iii national congress iscca, rome, 8-10 november2018 roles and … · 2018. 12. 4. · roles and...
TRANSCRIPT
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Roles and functions of γδγδγδγδ T cells in farm animals: an immune riddle.
Rome, 9 November 2018
Massimo Amadori
Laboratory of Cellular Immunology, Istituto Zooprofilattico Sperimentale della
Lombardia e dell’Emilia-Romagna, Brescia, Italy.
III National Congress ISCCA,
Rome, 8-10 november 2018
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Large Animal Models• Great advances in immunology: usually underpinned by
experiments carried out in animal models and inbred lines of mice.
• Corresponding knock-out or knock-in derivatives. • Yet, laboratory mice will never provide all the answers to
fully understand immunological processes.
• Large animal models offer unique biological and experimental advantages of great value to the understanding of biological and immunological processes.
• Example: identification of B cells in farm animals has contributed significantly to a better understanding of immunity.
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Why bother about farm animals in biomedical research ?
• The immune system of pigs more closely resembles humansfor >80% of analyzed parameters, as opposed to
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Peculiarities of γδγδγδγδ T cells
1. TcR binds to Ag directly, no MHC context (CDR1/2 regions)
2. Recognition of «unconventional» antigens
3. Overlapping of innate and adaptive immune functions (γδTcR, TLRs, scavenger receptors, NK receptors)
4. Regulatory networks
5. Wound repair
6. Antigen-presenting functions
7. Response to stress antigens
8. Little if any memory (see results in γδ KO mice) 9. CDR3 engagement: single chain (γ or δ)10. Tissue-specific oligoclonality (V gene repertoire /expression !)
αβαβαβαβ T cells = adaptive immunity γδγδγδγδ T cells = immune regulation, surveillance , homeostasis
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Difference between γδγδγδγδ «high» and «low» species: the pig model
Prevalence of γδ T cells shows a negative correlation with: (A) TcR αβαβαβαβdiversity and (B) Ig genes diversity
Young pigs, cattle, sheep present high prevalence of γδ T cells in peripheral bloodmononuclear cells.
This prevalence substantially decreases in older animals
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Peculiarities of γδγδγδγδ T cellsin order Artiodactyla
• «Null», CD4-/CD8- /CD5+ T cells are a major 3rd population, mainly expressing the γδγδγδγδ TcR and, often, a unique surfacereceptor in ruminants: WC1 (cattle) / T19 in sheep.
• CD2/CD8αααααααα define 3 major sub-populations of γδ T cells (big difference between spleen and circulating γδ T cells )
• In pigs, CD2 defines 2 lineages:• CD2- cells: terminally differentiated. They can acquire neither
CD2 nor CD8.
• They down-regulate TcR γδ after in vitro stimulation.
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WC1 properties
• WC1: SRCR superfamily, similar to CD163 and CD5
• 205(N4) - 215 (N3) and 300 kDa bands under reducingconditions (mAb CC15)
• Multiple group B SRCR domains, like CD5 and CD6
• Alternative splicing: secreted and membrane forms
• Immunocompetence. WC1+ γδ T cells: first T cells respondingto Leptospira vaccines
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Age-related prevalence of WC1+ γδγδγδγδ T cells in cattle
Compared to calves, far fewer WC1+ γδ T cells in PBMC of adult cattle, like all γδ T cells in humans (stressfulexperiences, race).
Anti-bovine WC1 mAb CC101 cross-reacts with a porcine γδγδγδγδ T cell subset (shorter, primitive WC1 form, 6 SRCR domains).
Calf, mAb CC15Cow, mAb IL-A29
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WC1 as both PRR and TcR co-receptor
• Ruminant WC1: scavenger receptor, an outright PRR.
• WC1 binds to Leptospira and supports γδ TcR function: sameas e.g. CD8 in αβ T cells. Crosslinking studies and knock-down experiments.
• WC1+ γδγδγδγδ T cells: pro-inflammatory , IFN-γ+, promoting IgG2 Ab (except WC1.2+).
• WC1+ γδγδγδγδ T cells : blood and skin, minor component in gutand spleen.
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Effector functions of WC1
• WC1 binding underlies activation by bacterial PAMPsfollowing low-affinity interaction with TcR.
• WC1 diversity (137 SRCR regions) adds diversity to γδγδγδγδTcR
• Soluble WC1 SRCRs inhibit Leptospira growthdose/dependent.
• Two synergic, effector mechanisms !! Humans: CD5/CD6/CD163
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A way to diversity: WC1 and γδγδγδγδ TcR• 13 genes in two loci of chromosome 5, each coding for 11 SRCR. • 2 sub-pop.: WC1.1 and WC1.2 (mAb to SRCR a1). Same Vγ genes,
diverse Vδ.• Different WC1 molecules are correlated with responses to
bacteria: e.g. WC1.1 / Leptospira, WC1.2/Anaplasma
• Phylogenetic evolution: WC1 genes selected for expression with• γδγδγδγδ TcR• WC1 gene expression: stable, like CD4 and CD8.
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γδγδγδγδ T cell responses to mycobacterial infections.
• γδ T cells: associated to granulomas, WC1+ first. IDT ! • Kinetics: crucial ! Granuloma organization !! BCG vaccine!• Final layout of WC1+ (external) and WC1- (internal).
• IFN-γγγγ (Th1) response, preceding the CD4 response. • WC1+ require priming (e.g. MAP vaccination), WC1- no!• MAP: stepwise increase of WC1- intraepithelial T cells.
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γγγγδδδδ T cell control over response !(Guzman et al., 2014)
• Some γδ T cells spontaneously secrete IL-10 • They proliferate in response to IL-10, TGF-β, and contact
with APCs.
• IL-10–expressing γδ T cells inhibit Ag-specific and nonspecific proliferation of CD4+ and CD8+ T cells in vitro.
• Instead, CD4+CD25high Foxp3+ T cells are neither anergicnor suppressive.
• Bovine γδ T cells are probably the major regulatory T cell subset in peripheral blood, even in TB cases.
• Pregnancy (sheep) ??
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γδγδγδγδ T cells and non-conventionalantigens
• Non-protein Ag like LAM recognized by TcR γδ• WC1+ Posphoantigens (IPP) but…..Vγγγγ9Vδδδδ2 counterpart
still lacking (maybe Vγ4/Cγ5) (TB model ?)
• Pig γδ T cells activated by alkylamine-like molecules likehuman γδ T cells (Summerfield and Saalmueller, 1998), molecules acting on IPP levels.
• Butyrophilin 3A1 receptor / Vγ9Vδ2: undefined in ruminantsbut present in many other species.
• Response to pAg: conserved in the phylogenetic evolution.
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Lipopeptides of M. bovisare crucial for protection !
1. Hydrophobic antigens of M. bovis BCG (CMEbcg): isolatedby chloroform-methanol extraction.
2. CMEbcg contained lipids and lipopeptides and had a lowcontent of high molecular weight protein.
3. Both in BCG vaccinated and M. bovis challenged calves, CMEbcg stimulated polyfunctional T-cells that producedIFN-γ, IL-12, IL-17 and IL-22.
4. The CMEbcg specific CD3+ T-cell proliferative responsefollowing BCG vaccination was the best predictor of protection against subsequent M. bovis challenge.
5. CMEbcg expanded T-cells killed CMEbcg loadedmonocytes. Lipopeptides were the immunodominantantigens in CMEbcg, also stimulating CD4+T-cells via MHC class II.
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Bovine γδγδγδγδ T cells and viral infectionsIn the 90’ our group reported:
−γδ T cells from FMD-vaccinated cattle cause dramatic yieldreduction of FMDV in a MHC-independent manner.
−γδ T cells infiltrate tongue and palate mucosae, and evenmore in FMD-vaccinated cattle
- BHV 1-infected cattle show a large increase in γδ T cells in PBMC during the first days of infection.
Toka et al. ( 2011): after FMD infection CD25++, CD62L±, CD45RO±, IFN-γ++ .- WC1+ cells: NK-like properties (perforin++, CD335++)
Administration of IFN-γ in vivo in BLV-infected cattle: increase of the γδ T cells which suppress BLV replication.
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Binding of bovine 611p γδγδγδγδ T cellsto target cells (Amadori et al., 1992)
A) Pi3-infected
primary FBK
cells
B) Doublet with a
BS-BEK cell
(established cell
line)
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γγγγδδδδ T cells and Innate immune responses to viruses
• Potent FMD vaccines induce protection of pigs in 4 dayswithout detectable antibodies (Barnett et al., 2002)
• Pig γδ T cells are strongly activated by exposure to FMDV antigens (Takamatsu et al., 2006)
• High proliferative responses to FMDV of PBMC from naive pigs after removal of plastic-adherent cells
• Role of non-structural viral proteins and/or stress proteins
• CD2+CD8+ γδγδγδγδ T cells are proliferating• No proliferation after depletion of such cells.
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Alarmins: histones secreted by FMDV O1-infected BHK-21 cells. A role for recognition
by γδγδγδγδ T cells? (Amadori et al., 1999)
Buffer 30’ k30 120’ k120’ 240’ k240’ 360 k360’
1 2 3 4 5 6 7 8
Histones secreted by virus-infected cells. K : non-infected control cells
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γγγγδδδδ T cells and the lymphoid stress-surveillance response (Hayday A.C., 2009)
There is a network of lymphocyte populations (mainly γδγδγδγδT cells)
They recognize neo-antigens like MIC on stressed cells
(Hayday A.C., 2009), i.e. cells exposed to events as
diverse as heat shock, infections, DNA damage, etc.
Role of γδγδγδγδ T cell surveillance at mucosal sites !!
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Responses of myeloidand lymphoid cells
Also in cattle, MIC proteins are ligands for the activating NK cell receptor NKG2D, expressed on NK cells, CD8+ αβand γδγδγδγδ T cells (Guzman et al., 2010).In pigs, γδ T cells may express NKG2D for recognition of MIC2 (homologue of human MIC) (Chardon et al., 2000).
PAMPs and DAMPs
are recognized
Neoantigens are
recognized
Myeloid cells
Lymphoid cells
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Lymphoid cell responses complement stress recognition by myeloid cells
Challenges to homeostasis (acidosis, osmolarity changes, hypoxia, ROS, ATP/AMP, a.a.) NEFA TLR4
inflammasome IL-1β / IL18
P38 MAPK
TLR ligands,
cytokines,
physico-
chemical
stressors
Pi3 / Akt /
mTOR
Expression of IL-12 and
IL-10 in myeloid cells
Regulation of pro and
anti-inflammatory
responses in tissues
eIF2α
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γγγγδδδδ T cells and metabolic stressin ruminants (Trevisi et al., 2018)
• Forestomachs can receive and elaborate signals for the immune cells infiltrating the rumen fluids.
• They participate in a cross-talk with the lymphoid tissues in the oral cavity, thus promoting regulatory actions at both regional and systemic.
• Our group (Trevisi et al., 2018) found a positive correlation between some inflammatory markers (e.g. paraoxonase, a negative acute phase protein) in blood and WC1+ γδγδγδγδ T cells infiltrating rumen fluids (p=0.0005).
• Instead, B cells showed a negative correlation (p< 0.0001), i.e. B cells would be correlated with APP-.
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γγγγδδδδ T cells and APC functionsin cattle
• CD28/B7 interaction is pivotal to αβαβαβαβ, but not γδ T cells!• Bovine γδ T cells do not express the CD28 gene.• Yet, activated bovine γδ T cells are MHC II++, CD13+,
CD80+, ingest and process exogenous proteins.
• γδ T cell lines primed with BRSV or OVA promoteproliferation of sorted CD4 T cells (Collins et al., 1998).
• No response of CD4 T cells from naive cattle.
• Cytokines (IL-1, IL-4, IFN-γ, TNF-α, GM-CSF) contribute to activation of DCs in tissues.
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APC functions in pigs
• Pig γδ T cells present OVA to CD4 T cells from OVA-immunized inbred pigs (Takamatsu et al., 2002)
• Proliferation blocked by depletion of γδ T cells and mAbto CD4 and MHC II, or chloroquine
• APC functions of γδ T cells observed in humans(Brandes et al., 2005) but not in mice !
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The main role of γδγδγδγδ T cells
Kalyan S. and Kabelitz D., Cellular & Molecular Immunology (2013) 10, 21–29
The effective recognition of the 4
entities dictates the need for high
vs. low prevalence of γδγδγδγδ T cells in blood and tissues
γδγδγδγδ T cells to differentiate:- Friend from foe .
- Pathological from benign.
1. Missing self: recognized by
NK cells
2. Dangerous non-self:
recognized by αβ αβ αβ αβ T cells3. Safe non-self: recognized by
γδγδγδγδ T cells4. Distressed self: recognized by
γδγδγδγδ T cells
Response guided by level of distress !!
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Conclusions• The adaptive immune system has evolved antigen
receptor diversity in T lymphocytes to cope with a large variety of pathogens and non-self antigens.
• Depending on phylogenetic evolution and environmental infectious pressure, the process hasdeveloped differently in distinct classes of SubphylumVertebrata.
• γδ T cells are probably a contact point of innate and adaptive immunity.
• In order Artiodactyla, there was an evolutionaryadvantage of keeping a high prevalence of γδ T cells, asopposed to humans and rodents.
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Thank you
for the attention !