PLANIFICACIÓN DE LAS RESPUESTAS, SEGUIMIENTO Y CONTROL DE

The fraction of T and B lymphocytes, monocytes and polymorphonuclear cells in MNC suspensions was assessed by immunocytochemistry of cytospins (2.9). As some of the procedures used involved the enrichment of cell types based on adherence, the proportions of different cell types in nylon wool non adherent cell and plastic non-adherent cell populations were also enumerated using immunohistochemsitry. In order to identify Tasmanian devil NK cells, both immunohistochemistry and Giemsa staining techniques were used (2.9).

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Table 4.1. Percentages of individual cell types in Tasmanian devil peripheral blood mononuclear cell, nylon wool filtered and plastic non adherent cell populations analysed by immunohistochemistry

4.2.1.1

Cell types present in total mononuclear cell suspensions

T lymphocytes were identified with an antibody against an intracellular CD3ε epitope. The T lymphocytes of Tasmanian devils were similar in appearance to their human equivalent, with large round nuclei and scant cytoplasm. They were the most abundant cell type in the majority of samples, representing an average of 55% of the total MNC population (Table 4.1). CD3ε protein was strongly expressed in T lymphocytes in a distribution consistent with membrane association as the staining was evenly spread throughout the cell, and across the nucleus, and appeared to be stronger at the cell periphery (Fig. 4.1a). Some larger lymphocytes expressed lower levels of CD3. These may have represented naive lymphoblasts or Natural Killer T cells (Fig. 4.1a).

Cell type Labelling Morphology Mean % presence ± Standard deviation

Mononuclear Cells Nylon non adherent cells Plastic non adherent cells

T lymphocyte CD3+ Round nucleus, scanty

cytoplasm 55 ± 8 73 ± 7 76 ± 12

B lymphocyte MHCII + Round nucleus, scanty

cytoplasm 33 ± 8 9 ± 6 13 ± 4

Monocyte MHCII +

Large cell, bean-shaped nucleus, abundant cytoplasm

5 ± 3 4 ± 2 1 ± 1

NK like CD3-/MHCII-

Large lymphocyte, round nucleus, scanty cytoplasm,

4 ± 1 5 ± 2 3 ± 2

Neutrophil CD3-/MHCII- Large cell, multi-lobar

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Figure 4.1. Appearance and Immunohistochemistry labelling patterns of normal peripheral blood mononuclear cells from Tasmanian devils.

Panel a: CD3 labelled MNC cytospins showing positive staining of T lymphocytes. Images were taken at 1000x magnification. Cells annotated with solid arrowheads represent T lymphocytes with normal morphology. Faintly stained cells annotated with open arrowheads are likely to be naive T lymphoblasts or NKT cells.

Panel b: CD3 labelled mononuclear cell cytospins showing negative staining of neutrophils. Images were taken at 1000x magnification. Cells annotated with solid arrowheads represent neutrophils with normal morphology as large cells characterised by a multilobar or circular nucleus.

Panel c: MHC class II labelled mononuclear cell cytospins showing positive staining of B lymphocytes. Images were taken at 1000x magnification. Cells annotated with solid arrowheads represent B lymphocytes with normal morphology.

Panel d: MHC class II labelled mononuclear cell cytospins showing positive staining of monocytes. Images were taken at 1000x magnification. Cells annotated with solid arrowheads represent monocytes with normal morphology characterised as large, MHC II positive cells with round or bean-shaped nuclei. a. c. b.

C

D

3

labeling

M

H

C

I

I

labeling

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Polymorphonuclear cells (neutrophils) were identified as large cells which had multi-lobar nuclei and did not express CD3ε or MHC II, (Fig. 4.1b). Although contamination with neutrophils was generally low when using histopaque gradient centrifugation, this cell type was found in all Tasmanian devil MNC samples. The majority of these contaminating neutrophils in the cytospins had ring-shaped nuclei, suggesting they were likely to be immature. Neutrophils comprised on average 7% of the total cell population.

B cells were identified as lymphocytes which expressed MHC II. The distribution was consistent with a mainly cytoplasmic protein with strong expression outside, rather than across, the nucleus, and no accumulation at the cell periphery (Fig. 4.1c). This type comprised, on average, 33% of the MNC population in Tasmanian devils (Table 4.1). Monocytes in MNC samples also strongly expressed MHC II but could be distinguished from B lymphocytes as they were larger and had distinct bi-lobar nuclei (Fig. 4.1d). This cell type comprised, on average, 5% of the total MNC population (Table 4.1).

4.2.1.2

Cell types in differentiated cell suspensions

Filtration of MNC through nylon wool depleted B lymphocytes in the MNC suspensions, reducing their percentage to an average of 9% compared to 33% in total MNC suspensions. Consequently, the percentage of T lymphocytes within the nylon non-adherent fractions increased, to an average of 73% (Table 1). Nylon wool filtration did not substantially affect the proportion of monocytes present in the cell suspensions indicating that, similar to other species, Tasmanian devil monocytes are nylon wool non-adherent. Nylon wool filtration increased the percentage of contaminating neutrophils in the population to an average of 13% (Table 1). However, there was substantial variation in neutrophil numbers between samples. Several attempts were made to elute the nylon wool adherent cells from the columns. However, recovery was consistently poor, with low numbers and viability in each sample. Consequently, the cell populations present in the nylon adherent population could not be analysed.

Plastic adherence substantially decreased the monocyte fraction to an average of 1% of the total population. The effect of plastic adherence on lymphocyte populations was similar to that of nylon wool, increasing T lymphocyte presence to an average of 76% and reducing B lymphocyte numbers to 13% (Table 1). The percentage of neutrophils increased slightly in plastic adherent cells compared to total MNC, with an average of 4%.

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4.2.1.1

Evidence for the physical presence of Natural Killer-like cells in the

peripheral blood of Tasmanian devils

Natural Killer cells may be an important cytotoxic effector cell type in the response against both K562 cells and DFTD cells. However, the absence of specific markers has hampered efforts to identify this cell type in Tasmanian devils. In other species, NK cells can be histologically identified as large lymphocytes (compared to T or B cells) with prominent cytotoxic granules in the cytoplasm (large granular lymphocytes). Giemsa staining is commonly used to identify NK cells, as it can stain the acidic cytotoxic granules of NK cells. Therefore, the cytospins of Tasmanian devil MNC were Giemsa stained to provide further evidence for the presence of NK-like cells in Tasmanian devil peripheral blood. NK-like cells were defined as cells with a similar appearance to T or B lymphocytes but a larger diameter and the presence of azurophilic (darkly stained) granules in the cytoplasm. Large granular lymphocytes matching this description were identified in all Giemsa-stained MNC preparations examined (Fig. 4.2a and b). NK cells can also be identified as large CD3-/MHC II- using immunohistochemistry. NK-like cells were identified in Tasmanian devil MNC cytospins (Fig. 4.2c and d) and accounted for on average 4% of the total MNC population (Table 4.1). Some CD3-/MHC II- cells in the MNC cytospins had abundant cytoplasm, a typical morphological characteristic of plasma cells rather than NK cells, and were not included in this enumeration.

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Figure 4.2. Identification of Natural Killer-like cells in Tasmanian devils.

Panels a and b show Giemsa stained MNC cytospins. The presence of large granular lymphocytes consistent with the morphological appearance of NK cells is indicated with solid black arrowheads. Panels c and d show immunohistochemistry double-staining with CD3 and MHC II. The presence of negatively stained large lymphocytes consistent with the phenotype of NK like cells is indicated with solid black arrowheads. The typical morphology of a plasma cell (also negative for both markers) is indicated with an open arrowhead.

4.2.2

Evidence for antibody-dependent cell mediated cytotoxicity (ADCC)