Immune Perturbation In Patients With Tgfbeta Pathway Defects
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857 Immune Perturbation In Patients With Tgfbeta Pathway Defects Dr. Dat Q. Tran, MD 1 , Mrs. Ellen Regalado 2 , Dr. Dianna Milewicz 2 ; 1 University of Texas Medical School at Houston, Houston, TX, 2 UTHealth. RATIONALE: Knowledge of TGFb regulation of the immune system stems predominantly from animal and in vitro studies. Heterozygous mutations in TGFbR1, TGFbR2 and SMAD3 have been associated with familial thoracic aortic aneurysms and aortic dissections (TAAD). These patients offer an opportunity to study their immune development when the TGFbeta pathway is defective. METHODS: Flow cytometry was used to analyze PBMC from patients with TAAD (n59) and age-matched healthy controls (HC, n58). Th1 and Th17 were determined with intracellular cytokine staining for IFNg and IL17A. Foxp3+ Tregs were detected with anti-Foxp3 (259D). CD19+ were analyzed for naive (IgD+CD27-), unswitched (IgD+CD27+) and switched memory (IgD-CD27+). Plasmacytoid (CD303+pDC) and myeloid (CD1c+mDC) were defined within lineage-1 negative population. RESULTS: %CD3-CD16+NK, CD3+CD16+NKT, CD4+, CD8+ and CD4+CD45RA+ in TAAD were similar to HC. Average %CD19+ (20.8vs7.3, p50.006) and naive B cells (81.3vs66.6, p50.004) were higher in TAAD. The unswitched were similar but the switched B cells were lower (8.6vs15.5, p50.01). While the %Tregs were similar, there was a remarkable reduction (1/2-3 folds) in Foxp3 concentration based on median fluorescence intensity of Foxp3 in TAAD. There was a significant reduction of %Th17 (0.14vs0.61, p50.01), while the Th1 were similar. % pDC (9.4vs24.1, p50.009) and %mDC (11.4vs17.9, p50.01) were also lower in TAAD. CONCLUSIONS: These results demonstrate for the first time in humans of the involvement of TGFbeta signaling in B cells, DCs, Th17 and Treg development. Further studies and monitoring of the clinical effects of these immunological perturbations in these TAAD patients are needed to appreciate the impact of their underlying disease. 858 Chemokine Receptors On Regulatory T Cell Surface, Surrogate Markers For Intracellular Th1 and Th2 Cytokines Mr. Satoru Watanabe 1,2 , Dr. Yoshiyuki Yamada 1 , Prof. Hirokazu Murakami 2 ; 1 Gunma Children’s Medical Center, Shibukawa, Gunma, Japan, 2 Gunma University Faculty of Medicine School of Health Science, Maebashi, Gunma, Japan. RATIONALE: To identify T-helper-1 (Th1) and T-helper-2 (Th2) cells, cytoplasmic cytokine staining is widely used. However, this method is complicated and time-consuming. Chemokine receptors and CRTH2 have been reported to function as surrogate markers for cytoplasmic Th1 and Th2 cytokines. Regulatory T cells (Tregs) may affect such surrogate marker analysis, as they share several chemokine receptors with Th1 and Th2 cells. The aim of this study was to determine better surrogate markers for cytoplasmic Th1 and Th2 cytokine staining. METHODS: With institutional review board approval, ten healthy volun- teers (5 males) were included. Surface and cytoplasmic markers of CD4 + lymphocytes were analyzed by flow cytometry. Th1, Th2, and Tregs were determined as being IFN-g + , IL-4 + /IL-13 + , and CD4 + /CD25 + / FoxP3 + cells, respectively. RESULTS: The percentage of Th1 cells significantly correlated with that of CXCR3 + and CCR5 + cells (r s 5 0.66 and 0.82, respectively). There was a significant correlation between the percentage of Th2 and CRTH2 + cells (r s 5 0.93), No correlation was observed between the percentage of Th2 and CCR3 + , CCR4 + , CCR7 + , or CCR8 + cells. In Th1-related chemokine recep- tors, a higher frequency of CXCR3 + than of CCR5 + Tregs was observed, whereas, among Th2-related chemokine receptors, the percentage of CCR4 + and CCR7 + Tregs was higher than those of CCR8 + and CRTH2 + . CONCLUSIONS: Surface CCR5 and CRTH2 can substitute for cyto- plasmic IFN-g and IL-4/IL-13 staining, respectively. In contrast, correla- tions between intracellular Th1 or Th2 cytokines and other surface chemokine receptors may be impaired due to their higher frequency on the surface of Tregs. 859 IL-4 Signaling Attenuates gd T Cell IL-17A Protein Expression Melissa T. Harintho, BS 1 , Dr. Dawn C. Newcomb Baker, PhD 2 , Jacqueline-Yvonne Cephus, BS 2 , Kasia Goleniewska 3 , Dr. R. Stokes Peebles, Jr, MD, FAAAAI 2 ; 1 Department of Pathology, Microbiology, and Immunology, Vanderbilt University School of Medi- cine, Nashville, TN, 2 Division of Allergy, Pulmonary and Critical Care Medicine, Vanderbilt University School of Medicine, Nashville, TN, 3 Al- lergy, Pulmonary, and Critical Care Medicine, Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN. RATIONALE: gd T cells reside at the interface of epithelial-environ- mental interfaces such as the respiratory and gastrointestinal tracts. gd T cells are potent producers of IL-17A which is important in antibacterial and antifungal immunity. Patients with asthma are at increased risk for invasive bacterial infections and bacterial pneumonia. There is an increase in CD4+ Th2 cells in the airway of asthma patients and Th2 cells produce IL-4. IL-4 negatively regulates IL-17A expression from CD4+ Th17 cells; however, the effect of IL-4 on IL-17A expression by gd T cells is unknown. We therefore hypothesized that IL-4 attenuates gd T cell IL-17A protein expression. METHODS: gd T cells were purified from the spleens of BALB/c mice. To induce IL-17 protein expression, gd T cells were cultured with IL-1b and IL-23 for 3 days in the presence or absence of IL-4 (10 ng/mL). IL- 17A, IL-17F, and IL-22 protein expression was determined by ELISA. RESULTS: We found that IL-4 significantly decreased IL-17A protein expression in gd T cells (35.96 1.6 ng/ml to 10.56 0.4 ng/mL, p<0.05, n55). We also looked at IL-17F and IL-22 protein expression and found that IL-4 significantly decreased protein expression of these IL-17 cytokine family members in gd T cells (21.96 2.5 ng/mL to 10.36 0.5 ng/mL and 10.46 0.2 ng/mL to 2.66 0.03 ng/mL, p<0.05, n55). CONCLUSIONS: IL-4 inhibition of IL-17 cytokine family protein expression by gd T cells is a possible explanation for why asthmatic patients are at higher risk for bacterial pneumonia. 860 Influence Of Dietary Fiber On Cellular Immunity In Experimental Vitamin Deficiency Dr. Roman Khanferyan, MD, PhD 1 , Dr. E. N. Trushina 2 , O. K. Musta- fina 3 , V. M. Kodentzova 2 , Prof. Lawrence M. DuBuske, MD, FAAAAI 4 ; 1 Insitute of Nutrition, Russian Academy of medical Sciences, Moscow, Russia, 2 Insitute of Nutrition Russian Academy of Sciences, Russia, 3 Institute of Nutrition Russian Academy of Sciences, Russia, 4 George Washington University School of Medicine, DC. RATIONALE: Vitamin deficiency leads to immunodeficiency which can be corrected by enrichment of the diet by vitamins. Foods enriched with food fiber (FF) may influence this process. This study assesses the influence of FF of wheat bran on cellular immunity in the a rat experimental model of poly-vitamin deficiency. METHODS: 48 Inbred Vistar rats with an initial body weight of (58.160.5 g) were divided into 6 experimental diet groups each with specific vitamin deficiency and FF enrichment. The control group was on standard diet with 100% of vitamin enrichment during all 35 days of experiment. The expression of CD45+, CD3+, CD4+, CD8+, CD161a+ receptors of rat blood mononuclear cells was assessed by direct immunofluorescence using a panel of monoclonal antibodies (IO Test, Beckman Coulter, USA). RESULTS: Enrichment of the control diet (100% vitamins) with FF led to decrease in the number of CD3+ lymphocytes but also increased numbers of cytotoxic T-lymphocytes (CD3+ CD8+). Marked suppression of cellular immunity with decreases in the number of CD3+ cells and NK-cells was seen in the group having low levels (20%) of vitamins and maximal consumable FF levels. CONCLUSIONS: Diets high in fiber may lead to suppression of cellular immunity in situations where vitamin levels are low, suggesting that vitamin supplementation may have to be considered when very high fiber diets are employed to avoid immune suppression. If high fiber diets are to be employed, vitamin supplementation may prevent adverse impact on immune responses. J ALLERGY CLIN IMMUNOL FEBRUARY 2014 AB248 Abstracts TUESDAY
Transcript of Immune Perturbation In Patients With Tgfbeta Pathway Defects
J ALLERGY CLIN IMMUNOL
FEBRUARY 2014
AB248 Abstracts
TUESDAY
857 Immune Perturbation In Patients With Tgfbeta PathwayDefects
Dr. Dat Q. Tran, MD1, Mrs. Ellen Regalado2, Dr. Dianna Milewicz2;1University of Texas Medical School at Houston, Houston, TX,2UTHealth.
RATIONALE: Knowledge of TGFb regulation of the immune system
stems predominantly from animal and in vitro studies. Heterozygous
mutations in TGFbR1, TGFbR2 and SMAD3 have been associated with
familial thoracic aortic aneurysms and aortic dissections (TAAD). These
patients offer an opportunity to study their immune development when the
TGFbeta pathway is defective.
METHODS: Flow cytometry was used to analyze PBMC from patients
with TAAD (n59) and age-matched healthy controls (HC, n58). Th1 and
Th17 were determined with intracellular cytokine staining for IFNg and
IL17A. Foxp3+ Tregs were detected with anti-Foxp3 (259D). CD19+ were
analyzed for naive (IgD+CD27-), unswitched (IgD+CD27+) and switched
memory (IgD-CD27+). Plasmacytoid (CD303+pDC) and myeloid
(CD1c+mDC) were defined within lineage-1 negative population.
RESULTS: %CD3-CD16+NK, CD3+CD16+NKT, CD4+, CD8+ and
CD4+CD45RA+ in TAAD were similar to HC. Average %CD19+
(20.8vs7.3, p50.006) and naive B cells (81.3vs66.6, p50.004) were
higher in TAAD. The unswitched were similar but the switched B cells
were lower (8.6vs15.5, p50.01).While the%Tregs were similar, therewas
a remarkable reduction (1/2-3 folds) in Foxp3 concentration based on
median fluorescence intensity of Foxp3 in TAAD. There was a significant
reduction of %Th17 (0.14vs0.61, p50.01), while the Th1 were similar. %
pDC (9.4vs24.1, p50.009) and %mDC (11.4vs17.9, p50.01) were also
lower in TAAD.
CONCLUSIONS: These results demonstrate for the first time in humans
of the involvement of TGFbeta signaling in B cells, DCs, Th17 and Treg
development. Further studies andmonitoring of the clinical effects of these
immunological perturbations in these TAAD patients are needed to
appreciate the impact of their underlying disease.
858 Chemokine Receptors On Regulatory T Cell Surface,Surrogate Markers For Intracellular Th1 and Th2 Cytokines
Mr. Satoru Watanabe1,2, Dr. Yoshiyuki Yamada1, Prof. Hirokazu
Murakami2; 1Gunma Children’s Medical Center, Shibukawa, Gunma,
Japan, 2Gunma University Faculty of Medicine School of Health Science,
Maebashi, Gunma, Japan.
RATIONALE: To identify T-helper-1 (Th1) and T-helper-2 (Th2) cells,
cytoplasmic cytokine staining is widely used. However, this method is
complicated and time-consuming. Chemokine receptors and CRTH2 have
been reported to function as surrogate markers for cytoplasmic Th1 and
Th2 cytokines. Regulatory T cells (Tregs) may affect such surrogate
marker analysis, as they share several chemokine receptors with Th1 and
Th2 cells. The aim of this study was to determine better surrogate markers
for cytoplasmic Th1 and Th2 cytokine staining.
METHODS: With institutional review board approval, ten healthy volun-
teers (5 males) were included. Surface and cytoplasmic markers of CD4+
lymphocytes were analyzed by flow cytometry. Th1, Th2, and Tregs
were determined as being IFN-g+, IL-4+/IL-13+, and CD4+/CD25+/
FoxP3+ cells, respectively.
RESULTS: The percentage of Th1 cells significantly correlated with that
of CXCR3+ andCCR5+ cells (rs5 0.66 and 0.82, respectively). Therewas a
significant correlation between the percentage of Th2 and CRTH2+ cells (rs5 0.93), No correlation was observed between the percentage of Th2 and
CCR3+, CCR4+, CCR7+, or CCR8+ cells. In Th1-related chemokine recep-
tors, a higher frequency of CXCR3+ than of CCR5+ Tregs was observed,
whereas, among Th2-related chemokine receptors, the percentage of
CCR4+ and CCR7+ Tregs was higher than those of CCR8+ and CRTH2+.
CONCLUSIONS: Surface CCR5 and CRTH2 can substitute for cyto-
plasmic IFN-g and IL-4/IL-13 staining, respectively. In contrast, correla-
tions between intracellular Th1 or Th2 cytokines and other surface
chemokine receptors may be impaired due to their higher frequency on
the surface of Tregs.
859 IL-4 Signaling Attenuates gd T Cell IL-17A Protein ExpressionMelissa T. Harintho, BS1, Dr. Dawn C. Newcomb
Baker, PhD2, Jacqueline-Yvonne Cephus, BS2, Kasia Goleniewska3,
Dr. R. Stokes Peebles, Jr, MD, FAAAAI2; 1Department of Pathology,
Microbiology, and Immunology, Vanderbilt University School of Medi-
cine, Nashville, TN, 2Division of Allergy, Pulmonary and Critical Care
Medicine, Vanderbilt University School of Medicine, Nashville, TN, 3Al-
lergy, Pulmonary, and Critical Care Medicine, Department of Medicine,
Vanderbilt University School of Medicine, Nashville, TN.
RATIONALE: gd T cells reside at the interface of epithelial-environ-
mental interfaces such as the respiratory and gastrointestinal tracts. gd T
cells are potent producers of IL-17Awhich is important in antibacterial and
antifungal immunity. Patients with asthma are at increased risk for invasive
bacterial infections and bacterial pneumonia. There is an increase in CD4+
Th2 cells in the airway of asthma patients and Th2 cells produce IL-4. IL-4
negatively regulates IL-17A expression from CD4+ Th17 cells; however,
the effect of IL-4 on IL-17A expression by gd T cells is unknown. We
therefore hypothesized that IL-4 attenuates gd T cell IL-17A protein
expression.
METHODS: gd T cells were purified from the spleens of BALB/c mice.
To induce IL-17 protein expression, gd T cells were cultured with IL-1b
and IL-23 for 3 days in the presence or absence of IL-4 (10 ng/mL). IL-
17A, IL-17F, and IL-22 protein expression was determined by ELISA.
RESULTS: We found that IL-4 significantly decreased IL-17A protein
expression in gd T cells (35.96 1.6 ng/ml to 10.56 0.4 ng/mL, p<0.05,
n55). We also looked at IL-17F and IL-22 protein expression and found
that IL-4 significantly decreased protein expression of these IL-17 cytokine
family members in gd T cells (21.96 2.5 ng/mL to 10.36 0.5 ng/mL and
10.46 0.2 ng/mL to 2.66 0.03 ng/mL, p<0.05, n55).
CONCLUSIONS: IL-4 inhibition of IL-17 cytokine family protein
expression by gd T cells is a possible explanation for why asthmatic
patients are at higher risk for bacterial pneumonia.
860 Influence Of Dietary Fiber On Cellular Immunity InExperimental Vitamin Deficiency
Dr. Roman Khanferyan, MD, PhD1, Dr. E. N. Trushina2, O. K. Musta-
fina3, V. M. Kodentzova2, Prof. Lawrence M. DuBuske, MD, FAAAAI4;1Insitute of Nutrition, Russian Academy of medical Sciences, Moscow,
Russia, 2Insitute of Nutrition Russian Academy of Sciences, Russia,3Institute of Nutrition Russian Academy of Sciences, Russia, 4George
Washington University School of Medicine, DC.
RATIONALE: Vitamin deficiency leads to immunodeficiency which can
be corrected by enrichment of the diet by vitamins. Foods enriched with
food fiber (FF) may influence this process. This study assesses the
influence of FF ofwheat bran on cellular immunity in the a rat experimental
model of poly-vitamin deficiency.
METHODS: 48 Inbred Vistar rats with an initial body weight of
(58.160.5 g) were divided into 6 experimental diet groups each with
specific vitamin deficiency and FF enrichment. The control group was on
standard diet with 100% of vitamin enrichment during all 35 days of
experiment. The expression of CD45+, CD3+, CD4+, CD8+, CD161a+
receptors of rat blood mononuclear cells was assessed by direct
immunofluorescence using a panel of monoclonal antibodies (IO Test,
Beckman Coulter, USA).
RESULTS: Enrichment of the control diet (100% vitamins) with FF led to
decrease in the number of CD3+ lymphocytes but also increased numbers
of cytotoxic T-lymphocytes (CD3+CD8+).Marked suppression of cellular
immunity with decreases in the number of CD3+ cells and NK-cells was
seen in the group having low levels (20%) of vitamins and maximal
consumable FF levels.
CONCLUSIONS: Diets high in fiber may lead to suppression of cellular
immunity in situations where vitamin levels are low, suggesting that
vitamin supplementation may have to be considered when very high fiber
diets are employed to avoid immune suppression. If high fiber diets are to
be employed, vitamin supplementation may prevent adverse impact on
immune responses.
