JVI Accepts, published online ahead of print on 26 March 2014 J. Virol. doi:10.1128/JVI.00590-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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Coregulatory Interactions between CD8α DCs, LAT, and PD-1 Contribute to higher HSV-1

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Latency

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Kevin R. Mott, Sariah J. Allen, Mandana Zandian, and Homayon Ghiasi*

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Center for Neurobiology and Vaccine Development, Ophthalmology Research, Department of

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Surgery, Cedars-Sinai Burns & Allen Research Institute, CSMC - D2066, 8700 Beverly Blvd.,

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Los Angeles, CA.

*Corresponding author. Center for Neurobiology and Vaccine Development - D2066, Cedars-Sinai Burns and Allen Research Institute, 8700 Beverly Blvd., Los Angeles, CA 90048

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PHONE: (310) 248-8582

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Email: [email protected]

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Running title: Factors contributing to HSV-1 latency.

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Keywords: dendritic cells, HSV-1, depletion, T cells, exhaustion

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Summary The latency-associated transcript (LAT) of HSV-1, CD8α+ dendritic cells (DCs), and

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programmed death-1 (PD-1) have all been implicated in the HSV-1 latency-reactivation cycle.

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It is not known, however, whether an interaction between LAT and CD8α+ DCs regulates

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latency and T-cell exhaustion. To address this question, we used LAT(+) and LAT(-)viruses.

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Depletion of DCs in mice ocularly infected with LAT(+) virus resulted in a reduction in the

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number of T cells expressing PD-1 in the trigeminal ganglia (TG), whereas depletion of DCs in

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mice similarly infected with LAT(-) virus did not alter PD-1 expression. CD8α+ DCs, but not

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CD4+ DCs, infected with LAT(+) virus had higher levels of ICP-0, ICP-4, TK, and PD-L1

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transcripts than those infected with LAT(-) virus. Coculture of infected bone marrow (BM)

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derived DCs from wild-type (WT) mice, but not infected DCs from CD8α-/- mice, with WT naive T

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cells contributed to an increase in PD-1 expression. Transfer of bone marrow from WT mice, but

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not from CD8α-/- mice, to recipient Rag1-/- mice increased the number of latent viral genomes in

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reconstituted mice infected with the LAT(+) virus. Collectively, this data indicated that a

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reduction in latency correlated with a decline in the levels of CD8α+ DCs and PD-1 expression.

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In summary, our results demonstrate an interaction among LAT, PD-1, and CD11cCD8α+ cells

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that regulates latency in the TG of HSV-1 infected mice.

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Importance

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Very little is known regarding the inter-relationship of LAT, PD-1, and CD8α+ DCs, and how

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such interactions might contribute to relative number of latent viral genomes. We have shown here

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that: (1) In both in vivo and in vitro studies, deficiency of CD8α+ DCs significantly reduced T-cell

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exhaustion in the presence of LAT(+) virus but not LAT(-) virus; (2) HSV-1 infectivity was

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significantly lower in the LAT(-)-infected DCs than their LAT(+)-infected counterparts; and (3)

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Adoptive transfer of bone marrow (BM) from WT but not CD8α-/- mice to recipient Rag1-/- mice

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restored latency to the WT mice level following infection with LAT(+) virus.

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These studies point to a key role for CD8α+ DCs in T-cell exhaustion in the presence of LAT,

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which leads to higher numbers of latent viral genomes. Thus, altering this negative function of

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CD8α+ DCs can potentially be used to generate a more effective vaccine against HSV infection.

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INTRODUCTION A characteristic feature of infection with HSV-1 is the ability of the virus to establish

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latency in sensory neurons of an infected host (1-4). Individuals who have acquired a latent

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infection are subject to episodic recurrences and serve as permanent carriers who are

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intermittently infectious (5-7). The recurrences are caused by reactivation of the virus, which

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results in its transit back to the original site of infection (8, 9). More than 80 HSV-1 genes are

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expressed in neurons during lytic infection. This expression of HSV-1 genes is drastically

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curtailed during latency. Indeed, the latency-associated transcript (LAT) is the only gene product

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consistently detected in abundance during latency in infected mice, rabbits, and humans (1, 2,

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4, 10, 11).

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Using LAT(+) and LAT(-) viruses, we recently demonstrated that the presence of LAT

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leads to the generation of dysfunctional T-cell responses in the trigeminal ganglia (TG) of

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latently infected mice (12). Both LAT expression and enhanced latency correlated with

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increased mRNA levels of CD8 and the inhibitory receptor programmed death-1 (PD-1) in the

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TG. These results suggested that TG that are latently infected with LAT(+) virus contain both

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more CD8+ T cells and more CD8+ T cells expressing the exhaustion marker, PD-1, than TG

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that are latently infected with LAT(-) virus. This was confirmed by flow cytometry analyses of

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expression of CD3/CD8/PD-1, HSV-1 gC, the gB498-505-specific CD8+ T-cell pentamer, IL-2, IFN-

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γ, and TNF-α. The functional significance of PD-1 and its ligands in HSV-1 latency was

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indicated by the significantly lower levels of HSV-1 latency in mice that were deficient in PD-1 or

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PD-L1 as compared to WT mice. The levels of HSV-1 latency were unaffected in PD-L2-deficent

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mice. We also have shown that latency is enhanced by immunization of infected WT mice with

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FMS-like tyrosine kinase 3 ligand (Flt3L) DNA, which increases the number of dendritic cells

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(DCs) (13, 14). Conversely, depletion of DCs was associated with reduced latency. Latency

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was also significantly lower in infected Flt3L-/- and CD8-/- mice than infected WT mice.

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Interestingly, however, although immunization of Flt3L-/- mice with Flt3L DNA enhanced latency, 4

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immunization of CD8-/- mice with Flt3L DNA did not. Transfer experiments using DCs expanded

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ex vivo in the presence of Flt3L or granulocyte-macrophage colony-stimulating factor (GM-CSF)

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suggested that enhanced latency was associated with the presence of lymphoid-related

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(CD11c+CD8α+) DCs, whereas reduced latency was associated with myeloid-related

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(CD11c+CD8α-) DCs. Modulation of DC numbers by Flt3L DNA immunization or by depletion

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did not, however, alter acute virus replication in the eye and TG or eye disease in ocularly-

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infected mice (13, 14). It has been shown that CD8+ T cells infiltrate TG by 3 days post ocular

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infection and it has been postulated that they act to inhibit HSV-1 reactivation (15). During

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latency, a subset of CD8+ T cells remain in direct contact with infected neurons (16). These

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cells can block HSV-1 reactivation from latency in ex vivo cultures of TG from latently-infected

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mice (15, 16). These results suggest a role for CD8+ T cells in HSV-1 latency, while our

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published studies indicate an involvement of DCs in the efficiency by which HSV-1 establishes

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latency (13, 14). Using a combination of knockout mouse and adoptive transfer approaches, we

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have now demonstrated clearly that CD8α+ DCs, rather than CD8+ T cells, are responsible for

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enhanced HSV-1 latency (17).

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Collectively our published studies show that: (1) Mice latently infected with WT HSV-1 have

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higher levels of LAT RNA, CD8 mRNA and PD-1 mRNA in the TG than in mice depleted of DCs (13,

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14, 18); (2) HSV-1 latency was significantly lower in PD-1-/- and PD-L1-/- mice as compared to WT

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mice (12); and (3) The presence of CD11c+CD8α+ directly increased the number of HSV-1 latent

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viral genomes in mice TG (13, 14). However, very little is known regarding the inter-relationship of

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LAT, PD-1, and CD8α+ DCs, and how such interactions might contribute to latency. We therefore

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undertook studies to test the hypothesis that CD11c+CD8α+ DCs in the presence of LAT are

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harboring more non-productive virus, which contributes to an increase in T-cell exhaustion and,

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thus, enhances latency. We report here that deficiency of CD8α+ DCs significantly reduced T-cell

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exhaustion in LAT(+) infected mice but not in LAT(-) infected mice. Additionally, in vitro assays

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revealed that incubation of HSV-1-infected WT DCs, but not similarly infected CD8α- DCs, with T

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cells isolated from naive WT mice promoted T-cell exhaustion. Furthermore, adoptive transfer of

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bone marrow (BM) from WT but not CD8α-/- mice to recipient Rag1-/- mice restored latency to the

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WT mice level following infection with LAT(+) virus. These studies point to a key role for CD8α+

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DCs in T-cell exhaustion in the presence of LAT, which leads to higher levels of virus.

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Materials and Methods

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Virus and mice. Plaque-purified virulent HSV-1 strains McKrae (WT, LAT(+)) and

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dLAT2903 (LAT(-)) were grown in rabbit skin (RS) cell monolayers in minimal essential medium

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(MEM) containing 5% fetal calf serum (FCS), as we described previously (19, 20). Throughout

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the study, McKrae (wild type) and dLAT2903 viruses are referred to as LAT(+) and LAT(-),

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respectively. dLAT2903 was derived from McKrae in which both copies of the LAT promoter

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(one in each viral long repeat) and the first 1,667 nucleotides (nt) of the LAT transcript were

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deleted (20).

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WT C57BL/6, C57BL/6-CD8α-/-, C57BL/6-β2m-/-, C57BL/6-DTR, C57BL/6-PD-L1-/-, and

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C57BL/6-Rag1-/- were used. All animal procedures adhered to the Association for Research in

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Vision and Ophthalmology (ARVO) statement for the Use of Animals in Ophthalmic and Vision

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Research and were conducted according to institutional animal care and use guidelines.

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Ocular infection. Mice were infected ocularly with 2 x 105 PFU per eye of LAT(+) or

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LAT(-) virus. Each virus was suspended in 2 μl of tissue culture media and administered as an

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eye drop without prior corneal scarification.

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DC culture. Six week-old mice were used as a source of bone marrow (BM) for the

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generation of mouse DCs in culture. BM cells were isolated by flushing femurs with PBS.

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Pelleted cells were briefly resuspended in red blood lysing buffer (Sigma-Aldrich, St. Louis, MO)

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to lyse red blood cells and stabilized by adding complete medium (RPMI 1640, 10% fetal bovine

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serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine). The cells were

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centrifuged and resuspended in complete medium supplemented with GM-CSF (100 ng/ml;

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Peprotech, NJ) to enhance replication of DCs (21). Afterwards, cells were plated in non-tissue

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culture plastic Petri dishes (1 bone per 10 cm dish) for 5 days at 37oC with CO2. After 5 days,

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the media was removed and the adherent cells were recovered by incubating them for 5 min at

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37oC with Versene (Invitrogen, San Diego, CA). Cells were washed, counted, and plated onto

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tissue-culture dishes for use the following day.

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Infection of DCs. Monolayers of DCs from the different strains of mice described above

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were infected with 1 PFU/cell of LAT(+) or LAT(-) virus. One hr after infection at 37oC, virus was

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removed and the infected cells were washed three times with fresh media and infected cells

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were incubated with growth medium for 24 hr. Infected DCs were used for the following studies:

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A) To measure differences in the infectivity of WT, β2m-/-, CD8α-/-, or PD-L1-/- DCs following

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infection with LAT(+) or LAT(-). Infected DCs monolayers were harvested and the presence of

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gB transcript was determined by qRT-PCR. B) To measure the differences in the infectivity of

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CD8α+ DCs versus CD4+ DCs, infected DCs were harvested and were separated into CD4+

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DCs or CD8α+ DCs as described below. The presence of ICP0, ICP4, TK, and PD-L1

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transcripts in each cell population were determined by qRT-PCR as described below. C) To

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measure the activation state of DCs following HSV-1 infection, DCs isolated from WT mice were

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mock-infected or infected with 1 PFU/cell of HSV-1 strain McKrae for 24 or 48 hr. Mock-infected

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and infected DCs were stained and flow cytometric analyses were performed as described

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below. D) Finally, to determine if DCs contributed to T cell exhaustion, mock-infected or HSV-1

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infected DCs from WT or CD8α-/- mice were co-cultured with different ratios of naive T cells from

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WT mice for 24, 48, 72, or 96 hr. The monolayers were harvested at various times post co-

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cultivation and stained for flow cytometric analyses as described below.

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Isolation of CD4+ and CD8α+ DCs. Cultured DCs were grown as above, harvested on

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day 5 and infected with 1 PFU/cell of LAT(+) or LAT(-) HSV-1 for 24 hr. After 24 hr PI, isolation

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of CD8α+ and CD4+ DCs was performed using a two-step magnetic cell sorting kit (Kit # 130-

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091-169 and 130-091-262, Miltenyi Biotec, Auburn CA) per the manufacturer’s protocol. Briefly,

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harvested cells were directly labeled with CD8 Microbeads and CD8α+ DCs were isolated by

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positive selection from the enriched DC fraction. The magnetically-labeled CD8α+ DCs were

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retained on a column and eluted after removal of the column from the magnetic field.

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Subsequently, the remaining enriched DCs that passed through the column were directly

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labeled with CD4 Microbeads, and CD4+ DCs were isolated by the same method of positive

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selection in a magnetic field.

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Adoptive transfer of BM to recipient mice. BM from WT C57BL/6 or CD8α-/- mice

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was isolated and each recipient Rag1-/- mouse received BM from one donor mouse

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intravenously (IV). Two weeks post-transfer, the Rag1-/- recipient mice were ocularly infected

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with LAT(+) virus. TG from infected mice were isolated for detection of LAT RNA and gB DNA

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on day 30 PI.

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Depletion of DCs. C57BL/6-DTR mice were depleted of their DCs using 100 ng of

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diphtheria toxin (DT) in 100 μl of PBS via an IP route 24 hr prior to ocular infection and at days

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2, 5, 10, and 14 PI. As a negative control, a subset of mice was similarly injected with PBS

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alone, and is therefore referred to as mock-depleted. Efficiency of DC depletion in corneas and

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spleens was monitored by flow cytometric analysis before ocular infection and at day 5 PI. After

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the first depletion, more than 90% of DCs had been depleted.

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Confocal Microscopy and Image Analysis. DCs isolated from different strains of mice

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were grown on Lab-Tex chamber slides (Sigma-Alderich) as we described previously (22). Briefly,

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cells were fixed by incubating slides in methanol for 10 min followed by acetone for 5 min at -20oC.

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Afterwards, slides were rinsed three times for 5 min each at ambient temperature in PBS containing

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0.05% v/v Tween-20 (PBS-T). Slides were then blocked for 30 min at ambient temperature in PBS-T

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containing 1% w/v BSA. Rat anti-CD8α (clone 53-6.7, eBioscience, San Diego, CA), rat anti-CD4

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(clone Gk1.5, eBioscience), rat anti-CD8β (clone YTS156.7.7, BioLegend), and hamster anti-CD11c

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(clone HL3, BD Biosciences) were used for IHC. Immunostaining was done using CD11c/CD4,

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CD11c /CD8α, or CD11c /CD8β antibody combinations and staining for 1 hr at 25°C. After three

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rinses for 5 min each in PBS, slides were incubated for 1 hr at 25°C with secondary antibodies

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labeled with anti-hamster FITC or anti-Rat TRITC (Invitrogen). Slides were washed three times with

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PBS, air-dried and mounted with Prolong Gold DAPI mounting media (Invitrogen). Images were

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captured at 1024 x 1024 pixels (original magnification = 20X) in independent fluorescence channels

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using a Nikon C1 eclipse inverted confocal microscope.

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Flow Cytometric Analysis. Infected or mock-infected cells were harvested at various times

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PI and stained with anti-CD3, anti-CD4, anti-CD8α, anti-PD-1, and anti-CD11c obtained from BD

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PharMingen (San Diego, CA) and BioLegend (San Diego, CA), and then analyzed by flow cytometry

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as we previously described (23).

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DNA extraction and PCR analysis for HSV-1 genomic DNA. DNA was isolated from

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homogenized individual TG using the commercially available DNeasy Blood &Tissue Kit

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(Qiagen, Stanford, CA) according to the manufacturer's instructions. PCR analyses were done

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using gB specific primers (Forward - 5'-AACGCGACGCACATCAAG-3'; Reverse - 5'-

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CTGGTACGCGATCAGAAAGC-3'; and Probe - 5'-FAM-CAGCCGCAGTACTACC-3'). The

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amplicon length for this primer set was 72 bp. Relative copy numbers for gB DNA were

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calculated using standard curves generated from the plasmid pAc-gB1 (24). In all experiments,

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GAPDH was used for normalization of transcripts.

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RNA Extraction, cDNA Synthesis, and TaqMan RT-PCR. RNA was extracted from

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latent TG or infected DCs as we have described previously (22, 25, 26). Following RNA

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extraction, 1 μg of total RNA was reverse-transcribed using random hexamer primers and

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Murine Leukemia Virus (MuLV) Reverse Transcriptase from the High Capacity cDNA Reverse

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Transcription Kit (Applied Biosystems, Foster City, CA), in accordance with the manufacturer's

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recommendations. The levels of various transcripts were evaluated using commercially

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available TaqMan Gene Expression Assays (Applied Biosystems) with optimized primer and

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probe concentrations. Primer-probe sets consisted of two unlabeled PCR primers and the

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FAM

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dye-labeled TaqMan MGB probe formulated into a single mixture. Additionally, all

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cellular amplicons included an intron-exon junction to eliminate signal from genomic DNA

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contamination. The HSV-1 ICP0, ICP4, TK, gB, LAT, and PD-L1 primers and probe used were

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as follows: 1) ICP0: forward primer, 5'- CGGACACGGAACTGTTCGA-3'; reverse primer, 5'-

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CGCCCCCGCAACTG-3'; and probe, 5'-FAM-CCCCATCCACGCCCTG-3' -- Amplicon length =

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111 bp; 2) ICP4: forward primer, 5'- GCGTCGTCGAGGTCGT-3'; reverse primer, 5'-

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CGCGGAGACGGAGGAG-3'; and probe, 5'-FAM-CACGACCCCGACCACC-3' -- Amplicon

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length = 69 bp; 3) TK: forward primer, 5'- CAGTAGCGTGGGCATTTTCTG-3'; reverse primer,

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5'-CCTCGCCGGCAACAAAA-3'; and probe, 5'-FAM-CTCCAGGCGGACTTC-3' -- Amplicon

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length = 59 bp; 4) gB: forward primer, 5'-AACGCGACGCACATCAAG-3', reverse primer, 5'-

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CTGGTACGCGATCAGAAAGC-3'; and probe, 5'-FAM-CAGCCGCAGTACTACC-3' – Amplicon

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length = 72 bp; 5)LAT: forward primer 5'-GGGTGGGCTCGTGTTACAG-3'; reverse primer, 5'-

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GGACGGGTAAGTAACAGAGTCTCTA-3'; and probe, 5'- FAM-ACACCAGCCCGTTCTTT-3'–

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Amplicon Length =81 bp, corresponding to LAT nts 119553-119634; and 6) PD-L1- ABI ASSAY

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I.D. Mm00452054_m1 – Amplicon length = 77 bp. As an internal control, a set of GAPDH

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primers from Applied Biosystems (ASSAY I.D. m999999.15_G1 - Amplicon Length = 107 bp)

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was used.

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Quantitative real-time PCR (qRT-PCR) was performed using an ABI PRISM 7900HT

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Sequence Detection System (Applied Biosystems, Foster City, CA) in 384-well plates as we

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described previously (25, 26). Real-time PCR was performed in triplicate for each tissue

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sample. The threshold cycle (Ct) values, which represents the PCR cycle at which there is a

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noticeable increase in the reporter fluorescence above baseline, were determined using SDS

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2.2 Software. GAPDH transcript was used for normalization purposes.

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Statistical analysis. Student’s t test and chi-squared tests were performed using the

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computer program Instat (GraphPad, San Diego). Results were considered statistically

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significant when the P value was < 0.05.

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RESULTS

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DCs play an indispensable role in regulating T-cell exhaustion in mice latently

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infected with HSV-1. We have shown that depletion of DCs reduces the number of HSV-1

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latent genomes in ocularly infected mice (13, 14) and that a decrease in latency correlates with

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a decrease in T-cell exhaustion in the TG of latently infected mice (12). However, very little is

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known regarding the role of DCs in T-cell exhaustion. As a proof-of-principal experiment, we

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depleted DCs in diphtheria toxin receptor (DTR) transgenic mice by treatment with DT one day

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before ocular infection with LAT(+) or LAT(-) HSV-1 virus and at various times post-infection

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(PI). As controls, DTR mice were mock-depleted of DCs and ocularly infected; DTR mice were

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both mock-depleted and mock-infected (designated “naive”); and WT C57BL/6 mice were

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infected with the viruses. Since 30 days PI is accepted as a point in time by which latency has

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been established (27), the TG were isolated from individual mice at day 30 PI and single-cell

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suspensions of the TG from each mouse were stained for CD3, CD8, and PD-1. The

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percentages of CD3+CD8+PD-1+ T cells in the TG of DC-depleted, mock-depleted, and WT mice

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infected with LAT(+) virus, as well as naive mice are shown in Figure 1A. As would be

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expected, there were few CD3+CD8+PD-1+ T cells in the TG from naive mice and the highest

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numbers of CD3+CD8+PD-1+ cells were found in the TG of the WT and mock-depleted DTR

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control mice. The numbers of CD3+CD8+PD-1+ cells in the TG of the DC-depleted mice was

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significantly lower than in these two virus-infected controls (Figure 1A). Moreover, the numbers

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of CD3+CD8+PD-1+ cells in the TG of DC-depleted mice that were infected with LAT(-) virus did

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not differ significantly from the numbers of CD3+CD8+PD-1+ cells in the TG of mock-depleted

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controls (Figure 1B). These results are consistent with a previously reported study for the

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number of CD8-positive T cells in TG of mice infected with LAT(-) virus versus LAT(+) virus (12).

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We next analyzed the kinetics of CD3/CD8/PD-1 expression during both lytic (days 5 and

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10) and latent (day 30) in the TG of DC-depleted or mock-depleted DTR mice infected with

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LAT(+) virus (Figure 1C). During both lytic and latent phases of HSV-1 infection, the numbers of 12

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CD3+CD8+PD-1+ T cells were significantly lower in the TG of DC-depleted mice than in the TG

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of the mock-depleted mice. In both groups, the numbers of CD3+CD8+PD-1+ cells increased

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through day 10 PI and had declined by day 30 PI. Similar to this study, previously it was shown

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that by 3 days PI, CD8+ T cells begin infiltrating the TG, peaking at 14 days (virus clear by day

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11 PI), declined significantly after clearance of lytic infection and persisting as latent virus (12,

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16, 27-29). In addition, in line with our results, previously it was shown that the level of PD-1

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expression during early stage of human hepatitis C virus (HCV) infection is significantly higher

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from subjects who progress to chronic HCV infection than from those who clear infection (30).

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Furthermore, during acute HCV infection, the loss of PD-1 expression by CD8+ T cells was

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shown to correlate with viral clearance (31). These data support our overall hypothesis that the

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presence of LAT and DCs affects the level of T-cell exhaustion during both lytic and latent

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cycles of HSV-1 infection.

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Expression of CD4 and CD8α transcripts in BM-derived DCs isolated from different

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strains of mice before HSV-1 infection. Recently, we reported that the absence of CD8α+ DCs

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correlated with reduced latency (17) . To determine if lower levels of latency associated with a

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reduction in CD8α+ transcripts, we quantified CD4 and CD8α transcripts in BM-derived DCs isolated

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from WT, β2m-/-, PD-L1-/-, and CD8α-/- naive mice. The CD4 copy numbers were similar in the DCs

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from all four strains of mice (Figure 2A, P>0.05). Although the copy numbers of CD8α transcripts in

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DCs isolated from WT mice were similar to the copy numbers found in β2m-/- mice, the copy numbers

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of the CD8α transcripts were significantly lower in the DCs isolated from the PD-L1-/- mice (Figure

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2B, P