Ignite Creation Date:
2024-05-05 @ 11:17 PM
Last Modification Date:
2024-10-26 @ 10:31 AM
Study NCT ID:
NCT01291381
Status:
COMPLETED
Last Update Posted:
2011-02-08
First Post:
2011-01-25
Brief Title:
Distinct Response of CD4CD25Foxp3 and IL-10-secreting Type I T Regulatory Cells to Cluster Specific Immunotherapy in Allergic Rhinitis Children
Sponsor:
Beijing Tongren Hospital
Organization:
Beijing Tongren Hospital
Study Overview
Official Title:
Regulatory T Cells in the Response to SIT in Allergic Rhinitis
Status:
COMPLETED
Status Verified Date:
2009-01
Last Known Status:
None
Delayed Posting:
No
If Stopped, Why?:
Not Stopped
Has Expanded Access:
False
If Expanded Access, NCT#:
N/A
Has Expanded Access, NCT# Status:
N/A
Acronym:
None
Brief Summary:
While allergen specific immunotherapy SIT is highly effective for allergic diseases in children the underlying immunological mechanisms are unclear Regulatory T Treg cells may be crucial in induction of tolerance
Our aim was to investigate the role of CD4CD25Foxp3 T cells and IL-10-secreting type I T regulatory Tr1 cells in the response to one year of cluster SIT to Dermatophagoides pteronyssinus for allergic rhinitis in children
CD4CD25Foxp3regulatory T cells and IL-10-secreting type I T regulatory Tr1 cells were analyzed in children allergic to Dermatophagoides pteronyssinus during one year cluster specific immunotherapy SIT in a prospective and randomized study Peripheral blood mononuclear cells PBMCs were collected from 25 children receiving SIT and 21 receiving pharmacotherapy The frequencies of CD4CD25Foxp3 T cells and allergen-specific IL-10IL-4- IFN-γIL-4- IL-4IFN-γ-CD4 T cells were measured by flow cytometry Production of IL-4 IFN-r and IL-10 in supernatants from allergen-stimulated PBMC culture was measured by ELISA Finally the suppressive effect of CD4CD25highTreg cells from both groups was estimated
Our aim was to investigate the role of CD4CD25Foxp3 T cells and IL-10-secreting type I T regulatory Tr1 cells in the response to one year of cluster SIT to Dermatophagoides pteronyssinus for allergic rhinitis in children
CD4CD25Foxp3regulatory T cells and IL-10-secreting type I T regulatory Tr1 cells were analyzed in children allergic to Dermatophagoides pteronyssinus during one year cluster specific immunotherapy SIT in a prospective and randomized study Peripheral blood mononuclear cells PBMCs were collected from 25 children receiving SIT and 21 receiving pharmacotherapy The frequencies of CD4CD25Foxp3 T cells and allergen-specific IL-10IL-4- IFN-γIL-4- IL-4IFN-γ-CD4 T cells were measured by flow cytometry Production of IL-4 IFN-r and IL-10 in supernatants from allergen-stimulated PBMC culture was measured by ELISA Finally the suppressive effect of CD4CD25highTreg cells from both groups was estimated
Detailed Description:
Patients The design was a one-year randomized open-label parallel group study in which patients were randomized in a 11 ratio to receive SIT with Dermatophagoides pteronyssinus extract Alutard SQ ALK-Abello Hørsholm Denmark or to observe with standard pharmacotherapy control group
The study participants included 50 house dust mite HDM-allergic children aged 9-13 who received SIT with Dermatophagoides pteronyssinus Der p extract for one year These were compared with 21 HDM-allergic children aged 9-14 who served as controls All children were of Han nationality and were recruited from the allergy clinic of Beijing TongRen Hospital They had a history of HDM-induced moderate or severe rhino-conjunctivitis of at least three years duration Each patient also had a positive skin prick test SPT result for Der p ALK-Abello Hørsholm Denmark with a wheal diameter of at least 6 mm and were positive for specific immunoglobulin E IgE to Der p Pharmacia CAP System Pharmacia Diagnostics Uppsala Sweden with a RAST value of at least 07 kUL Children with a history of asthma or atopic dermatitis were excluded The children undergoing immunotherapy were matched to atopic donors for age sex SPT response to Der p total IgE and specific IgE to Der p levels Table 1 The study protocol was approved by our institutional review board for human studies and informed consent was obtained from all subjects
Immunotherapy protocol Children undergoing subcutaneous immunotherapy were given Dermatophagoides pteronyssinus extract Alutard SQ ALK-Abello Hørsholm Denmark according to a cluster protocol which we have described previously15 In the first six weeks children underwent visits for up-dosing receiving two injections one hour apart with an increasing dose every week The highest concentration given in the sixth week had an allergenic activity of 100000 SQml and contained 98 μgml Der p1 After week six the dosing interval was increased to one month and maintained until the end of the first year For patients randomized to non-SIT group persistent rhinitis was managed with pharmacotherapy including intranasal steroids and oral antihistamines Intranasal steroids were kept at the same dose during the study and antihistamines were used as required Blood samples were taken at the baseline and after one year treatment in both groups
Clinical evaluation Symptoms of AR children were assessed by the Total 5 Symptom Score T5SS which includes rhinorrhea sneezing nasal congestion and nasal and ocular pruritus Each symptom was scored on a scale of 0-3 0 none 3 severe Medication use was scored and recorded daily as described previously 16 Arbitrary scores were attributed to the drugs used 075 points for 1 puff of nasal corticosteroids and 1 points for 1 tablet of antihistamine Patients were instructed to use local steroids only plus antihistamines if they did not improve symptoms and to report each administration or variation of the initial drug therapy in the diary Patients were also instructed to stop drugs at least 7 days before blood sampling
Flow cytometry Peripheral blood was obtained by venous puncture and collected into preservative-free heparin Peripheral blood mononuclear cells PBMCs were isolated by means of Ficoll-Plaque Plus density gradient centrifugation Amersham Biosciences NJ USA For intracellular detection of cytokine PBMCs 2 106 cellsmL were stimulated with 10 μgmL Der p1 D pteronyssinus major allergen 1 Indoor Biotechnologies United Kingdom for 24 hours Twenty-five ngmL phorbol 12-myristate 13-acetate PMA 1 μgmL ionomycin and 17 μgmL monensine were added at 37 C for the final four hours of stimulation all from Sigma Missouri USA
For flow cytometry PBMCs were surface stained at room temperature for 20 minutes with anti-CD4-PerCP and anti-CD25-PE or isotype control mouse IgG1-PerCP or IgG1-PE all from Beckman Coulter CA USA For intracellular staining of Foxp3 cells were fixed and permeabilized with Foxp3 fixationpermeabilization buffer then stained with anti-human Foxp3-FITC or isotype control IgG1-FITC all from BioLegend CA USA For the intracellular cytokine analysis Der p1-stimulated PBMCs were surface stained with anti-CD3-APC and anti-CD8-PerCP and fixed with 4 paraformaldehydePBS After being permeabilized using FACS Permeabilizing Solution BD Pharmingen CA USA cells were incubated for 30 minutes at room temperature with anti-IL-4-PE plus anti-IFN-γ-FITC or anti-IL-10-PE plus anti-IL-4-FITC and each antibody was matched with a respective isotype IgG1 as a control all from BD Pharmingen Different T subsets were selected for detailed phenotypic analysis as follows 1 Th1 cells IFN-γIL-4-CD3CD8- T cells 2 Th2 cells IL-4IFN-γ-CD3CD8- T cells and 3 Tr1 cells IL-10IL-4-CD3CD8- T cells For each analysis at least 20000 events were collected Analysis was conducted using a FACSCalibur flow cytometer BD Biosciences NJ USA
ELISA PBMCs were resuspended at 2 106 cellsmL in RPMI-1640 medium and stimulated with 10 μgmL Der p1 D pteronyssinus major allergen 1 Indoor Biotechnologies United Kingdom Cultures were incubated for 6 days at 37 ºC in a humidified incubator containing 5 CO2 Supernatants from allergen-stimulated PBMCs cultures were assayed for the presence of IFN-γ IL-4 and IL-10 by means of ELISA RD Systems Minneapolis USA
Isolation of CD4CD25high Treg cells Heparinized blood was obtained from each of eight recruited SIT-treated children and eight AR controls for cell isolation and functional analysis After non-CD4 cells in the PBMCs were removed by LD column with a biotin-antibody cocktail and antibiotin microbeads CD4 T cells were isolated from the PBMCs by negative selection and CD4CD25 T cells were positively selected from the CD4 T cells Finally CD25- fractions were recovered using a MS-magnetic column using a human CD4CD25 regulatory T cell isolation kit according to the manufacturers instructions Miltenyi Biotec Germany Cells with the highest CD25 expression CD25high were selected through incubation with a limiting quantity of anti-CD25 antibody beads 2 μl of anti-CD25 beads107 cells17 The mean purity of the isolated CD4 was 95 93-97 and 76 72-88 for CD4CD25high T cells being Foxp3-positive
Suppressive capacity of CD4CD25high Treg cells To evaluate the suppressive capacity of CD4CD25high Treg cells cell proliferation and cytokine were analyzed CD4CD25- T cells responders alone or mixed CD4CD25- T cells and CD4CD25high T cells suppressors at a ratio of 21 were cultured in a final volume of 200 µl RPMI-1640 medium with 10 µgml Der p1 Cultures were incubated for 6 days at 37 ºC in a humidified incubator with 5 CO2 Autologous irradiated PBMCs 3000 rad were added at 2 105 as antigen-presenting cells to CD4CD25- T cell cultures In all cases triplicate medium wells were included as negative controls At day six 100 µl of supernatant was removed for Th1 and Th2 cytokine IFN-γ and IL-4 analysis with an ELISA kit RD Systems Minneapolis USA Cell proliferation of CD4CD25- T cells was then assessed using a WST-8 modified tetrazolium salt cell proliferation kit Cell Counting Kit-8 Dojin Japan according to the manufacturers protocols The proliferation result was expressed as absorbance at 450nm of the culture medium
Statistics Statistical significance between groups was analyzed using a Mann-Whitney test The significance of intra-group pre- and post-therapy changes were determined using a nonparametric Wilcoxon matched pairs test The correlation coefficient r was generated using the Pearson correlation All analyses were performed using SPSS version 130 statistical software A 5 significance level was used α 005 power 1- β equal to 90 All tests were 2-tailed and P values of less than 005 were considered statistically significant
The study participants included 50 house dust mite HDM-allergic children aged 9-13 who received SIT with Dermatophagoides pteronyssinus Der p extract for one year These were compared with 21 HDM-allergic children aged 9-14 who served as controls All children were of Han nationality and were recruited from the allergy clinic of Beijing TongRen Hospital They had a history of HDM-induced moderate or severe rhino-conjunctivitis of at least three years duration Each patient also had a positive skin prick test SPT result for Der p ALK-Abello Hørsholm Denmark with a wheal diameter of at least 6 mm and were positive for specific immunoglobulin E IgE to Der p Pharmacia CAP System Pharmacia Diagnostics Uppsala Sweden with a RAST value of at least 07 kUL Children with a history of asthma or atopic dermatitis were excluded The children undergoing immunotherapy were matched to atopic donors for age sex SPT response to Der p total IgE and specific IgE to Der p levels Table 1 The study protocol was approved by our institutional review board for human studies and informed consent was obtained from all subjects
Immunotherapy protocol Children undergoing subcutaneous immunotherapy were given Dermatophagoides pteronyssinus extract Alutard SQ ALK-Abello Hørsholm Denmark according to a cluster protocol which we have described previously15 In the first six weeks children underwent visits for up-dosing receiving two injections one hour apart with an increasing dose every week The highest concentration given in the sixth week had an allergenic activity of 100000 SQml and contained 98 μgml Der p1 After week six the dosing interval was increased to one month and maintained until the end of the first year For patients randomized to non-SIT group persistent rhinitis was managed with pharmacotherapy including intranasal steroids and oral antihistamines Intranasal steroids were kept at the same dose during the study and antihistamines were used as required Blood samples were taken at the baseline and after one year treatment in both groups
Clinical evaluation Symptoms of AR children were assessed by the Total 5 Symptom Score T5SS which includes rhinorrhea sneezing nasal congestion and nasal and ocular pruritus Each symptom was scored on a scale of 0-3 0 none 3 severe Medication use was scored and recorded daily as described previously 16 Arbitrary scores were attributed to the drugs used 075 points for 1 puff of nasal corticosteroids and 1 points for 1 tablet of antihistamine Patients were instructed to use local steroids only plus antihistamines if they did not improve symptoms and to report each administration or variation of the initial drug therapy in the diary Patients were also instructed to stop drugs at least 7 days before blood sampling
Flow cytometry Peripheral blood was obtained by venous puncture and collected into preservative-free heparin Peripheral blood mononuclear cells PBMCs were isolated by means of Ficoll-Plaque Plus density gradient centrifugation Amersham Biosciences NJ USA For intracellular detection of cytokine PBMCs 2 106 cellsmL were stimulated with 10 μgmL Der p1 D pteronyssinus major allergen 1 Indoor Biotechnologies United Kingdom for 24 hours Twenty-five ngmL phorbol 12-myristate 13-acetate PMA 1 μgmL ionomycin and 17 μgmL monensine were added at 37 C for the final four hours of stimulation all from Sigma Missouri USA
For flow cytometry PBMCs were surface stained at room temperature for 20 minutes with anti-CD4-PerCP and anti-CD25-PE or isotype control mouse IgG1-PerCP or IgG1-PE all from Beckman Coulter CA USA For intracellular staining of Foxp3 cells were fixed and permeabilized with Foxp3 fixationpermeabilization buffer then stained with anti-human Foxp3-FITC or isotype control IgG1-FITC all from BioLegend CA USA For the intracellular cytokine analysis Der p1-stimulated PBMCs were surface stained with anti-CD3-APC and anti-CD8-PerCP and fixed with 4 paraformaldehydePBS After being permeabilized using FACS Permeabilizing Solution BD Pharmingen CA USA cells were incubated for 30 minutes at room temperature with anti-IL-4-PE plus anti-IFN-γ-FITC or anti-IL-10-PE plus anti-IL-4-FITC and each antibody was matched with a respective isotype IgG1 as a control all from BD Pharmingen Different T subsets were selected for detailed phenotypic analysis as follows 1 Th1 cells IFN-γIL-4-CD3CD8- T cells 2 Th2 cells IL-4IFN-γ-CD3CD8- T cells and 3 Tr1 cells IL-10IL-4-CD3CD8- T cells For each analysis at least 20000 events were collected Analysis was conducted using a FACSCalibur flow cytometer BD Biosciences NJ USA
ELISA PBMCs were resuspended at 2 106 cellsmL in RPMI-1640 medium and stimulated with 10 μgmL Der p1 D pteronyssinus major allergen 1 Indoor Biotechnologies United Kingdom Cultures were incubated for 6 days at 37 ºC in a humidified incubator containing 5 CO2 Supernatants from allergen-stimulated PBMCs cultures were assayed for the presence of IFN-γ IL-4 and IL-10 by means of ELISA RD Systems Minneapolis USA
Isolation of CD4CD25high Treg cells Heparinized blood was obtained from each of eight recruited SIT-treated children and eight AR controls for cell isolation and functional analysis After non-CD4 cells in the PBMCs were removed by LD column with a biotin-antibody cocktail and antibiotin microbeads CD4 T cells were isolated from the PBMCs by negative selection and CD4CD25 T cells were positively selected from the CD4 T cells Finally CD25- fractions were recovered using a MS-magnetic column using a human CD4CD25 regulatory T cell isolation kit according to the manufacturers instructions Miltenyi Biotec Germany Cells with the highest CD25 expression CD25high were selected through incubation with a limiting quantity of anti-CD25 antibody beads 2 μl of anti-CD25 beads107 cells17 The mean purity of the isolated CD4 was 95 93-97 and 76 72-88 for CD4CD25high T cells being Foxp3-positive
Suppressive capacity of CD4CD25high Treg cells To evaluate the suppressive capacity of CD4CD25high Treg cells cell proliferation and cytokine were analyzed CD4CD25- T cells responders alone or mixed CD4CD25- T cells and CD4CD25high T cells suppressors at a ratio of 21 were cultured in a final volume of 200 µl RPMI-1640 medium with 10 µgml Der p1 Cultures were incubated for 6 days at 37 ºC in a humidified incubator with 5 CO2 Autologous irradiated PBMCs 3000 rad were added at 2 105 as antigen-presenting cells to CD4CD25- T cell cultures In all cases triplicate medium wells were included as negative controls At day six 100 µl of supernatant was removed for Th1 and Th2 cytokine IFN-γ and IL-4 analysis with an ELISA kit RD Systems Minneapolis USA Cell proliferation of CD4CD25- T cells was then assessed using a WST-8 modified tetrazolium salt cell proliferation kit Cell Counting Kit-8 Dojin Japan according to the manufacturers protocols The proliferation result was expressed as absorbance at 450nm of the culture medium
Statistics Statistical significance between groups was analyzed using a Mann-Whitney test The significance of intra-group pre- and post-therapy changes were determined using a nonparametric Wilcoxon matched pairs test The correlation coefficient r was generated using the Pearson correlation All analyses were performed using SPSS version 130 statistical software A 5 significance level was used α 005 power 1- β equal to 90 All tests were 2-tailed and P values of less than 005 were considered statistically significant
Study Oversight
Has Oversight DMC:
None
Is a FDA Regulated Drug?:
None
Is a FDA Regulated Device?:
None
Is an Unapproved Device?:
None
Is a PPSD?:
None
Is a US Export?:
None
Is an FDA AA801 Violation?:
None
Secondary IDs
| Secondary ID | Type | Domain | Link |
|---|---|---|---|
| 81025007 | OTHER_GRANT | the National Science Fund | None |