Capítulo 2. Diseño del Hardware
2.1 Sistema empotrado
2.1.2 Módulo PWM
Observation
Betty 1.7 x106 LAK + 104 DFTD 170:1 no tumour
(day 112)
Grommit 2.4 x106 LAK + 104 DFTD 240:1 no tumour
(day 103)
Lotti 4.9 x106 LAK + 104 DFTD 490:1 no tumour
(day 136)
Maydin 7.4 x105 LAK + 104 DFTD 74:1 no tumour
(day 106)
Phil 2.2 x106 LAK + 104 DFTD 220:1 no tumour
( day 93)
Elsie 2 x106 LAK + 2 x 104 DFTD 100:1 no tumour
(day 46)
Elsie 2 x106 LAK + 2 x 104 DFTD 100:1 no tumour
(day 53)
Control 104 DFTD cells only DFTD tumour
(day 75)
Control 104 DFTD cells only no tumour
(day 99)
Control 2 x 104 DFTD cells only DFTD tumour
(day 53)
Control 2 x 104 DFTD cells only no tumour
5-12 Two problems hindering the in vivo LAK cell experiments were the health issues associated with the ageing NOD/SCID mice and the failure to reliably establish DFTD tumours in the control mice. To overcome this, the number of DFTD cells injected was increased to 5 x 104. This would shorten the duration to tumour
development and consequently the age of the mice. But it also reduced the LAK cell to DFTD cell ratio to 20:1.
PBMNC from a single Tasmanian devil were activated with Con A sup for 48 hours and co-injected with DFTD cells into three NOD/SCID mice. Seven control mice received DFTD cells only. The mice were monitored for up to 52 days at which time all three LAK cell treated mice had developed tumours. At necropsy the control mice were examined and five of seven had developed tumours (Table 5-2).
Increasing the number of injected DFTD cells increased the proportion of control mice developing tumours within a reasonable timeframe but the LAK cells failed to provide protection (Table 5-2). This may have been the result of using a 20:1 ratio, a failure to activate the cells during stimulation with Con A sup, or biological variability associated with the Tasmanian devil PBMNC source.
5-13
Table 5-2. Adoptive transfer into NOD/SCID mice of LAK cells at 20:1 Tasmanian devil LAK cell to DFTD cell ratio
Tasmanian devil donor
Injection
(number of LAK cells and DFTD cells)
LAK : DFTD cell ratio
Observation
Carlotta 106 LAK cells + 5x104 DFTD cells 20:1 Tumour
(Day 52)
Carlotta 106 LAK cells + 5x104 DFTD cells 20:1 Tumour
(Day 52)
Carlotta 106 LAK cells + 5x104 DFTD cells 20:1 Tumour
(Day 52)
Control 5x104 DFTD cells only Tumour
(Day 52)
Control 5x104 DFTD cells only Tumour
(Day 52)
Control 5x104 DFTD cells only Tumour
(Day 39)
Control 5x104 DFTD cells only Tumour
(day 42)
Control 5x104 DFTD cells only Tumour
(day 46)
Control 5x104 DFTD cells only No tumour
(Day 52)
Control 5x104 DFTD cells only No tumour
5-14 Since 20:1 LAK cell to DFTD cell ratio did not appear to be protective the ratio was increased. PBMNC from four devils were activated with Con A sup for 48 hours and co-injected with 5 x 104 DFTD cells at LAK cell to DFTD cell ratios ranging from 50:1 to 80:1. By day 74 none of the six LAK cell treated mice had established DFTD tumours but two of four control mice had developed tumours (Table 5-3).
Table 5-3. Adoptive transfer into NOD/SCID mice of LAK cells at 50:1 to 80:1 Tasmanian devil LAK cell to DFTD cell ratios
Tasmanian devil donor
Injection
(number of LAK cells and DFTD cells)
LAK : DFTD cell ratio
Observation
Bangles 4.1 x 106 LAK cells + 5x104 DFTD cells 80:1 No tumour
(Day 68)
Bangles 4.1 x 106 LAK cells + 5x104 DFTD cells 80:1 No tumour
(Day 74)
Floyd 3.3 x 106 LAK cells + 5x104 DFTD cells 60:1 No tumour
(Day 74)
Cory 3 x 106 LAK cells + 5x104 DFTD cells 60:1 No tumour
(Day 74)
Cory 3 x 106 LAK cells + 5x104 DFTD cells 60:1 No tumour
(Day 74)
Andrea 2.5 x 106 LAK cells + 5x104 DFTD cells 50:1 No tumour
(Day 59)
Control 5x104 DFTD cells only Tumour
(Day 74)
Control 5x104 DFTD cells only Tumour
(Day 74)
Control 5x104 DFTD cells only No tumour
(Day 74)
Control 5x104 DFTD cells only No tumour
5-15 As two of the four control mice had developed tumours the next trial used 105 DFTD cells and the LAK cells were used at ratios ranging from 7:1 to 22:1. After 45 days two of the four LAK cell treated mice had established DFTD tumours compared to five of the six control mice. Within the LAK cell treated group of mice there was no clear evidence of LAK cell dose-dependent response as 13:1 did not develop a tumour but 20:1 did (Table 5-4).
Table 5-4. Adoptive transfer into NOD/SCID mice of LAK cells at 7:1 to 22:1 Tasmanian devil LAK cell to DFTD cell ratios
Tasmanian devil donor
Injection
(number of LAK cells and DFTD cells)
LAK : DFTD cell ratio
Observation
Sedate Ed 2 x 106 LAK cells + 105 DFTD cells 20:1 Tumour
(day 45)
Sedate Ed 7 x 105 LAK cells + 105 DFTD cells 7:1 Tumour
(day 45)
Sedate Ed 2.2 x 106 LAK cells + 105 DFTD cells 22:1 No tumour
(day 45)
Sedate Ed 1.3 x 106 LAK cells + 105 DFTD cells 13:1 No tumour
(day 45)
Control 105 DFTD cells Tumour
(day 45)
Control 105 DFTD cells Tumour
(day 45)
Control 105 DFTD cells Tumour
(day 45)
Control 105 DFTD cells Tumour
(day 45)
Control 105 DFTD cells Tumour
(day 45)
Control 105 DFTD cells No tumour
5-16 Combining all the data from Tables 5-1 to 5-4 into a summary table highlighted the need for high LAK cell to DFTD cell ratios to protect mice from tumour development as well as the need for sufficient DFTD target cells to establish tumours in control mice. Evaluation of the combined LAK cell adoptive transfer trials revealed a 50:1 LAK cell to DFTD ratio was protective in 13 of 13 mice while a ratio of 20:1 was not protective with 5 of 6 mice developing tumours. Lowering the number of DFTD target cells appeared to decrease the proportion of control mice which established DFTD tumours in a dose-dependent manner (Table 5-5).
Table 5-5. Summary table of LAK cell adoptive transfer trials
Summary Table
LAK cell : DFTD cell ratio Tumour development
≥ 50:1 0 of 13
mice developed tumours
≤ 20:1 5 of 6
mice developed tumours
1 x 104 DFTD cells only controls 2 of 4
mice developed tumours
5 x 104 DFTD cells only controls 7 of 11
mice developed tumours
1 x 106 DFTD cells only controls 5 of 6
5-17
5.3
Discussion
The first chapter revealed that DFTD cells are immunogenic and can be killed by the murine immune system. This chapter evaluates ways of stimulating Tasmanian devil lymphocytes into cytotoxic cells capable of killing DFTD cells. Adoptive cell transfer experiments were then used to evaluate if in vitro activated Tasmanian devil
lymphocytes could provide adoptive protection in vivo. However, the endangered status of the Tasmanian devil limited access for research purposes. As a
consequence a suitable alternative was required; hence the in vivo work was
conducted in NOD/SCID mice. As discussed in the second chapter, these mice had proven suitable for adoptive transfer using immune cells from BALB/c mice. In this chapter adoptive cell transfer from Tasmanian devils into NOD/SCID mice is evaluated.
5.3.1 Overcoming NK resistance of DFTD through LAK cell activation
Recent research has revealed that DFTD cells downregulate MHC expression (Siddle and Kaufman 2013) and thereby avoid immunosurveillance and destruction by MHC-restricted lymphocytes. The lack of MHC should make the DFTD cells targets for killing by non-MHC restricted lymphocytes such as NK cells (Siddle and Kaufman 2013). The development of tumours indicates that NK cells do not kill DFTD cells in vivo. It is likely that DFTD tumours are NK-resistant.
NK-resistant cancers are well documented with human patients. One way to overcome NK-resistance in humans involves in vitro activation of the patient’s lymphocytes through stimulation with cytokines or mitogens. This activates the cells to become lymphokine activated killer (LAK) cells, cytokine induced killer (CIK) cells or mitogen activated killer (MAK) cells respectively (Qian et al 2014). These cells have the capacity to kill NK resistant tumours in vivo when reintroduced to the patient.
The division into the three categories of LAK, CIK and MAK cells is artificial and not without some overlap. The term LAK cells is the original term from the 1980’s used to describe lymphocytes activated by cytokines (at that time referred to as
lymphokines) and cytotoxicity was attributed to NK cells activated by IL-2 (Grimm et al 1982, Herberman et al 1987). CIK cells is a term first appearing in the literature in
5-18 the 1990’s and then recently identified population of cells with both T cell and NK cell markers (NKT cells) were attributed as the main cytotoxic cells (Lu and Negrin
1994). MAK cells refer to lymphocytes activated by mitogens such as concanavalin A (Qian et al 2014). MAK cells include NKT cells, NK cells and monocytes activated by cytokines produced in response to mitogen stimulation converting them into killer cells (Qian et al 2014). Considering that the lymphocytes activated by these three methods are usually a mixed population sourced from PBMNC the effector cells should not be looked at in isolation but rather in synergy. It is possible to enrich specific populations of cells to ascertain the specific role of each. This could mislead rather than enlighten since each cell type produces cytokines that either promote or inhibit other cell types as a cascade of events. In this thesis the term LAK cell has been used to describe the population of Tasmanian devil lymphocytes stimulated with cytokines or mitogens.
When lymphocytes from peripheral blood were cultured with DFTD cells there was no evidence for cytotoxicity. As there is evidence for NK cells in peripheral blood (Brown et al 2011) the lack of cytotoxicity supports the concept that DFTD cells are NK resistant. But when peripheral blood lymphocytes were stimulated with either concanavalin A or Con A sup, the activated cells demonstrated a dose-dependent cytotoxic response. The significance of this observation is that activated cytotoxic cells have the capacity to kill DFTD cells. In chapter three DFTD cells were shown not to be resistant to apoptosis. Consequently NK resistance is due to a failure of recognition and subsequent activation of cytotoxic cells.
The stimulation of Tasmanian devil lymphocytes to become LAK cells was tested with different incubation times and concanavalin A concentrations. Stimulation with concanavalin A for 48 hours consistently induced cytotoxicity and 5 µg/ml was as equally effective as 25 µg/ml of concanavalin A. The lower concentration had the advantage that it had less toxic effects on lymphocytes. Hence 5 µg/ml was used in future experiments.
Lymphocytes can be directly activated by cytokines (Choi et al 2012, Qian et al 2014) such as those produced during concanavalin A stimulation. Once the
cytokines have been produced there is no continuing need for concanavalin A and the supernatant from the culture medium (Con A sup) can also promote activation,
5-19 even with the remaining concanavalin A inactivated (Fidler et al 1976, Palacios 1982). The addition of supernatant obtained from concanavalin A stimulated
lymphocytes (Con A sup) at a final concentration of 10% to the culture medium was sufficient to activate Tasmanian devil lymphocytes into LAK cells.
Consequently there were two means of activating Tasmanian devil lymphocytes into cytotoxic cells capable of killing DFTD cells. Concanavalin A allowed precise
conditions to be reproduced whereas different preparations of Con A sup varied between batches, most likely due to different levels of the cytokines produced. This source of variability added to inter-devil variability in the levels of cytotoxicity with the lymphocytes obtained from different Tasmanian devils. Inter-patient and inter-
experimental variability with LAK cells trials have also been reported in human trials (Qian et al 2014).
The ability to activate Tasmanian devil lymphocytes to kill DFTD cells is a significant milestone towards development of a treatment or vaccine against DFTD. It reveals that DFTD cells can be killed by in vitro activated PBMNC cells. It also highlights that effective cytotoxic cells can be extracted from blood. For devils to induce cytotoxicity following vaccination it would require cytotoxic cells to be present in secondary lymphoid organs such as spleen and lymph nodes. This was investigated with devils that had been euthanised for ethical reasons. Following activation, cytotoxicity was identified in cells extracted from lymph nodes and the spleen. This important finding reveals that Tasmanian devils have a competent immune system that contains cells with the capacity to kill DFTD cells in secondary lymphoid organs.
The failure of their immune recognition (due to MHC downregulation) can be overcome by activated lymphocytes in vitro. LAK and MAK cell therapy is when in vitro activated lymphocytes are introduced into a patient to target NK resistant tumours. This may be a suitable approach to overcome the lack of recognition of DFTD cells by Tasmanian devils. To evaluate the efficacy of these approaches a reliable supply of lymphocytes was required for experiments. As a consequence, evaluation of different lymphocyte sources was undertaken. The level of killing by PBMNC was as effective as those obtained from the spleen and lymph nodes. Any of these sources would be suitable to perform further cytotoxicity experiments. For ethical reasons spleens and lymph nodes could only be obtained at necropsy.
5-20 Therefore PBMNC are the preferred source for lymphocytes but spleens and lymph nodes can be collected opportunistically at necropsy to augment supply.
A large number of lymphocytes can be obtained at necropsy from the blood, spleen and lymph nodes. It is not practical to use all of these cells at time of harvest. A possible solution would be cryopreserving cells for later use. It was unknown if Tasmanian devil lymphocytes could be cryopreserved, thawed and remain
functional. As a consequence this was evaluated using lymphocytes sourced from blood, spleen and lymph nodes. Tasmanian devil lymphocytes samples were
cryopreserved, thawed and tested for viability with dye exclusion. Other laboratories report between 50 to 70% recovery rate of frozen cells from humans (Jewett et al 1976, Kleeberger et al 1999) and similar results were obtained with the Tasmanian devil lymphocytes. Noteworthy is that platelet contamination negatively impacts human lymphocyte cryopreservation (Strong et al 1975) and Tasmanian devil lymphocytes have proven difficult to isolate without red blood cell and platelet contamination.
Cryopreservation of Tasmanian devil lymphocytes was possible and would provide a readily accessible source for experiments. Cryopreserved samples can be best for longitudinal studies (Jewett et al 1976) and frozen human lymphocytes demonstrate all the characteristics of fresh cells (Strong et al 1975) and can be preserved for at least 12 years (Kleeberger et al 1999). This has facilitated the application of newly developed assays to specimens in repositories to measure markers not available at the time of collection (Kleeberger et al 1999). Of important significance to the LAK and MAK cell therapy with DFTD is the observation that cryopreserved human cells have decreased responses to some specific antigens but no significant difference in response to concanavalin A (Jewett et al 1976).
When human samples are cryopreserved there is some shift in the subpopulations recovered (Jewett et al 1976, Strong et al 1975). The impact this would have on cytotoxic cells in Tasmanian devil lymphocytes was unknown. Consequently,
functional cytotoxicity assays were used to compare thawed cells and fresh cells for their killing of DFTD cells. The observation that following activation thawed
Tasmanian devil lymphocytes killed DFTD cells equally well as fresh lymphocytes reveals the cytotoxic cells and their function were not impacted by cryopreservation.