Naïl Benallegue
1,2,3,4, Bryan Nicol
1,2,3,4, Séverine Bézie
1,2,3, Hadrien Regue
1,2,3, Nadège Vimont
1,2,3, Léa Flippe
1,2,3,
Alexandra Garcia
1,2,3, David A. Laplaud
1,2,3,4and Carole Guillonneau
1,2,3,4.
1Center for Research in Transplantation and Immunology, INSERM, Université de Nantes, Nantes, France; 2Institut de Transplantation Urologie Néphrologie (ITUN), CHU Nantes, Nantes, France; 3LabEx
IGO “Immunotherapy, Graft, Oncology”, Nantes, France; 4co-authors
Alteration of CD8
+
CD45RC
int/neg
regulatory T cells functions in
Multiple Sclerosis and correlates with disease severity
Autoimmune diseases can develop following pathological activation of autoreactive effector cells and/or, alternatively, after weakening of
self-protective regulatory mechanisms. Most of the studies have focused on CD4+ Tregs and the role of CD8+ Tregs in Multiple Sclerosis
(MS) remains largely unexplored. We previously reported the suppressive properties of rat and human CD8+CD45RCint/neg Treg cells,
expressing Foxp3 and acting through IFNg, TGFb and IL34 cytokines (Guillonneau, JCI, 2007; Bézie, JCI, 2015, Bézie, Front. Immunol.,
2018). Thus, their potency of suppression make them strong candidates of disruptive immune tolerance, especially in MS where CD8+ T
cells play a major role. Thus, the overreaching goal of this study is to define the role of CD8+ regulatory T cell in MS pathogenesis
B
ACKGROUND56 untreated relapsing-remitting MS patients and 52 age- and gender-matched healthy volunteers (HV) were recruited.
•Tregs were defined as CD3+CD8+ (or CD4-) CD161lowVα7-CD45RCint/neg T cells. MAIT cells were excluded from all analysis.
•Staining: T cells were stimulated or not 5h with PMA+ionomycin including 4h with BFA before FACS depending on the marker investigated. •Transcriptomic analysis: 3’-Digital Gene Expression Sequencing was performed on unstimulated cells. Analysis was performed using R packages and KEGG, Reactome and Gene Ontology databases for Gene Set Enrichment Analysis (GSEA).
M
ATERIAL& M
ETHODS1 :1 :0 4 :4 :1 2 :2 :1 1 :1 :1 1 :1 :2 1 :1 :4 0 5 0 1 0 0 1 5 0 r e s p o n d e r : s t i m u l a t o r : s u p p r e s s o r c e l l s r a t i o r e la ti v e p r o p o r ti o n o f d iv id in g C D 4 + C D 2 5 - T c e ll s H V , n = 3 M S S S < 3 . 5 , n = 2 M S S S > 3 . 5 , n = 5 N S 1:1 :0 4:4 :1 2:2 :1 1:1 :1 1:1 :2 1:1 :4 0 2 0 4 0 6 0 8 0 1 0 0 r e s p o n d e r : s t i m u l a t o r : s u p p r e s s o r c e l l s r a t i o r e la ti v e p r o p o r ti o n o f d iv id in g C D 4 + C D 2 5 - T c e ll s M S S S < 3 . 5 , n = 1 3 M S S S > 3 . 5 , n = 1 1 N S Regulation of Inflammatory response Cytokine-mediated Signaling Pathway
CD8+CD45RC
INTT
REGS FREQUENCY IN BLOOD IS REDUCED DURING EXACERBATION WHEREASCD8+CD45RC
NEGT
REGS ARE MORE FREQUENT WITH ENHANCED SUPPRESSIVE FUNCTIONSCD8+CD45RCint CD8+CD45RCneg CD8+CD45RCint/neg 0 2 0 4 0 6 0 8 0 1 0 0 C D 8 + C D 4 5 R C in t /n e g (% o f n o n M A IT C D 8 + T c e ll s ) 0 2 0 4 0 6 0 8 0 1 0 0 C D 8 + C D 4 5 R C in t (% o f n o n M A IT C D 8 + T c e ll s ) p = 0 . 0 5 1 * * H V , n = 3 4 M S , n = 3 2 M S r e la p s e , n = 8 0 2 0 4 0 6 0 8 0 1 0 0 C D 8 + C D 4 5 R C n e g (% o f n o n M A IT C D 8 + T c e ll s ) * H V , n = 3 4 M S , n = 3 2 M S r e la p s e , n = 8 1:1 :0 4:4 :1 2:2 :1 1:1 :1 1:1 :2 1:1 :4 0 2 0 4 0 6 0 8 0 1 0 0 r e s p o n d e r : s t i m u l a t o r : s u p p r e s s o r c e l l s r a t i o r e la ti v e p r o p o r ti o n o f d iv id in g C D 4 + C D 2 5 - T c e ll s * H V , n = 2 2 A c u t e e x a c e r b a t io n , n = 5 M S , n = 1 9 * 1:1 :0 4:4 :1 2:2 :1 1:1 :1 1:1 :2 1:1 :4 0 2 0 4 0 6 0 8 0 1 0 0 r e s p o n d e r : s t i m u l a t o r : s u p p r e s s o r c e l l s r a t i o r e la ti v e p r o p o r ti o n o f d iv id in g C D 4 + C D 2 5 - T c e ll s H V n = 2 4 A c u t e e x a c e r b a t io n , n = 5 M S , n = 2 3 Suppression assay Tregs CFSE CD4+CD25 -T cells allogeneic APCs 5 days CD8+CD45RCint CD8+CD45RCneg
I
N SEVERE PATIENTS WITH A HIGHERM
ULTIPLES
CLEROSISS
EVERITYS
CORE, CD8+CD45RC
INTT
REGS FUNCTION IS IMPAIRED CD45RC 1:1 :0 4:4 :1 2:2 :1 1:1 :1 1:1 :2 1:1 :4 0 2 0 4 0 6 0 8 0 1 0 0 r e s p o n d e r : s t i m u l a t o r : s u p p r e s s o r c e l l s r a t i o r e la ti v e p r o p o r ti o n o f d iv id in g C D 4 + C D 2 5 - T c e ll s M S S S < 3 . 5 , n = 1 2 M S S S > 3 . 5 , n = 1 1 * *Suppression assay Transcriptomic Analysis (GSEA)
Immune response pathways are overrepresented in
CD8+CD45RCint of MSSS<3,5
patients as compared to MSSS>3,5 patients
Flow Cytometry Analysis
For the first time, we demonstrate an impairment of CD8+CD45RC
intTregs in MS.
We propose to define two populations of CD8+CD45RC Tregs:
• CD8+CD45RCint are dysfunctional in severe MS patients and less frequent during exacerbations
• CD8+CD45RCneg Tregs react properly to inflammation with enhanced regulatory functions during exacerbations
We suggest CD8
+CD45RC
int/negT cells and subsets may be potential therapeutic targets and prognostic tools in MS
C
ONCLUSIONAuthors have no financial disclosures that would be a potential conflict of interest with this presentation
CD8+CD45RCint
CD8+CD45RCneg
CD8+CD45RChigh
PD-1, and IFNg expression
are decreased in CD8+CD45RCint F o x P 3 C D 2 5 C D 1 2 7 2 B 4 B T L A P D -1 T IM 3 C T L A -4 C D 1 6 0 L A G 3 K L R G 1 C D 1 0 3 C D 1 5 s C D 1 2 2 C X C R 3 M -C S F C D 2 8 C D 1 5 4 G IT R IN F g IL -1 0 0 2 0 4 0 6 0 8 0 1 0 0 % i n n o n M A IT C D 8 + T c e ll s C D 8 + C D 4 5 R C i n t C D 8 + C D 4 5 R C n e g * * * * * * * *
