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

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.

Why bother about farm animals in biomedical research ?

• The immune system of pigs more closely resembles humansfor >80% of analyzed parameters, as opposed to

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

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

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.

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

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

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.

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

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.

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.

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) ??

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.

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.

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.

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)

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.

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

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 !!

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

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

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-.

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.

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 !

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 !!

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.

Thank you

for the attention !