Presentation

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

T cell differentiation : molecular and functional characteristics

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Zhu and Paul. Blood 2008

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

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

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

Methods in the paper For comparison: Typical strategy for stimulating and studying naïve murine T cells:

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3 days : aCD3 + aCD28 + IL-2, then 2 days IL-2 : effector T cells generated, can assess cytokine production

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3 days aCD3+/- aCD28 : CFSE or thymidine incorporation to assess proliferation

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o/n aCD3 stimulation : assess surface CD69 expression to assess early activation

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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)

Methods in the paper • • • •

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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

Methods in the paper • Co-culture assay • •

3x 104 monocytes + 105 T cells + antigen x 36 hours (supply TCR stimulus + costimulation from monocytes)

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Normal skin histology QuickTime™ and a decompressor are needed to see this picture.

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histopathology • • •

Eczema Spongiosis (dermal edema produced by inflamm mediators) Infiltrating T cells, eosinophils (H&E staining: pink), granulocytes – (eos are drawn by Th2 cytokines)

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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)

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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

QuickTime™ and a decompressor needed to see this picture. 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?

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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