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GEMS Journal Club 8/29/12

GEMS Journal Club 8/29/12. T cell polarization: Eyerich et al. NEJM 7/21/11. T cell differentiation : molecular and functional characteristics. Zhu and Paul. Blood 2008. Psoriasis and eczema : classic examples of Th1/17 and Th2 autoimmune skin diseases respectively.

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GEMS Journal Club 8/29/12

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  1. GEMS Journal Club 8/29/12 T cell polarization: Eyerich et al. NEJM 7/21/11

  2. T cell differentiation : molecular and functional characteristics Zhu and Paul. Blood 2008

  3. Psoriasis and eczema : classic examples of Th1/17 and Th2 autoimmune skin diseases respectively • Skin diseases associated with other disease manifestations • Psoriasis and psoriatic arthritis • Eczema and asthma • Mouse models are unsatisfactory… • Are these diseases driven by a specific antigen? • Appeal of this report : • Rare simultaneous occurance of antagonistic diseases of skin • translational study of real human disease with some mechanistic insights • Atypical paper for journal club : • lots of clinical content, not conclusive • But : very nice study of T cell-driven immunopathology in the skin

  4. Methods in the paper • T cell lines derived from skin biopsy • Co-culture assay • intracellular cytokine staining • ELISA for cytokine production • CFSE dilution and H3 incorporation • CD69 upregulation

  5. Methods in the paper • T cell lines derived from skin biopsy • Co-culture assay • intracellular cytokine staining • ELISA for cytokine production • CFSE dilution and H3 incorporation • CD69 upregulation

  6. Methods in the paper For comparison: Typical strategy for stimulating and studying naïve murine T cells: • 3 days : aCD3 + aCD28 + IL-2, • then 2 days IL-2 : • effector T cells generated, • can assess cytokine production • 3 days aCD3+/- aCD28 : • CFSE or thymidine incorporation to assess proliferation • o/n aCD3 stimulation : • assess surface CD69 expression to assess early activation • direct ex vivo isolation and restimulation of effector T cells to detect polarization • In each case, can stimulate with antigen + APC, with aCD3 Ab, or with PMA/io • Latter two methods give polyclonal stimulation (all TCRs)

  7. Methods in the paper • T cell lines derived from skin biopsy biopsy specimens were cultured in complete media x 2 days • D2 Migrated cells collected and grown in complete media • D6 clone by limiting dilution (0.6 cells/well in 96-well U-bottom microplates) (+media, IL-2, PHA, feeder PBMCs) • Fresh medium containing IL-2 was added 3 times a week, and clones were restimulated with irradiated feeder PBMCs • IL-2 required for T cell survival / activation • Irradiated PBMCs likely supply costimulation • PHA provides a TCR stimulus equivalent to giving a-CD3, a-CD28, and IL-2 : more typical for in vitro T cell expansion

  8. Methods in the paper • Co-culture assay • 3x 104 monocytes + 105 T cells + antigen x 36 hours • (supply TCR stimulus + costimulation from monocytes)

  9. Normal skin histology

  10. histopathology • Eczema • Spongiosis (dermal edema produced by inflamm mediators) • Infiltrating T cells, eosinophils (H&E staining: pink), granulocytes • (eos are drawn by Th2 cytokines) • Psoriasis: • Acanthosis : hyperplasia and thickening of the stratum spinosum of the epidermis • Elongated rete ridges (epidermial outpouching into dermis) • Neutrophilic microabscesses • (notable given Th17 cytokines recruit PMNs)

  11. A word about therapy used in this paper : -TNFa inhibitors effective therapy for: RA, psoriasis psoriatic arthritis other spondyloarthropathies -ustekinumab = anti-IL 12 p40 subunit Ab (block Th1 and Th17 induction) used in psoriasis but not other rheum dz so far -cyclosporine: calcineurin inhibitor - blocks Ca signaling ds of TCR in T cells. Not specific to T cell subsets used mostly in transplant immunology

  12. Filaggrin : important for function of epidermal barrier Truncation mutations in filaggrin strongly associated with severe eczema… “lower expression” in eczema than psoriasis : ? Driven by cytokines?

  13. Conclusions : -antigen-specific T cells are recruited to ATP sites and drive eczema even in patients with psoriasis. Argues that antigen-specific T cells play a role in setting up pathology -skin lesions are autonomous : local cytokine / inflammatory milieu -ACD and psoriasis coexist more than eczema and psoriasis : antagonistic T cell polarization in latter -bacterial colonization as related to local cytokine production staph aureus is seen in eczema but not psoriasis less staph in lesions where Th17 cells are concentrated

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