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 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: –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)

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

Methods in the paper Co-culture assay 3x 10 4 monocytes T cells + antigen x 36 hours (supply TCR stimulus + costimulation from monocytes)

Normal skin histology

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)

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

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?

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