Initial Characterization of Skin Immune Cell Populations in Juvenile Göttingen Minipigs L. Allais, C. Priou, O. Barneoud-Rousset, P. Michel, F. Condevaux, N. Ravas, and E. Lafond Charles River, Safety Assessment, St Germain Nuelles, France

CONCLUSIONS

1 INTRODUCTION

The development of new pharmaceuticals benefits from continuous advances in biomedical research for both adults and children. Safety evaluation of new pediatric medicines is performed by the conduct of toxicology studies using juvenile animals. The minipig is now considered as a useful alternative non-rodent species for safety testing of pharmaceuticals. Human parallels in many features of its anatomy, physiology and biochemistry make the minipig a good model for man. For use in juvenile toxicology studies, the development of main organs or systems, including the immune system, of the minipig still requires further characterization. Although the immune system of the adult pig has been studied, particularly in relation to different infectious diseases in pigs and xenotransplantations, the development of the immune system in juvenile Göttingen minipigs is still unknown or was evaluated with very limited immune developmental endpoints. The immune system could evolve, during the animal development, into specialized properties at a particular anatomic location, especially in epithelial barrier tissues. The skin is the largest and most exposed organ of the mammalian body with various protection properties, the immune protection being one of its most important functions throughout life acting as a first line of immunological defense and involving both innate and adaptive responses. The objective of this preliminary study was to identify the main epidermis and dermis immune cells (including Langherans cells, dermal dendritic cells, macrophages, CD4/CD8 T cells and αβ/γδ T cells) in the juvenile Göttingen minipig using flow cytometry and immunohistofluorescence (IHF) methods. The proportions of T, B and NK cells analyzed in the dermis and epidermis were also compared with the same populations in selected lymphoid organs and in blood from the same animals. This work was performed at different ages from 3 days to 6 months after birth. In addition, a comparative analysis between healthy skin and chemically-induced inflamed skin was further investigated in 2-month and 6-month old minipigs.

RESULTS 3.1 Preliminary comparative analysis of T, NK and B cells distribution between blood, skin and lymphoid organs/tissues across age (Figure 2): - For each biological matrix, the distribution of T, NK and B cells is generally comparable

from PND3 to PNM6 Göttingen minipigs . - The proportion of γδ-T cells is higher in blood than in the other matrices. - The proportions of NK and B cells are higher in the secondary lymphoid

organs/tissues (spleen an lymph nodes) than in the other matrices. - Interindividual variations are noted, especially for the skin populations.

3.2 Comparative analysis of αβ-T cell subsets (Figure 3 left): - Proportion of double positive CD4+CD8+ cells (memory T cells) is higher in the

thymus than in other matrices and is increasing with age. - CD4+ (T-helper) cells are in higher proportion than CD8+ cells (T-cytotoxic) in

blood and secondary lymphoid organs on each occasion. 3.3 Comparative analysis of γδ-T cell subsets (Figure 3 right): - Proportion of CD2+CD8- cells is higher in the thymus than in other matrices with a

trend to an increase with age . 3.4 Preliminary comparative analysis of macrophages and dendritic cells between control and inflamed skins (Figure 4 and Figure 5): - Separation between macrophages and dendritic cells was difficult since these two

populations share similar cell surface markers (CD172, CD163). - There was in general no evidence of differences in the proportion of target cells

between the control and inflamed skin from PNM2 and PNM6 minipigs.

Abstract Final ID 2779 / P253

2 MATERIALS AND METHODS On each occasion, i.e. PND (Post-Natal Day) 3 and 7, PNW (Post-Natal Week) 2 and 4, and PNM (Post-Natal Month) 2, 4* and 6, the selected animals were euthanized after blood sampling and necropsied. Skin and selected lymphoid organs/tissues (thymus, spleen and axilla-inguinal/iliac lymph nodes) were collected and prepared, as follows: - frozen for IHF staining and numeration using a Fluorescent Microscope

(Olympus BX51-P) - or immediately processed in cell suspensions for analysis using

FACSanto I cytometer (BD Biosciences) and FACSDiva software (BD Biosciences). The epidermis was separated from the dermis after overnight digestion with Dispase II (figure 1).

In addition, for PNM2 and PNM6 animals, skin inflammation was enhanced by dermal application of 4% 1-Chloro-2,4-dinitrobenzene on the left flank 24 hours before skin collection and analysis. The right flank was used as negative control (vehicle: acetone/olive oil 1:3).

Both cytometry analysis and IHF methods confirmed the presence of the main immune cell populations in the epidermis and/or dermis of 3-day old minipigs (PND3). There were no major differences in the proportion of immune cells across different ages. However, one of the limits of this preliminary investigation is the small number of animals analyzed on each occasion. The next study will include at least 3 males and 3 females per occasion for a more comprehensive analysis. The conventional dendritic cells (cDC1 and cDC2) will be further characterized using additional cell surface markers (such as MHCII and CD1). Inflammatory dendritic cells will also be further defined after separation from macrophages. In addition to cell phenotyping, the immune response of the Göttingen minipig will be evaluated using an immunomodulator and in-vivo / ex-vivo functional tests. The authors would like to thank Charles River Scientific and Operations groups for their direct or indirect contribution to this work.

Figure 1: Experimental design. Preparation of skin (dermis and epidermis), blood and selected lymphoid organs/tisues (thymus, spleen, LN (axillo-inguinal/iliac lymph nodes)) for IHF staining or cytometry analysis.

Table 1: Selected markers used to identify the immune cells in the skin, blood and/or selected lymphoid organs/tissues.

Ten piglets, 5 males and 5 females, were born in our animal facilities from two Gottingen sows (Ellegaard, Denmark) and were raised with their respective mother under optimal housing conditions until weaning, i.e. 4 weeks after birth. After weaning, the animals were group-housed per gender. The study design was approved by the Animal Ethical Committee and conducted in compliance with the European Animal Welfare Guidelines in an AAALAC accredited Test Facility.

Figure 2: Cytometry analysis. Relative proportions (% of CD45+ cells) of immune cells: NK cells (purple), B cells (green), αβ-TCR T cells (blue) and γδ-TCR T cells (red) in blood, skin and selected lymphoid organs/tissues in the Göttingen minipig across age (N=1 on PND3; N=1 on PND7; N=2 on PNW2; N=2 on PNW4; N=2 on PNM2 and N=1 on PNM6).

Figure 4: Cytometry analysis. % CD172-CADM1+ (including conventional dendritic cells CDc1), % CD172+CADM1- (including conventional dendritic cells CDc2) and % CD172+CADM1+ subsets (to be further characterized in CD172+CADM1low and CD172+ CADM1high). Comparison between control and inflamed skin and between 2-month old (PNM2; N=2) and 6-month old (PNM6; N=1) minipigs.

Figure 6: IHF staining analysis. Skin CD135+ (Flt3) cells (A), CADM1+ cells (B), double positive conventional dendritic cells (C) and CD207+ representing Langherans cells (D and E) from PNM6 minipig. Skin CD135+ cells (F), CADM1+ cells (G), double positive conventional dendritic cells (H) and CD207+ representing Langherans cells (I and J) from PND3 minipig. Skin γδ-TCR cells (K), CD3+T-cells (L) and double positive (N) in PNM6 minipig. Skin γδ-TCR cells (O), CD3+T-cells (P) in PND3 minipig. DAPI stained skin cells in PNM6 minipig (M) and PND3 (Q) minipig. D: Dermis; E: Epidermis.

Figure 5: IHF staining analysis. Numeration of positive CD163+ or CD172+ cells in 5 areas/slide from skin samples collected from PNM2 and PNM6 minipigs. Comparison between negative control and inflamed skins for each age.

Figure 3: Cytometry analysis. Left: % CD4+ cells ( T-helper cell), CD8+ (T-cytotoxic cells), double C4+CD8+ T cellls (memory T cells) and CD4-CD8- (naive T cells) in the αβ-TCR population. Right: % CD2+CD8-,CD2+CD8+ and CD2-

CD8- subsets in the γδ-TCR population. (N=1 on PND3; N=1 on PND7; N=2 on PNW2; N=2 on PNW4; N=2 on PNM2 and N=1 on PNM6). PN

M6

PND

3 PN

D3

PNM

6 Merge PNM6

3 4

* Data from PNM4 minipig not included (technical issues during analysis)

5

The authors have no conflict of interest to declare.

REFERENCES Debeer, S. et al. Comparative histology and immunohistochemistry of porcine versus human skin. Eur. J. Dermatology 23, 456–466 (2013). Marquet, F. et al. Characterization of dendritic cells subpopulations in skin and afferent lymph in the swine model. PLoS One 6, 1–8 (2011). Summerfield, A., Meurens, F. & Ricklin, M. E. The immunology of the porcine skin and its value as a model for human skin. Mol. Immunol. 66, 14–21 (2015).