# Evaluation of innate lymphoid cells, mucosal-associated invariant T cells and helper T cells in human autoimmune uveitis

Paul Decker, Thomas Moulinet, Rolland Jaussaud, Maxime Cravat, Jérémy Corriger, Marcelo de Carvalho-Bittencourt

<figure><img src="image_1.png" /></figure>

## ► To cite this version:

Paul Decker, Thomas Moulinet, Rolland Jaussaud, Maxime Cravat, Jérémy Corriger, et al.. Evaluation of innate lymphoid cells, mucosal-associated invariant T cells and helper T cells in human autoimmune uveitis. 52nd Annual Meeting of the French Society for Immunology, Nov 2019, Nantes, France. hal-02986074

HAL Id: hal-02986074

https://hal.univ-lorraine.fr/hal-02986074

Submitted on 2 Nov 2020

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
# Evaluation of innate lymphoid cells, mucosal-associated invariant T cells and helper T cells in human autoimmune uveitis

<figure><img src="image_3.png" /></figure>

UNIVERSITÉ
DE LORRAINE

<figure><img src="image_4.png" /></figure>

P. Decker<fnref n="1" />, T. Moulinet<fnref n="2" />, R. Jaussaud<fnref n="2" />, M. Cravat<fnref n="1" />, J. Corriger<fnref n="1" />, M. De Carvalho Bittencourt<fnref n="1" />,<fnref n="3" />

IMoPA
Ingénierie Moléculaire & Physiopathologie Articulaire

CHRU
NANCY

## INTRODUCTION AND OBJECTIVE

Up to 50 % of uveitis are classified as « idiopathics ». Uveitis association with Th1 and Th17-mediated systemic inflammatory disorders - such as IBD and spondylarthritis - and immunosuppressive agents efficacy suggest underlying autoimmune mechanisms. Specific T cell responses to retinal antigens (IRBP and S-arrestin) have been identified in patients with idiopathic uveitis. A transgenic murine model of spontaneous autoimmune uveitis recently showed that specific retinal antigen T cells were initially activated in the intestine by microbiota extracts before they migrated and entered into the eye (1).

**Innate lymphoid cells (ILC) and mucosal-associated invariant T cells (MAIT) are innate immune cells recently discovered and particularly represented in mucosal tissues.**
These cells have never been evaluated in human uveitis.

Objective → To study **circulating ILC, MAIT and T lymphocytes** in **human autoimmune uveitis**

## Functional evaluation of T cells: polyclonal stimulation and identification of S-arrestin specific T cells

<caption>FLOW CYTOMETRY</caption>

### ICS

**PBMC stimulation (1x10⁶ cells)**

PMA/ionomycin
or

S-arrestin peptides

**Conditions:**

* Negative control

* Overnight incubation (14-16h)

* 37 °C, 5% CO₂

<figure><img src="image_5.png" /></figure>

IFN-γ
TNF-α
IL-17

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

S-arrestin overlapping peptides pool
(Genscript)
-15 AA length
- 5 AA overlap

**Gating:**

* lymphoid cells (SS/FS)

* singlets

* CD3 +

* viability stain 510 -

<figure><img src="image_8.png" /></figure>

S-Ag

PMA iono

### PROLIFERATION

**CFSE-labeled PBMC stimulation (2x10⁵ cells)**

S-arrestin peptides
or

Tetanus anatoxin

**Conditions:**

* Negative control

* 7 days incubation

* 37 °C, 5% CO₂

<figure><img src="image_9.png" /></figure>

NEG

S-Ag

TET

**Inclusion criteria:**
Active « idiopathic » uveitis
(i.e. no infection, no cancer)
+
No prior corticosteroid treatment

<figure><img src="image_10.png" /></figure>

Whole blood

<figure><img src="image_11.png" /></figure>

Lymphocyte separation medium
Density gradient

<figure><img src="image_12.png" /></figure>

PBMC

## RESULTS

## Proportions of circulating monofunctional IL-17+ CD4+ T cells and S-arrestin specific T cells

<figure><img src="image_13.png" /></figure>

▲ controls

○ patients

**Elevation of Th17 cells in uveitis patients compared to controls at diagnosis**

**Detection of S-arrestin specific lymphocyte responses**

(IFN-γ production and/or proliferation) in **4/15 patients**

<table><thead><tr><th rowspan="2"></th><th colspan="3">ICS</th><th colspan="3">CFSE</th></tr><tr><th>Δ CD3 IFN-γ+ (ratio)</th><th>Δ CD4 IFN-γ+ (ratio)</th><th>Δ CD8 IFN-γ+ (ratio)</th><th>Δ CD3+ (index)</th><th>Δ CD4+ (index)</th><th>Δ CD8+ (index)</th></tr></thead><tbody><tr><td>#4</td><td>0,05 % (1,71)</td><td>0,04 % (2)</td><td>0,03 % (1,75)</td><td>1,8 %</td><td>2,7 % (2,26)</td><td>0,03 %</td></tr><tr><td>#5</td><td>0 %</td><td>0 %</td><td>0 %</td><td>0,3 %</td><td>0,5 % (2,01)</td><td>0 %</td></tr><tr><td>#9</td><td>0,03 % (1,67)</td><td>0,03 % (4)</td><td>0,01 %</td><td>0 %</td><td>0 %</td><td>0 %</td></tr><tr><td>#15</td><td>0 %</td><td>0 %</td><td>0,01 %</td><td>2,7 % (1,77)</td><td>3,7 % (1,82)</td><td>1,8 (0,75)</td></tr></tbody></table>

## Proportions and absolute numbers of circulating ILC and MAIT cells

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /></figure>

**Diminution of ILC 3 in uveitis patients compared to controls at diagnosis**
**Elevation of ILC 3 after 3 months of corticosteroids in uveitis patients (no clinical activity detected)**

<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /></figure>

UVEDAI score

**Elevation of CCR6+ ILC 3 proportions in uveitis patients at diagnosis**

**Total ILC were negatively correlated with clinical activity in uveitis patients**

<figure><img src="image_19.png" /></figure>

<figure><img src="image_20.png" /></figure>

<figure><img src="image_21.png" /></figure>

**Diminution of MAIT in uveitis patients compared to controls at diagnosis**

## DISCUSSION

* Confirmation of global Th17 cellular responses in human autoimmune uveitis

* In 4/15 patients, retinal antigen-specific lymphocyte responses were detected at the time of diagnosis

* This is the first description of an **altered distribution of circulating ILC and MAIT**

correlated with clinical activity in human uveitis

implication of ILC and MAIT in uveitis pathogenesis ?

* Intestinal recruitment of CCR6+ ILC (LTi? ILCP?) could putatively participate in Th1- and Th17-specific retinal antigens responses initiation or maintenance

* Our results need to be confirmed in a larger cohort and further investigations

are needed to study **ILC and MAIT interactions with T cells** and determine whether they could represent **new therapeutic targets** in the future

## MATERIAL & METHODS

### ILC and MAIT identification

<figure><img src="image_22.png" /><figcaption>FLOW CYTOMETRY</figcaption></figure>

### ILC

Navios™ 3L 10C
(Beckman Coulter)

**Pool lineage:**

<table><tr><td>CD2</td><td>-</td><td>CD19</td></tr><tr><td>CD3</td><td>-</td><td>CD20</td></tr><tr><td>CD8</td><td>-</td><td>CD33</td></tr><tr><td>CD14</td><td>-</td><td>CD34</td></tr><tr><td>CD15</td><td>-</td><td>CD56</td></tr><tr><td>CD16</td><td>-</td><td>CD123</td></tr></table>

**Gating:**

* lymphoid cells (SS/FS)

* singlets

<figure><img src="image_23.png" /></figure>

### MAIT

**Gating:**

* lymphoid cells (SS/FS)

* singlets

* CD3 +

<figure><img src="image_24.png" /></figure>

## Patients characteristics

<table><thead><tr><th></th><th>Healthy volunteers (n=10)</th><th>Uveitis patients (n=15)</th><th>p-value</th></tr></thead><tbody><tr><td>Age, years</td><td>54 [33-60]</td><td>47 [42-69]</td><td>0,53</td></tr><tr><td>Sex ratio (F/M)</td><td>5/5</td><td>9/6</td><td>0,7</td></tr><tr><td>Bilateral uveitis, n (%)</td><td></td><td>8/15 (53 %)</td><td></td></tr><tr><td>Localization, n (%)</td><td></td><td></td><td></td></tr><tr><td>Anterior</td><td></td><td>2/15 (13 %)</td><td></td></tr><tr><td>Posterior</td><td></td><td>5/15 (33 %)</td><td></td></tr><tr><td>Panuveitis</td><td></td><td>8/15 (53 %)</td><td></td></tr><tr><td>Anterior chamber cells (RE/LE)</td><td></td><td>0 [0-1]/0 [0-1]</td><td></td></tr><tr><td>Vitreous haze (RE/LE)</td><td></td><td>0 [0-1]/0 [0-1]</td><td></td></tr><tr><td>Papillitis, n (%)</td><td></td><td>8/15 (53 %)</td><td></td></tr><tr><td>Retinal and/or choroidal lesions, n (%)</td><td></td><td>1/15 (7 %)</td><td></td></tr><tr><td>Retinal vasculitis, n (%)</td><td></td><td>5/15 (33 %)</td><td></td></tr><tr><td>Macular edema, n (%)</td><td></td><td>5/15 (33 %)</td><td></td></tr><tr><td>Visual acuity (RE/LE)</td><td></td><td></td><td></td></tr><tr><td>Monoyer</td><td></td><td>5 [2-10]/9 [4-10]</td><td></td></tr><tr><td>Parinaud</td><td></td><td>2 [2-3,5]/2 [2-5,5]</td><td></td></tr><tr><td>UVEDAI score</td><td></td><td>2,77 [0,38-3,75]</td><td></td></tr><tr><td>Leucocytes, nb/mm3</td><td>5,85 [4,96-6,34]</td><td>7,07 [5,99-8,5]</td><td>0,03</td></tr><tr><td>Lymphocytes, nb/mm3</td><td>1,78 [1,69-1,88]</td><td>1,94 [1,61-2,3]</td><td>0,8</td></tr><tr><td>Neutrophils, nb/mm3</td><td>3,27 [2,91-3,47]</td><td>4,1 [3,27-5,73]</td><td>0,08</td></tr><tr><td>C-reactive protein (CRP), mg/l</td><td></td><td>1,1 [0,1-5,9]</td><td></td></tr><tr><td>Oral corticosteroid regimen, n (%)</td><td></td><td>12/15 (80 %)</td><td></td></tr></tbody></table>

RE: right eye, LE: left eye

<footnote n="1">¹ UMR 7365 IMoPA, Université Lorraine/CNRS, Vandoeuvre-lès-Nancy, France</footnote>

<footnote n="2">² Département de médecine interne, CHRU Nancy, Vandoeuvre-lès-Nancy, France</footnote>

<footnote n="3">³ Laboratoire d'Immunologie, CHRU Nancy, Vandoeuvre-lès-Nancy, France</footnote>

<footnote>*p < 0,05, **p < 0,01, ****p < 0,0001, ns: not significant</footnote>