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Tolerance and immunity after sequential lung and bone marrow transplantation from an unrelated cadaveric donor  Paul Szabolcs, MD, Rebecca H. Buckley,

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Presentation on theme: "Tolerance and immunity after sequential lung and bone marrow transplantation from an unrelated cadaveric donor  Paul Szabolcs, MD, Rebecca H. Buckley,"— Presentation transcript:

1 Tolerance and immunity after sequential lung and bone marrow transplantation from an unrelated cadaveric donor  Paul Szabolcs, MD, Rebecca H. Buckley, MD, Robert Duane Davis, MD, Jerelyn Moffet, PA, Judith Voynow, MD, Jeyaraj Antony, PhD, Xiaohua Chen, PhD, Gregory D. Sempowski, PhD, David W. Zaas, MD  Journal of Allergy and Clinical Immunology  Volume 135, Issue 2, Pages e3 (February 2015) DOI: /j.jaci Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 A, T-cell immunity as assessed by mitogen responses on a chronological scale. The y-axis shows counts per minute (CPM) following3 H-thymidine incorporation and measured as previously described.6 PBMCs were stimulated with PHA, pokeweed mitogen (PWM), concanavalin A (ConA), or medium alone as negative control. B, Lymphocyte reconstitution. Absolute numbers of lymphocyte subsets per μL/mm3 were measured as previously described7 from whole blood. C, Tempo of thymopoiesis is depicted by the absolute number of CD4+ T cells over time that display the combinatorial markers of “naive” T-cell immunophenotype (CD45RA/CD62L coexpression). D, Pulmonary function tests before and after BOLT. FEV1 and forced expiratory flow rates at 25% to 75% of FVC (FEF25-75) as measured by standard spirometry. Journal of Allergy and Clinical Immunology  , e3DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 Proliferative responses and cytokine secretion of donor T cells. Negatively selected (Easysep, Stem Cell Technologies, Vancouver, Calif) >95% pure CD3+ T cells (1 × 10−5 cells/well) isolated from the recipient (100% donor) 1 year after BMT (in vivo tacrolimus levels ∼3-4 ng/dL range) were cocultured for 5 days with 1 × 10−4 cells/well of EBV/LCL from the indicated sources. To analyze restriction elements in alloreactivity, EBV/LCLs were opsonized at 2 μg/mL with MHC class-I or class-II–blocking antibodies (clones W632 and L243, Bio-Legend Leaf, San Diego, Calif) before coculture. The impact of regulatory T-cell removal was assayed by depleting CD25+ cells among responders with IL-2 immunotoxin as previously described.7 Lung/marrow donor-derived EBV/LCL was not available to be tested as antigen-presenting cells. Culture supernatant was collected and cryopreserved for batched cytokine analysis by luminex assay on a Bio-Plex 200 system (Bio-Rad, Hercules, Calif) (see Table E2). Immediately thereafter, T-cell proliferation was quantitated (counts per minute) by3 H-thymidine incorporation7 and depicted on the y-axis. Significance was tested as indicated by paired 2-tailed Student t test. Journal of Allergy and Clinical Immunology  , e3DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig E1 A and B, T-cell receptor (TCR) Vβ repertoire. Before BMT there was a severely restricted and oligoclonal pattern (A) that is in stark contrast to the polyclonal TCR diversity displayed 18 months post-BMT shown on panel B reflecting robust thymopoiesis. The single monoclonal peak of Jurkat cell line in the Vβ8 family serves as internal control. TCRVβ spectratyping was performed as previously published.8,9 Journal of Allergy and Clinical Immunology  , e3DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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