JC, Hurtado C Llombart-Cussac A and Cuéllar C).

Funding:

The authors did not receive support from any organization for the submitted work.

Availability of data and materials

Data sharing not applicable to this article as no datasets were generated or analyzed during the current study.

Coulombe F, Behr MA (2009) Crohn’s disease as an immune deficiency? Lancet 374:769–770
Torres J, Mehandru S, Colombel JF et al (2017) Crohn's disease. Lancet 389(10080):1741–1755
Adams EJ, Gu S, Luoma AM (2015) Human gamma delta T cells: Evolution and ligand recognition. Cell Immunol 296:31–40
Andreu-Ballester JC, Amigó-García V, Catalán-Serra I et al (2011) Deficit of Gammadelta T Lymphocytes in the Peripheral Blood of Patients with Crohn's Disease. Dig Dis Sci 56:2613–2622
Catalan-Serra I, Sandvik AK, Bruland T et al (2017) Gammadelta T Cells in Crohn's Disease: A New Player in the Disease Pathogenesis? J Crohns Colitis 11:1135–1145
Rochman Y, Spolski R, Leonard WJ (2009) New insights into the regulation of T cells by gamma(c) family cytokines. Nat Rev Immunol 9:480–490
Chen D, Tang TX, Deng H et al (2021) Interleukin-7 Biology and Its Effects on Immune Cells: Mediator of Generation, Differentiation, Survival, and Homeostasis. Front Immunol 12:747324
Maki K, Sunaga S, Komagata Y et al (1996) Interleukin 7 receptor-deficient mice lack gammadelta T cells. Proc Natl Acad Sci U S A 93:7172–7177
Laky K, Lefrançois L, von Freeden-Jeffry U et al (1998) The role of IL-7 in thymic and extrathymic development of TCR γδ cells. J Immunol 161:707–713
Laky K, Lefrançois L, Lingenheld EG et al (2000) Enterocyte expression of interleukin 7 induces development of gammadelta T cells and Peyer's patches. J Exp Med 191:1569–1580
Andreu-Ballester JC, Pérez-Griera J, Garcia-Ballesteros C et al (2012) Deficit of interleukin-7 in serum of patients with Crohn's disease. Inflamm Bowel Dis. Feb 7
Belarif L, Danger R, Kermarrec L et al (2019) IL-7 receptor influences anti-TNF responsiveness and T cell gut homing in inflammatory bowel disease. J Clin Invest 129:1910–1925
Ina K, Itoh J, Fukushima K et al (1999) Resistance of Crohn’s disease T cells to multiple apoptotic signals is associated with a Bcl-2/Bax mucosal imbalance. J Immunol 163:1081–1090
Boirivant M, Marini M, Di Felice G et al (1999) Lamina propria T cells in Crohn’s disease and other gastrointestinal inflammation show defective CD2 pathway-induced apoptosis. Gastroenterology 116:557–565
Atreya R, Mudter J, Finotto S et al (2000) Blockade of interleukin 6 trans signaling suppresses T-cell resistance against apoptosis in chronic intestinal inflammation: evidence in Crohn disease and experimental colitis in vivo. Nat Med 6:583–588
Itoh J, de La Motte C, Strong SA et al (2001) Decreased Bax expression by mucosal T cells favours resistance to apoptosis in Crohn’s disease. Gut 49:35–41
Sturm A, Leite AZ, Danese S et al (2004) Divergent cell cycle kinetics underlie the distinct functional capacity of mucosal T cells in Crohn’s disease and ulcerative colitis. Gut 53:1624–1631
Doering J, Begue B, Lentze MJ et al (2004) Induction of T lymphocyte apoptosis by sulphasalazine in patients with Crohn’s disease. Gut 53:1632–1638
Lennard-Jones JE (1989) Classification of inflammatory bowel disease. Scand J Gastroenterol Suppl 170:2–6 discussion 16 – 9
Best WR, Becktel JM, Singleton JW et al (1976) Development of a Crohn's disease activity index. National Cooperative Crohn's Disease Study. Gastroenterology 70:439–444
Matsuo Y, Tsujimura T, Drexler HG (2021) Proposal for the designation of the natural killer antigens-positive γδ T-cell subset as γδ NKT-cells: nomenclature based on immunoprofile. Hum Cell 34:1278–1279
Andreu-Ballester JC, Tormo-Calandín C, Garcia-Ballesteros C et al (2013) Association of γδ T cells with disease severity and mortality in septic patients. Clin Vaccine Immunol 20:738–746
Andreu-Ballester JC, Galindo-Regal L, Hidalgo-Coloma J et al (2020) Differences in circulating γδ T cells in patients with primary colon cancer and relation with prognostic factors. PLoS ONE 15(12):e0243545
Andreu-Ballester JC, Arribas MA, Rico M et al (2022) Changes of CD3 + CD56 + γδ T cell number and apoptosis during hospital admission are related to mortality in septic patients. Clin Immunol 236:108956
Yada S, Nukina H, Kishihara K et al (2001) IL-7 prevents both caspase-dependent and -independent pathways that lead to the spontaneous apoptosis of i-IEL. Cell Immunol 208:88–95
Barata JT, Durum SK, Seddon B (2019) Flip the coin: IL-7 and IL-7R in health and disease. Nat Immunol 20:1584–1593
Leonard WJ, Lin JX, O'Shea JJ (2019) The γ_c Family of Cytokines: Basic Biology to Therapeutic Ramifications. Immunity 50:832–850
Huang H, Fang M, Jostins L et al (2017) Fine-mapping inflammatory bowel disease loci to single-variant resolution. Nature 547(7662):173–178
Andreu-Ballester JC, Garcia-Ballesteros C, Amigo V et al (2013) Microsporidia and its relation to Crohn's disease. A retrospective study. PLoS ONE 8(4):e62107

Supplementary File 1 is not available with this version.

Supplementary file 1. Gating strategy for T cell subsets in healthy subjects (A) vs patients with Crohn's disease (B). Cells positive for CD45 were first selected, total lymphocytes. From total lymphocytes we differentiated αβ and γδ T cell subpopulations, and from these we face the expression of CD4 against CD8. Gating strategy CD56+ T cell subsets in healthy controls (C) vs patients with Crohn's disease (D). After selection of lymphocyte gate, we select CD56+ cells based on SSC and CD56+ expression. We differentiated CD3+CD56+αβ (panel C and E) and CD3+CD56+γδ+ (panel D and F) T cell subsets in CD3 vs αβ/γδ dot plot.

No competing interests reported.

SupplementaryTable1.docx
Supplementary Table 1: Primers sequences of IL-7 and IL-7 receptor for qPCR.
SupplementaryTable2.docx.docx
Supplementary Table 2: Antibody used in Western-Blot.
SupplementaryTable3.docx
Supplementary Table 3. Clinical characteristics of Crohn’s disease patients (n=80).
SupplementaryTable4.docx
Supplementary Table 4: Treatment in Crohn's disease patients according to clinical scenarios.

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Decreased gene expression of common γ-chain receptor (CD132) in tissues of Crohn’s disease patients

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Abstract

Background.

Methods

Results

Conclusion

Figures

1. INTRODUCTION

2. METHODS

2.1. Study population

2.2. Tissue sampling of intestinal biopsies

2.3. Cell isolation for analysis of γδ and αβ T cells and apoptosis evaluation

2.3.1. Blood sample analysis

2.3.2. Functional analysis of γδ and αβ T cells

2.3.3. Apoptosis evaluation

2.3.4. IL-7 in peripheral blood.

2.3.5.1. RNA extraction

2.3.5.2. Reverse Transcription-Polymerase Chain Reaction

2.3.6. Analysis of IL-7 and caspase-3 by Western-Blot.

2.4. Statistical Analysis

3. RESULTS

3.2. Relationship between the number and apoptosis of αβ and γδ T cells.

3.3. IL-7 gene expression and IL-7 protein.

3.4. Expression of Caspase 3 in tissues and its relationship with IL-7 and γδ T cells.

3.5. Percentages of αβ and γδ T cells in tissues.

3.6. IL-7 receptor expression: IL-7 receptor α (CD127) and common γ-chain receptor (CD132).

4. DISCUSSION

5. Conclusions

Declarations

References

Supplementary File

Additional Declarations

Supplementary Files

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