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