Tregs that accumulate in the encephalomyocarditis virus-infected mouse brain

232 Pages·2017·7.47 MB·English

Tregs that accumulate in the encephalomyocarditis virus-infected mouse brain:
Origin, compartmentalization, function, and gene signature
Sarah Puhr

Submitted in partial fulfillment
of the requirements
for the degree of Doctor of Philosophy
under the Executive Committee

of the Graduate School of Arts and Sciences

COLUMBIA UNIVERSITY
2017
© 2017
Sarah Puhr
All Rights Reserved
ABSTRACT
Tregs that accumulate in the encephalomyocarditis virus-infected mouse brain:
Origin, compartmentalization, function, and gene signature
Sarah Puhr
It is well recognized that regulatory T cells (Tregs) are immunosuppressive, by
which they prevent systemic autoimmunity throughout life. Beyond this stereotypical
function, however, a growing body of evidence demonstrates that Tregs in distinct
tissues, including the visceral adipose tissue, dystrophic muscle, the flu-infected lung,
and wounded skin can acquire unique functions directed by their local environment.
Tregs in these tissues can employ a wide variety of mechanisms to accumulate and
acquire tissue-specific function, including conversion from conventional T cells,
canonical T cell receptor (TCR)-dependent expansion and non-canonical, TCR-
independent, cytokine-dependent expansion. Intriguingly, the niche-specific function of
tissue Tregs can be independent of, and mutually exclusive of, their immunosuppressive
capacity. Together, this recent literature reveals that Tregs can accumulate in discrete
tissue sites through non-canonical mechanisms, and in response to niche-specific cues
can acquire distinct functions, which distinguish them from their peripheral, lymphoid
Treg counterparts. Other tissue Treg populations remain to be identified and
characterized. Moreover, it is unknown whether other tissue Tregs rely on non-canonical
mechanisms of accumulation, and exhibit functions distinct from the typical Treg
immunosuppressive role.
Tregs are known to accumulate in the CNS during infection, injury and
inflammation. The CNS is an organ with distinctive architecture that maintains a
regulated interaction with the peripheral immune system due to its critical function and
poor regenerative capacity. While it is known that Tregs broadly protect against
excessive tissue pathology in the diseased CNS, the origin, localization, function,
mechanism of accumulation, and gene signature of CNS-infiltrating Tregs have not been
studied, likely due to the challenge of isolating these rare cells and distinguishing them
from circulating cells left over after perfusion.
Here, we establish a safe model of CNS infection using encephalomyocarditis
virus and employ a series of methods to locate, monitor and isolate CNS-infiltrating
Tregs free from contamination from the circulation. We show that a distinct population of
thymus-derived Tregs accumulates within the cerebrospinal fluid (CSF) of the EMCV-
infected CNS, independently of lymph node priming. Tregs function in this unique niche
to limit excessive tissue pathology. While CNS Tregs maintain expression of core Treg
signature genes, including FoxP3, their global transcriptome is more similar to that of
conventional T cells (Tcons) harvested from the infected CNS than to that of peripheral
Tregs. Bioinformatics analysis reveals that genes shared by CNS Tcons and CNS Tregs
are also shared by Tregs and Tcons from injured muscle and from the visceral adipose
tissue of aged mice, indicating that tissue inflammation and injury, rather than viral
infection per se, contribute to CNS Treg accumulation, function and phenotype.
Additionally, we observe that CNS Treg accumulation during infection is
associated with a simultaneous increase in meningeal/choroid plexus dendritic cells
(m/chDCs), which are professional antigen presenting cells that localize to the gates of
the CNS. Splenic cDC and peripheral lymphoid Treg homeostasis are linked, and both
populations can be artificially increased by treatment with the DC-poietin and adjuvant,
Ftlt3L. Therefore, we hypothesized that CNS Tregs and m/chDCs may also be linked and
could also be manipulated by Flt3L treatment. Indeed, treatment with Flt3L in
conjunction with EMCV infection results in enhanced CNS Treg and m/chDC
accumulation, independent of Flt3 receptor expression on Tregs. In an effort to determine
if dendritic cells mediate CNS Treg increase during infection, we turned to a DC-ablative
mouse model in which all CD11c-expressing cells express the catalytic subunit of
diphtheria toxin and are depleted. Surprisingly, while splenic cDCs are completely
abrogated in these mice, a portion of m/chDCs persists, unaffected. Moreover, CNS
Tregs accumulate normally in these mice during infection. This data suggests an
unappreciated heterogeneity in m/chDCs, and indicates that those that remain unaffected
in these mice may mediate CNS Treg accumulation during infection. While
characterizing m/chDC heterogeneity, we found that m/chDCs comprise three distinct
subsets with unknown potential. Whereas m/chDCs were previously considered to be a
homogeneous, CD45hiB220-CD11c+MHCII+ population, we have found them to contain
three subsets, distinguishable by IRF8 and FcR-γ expression. This finding paves the way
for further study of the origin, localization, and division of labor between these three
m/chDC subsets.
In summary, our studies clarify the distinct compartmentalization, lymph node-
independent accumulation, and inflammation-associated gene signature of CNS Tregs.
Most importantly, these findings have implications for neuro-immune cross-talk,
particularly at the interface of the CSF and brain parenchyma. That is, neural progenitors
extend their apical domains into the CSF of the ventricles, and therefore may be subject
to regulation by CSF-borne Tregs. Further, while many studies have focused on the
differences between tissue Treg subsets, we find a core set of genes expressed by CNS
Tregs, injured muscle Tregs and VAT Tregs. This data suggests that common
mechanisms may be used for therapeutic manipulation of these cells
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TABLE OF CONTENTS
List of Figures ---------------------------------------------------------------------------------------- iv
List of Tables ---------------------------------------------------------------------------------------- vii

Chapter I. Introduction ---------------------------------------------------------------------------- 1
1. Regulatory T cells: seminal findings revealed their
canonical immunosuppressive role ---------------------------------------------------------------- 2
2. Tissue Tregs, directed by their niche, can perform functions independent and mutually
exclusive of their canonical immunosuppressive role ----------------------------------------- 14
3. Tregs accumulate in the central nervous system—a unique organ with distinct
architecture that counteracts the passage of immune cells in the steady state ------------- 22
4. Tregs accumulate in the central nervous system during infection, inflammation and
injury but their origin, localization, mechanism of accumulation, and gene signature are
undefined --------------------------------------------------------------------------------------------- 32
5. Encephalomyocarditis virus exhibits CNS tropism and can result in lethal hind-limb
paralysis mediated by CD4+ T cells ------------------------------------------------------------- 35
6. Summary and Research Goals ----------------------------------------------------------------- 40

Chapter II: Materials and Methods ----------------------------------------------------------- 42
1. Mice ------------------------------------------------------------------------------------------------ 42
2. Virus preparation and infection ---------------------------------------------------------------- 42
3. Plaque assays ------------------------------------------------------------------------------------- 44
4. Antibodies used ---------------------------------------------------------------------------------- 45
i
5. Cell preparation, flow cytometry and sorting ------------------------------------------------ 46
6. CSF isolation and staining ---------------------------------------------------------------------- 49
7. Adoptive transfers ------------------------------------------------------------------------------- 50
8. Treg Depletion ----------------------------------------------------------------------------------- 50
9. Flt3L treatment ----------------------------------------------------------------------------------- 51
10. Histology and immunofluorescence microscopy ------------------------------------------ 51
11. Microarray --------------------------------------------------------------------------------------- 53
12. RNAseq ------------------------------------------------------------------------------------------ 53
13. Statistics ----------------------------------------------------------------------------------------- 55

Chapter III. Elucidating the function, origin, localization, mechanism of
accumulation and gene signature of regulatory T cells that accumulate in the CNS
during EMCV infection -------------------------------------------------------------------------- 57
1. Introduction --------------------------------------------------------------------------------------- 58
2. Results --------------------------------------------------------------------------------------------- 61
3. Discussion ---------------------------------------------------------------------------------------- 83
4. Figures --------------------------------------------------------------------------------------------- 85
5. Tables --------------------------------------------------------------------------------------------- 121

Chapter IV. Difficulty in ablating meningeal/choroid plexus dendritic cells while
studying their role in EMCV-induced CNS Treg expansion reveals unappreciated
heterogeneity in m/chDCs ---------------------------------------------------------------------- 130
1. Introduction -------------------------------------------------------------------------------------- 131
ii
2. Results -------------------------------------------------------------------------------------------- 134
3. Discussion --------------------------------------------------------------------------------------- 140
4. Figures -------------------------------------------------------------------------------------------- 143

Chapter V. Discussion --------------------------------------------------------------------------- 161
1. Results Summary ------------------------------------------------------------------------------- 161
2. The potential for CNS Treg and neuronal cross-talk at the CSF barrier ---------------- 162
3. Does lymph node-independent CNS Treg accumulation during viral infection represent
a non-canonical mechanism of accumulation? ------------------------------------------------ 165
4. CNS T cells share a gene signature with T cells entering inflamed tissue sites, but
uniquely express the thyroxine binding protein, transthyretin ------------------------------ 168
5. M/chDCs are a heterogeneous population with possible roles in mediating
CNS Treg increase during infection, providing a tool for manipulating Tregs
during CNS disease -------------------------------------------------------------------------------- 171

References ------------------------------------------------------------------------------------------ 176

Appendix: Generating a recombinant EMCV expressing a FLAG-tagged surrogate
antigen ---------------------------------------------------------------------------------------------- 199
1. Introduction and Approach -------------------------------------------------------------------- 199
2. Results -------------------------------------------------------------------------------------------- 200
3. Discussion --------------------------------------------------------------------------------------- 203
4. Figures -------------------------------------------------------------------------------------------- 205
iii