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Revertant T lymphocytes in a patient with Wiskott-Aldrich syndrome: Analysis of function and distribution in lymphoid organs  Sara Trifari, PhD, Samantha.

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Presentation on theme: "Revertant T lymphocytes in a patient with Wiskott-Aldrich syndrome: Analysis of function and distribution in lymphoid organs  Sara Trifari, PhD, Samantha."— Presentation transcript:

1 Revertant T lymphocytes in a patient with Wiskott-Aldrich syndrome: Analysis of function and distribution in lymphoid organs  Sara Trifari, PhD, Samantha Scaramuzza, PhD, Marco Catucci, MS, Maurilio Ponzoni, MD, Luca Mollica, PhD, Robert Chiesa, MD, Federica Cattaneo, MD, Fanny Lafouresse, MS, Ronan Calvez, PhD, William Vermi, MD, Daniela Medicina, MS, Maria Carmina Castiello, MS, Francesco Marangoni, PhD, Marita Bosticardo, PhD, Claudio Doglioni, MD, Maurizio Caniglia, MD, Alessandro Aiuti, MD, Anna Villa, MD, Maria-Grazia Roncarolo, MD, Loïc Dupré, PhD  Journal of Allergy and Clinical Immunology  Volume 125, Issue 2, Pages e8 (February 2010) DOI: /j.jaci Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 Characterization of revertant WASP expression caused by secondary site mutation. A, Flow-cytometry analysis of WASP expression in patient W4 (age 25 years) and HD PBMCs, in combination with CD3, CD19, CD16, and CD14 stainings. B, Nucleotide and aa sequences of W4 germ-line (W4) and secondary (W4R) mutations. Flow-cytometry (C) and Western blot (D) analysis of WASP expression in CD4+ T-cell lines from 1 HD and patients W4, W2, and W6. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 WASP subcellular localization. HD, patient W4, and patient W2 CD4+ T cells stimulated with CXCL12 (A) or superantigen (SAg)-pulsed APC (B) and costained for WASP, F-actin, α-tubulin, or CD2. Differential interference contrast (DIC) is shown in parallel. Empty arrows indicate WASP-negative cells. Filled arrows indicate CXCL12-induced lamellipodial protrusion enriched in WASP and F-actin (A) or immunologic synapses enriched in WASP, F-actin, and CD2 (B). Results representative of 3 independent experiments. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig 3 Expansion and TCR diversity of revertant T cells. A, Flow-cytometry analysis of WASP expression in patient W4 peripheral blood CD4+ and CD8+ T cells along time (age years) and in a T-cell line stimulated and expanded in vitro (from PBMCs obtained at age 25 years). B, Size distribution of the major TCRβ rearrangements (Vβ-Jβ1/2.2/2.6/2.7 ; Vβ-Jβ2.1/2.3/2.4/2.5 ; Dβ-Jβ, as indicated by green and blue) analyzed on peripheral blood T cells from a HD and on sorted WASP+ and WASP- peripheral blood T cells from patient W4. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig 4 Proliferation and cytokine production. A, Anti-CD3/CD28 mAb–driven proliferation in counts per minute (cpm) of T cell lines from HDs and patients W4, W2, and W10. B, Anti-CD3/CD28 mAb–driven production of IL-2 and IFN-γ in peripheral blood CD4+ T cells from a HD and patients W4 and W31. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig 5 Analysis of T-cell clones. A, Flow-cytometry analysis of WASP expression in representative CD4+ T-cell clones with corresponding sequence (1 clone from a HD; 1 WASP- and 1 WASP+ clone from patient W4, age 23 years). B, Anti-CD3/CD28 mAb–driven proliferation in counts per minute (cpm) of CD4+ T-cell clones from 1 HD and patient W4 (divided according to WASP expression). The average of 3 different experiments in shown (∗P < .05; ∗∗P < .01; n.s., not significant). Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 Fig 6 Lymphoid tissue distribution of revertant WASP expression. Histologic analysis of WASP expression in lymphoid tissues from patient W4, including spleen (A and B) collected at age 10 years, and lymph node (C-E) and bone marrow (F and G) collected at age 23 years. Stainings for WASP (A, C, E, F, and G) and CD3 (B and D) on adjacent sections or Pax5 as costaining (E). Pictures taken with a ×10 (G) or ×20 (A-F) objective show representative areas of each tissue. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

8 Revertant WASP expression in CD4+ and CD8+ T-cell subsets from patient W4. PBMCs were purified from a blood sample collected when the patient was 24 years of age. Expression of CD8 and WASP (upper panels) or CD45RA and WASP (lower panels) was analyzed by flow cytometry in T cells gated on the basis of CD3 expression. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

9 Analysis of WASP expression in megakaryocytes
Analysis of WASP expression in megakaryocytes. Confocal microscopy analysis of megakaryocytes differentiated from CD34+ cells of a HD and the patient W4. Cells were colabelled with 4'-6-diamidino-2-phenylindole, dihydrochloride (DAPI; blue) to identify nuclei, the specific megakaryocyte marker CD41 (yellow), and WASP (red). Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

10 Structure modeling of the protein fraction surrounding the reversion event. A, Schematic representation of the average structure of MD simulations for the WT and for the W4R peptide. The residues are numbered according to internal enumeration of the model peptides (163→1). B, Secondary structure content of WT and W4R peptides computed during the picosecond (ps) MD simulations using the DSSP algorithm as implemented in the GROningen Machine for Chemical Simulation suite. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

11 Physiological lymphoid tissue distribution of WASP expression
Physiological lymphoid tissue distribution of WASP expression. Histologic analysis of WASP expression in normal human lymphoid tissues, including spleen (A), lymph node (B), and bone marrow (C). WASP-specific staining appears in brown, whereas hematoxylin counterstaining appears in blue. Pictures taken with a ×20 objective show representative areas of each tissue. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

12 Revertant WASP expression in CD4+CD161+ and CD4+CD161- T cells
Revertant WASP expression in CD4+CD161+ and CD4+CD161- T cells. PBMCs from a healthy donor and from patient W4 (age 27 years) were analyzed by flow cytometry for the expression of CD4, CD161, and WASP. Upper panels show the distribution of CD161 expression in CD4+ gated lymphocytes, and lower panels show the expression of WASP in the CD161-(red) and CD161+(blue) subpopulations. Max, Maximum. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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