4
Discussion
The porcine T-cell response against ASFV infection is largely not
understood. Moreover, knowledge about the responses of susceptibleSuidae subspecies outside of Africa, domestic pigs (Sus
scrofa domesticus ) and wild boar (Sus scrofa scrofa ), is scarce.
Similar to our first approach to analyze these differences upon
infection with the highly virulent ASFV strain “Armenia08” , we used a
multicolor flow cytometry platform to investigate T-cell responses in
domestic pigs and wild boar after infection with the moderately virulent
ASFV isolate, “Estonia2014”.
The most fundamental characteristics of an ongoing immune response are
local or systemic alterations in the composition of leukocytes in
tissues affected by disease. As one of the few known parameters of
protection against ASFV infection, the importance of (cytotoxic)
CD8α+ lymphocytes has been demonstrated, although to a
limited extent only. It has been shown that isolated porcine PBMC, afterin vivo priming with virulent ASFV, were able to specifically
lyse ASFV-infected cells in vitro . This was a first indication
of virus-specific cytotoxicity. However, PBMCs were not differentiated
and protection was not investigated. SLA I- and CD8-dependent lysis of
ASFV-infected target cells by PBMCs from ASFV-immune minipigs and
specific lysis of isolated CD8α+ but not
CD4+ T cells has also been shown. In another study,
antibody-dependent depletion of CD8α+ cells in
vivo in domestic pigs primed with the low virulent ASFV strain
“OUR/T88/3” resulted in loss of protection after homologous challenge
with the virulent ASFV strain “OUR/T88/1” . We could show that the
CD8α response in wild boar and domestic pigs during infection with
moderately virulent ASFV is based primarily on increases of
CD4–/CD8α+ and to a lesser extent
of CD4+/ CD8α+ (DP) T cells.
Interestingly, in our previous study with the highly virulent ASFV
“Armenia08”, the CD8α response was primarily based on DP T cells .
CD4+/ CD8α+ DP T cells are often
described to possess memory functions . ASFV-specific memory responses
can be excluded during experimental ASFV infection with naïve pigs.
However, ASFV-specific responses might play a role in field infections
since serological evidence for previous ASFV infections was found in
hunted animals . On the other hand, DP memory T cells in the spleen
might be activated independent of their cognate antigen by release of
IL‑15 or IL‑18 . Porcine DP T cells are also described to exhibit
effector functions, like cytotoxic responses or cytokine production .
However, in contrast to CD8αα+ or
CD8αβ+ αβ T cells, we only found proliferating DP T
cells in the spleen but not in other tissues. Moreover, the pronounced
loss of perforin in other cytotoxic T-cell populations was not found in
DP T cells. This might indicate that DP T cells are orchestrators of
systemic responses but do not take part in antiviral responses in
disease-affected tissue during moderately virulent ASFV infection.
Pigs belong to a group of mammals with relatively high frequencies of γδ
T cells. They can exert effector functions like cytokine production and
cytotoxicity, and are even able to present antigens to other lymphocytes
. The main effector population is characterized as
CD2+/CD8α+ . In the present study,
we found pronounced increases of effector γδ T-cell frequencies in
spleen, lung, and liver of infected wild boar but not domestic pigs.
Moreover, we detected T‑bet-dependent activation of γδ T cells in wild
boar only. This is in line with our previous findings during highly
virulent ASFV infection, where wild boar were found to have a
considerably stronger bias for γδ T-cell responses . This indicates a
profound dissimilarity in the antiviral responses of both subspecies and
might give an explanation for their different disease severity and
survival. Of note, this is in contrast to previous findings, where
higher frequencies of circulating γδ T cells correlated with increased
survival of infection with moderately virulent ASFV, independent of age
or virus dose . There might be some explanations for this discrepancy.
First, we detected increased lethality and heightened frequencies of
effector γδ T cells in wild boar, while the aforementioned study used
domestic pigs. While we were also not able to detect changes of γδ
T-cell frequencies in domestic pigs, this might be caused by different
ASFV strains used. Finally, the numbers of survivors that showed
correlations with γδ T-cell levels in the study by Post et al. were
relatively small. This underlines the need for in-depth research not
only during ASFV infection in general but also for the differences
between wild boar and domestic pigs. Moreover, since infection of
professional antigen-presenting cells alter their function , it would be
of interest to investigate whether γδ T cells take part in the antigen
presentation during ASFV infection.
We found regulatory T cells (Tregs) in both subspecies but higher
frequencies in wild boar. The role of Tregs during ASFV infection is
largely unexplored. However, previous studies showed that Tregs might
present a way of viral immune evasion because they were able to inhibit
specifically antiviral responses . Higher percentages of Tregs in wild
boar might therefore be an explanation for their higher disease burden
in this study and lethality previously . In a parallel study by Sehlet al. using histopathology from tissues of trial 2 of this
study, domestic pigs but not wild boar showed lymphohistiocytic
interstitial pneumonia even 10 dpi . This might be a sign for prolonged
pro-inflammatory responses in domestic pigs in contrast to wild boar.
This is in line with our findings of higher Treg frequencies in wild
boar. Moreover, this indicates that pro-inflammatory responses are able
to counteract ASFV infection, as long as they are not downregulated too
early.
A porcine T-cell population that is still not well understood is
invariant Natural Killer T (iNKT) cells. We could previously show that
iNKT-cell frequencies significantly increased in some tissues during
infection with highly virulent ASFV . Although at the time we were not
able to investigate effector mechanisms or surface markers on iNKT cellsex vivo , our study provided first evidence that iNKT cells
participate in the antiviral response during ASFV infection. The fact,
that we were unable to find changes in the general iNKT-cell frequency
might be explained by the less virulent ASFV strain in this study.
Nevertheless, activation of iNKT cells was shown by significantly
increased frequencies of ICOS+ iNKT cells. ICOS is an
essential protein for iNKT-cell activation, homeostasis, and survival .
Some studies correlated ICOS expression on iNKT cells with
pro-inflammatory Th1 responses , while others described it as a marker
of effector iNKT cells . Increased expression of CD8α and CD4, as
previously established markers of maturation of porcine iNKT cells ,
underlines these findings. Interestingly, a role for NKT cells has
previously been suggested, as
CD3+/CD4−/CD8α+/CD5±/CD6−/CD11b+/CD16+cells expanded after in vitro stimulation of porcine PBMC with
ASFV . However, even though we and others could show that the phenotype
of iNKT cells differs from that finding , the significant alterations in
iNKT-cell frequency in our first study and our findings in this study
support the notion that iNKT cells take part in the antiviral response
against ASFV.
Besides analysis of the cellular composition of leukocytes in the
investigated tissues, effector functions are also pivotal to understand
the underlining immune mechanisms. Perforin is one of the major lytic
molecules used by cytotoxic lymphocytes to kill target cells . Instead
of direct cell lysis, cytotoxic lymphocytes can also induce apoptosis in
their target cells by death receptor-mediated pathways using Fas ligand
(FasL) or TRAIL . The significant and partially complete loss of
perforin 4 to 5 dpi in this study resembled the observed loss of
perforin we found during infection with the highly virulent ASFV strain
“Armenia08” . However, the perforin decrease was more pronounced
during infection with highly virulent ASFV, especially on a systemic
level, i.e. in the spleen. There are various explanations for the
perforin loss observed in both studies. Perforin-mediated killing is
thought to be the major pathway in the early cytotoxic response but can
be switched to Fas/FasL-mediated apoptosis induction with a complete
loss of perforin expression on RNA and protein level during the course
of infection . An effector molecule switch might be an explanation for
the observations in our study, however, this is not directly detectable
because antibodies against porcine FasL are still missing. Still, there
are some lines of evidence suggesting this might be the case. Expression
of viral homologues of the mammalian anti-apoptotic protein Bcl-2 and
also prevention of apoptosis by these viral homologues has been shown
for ASFV strains . Bcl-2 is also known to preferentially inhibit
perforin-mediated apoptosis but less Fas/FasL-mediated apoptosis,
depending on the cellular target . A switch from perforin-mediated to
Fas/FasL-mediated cytotoxic responses might therefore be beneficial and
protective and would be in line with the lower disease severity and
heightened survival of domestic pigs. Wild boar, in contrast, had higher
levels of perforin+ lymphocytes on average, indicating
that they might not have switched the cytotoxic pathway or at least not
to the extent that domestic pigs did. Given that inhibition of perforin
has been shown to protect from tissue damage during viral hepatitis ,
this might also be an explanation for the more severe inflammation and
tissue degradation in the liver of infected wild boar . On the other
hand, it cannot be excluded that we missed newly synthesized and
immediately secreted perforin, which is not detected by antibody clone
dG9 used in this study . However, missing detection due to immediate
secretion would still hint to a strong cytotoxic response. In this case,
the cytotoxic response would have been higher in domestic pigs because
the perforin loss was detected earlier and more pronounced than in wild
boar. Therefore, it can be hypothesized that the more pronounced and
earlier response in domestic pigs was beneficial and protective at least
during infection with moderately virulent ASFV. Wild boar, in contrast,
might not have been able to counter the infection because of their
impaired response, eventually leading to death as observed in previous
studies . Which explanation holds true, switch of cytotoxic pathways or
earlier and stronger cytotoxic response, has to be investigated in
future studies. It still has to be kept in mind that cytotoxic responses
are not only beneficial but might also cause immunopathology and thus,
contribute to disease burden .
In summary, we described the first comparative analysis of immune
responses of wild boar and domestic pigs during moderately virulent ASFV
infection. While more severe in wild boar, domestic pigs showed signs of
moderate disease and recovery of all animals. Overall, we found
comparable courses of immunity in both subspecies. Both developed a
heavily CD8α+-biased response with proliferation of
CD8αα+ and CD8αβ+ cells. However,
although their αβ T-cell responses were largely similar, wild boar
developed a more pronounced effector γδ T-cell response. We also found
only small signs of T-bet-dependent activation predominately in lungs
and liver of wild boar but none in domestic pigs. Moreover, we found a
distinct loss of perforin in cytotoxic T cells in domestic pigs and to a
lesser extent also in wild boar, similar to previous results during
infection with highly virulent ASFV “Armenia08”. Tregs appeared in
higher levels in wild boar. Finally, we were able show the first
description of functional iNKT-cell responses during ASFV infection.
With this data, our study paves the way for further in-depth analyses of
porcine immunity towards ASFV.