ABSTRACT: extensively investigated and only recently garnered attention.

ABSTRACT: A hallmark feature of follicular dendritic cells (FDCs)
is their ability to retain antigens and virions for a prolonged duration. FDCs
in the cervical lymph nodes (CLNs) are particularly relevant in elucidating
Human Immunodeficiency Virus (HIV) – 1 infection of the central nervous system
(CNS), which are the cerebrospinal fluid (CSF) draining lymph nodes (LNs). We
postulate that any potential HIV egress from the CNS is likely reflected in the
CLN viral archive. The CLN FDC viral reservoir like the other HIV reservoirs
would contribute to both low-level viremia and viral resurgence upon cessation
or failure of combined antiretroviral therapy (cART). Besides prolonged virion
retention on CLN FDCs, the suboptimal penetration of cART similar to other LNs
is another factor contributing to establishing this viral reservoir. Unlike the
peripheral LNs and FDCs within them, the CLN FDCs have not been extensively
investigated and only recently garnered attention. This interest in CLN FDCs
has been driven by detailed elucidation of the meningeal lymphatic system. As
the CSF drains through the meningeal lymphatics and nasal lymphatics via the
cribriform plate, CLN FDCs may acquire HIV after capturing them from T cells,
antigen presenting cells or virions in the CSF. In this review, we outline the
underlying mechanisms of viral accumulation on CLN FDCs and the potential
impact on viral resurgence or achieving a cure for HIV infection.

 

 

INTRODUCTION

The cervical lymph
nodes (CLNs) are a group of lymph nodes (LNs) in the neck region that are
located adjacent to the cervical region of the spinal cord and in close
proximity to the sternocleidomastoid muscle. Depending on the location of the
CLNs, they may be classified as, a) Superficial anterior CLNs; b) Superficial posterior
CLNs; c) Superior deep CLNs; or d) Inferior deep CLNs. The glymphatics and
meningeal lymphatic system connects the central nervous system with the CLNs 1; 2; 3; 4; 5; 6; 7; 8. More
importantly, T cells and antigen presenting cells (APCs) migrate with the
cerebrospinal fluid (CSF) as it drains along the nasal lymphatic path through
the cribriform plate and eventually access the CLNs (Figure 1A) 1. There is a network
of stromal cells within LNs and follicular dendritic cells (FDCs) are one of those
stromal cell types. FDCs were first identified as “antigen retaining reticular
cells” 9. Subsequently, FDCs have been
recognized for their unique ability to retain antigens for a prolonged duration
10. This property of FDCs is critical
for several immune functions, including germinal center (GC) formation and
long-term immune memory. FDCs develop from perivascular precursors of stromal
cell origin, which are seeded throughout the body. Their maturation requires
lymphotoxin and tumor necrosis factor (TNF) signaling via B cells 11. FDCs are found within the B-cell
follicles (BCFs) and GCs develop in the BCFs during T cell-dependent antibody
response 12. As the BCFs mature into GCs, FDCs
migrate into the light zone (Figure 1B).

Antigen
acquisition, processing and retention by FDCs impact immune response. FDCs
possess a unique ability to retain antigens from months to years 13. However, there
is inadequate experimental data demonstrating sustained antigen retention by
FDCs. In fact, most predictions are merely extrapolations based on the decay
rates. In addition to antigen retention, FDCs possess the unique ability to
retain Human Immunodeficiency Virus (HIV) – 1 (Figure 1C) 14 as the  FDC microenvironment is highly conducive for
active HIV infection 15. Some studies suggest combined
antiretroviral therapy (cART) can result in clearance of FDC-associated virus 16. On the other
hand, other studies suggest that cART does not diminish FDC HIV reservoir 17. Nonetheless, FDCs
are considered a lymphoid tissue viral reservoir responsible for low-level
viremia 18 and a source of resurgent virus upon
cessation of cART 19. Of note, FDC
retain HIV with divergent viral archive 20. Similar to the
FDCs in the peripheral LNs, the CLN FDCs are also a HIV/SIV viral reservoir. We
postulate that the FDC viral reservoir in the CSF draining CLNs represents HIV
egressing from the CNS.  In this review,
we will discuss how these CSF draining CLNs may acquire, accumulate and
transmit HIV. In addition, we present some recent advances in FDC-related HIV
research (Table 1).

 

CNS and CLN FDCs are important
components of HIV neuroimmunopathogenesis

HIV
enters the CNS very early during infection 21; 22. It
is believed that transmigration of infected monocytes/macrophages is the
predominant contributor of HIV CNS invasion 23. These HIV-infected
monocytes reside as long-lived myeloid cells within the CNS. Interestingly, HIV
infection of the CNS occurs within a few days to weeks based on studies with
SIV/SHIV macaque models 24. Once HIV enters
the CNS, it cannot be eliminated from infected
monocytes or microglia as these cells have a long lifespan and low turnover 25. The monocytes and
microglia support latent HIV infection 26; 27; 28; 29, thereby
establishing a viral reservoir with suboptimal penetration of cART 30; 31 across the blood
brain barrier (BBB).

Besides the CNS,
HIV persists in the LNs, spleen, gut-associated lymphoid tissue (GALT),
reproductive organs and lungs 32; 33. LNs are a known
reservoir of persistent HIV/SIV viral infection under suppressive cART 34; 35; 36; 37; 38; 39. Several unique
characteristics of the LNs contribute to HIV’s ability to persist in this
tissue. For example, LN tissue has a slower decay rate than in the peripheral
blood 36. Additionally,
the LN follicles contain FDCs that capture HIV virions on their cell surface in
immune complexes 40. FDCs in the peripheral LNs have
been characterized as another viral reservoir site 18; 41; 42. Of note, HIV
susceptible TFH cells are located in close proximity to FDCs, which within peripheral LNs have been shown to
trap virions in their native non-degraded state for months to years 43; 44; 45; 46 with a half-life of
approximately 2-3 months 47; 48. While FDC-trapped
virus does not replicate or evolve; however, it can infect nearby trafficking
cells 48; 49. Even during cART, replicating virus
persists and replenishes trapped stores of HIV 19; 50.

Until the
description of glymphatics and functional meningeal lymphatic system, CNS was
considered to be immune-privileged 3. With the elucidation of structural
and functional features of this CNS associated lymphatic system 1; 4; 51, it is now well accepted
that CNS undergoes constant immune surveillance in the meningeal compartment.
The meningeal lymphatic system, along with glymphatics presents a unique
connection between the CNS and CLNs. HIV may pass with CSF as virions, infected
T cells or APCs through the cribriform plate along the nasal lymphatic pathway
and access the CLNs. Lymph entering the CLNs through the afferent lymphatics is
channeled through the subscapular sinus (SCS) into the medulla. The
fibroblastic reticular cells (FRCs) conduits access afferent lymph and traverse
BCFs, where they intersect FDCs. FRC conduits continue into the cortex where
they terminate at high endothelial vessels or the medulla 52.

Recent focus on
elucidating the meningeal lymphatic system has tremendously enhanced our
understanding of immune surveillance in the CNS 1; 2; 3; 4; 5; 6; 7; 51. Lymphatic vessels were first
identified in the dura mater of rats 5. In some studies of the
meningeal lymphatics 1, the system has been
described as part of the CNS, while others have opposing conclusions 7. This is not surprising since lymphatic vessels are component of the
surrounding connective tissue that are included in the CNS. However, lymphatic
vessels can absorb CSF from adjacent subarachnoid space and brain interstitial
fluid via the glymphatics. Further detailed investigations are required to fully
understand the functionality of CSF drainage and how it might impact HIV
accumulation within CLNs.  

Circulating
conventional DCs (cDCs) are known to traffic into the CNS in response to
neuroinflammation 53; 54; 55; 56; 57; 58; 59; 60 during HIV/SIV
infection 61. Within
CNS,
cDCs act as both “carrier and bearer”
of HIV and contribute both to neuropathology as well as CNS reservoir. Recent
studies suggest that cDCs may capture HIV within the CNS and deliver it to
different compartments of CLNs including FDCs 43; 44; 62; 63. The CLN FDCs
would create a viral repository where virus can remain bound for prolonged
duration 46. The immune cell retrograde
transport studies 1; 4; 64; 65 provide clues
that cDCs upon encountering HIV virions within brain would migrate along the
meningeal lymphatic vessels to draining LNs (CLNs, near the brain stem) via
glymphatics delivering HIV particles to different compartments of CLNs
including FDCs as shown for peripheral LNs 43; 44; 62. It is important
to note that CLNs are the major site for systemic activation of CNS-specific T
cells. They receive input from the CNS in the form of antigens and cDCs 66. Within CLNs, HIV
viral particles could be transmitted to CD4+ T cells or trapped in FDCs, which
stabilize and protect HIV thus providing a long-term reservoir of infectious
HIV 18; 34; 36; 67. In addition,
FDCs activate CD4+ T cells within GCs and increase virus production in these
cells even in the presence of cART 42; 68; 69; 70; 71. Given the recent
detailed description of the functional meningeal lymphatic system 1, it is both
timely and significant to assess the involvement of deep CLNs in HIV neuropathogenesis.
Of note, additional mechanistic studies are required to determine if the CLN
FDC reservoir is an archive of CNS egressing virus.

 

Key Cellular Players in the CLN

CLNs like other LNs play a central role in the
development of adaptive immunity against pathogens and particularly the
generation of antigen-specific B cell responses in specialized areas within
GCs. LN pathology was recognized as an important consequence of HIV infection
since the beginning of the epidemic. Investigation into the structural and
functional alterations induced by HIV and SIV has further cemented the central
role that lymphoid tissue plays in HIV/SIV pathogenesis. The coexistence of
constant local inflammation, altered tissue architecture, and relative
exclusion of virus-specific CD8 T cells from GCs create a unique environment
for the virus evolution and establishment of viral reservoir in specific GC
cells, namely T-follicular helper CD4 T cells (TFH). A better
understanding of the biology of immune cells in HIV-infected LNs is a
prerequisite to attaining complete viral eradication 72.

FDCs are a subset
of DCs that are essential for GC formation and production of various types of
antibodies 73. They reside in
secondary lymphoid tissues such as spleen, tonsils, LNs, and follicles that
appear at extra-nodal sites 74. GCs of secondary lymphoid tissues
are composed of activated B cells, TFH cells and FDCs. In the GC
microenvironment, activated B cells communicate with FDCs by interacting with
an antigen on their surface and then present this antigen to TFH
cells. FDCs can select for B cells to re-enter the GC or exit with the help of
TFH cells 13. FDCs have a
unique ability to retain immune complexes on their dendritic processes. These
immune complexes consist of antigen-antibody complexes 75, which can retain infectious virions
for several months even in the presence of neutralizing antibodies or under
cART 34. FDCs interact
with TFH cells in GCs and these cells serve as a reservoir of
infectious virus. Surrounding GC T cells become highly susceptible to infection
with HIV X4 isolates 69. HIV production
increases to two-fold when viral particles are transferred from FDCs to
susceptible CD4+ T cells 42. FDCs can secrete inflammatory
cytokines 76 including tumor
necrosis factor alpha (TNF-a) and thereby contribute to enhanced transcription
in the LNs 42. Data has been generated to
understand the interactions of FDCs with their GC counterparts. However, underlying
mechanisms behind FDC reservoirs remain unclear and require further studies. Several
studies in model systems indicate that the HIV virions on FDCs remain
infectious 37. Specifically,
Fletcher et al. demonstrated that HIV virions in immune complexes on murine
FDCs remain infectious ex vivo for up
to nine months after being captured by FDCs 34. This
characteristic of the FDCs is particularly interesting because most of the
identified reservoirs of persistent virus are found in the integrated pro-viral
stage of the HIV replication cycle. It is important to note that current
approaches to eliminate persistent HIV have largely focused on elimination of
HIV pro-viral DNA. While the HIV/SIV lymphoid reservoir has been
well-characterized utilizing RNAscope and DNAscope methodologies 77, however the CLN
FDC HIV reservoir has only recently been characterized 40 and warrants further studies to
fully define.

TFH are a subset of CD4 T
lymphocytes. TFH play a key role in B-cell differentiation.  TFH cells assist B cells in the
production of antigen-specific antibodies. They are essential for memory B cell
activation, survival, and differentiation. Restriction of productive SIV
replication to these TFH cells in elite controller macaques and not
typical progressors has been demonstrated 78.
During HIV infection, cellular interactions
between FDCs, GC B-cells and TFH cells result in reservoir
establishment. TFH associated replication competent virus may be the
source of resurgent HIV after cART interruption or failure. As such, TFH
are increasingly recognized as another major CLN associated reservoir of HIV
infection 79; 80.
However, mechanisms by which these cells get infected remain unclear. TFH
express very little CCR5 and in macaque studies it has been shown that TFH
lacking CCR5 cells can be infected in
vivo with CCR5-tropic SIV 81; 82.
In both humans and macaques, TFH infection is almost certainly resulting
from infection of CCR5-expressing pre-TFH population 83.
Accelerating the TFH responses early during HIV/SIV infection is a
promising immunotherapeutic approach. However, because TFH cells are
infected during the HIV/SIV infection and represent a reservoir, this may
interfere with HIV vaccine strategy. Thus, TFH represent both good
and bad guys during HIV/SIV infection 84.

In cART-naïve as well as treated
individuals, TFH and GC B cells are elevated. In addition, there is
a direct correlation with the activated T-cell population in LNs 85.
In absence of cART, during chronic HIV infection, viral replication is
concentrated in secondary lymphoid follicles (SLF). TFH cells have
been shown to be highly permissive to HIV within SLF. They are the source of
replication competent HIV during latency 86. HIV vaccines are not strong inducers of neutralizing antibodies.
However, in a study of rhesus macaques immunized with HIV envelope glycoprotein
trimer there was a substantial production of HIV neutralizing antibodies 87. The high antibody titers had a strong correlation to GC B cells and TFH  87. These observations highlight the need to study more details of LNs,
since in the past HIV reservoir studies have frequently focused on peripheral
blood.

            Another
subset of T cells in SLF, follicular regulatory T (TFR) cells have been
described 88.
These cells share some phenotypic characteristics with TFH cells. Of
note, studies that demonstrated TFH permissiveness to HIV infection
also included TFR 89.
TFR express greater levels of CCR5 and CD4. They also support higher frequency
of viral replication. Expression of Ki67, a marker of proliferative capacity
appears to correlate with viral replication in these cells. As such, TFR differ
from TFH in their susceptibility to R5 HIV infection 89. Furthermore, recently, Natural killer (NK) cells have also
been shown to migrate into the LNs. The role of NK cells in LNs is not clear.
However, studies in African green monkeys demonstrated that these cells enter
and persist within the LNs. Their depletion with IL-15, resulted in an increase
in viral replication thereby suggesting a key role for NK cells to establish
and maintain this reservoir 90.

 

Eradication of FDC viral reservoir

A significant challenge to HIV
eradication is the elimination of viral reservoirs in GC TFH cells. GCs
are considered to be immune privileged for antiviral CD8+ T cells, however unselected
CD8+ T cells engineered to express CXCR5 directs them to viral sanctuaries 91.
CXCR5 expressing cytotoxic T cells are able to selectively enter BCFs and
eradicate infected TFH and B cells 92.
Certain population of SIV-specific CD8+ T cells expresses CXCR5 (C-X-C
chemokine receptor type 5, a chemokine receptor required for homing to GCs) and
expands in LNs following pathogenic SIV infection in a cohort of vaccinated
macaques 93.
Animals that exhibited greater control of SIV replication had a greater
expansion of these cells. The CXCR5+ SIV-specific CD8 T cells demonstrated
enhanced polyfunctionality, restricted expansion of antigen-pulsed TFH
cells in vitro, and possessed a
unique gene expression pattern related to TFH and Th2 cells. The
increase in CXCR5+ CD8 T cells was associated with the presence of higher
frequencies of SIV-specific CD8 T cells in the GC. Following TCR-driven
stimulation in vitro, CXCR5+ but not
CXCR5- CD8+ T cells generated both CXCR5+ as well as CXCR5- cells. However, the
addition of TGF-beta to CXCR5- CD8+ T cells induced a population of CXCR5+ CD8+
T cells, suggesting that this cytokine may be important in modulating these
CXCR5+ CD8+ T cells in vivo. Thus,
CXCR5+ CD8 T cells represent a unique subset of antiviral CD8+ T cells that
expand in LNs during chronic SIV infection and may play a significant role in
the control of pathogenic SIV infection 93.

An important milestone in purging FDC
reservoir was demonstrated by utilization of soluble complement receptor 2 or
CD21 94.
CD21 is necessary for HIV interaction with FDCs and B-cells 95.
In fact, interaction of HIV with FDC stabilizes the virus 67. Despite the stabilized interaction, Heesters and coworkers were able
to purge the FDCs of HIV virions by utilizing a soluble form of CD21 94.
Thus, intersecting CD21:C3d interactions significantly reduced recycling of
virions through the endosomal compartment. In addition, viral transmission to TFH
was diminished in in vitro studies 94.
An alternate approach to purge FDC HIV reservoir involved monoclonal antibodies
targeting CD21 96.
Thus, blocking CD21 interactions appears to be a promising strategy for
eradication of the FDC HIV reservoir.

 

Future Perspectives

Profound and
durable suppression of HIV by cART represents a major accomplishment in
HIV/AIDS research 97; 98. However, HIV
persists in patients despite long-term administration of cART 35. Withdrawing cART
invariably results in viral rebound 99; 100. One of the
major challenges with cART is to prevent virological failure. Studies in rhesus
macaque have demonstrated sustained virologic control with an antibody that
targets the gut-homing integrin a4b7 in combination with cART 101. While,
mechanisms underlying observed virologic suppression remain to be elucidated.

Progress
beyond a functional cure has been limited to some isolated instances such as
the case of a Berlin patient 102 or
experimental strategies involving CRISPR/Cas9 mediated excision of HIV 103; 104. Whether
these innovative strategies purge CNS and LN HIV reservoirs remain to be tested
since there is limited drug penetration in the CNS and LNs. Lifetime cART
treatment is associated with toxicity, residual chronic inflammation and the
accelerated onset of co-morbidities associated with aging. As such, better
strategies are needed to eliminate these complications such as immunotherapy in
combination with cART or any therapies, which include enhancement of innate
immunity.

The exact location
and uneven anatomic distribution of cell subsets that harbor HIV during cART represent
significant barriers to the development of HIV cure. Understanding viral
replication in lymphoid tissues may explain low level viremia during cART and
viral resurgence after treatment failure or interruption 105. Understanding LN viral reservoirs,
particularly CLN reservoirs of HIV may shed light on HIV egress from the CNS
and explain the need to purge these HIV reservoirs in order to achieve a
functional cure. This is important because even in the era of highly effective
cART allowing people to live their normal life spans 106; 107, HIV infection
continues to be a serious public health issue.