CD8+ T Cells Prevent Lethality from Neonatal Murine Roseolovirus Infection Swapneel J

CD8+ T Cells Prevent Lethality from Neonatal Murine Roseolovirus Infection Swapneel J. Patel and Wayne M. Yokoyama This information is current as J Immunol 2017; 199:3212-3221; Prepublished online 2 of September 26, 2021. October 2017; doi: 10.4049/jimmunol.1700982 http://www.jimmunol.org/content/199/9/3212 Downloaded from

References This article cites 27 articles, 11 of which you can access for free at: http://www.jimmunol.org/content/199/9/3212.full#ref-list-1

Why The JI? Submit online. http://www.jimmunol.org/

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication

*average by guest on September 26, 2021

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

CD8+ T Cells Prevent Lethality from Neonatal Murine Roseolovirus Infection

Swapneel J. Patel* and Wayne M. Yokoyama*,†

A recently described mouse homolog of the human roseoloviruses, murine roseolovirus (MRV), causes loss of peripheral and thymic CD4+ cells during neonatal infection of BALB/c mice. Despite signiﬁcant disruptions to the normal adaptive immune response, infected BALB/c mice reproducibly recover from infection, consistent with prior studies on a related virus, mouse thymic virus. In this article, we show that, in contrast to published studies on mouse thymic virus, MRV appears to robustly infect neonatal C57BL/6 (B6) mice, causing severe depletion of thymocytes and peripheral T cells. Moreover, B6 mice recovered from infection. We investigated the mechanism of thymocyte and T cell loss, determining that the major thymocyte subsets were infected with MRV; however, CD4+ and CD4+CD82 T cells showed increased apoptosis during infection. We found that CD8+ T cells populated MRV- + infected thymi. These CD8 T cells expressed markers of activation, had restricted TCR repertoire, and accumulated intracellular Downloaded from effector proteins, consistent with a cytotoxic lymphocyte phenotype and suggesting their involvement in viral clearance. Indeed, absence of CD8+ T cells prevented recovery from MRV infection and led to lethality in infected animals, whereas B cell–deﬁcient mice showed CD4+ T cell loss but recovered from infection without lethality. Thus, these results demonstrate that CD8+ T cells are required for protective immunity against a naturally occurring murine pathogen that infects the thymus and establish a novel infection model for MRV in B6 mice, providing the foundation for detailed future studies on MRV with the availability of

innumerable mutant mice on the B6 background. The Journal of Immunology, 2017, 199: 3212–3221. http://www.jimmunol.org/

nfection with the roseoloviruses, human herpesvirus (HHV) infection. A clearer understanding of the specific roles of each 6A, HHV6B, and HHV7, is virtually ubiquitous among hu- aspect of the immune system may aid in prevention of HHV6- I mans (1). Epidemiological and cross-sectional studies have dependent pathological consequences, but it has been challenging identified a myriad of conditions associated with viremia or se- to study the pathogenesis of human roseolovirus infections be- ropositivity (2–12). However, these studies are hampered by the cause these viruses are highly species specific and require ma- paucity of appropriate uninfected control individuals. nipulation of the host to infect mice for detailed experimental Primary HHV6 infection usually resolves, and the virus persists analysis (1). as a latent virus or as a chronic viral infection (13). HHV6 can also We recently identified that a naturally occurring murine beta- by guest on September 26, 2021 reactivate and cause viremia during immune suppression. The herpesvirus, murine roseolovirus (MRV), is a murine homolog of humoral and cytotoxic T lymphocyte responses to HHV6 have the human roseoloviruses (17). MRV phenotypically resembled been characterized (14). Neutralizing IgM Abs toward HHV6 mouse thymic virus (MTV; also known as mouse T lymphotropic develop during acute exanthem subitum (15). Virus-specific CD4+ virus and murid herpesvirus 3) because both viruses caused thy- and CD8+ T cells are present in adults without HHV6 viremia, mic “necrosis” in neonatal mice; however, genome sequence data presumably as a result of prior exposure to the virus (14, 16). for MTV are not publically available so we cannot definitely make However, the relative importance of these cell types during conclusions about the relationships between these two viruses. acute HHV6 infection or reactivation is unclear. Several clin- Phylogenetic analyses showed that MRV is more closely related to ical conditions arising after immune suppression are associated human roseoloviruses than to another naturally occurring murine with HHV6 reactivation, suggesting that active anti-HHV6 betaherpesvirus, murine CMV. Because MRV infects the neonatal surveillance contributes to controlling latent or chronic HHV6 thymus (17), and HHV6 can directly infect and lyse CD4+ T cells (18–20), MRV and human roseoloviruses may share common *Division of Rheumatology, Department of Medicine, Washington University School pathological mechanisms, potentially allowing insight into the of Medicine, St. Louis, MO 63110; and †Howard Hughes Medical Institute, Wash- human viruses by studying MRV. ington University School of Medicine, St. Louis, MO 63110 Prior studies of BALB/c mice infected with MTV indicate that ORCIDs: 0000-0001-6088-4029 (S.J.P.); 0000-0002-0566-7264 (W.M.Y.). recovery from infection can occur, but the mechanisms contrib- Received for publication July 7, 2017. Accepted for publication August 28, 2017. uting to these findings remained unknown (21). In this study, we This work was supported by National Institutes of Health Grant T32GM008200 (to found that neonatal infection of BALB/c mice with MRV leads to S.J.P.), the Howard Hughes Medical Institute (to W.M.Y.), and the Barnes-Jewish + Hospital Foundation (to W.M.Y.). severe CD4 thymocyte and T cell depletion, followed by re- Address correspondence and reprint requests to Dr. Wayne M. Yokoyama, Division covery, consistent with prior studies on MTV. In addition, in of Rheumatology, Campus Box 8045, Washington University School of Medicine, contrast to prior studies of MTV in which C57BL mice were Box 8045, 660 South Euclid Avenue, St. Louis, MO 63110. E-mail address: relatively resistant to infection (22), we found that C57BL/6 (B6) [email protected] mice are susceptible to infection, allowing us to address issues of Abbreviations used in this article: B6, C57BL/6; CD4 SP, CD4 single-positive; CD8 ISP, CD8 intermediate single-positive; CD8 SP, CD8 single-positive; DN, CD42 viral tropism and potential pathogenic mechanisms by use of CD82 double-negative; DP, CD41CD81 double-positive; HHV, human herpesvirus; genetic mutants available on the B6 genetic background. We MRV, murine roseolovirus; MTV, mouse thymic virus; P0, postnatal day 0; p.i., studied immune mechanisms controlling MRV infection and postinfection; TEM, transmission electron microscopy; WT, wild-type. found that CD8+ T cells, but not mature B cells, are crucial for Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$35.00 control of MRV infection. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1700982 The Journal of Immunology 3213

Materials and Methods crosis, demonstrating near-complete loss of CD4 single-positive Mice (CD4 SP) and CD4+CD8+ double-positive (DP) thymocytes.

2/2 2/2 Splenic T cell numbers were also significantly reduced, with B6 mice were purchased from Charles River Laboratories. Rag1 ,CD8 , + + and mMT mice were purchased from The Jackson Laboratory. Infected and near-complete loss of CD4 T cells and reductions in CD8 T cell uninfected mice were independently housed to avoid horizontal viral number. Furthermore, we assayed various organs at day 7 post- transmission. infection (p.i.) for viral copy number (Fig. 1C). As in BALB/c mice (17), viral titers were highest in the thymus of infected an- Viral isolation and infection imals, but MRV genomes also could be found in the spleen, liver, Viral stocks were produced by in vivo passaging, as previously described CNS, lung, kidney, and salivary gland. These results indicated that (18, 21, 23). A single viral stock was used for all experiments. For all MRV-dependent thymocyte depletion can reproducibly occur in infections, postnatal day 0 (P0) mice were inoculated i.p. with 100 mlofa 1:10 dilution of thawed MRV viral stock. B6 mice. Remarkably, we found that B6 mice infected with MRV re- Flow cytometry covered CD4 SP and DP thymocyte percentage and absolute Single-cell lymphocyte suspensions were prepared from thymi or spleens number at 7 wk p.i. (Fig. 2A, 2B). At this time, we also observed from infected animals and processed by manual disruption through a 70-mM that peripheral CD4+ and CD8+ T cell numbers were not signifi- filter. A total of 106–107 cells was stained with fixable viability dye cantly different between infected and uninfected control animals (eBioscience, San Diego, CA) before incubation in 2.4G2 supernatant (Fig. 2A, 2B). We surmised that this recovery was the result of (anti-FcgRII/III). Cells were stained with the following surface Abs (Affymetrix, Santa Clara, CA): anti-CD3ε (145-2C11), anti-CD4 (RM4-5), control of viral titers, because the observed viral titers at 7 wk p.i. anti-CD8 (53-6.7), anti-TCRb (H57-597), anti-CD45.2 (104) CD24 (M1/ were significantly lower in the thymus compared with 1 wk p.i. Downloaded from 69), CD8b (eBioH35-17.2), CD62L (MEL-14), CD44 (IM7), CD69 (Fig. 2C). We observed a similar trend in the spleens of infected (H1.2F3), KLRG1 (2F1), IFN-g (XMG1.2), granzyme B (GB11), and animals; however, the degree of reduction in viral titers was more mouse TCR Vb screening panel (BD Biosciences, San Diego, CA). modest (Fig. 2D). Annexin V staining was done following the manufacturer’s recommendations (BioLegend, San Diego, CA). Intracellular cytokine staining was Acute infection of thymocyte subsets conducted using a BD Cytofix/Cytoperm Fixation/Permeabilization Solu-

tion Kit (BioLegend). Samples were run by ﬂow cytometry using a Because we observed that MRV viral copies were highest in the http://www.jimmunol.org/ FACSCanto (BD Biosciences, San Jose, CA) and analyzed using FlowJo acutely infected thymus, we investigated which thymocyte subsets v10 (TreeStar, Ashland, OR). Statistical analysis was conducted using GraphPad Prism 6.01 (GraphPad, La Jolla, CA). were directly infected with MRV. Given the near-complete reduction of CD4+ cells during infection, we hypothesized that CD4+ cells Transmission electron microscopy were the major substrate for acute infection. Perhaps once all of the + Ultrastructural analysis was performed by the Molecular Microbiology CD4 cells were lost during infection, there would be no more Imaging Facility at Washington University. Cells were ﬁxed in 2% permissive cells to infect, leading to reduced titers and recovery from paraformaldehyde/2.5% glutaraldehyde in 100 mM phosphate buffer (pH 7.2) infection. To test this hypothesis, we harvested thymi from mice at for 1 h at room temperature. Samples were washed in sodium cacodylate buffer 6 d p.i., because by 7 d p.i. there were very few CD4 SP thymocytes.

andpostfixedin1%osmiumtetroxide(Polysciences) for 1 h. Samples were by guest on September 26, 2021 We FACS purified the CD4 SP, DP, CD8 single-positive (CD8 SP), rinsed extensively in dH2O prior to en bloc staining with 1% aqueous uranyl 2 2 acetate (Ted Pella, Redding, CA) for 1 h. Following several rinses in dH2O, and CD4 CD8 double-negative (DN) fractions, followed by samples were dehydrated in a graded series of ethanol and embedded in transmission electron microscopy (TEM) for identification of cells Eponate 12 resin (Ted Pella). Sections of 95 nm were cut with a Leica with visible herpesvirus virions (Fig. 3A). From single sections of Ultracut UCT Ultramicrotome (Leica Microsystems, Bannockburn, IL), sorted cells, we found that ∼20% of CD4 SP cells and 15% of DP stained with uranyl acetate and lead citrate, and viewed on a JEOL 1200 EX ∼ transmission electron microscope (JEOL USA, Peabody, MA) equipped with cells had visible virions (Fig. 3B). Surprisingly, 40% of CD8 SP an AMT 8 megapixel digital camera and AMT Image Capture Engine V602 and 25% of DN cells also had virions. We extended this analysis by software (Advanced Microscopy Techniques, Woburn, MA). quantifying the number of virions per infected cell and observed that , Viral genome copy quantification CD4 SP and DP cells had 5 virions per cell, but CD8 SP and DN cells had 10 and 15 virions per infected cell, respectively (Fig. 3C). Genomic DNA was prepared from mouse tissues using a Puregene ex- We confirmed these findings by conducting quantitative PCR on the traction kit (QIAGEN, Valencia, CA), following the manufacturer’s recommendations. Each quantitative PCR reaction used TaqMan Universal thymocyte subsets from day 5–6 MRV-infected neonates (Fig. 3D). PCR Master Mix, no AmpErase UNG (Life Technologies, Grand Island, MRV genome copies were present in CD4 SP and DP cells of in- NY), as per the manufacturer’s recommendations. MRV- and actin-specific fected thymi, but much higher levels were found in the CD8 SP and primers were as previously described (17). Reactions were run in triplicate, DN subsets. In sum, these data suggest that the high viral burden in and absolute quantification was determined by a standard curve. MRV-infected thymi is not due solely to high levels of MRV directly Statistical analyses infecting CD4 SP or DP cells; rather, CD8 SP and DN thymocytes appeared to carry more MRV per cell. Statistical analyses were performed using GraphPad Prism (GraphPad). Cell 2/2 number and frequency were compared using unpaired two-tailed t tests. We also infected Rag1 mice, in which thymocytes are Viral titers were compared using unpaired two-tailed t tests on log10 rel- arrested at the DN3 stage of development because of a lack of ative genome copies. Survival analyses were conducted using the log-rank productive TCRb rearrangements; thus, they do not possess DP, , , Mantel–Cox test. Statistical significance was defined as *p 0.05, **p CD4 SP, or CD8 SP cells (Fig. 3E, 3F). This experiment allowed 0.01, ***p , 0.001, and ****p , 0.0001. us to test whether MRV could infect cells other than these cell types. We found that, at day 10 p.i., infected thymi from Rag12/2 Results mice had MRV levels comparable to B6 control mice. Although MRV infection and recovery in B6 mice Rag12/2 mice lack peripheral T cells, they also had significantly Prior studies investigating infection of B6 mice with MTV have higher viral titers in the spleen compared with B6 control mice. shown that neonatal B6 mice, in contrast to BALB/c mice, were These findings support the notion that MRV has tropism beyond resistant to thymic necrosis (22). We repeated similar experiments thymocytes and T cells. in B6 neonates, infecting them with MRV at P0, within 24 h after These results in wild-type (WT) B6 mice were rather unex- birth (Fig. 1A, 1B). Infected B6 neonates had severe thymic ne- pected, because absolute numbers of CD8 SP and DN thymocytes 3214 CD8 CONTROL OF MRV Downloaded from

FIGURE 1. B6 mice have CD4+ T cell depletion after neonatal MRV infection. P0 B6 mice were infected i.p. with MRV. (A and B) Flow cytometry on thymus and spleen 10 d p.i. with MRV. Thymus http://www.jimmunol.org/ cells were gated on live lymphocytes, and spleen cells were gated on live CD3+TCRb+ lymphocytes with dot plots (A), frequency, and absolute quantiﬁcation (B). Open circles represent uninfected mice; ﬁlled circles represent infected mice. Data are representative of three independent experiments with n = 6–8 mice per experiment. (C) Ratio of MRV/actin genome copy numbers at 1 wk p.i. Data are representative of two independent experiments with n = 4–6 mice per experiment. ****p , 0.0001, unpaired two-tailed t test. by guest on September 26, 2021

appeared to be only modestly impacted during acute MRV in- contrast, CD8 SP cells were relatively spared from apoptosis, even fection, and effectively rules out the possibility that MRV infection though they harbored virions, as assessed by TEM and viral ge- resolves because of complete depletion of CD4-expressing cells. nome copy number (Fig. 3A–D). We then sought to investigate the mechanism by which CD4 SP thymocytes and DP thymocytes were lost during MRV infection. Accumulation of CD8 SP thymocytes in MRV-infected thymi We hypothesized that these cells may be dying of apoptosis. Indeed, Reproducibly at day 10 p.i. in WT B6 mice, we observed that CD4 SP, DP, and DN fractions of the MRV-infected thymi had infected thymi have CD8-expressing cells that disproportionately increased percentages of early and late apoptotic cells (Fig. 4). In populated the thymus compared with the other major thymocyte The Journal of Immunology 3215 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 2. B6 mice recover from MRV infection. P0 B6 mice were infected i.p. with MRV. (A and B) Flow cytometry on thymus and spleen 7 wk p.i. with MRV. Thymus cells were gated on live lymphocytes, and spleen cells were gated on live CD3+TCRb+ lymphocytes with dot plots (A), frequency, and absolute quantification (B). Open circles represent uninfected mice; filled circles represent infected mice. Ratio of MRV/actin genome copy numbers at 1 or 7wkp.i.inthethymus(C) or the spleen (D). Data are representative of three independent experiments with n = 5 or 6 mice per experiment. *p , 0.05, ***p , 0.001, ****p , 0.0001, unpaired, two-tailed t test. subsets. We hypothesized that these cells may possess unique We next explored whether these CD8 SP cells could be effector properties that distinguish them from typical CD8 SP thymocytes. cells. The mean fluorescence intensity of TCRb on CD8 SP T cells In the absence of their immediate developmental precursor, DP was reduced in infected thymi, consistent with activation-induced cells, we reasoned that these cells might be CD8 intermediate TCR downregulation (Fig. 5D) (24). We further found that these single-positive (CD8 ISP) cells. However, by flow cytometry, we cells were positive for KLRG1, CD69, and CD44, whereas they found that the cells were HSAlow, suggesting that they were not had decreased expression of CD62L, compared with CD8 SP CD8 ISP cells (Fig. 5A, 5B). We also hypothesized that these cells thymocytes from uninfected mice, consistent with effector T cells might express TCRgd because of their prevalence during the (Fig. 5E, 5F). There was a stark reduction in naive CD8+ T cells neonatal and perinatal period. Instead, we found that these thymic and increase in the number of effector T cells in MRV-infected CD8 SP cells were CD8ab+TCRb+, resembling mature CD8+ thymi. We also observed enrichment of CD8 SP cells with T cells (Fig. 5C). TCRVb3, 6, 8.3, and 13, consistent with an Ag-restricted poly- 3216 CD8 CONTROL OF MRV Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 3. Thymocytes are directly infected with MRV. (A–D) P0 B6 mice were infected i.p. with MRV. (A) TEM of FACS-puriﬁed fractions. (A) Thymocytes were pregated on live lymphocytes. Representative images of sorted fractions from day 5–6 p.i. (B) Percentage of cells scored positive for the presence of herpesvirus virions by TEM (n = 100 per fraction). (C) Number of scored virions by TEM per infected cell. (D) Ratio of MRV/actin genome copy numbers from the indicated sorted fraction of thymus from mice 5–6 d p.i. Ratio of MRV/actin genome copy numbers from B6 or Rag12/2 mice at 10 d p.i. in the thymus (E) or the spleen (F). Data are pooled from three independent experiments. Cell number and frequency were compared using an unpaired two-tailed t test. Viral titers were compared using an unpaired two-tailed t test on log10 relative genome copies. ****p , 0.0001. clonal response (Fig. 6A, 6B). The CD8+ T cells also had in- thymus of infected WT B6 mice strongly suggested that they may creased expression of effector molecules, IFN-g and granzyme B, play a role in antiviral immunity. We found that CD82/2 neonates compared with uninfected control mice (Fig. 6C, 6D). Collec- could be infected with MRV, with CD4+ T cell depletion occurring tively, these data suggest that MRV infection elicits an effector by 1 wk p.i. However, unlike B6 and mMT mice, CD82/2 animals CD8+ T cell response in infected thymi, and their accumulation were unable to recover peripheral CD4+ T cells, and MRV in- may help to explain why there was no apparent depletion of CD8 fection was lethal to 80% of animals by day 50 p.i. (Fig. 7A, 7B). SP cells, despite infection of these cells and depletion of their DP In contrast, neonatal mice deﬁcient in mature B cells (mMT) in- thymocyte precursors (Fig. 3). fected with MRV showed near-complete loss of peripheral blood + + CD4 T cells at 1 and 2 wk p.i. Yet, by 3 wk p.i., mMT mice had CD8 T cells protect against MRV infection recovered normal complements of CD4+ T cells (Fig. 7C). Ad- The uncontrolled MRV infection in Rag12/2 mice and the pres- ditionally, infection of mMT mice was not lethal, because 100% of ence of activated CD8+ T cells with effector phenotype in the infected animals survived 5 mo p.i. (Fig. 7D). The Journal of Immunology 3217

FIGURE 4. CD4-expressing thymocytes un- dergo increased apoptosis after MRV infection. Downloaded from (A–C) Flow cytometry from thymi of mice on day 5.5 p.i. Early apoptotic cells were defined as Annexin V+Viability Dye2. Late apoptotic cells were defined as Annexin V+Viability Dye+. Open circles represent uninfected mice; filled circles represent infected mice. (D) Representa- tive flow cytometry. **p , 0.01, ***p , 0.001, http://www.jimmunol.org/ ****p , 0.0001, unpaired, two-tailed t test. by guest on September 26, 2021

Because CD4+ T cell depletion occurred in CD82/2 mice, it mice were relatively resistant to gross thymic necrosis; however, in appeared that this depletion was not dependent on direct killing by our studies with MRV, the viral titers and CD4+ cell loss were CD8+ T cells, suggesting that there was enhanced proliferation of comparable between BALB/c and B6 mice. The basis for these MRV. Indeed, we found that there was increased viral burden in differences between MRV and MTV is unclear, but the original 2 2 the thymus and spleen at 1 wk p.i. in CD8 / animals (Fig. 7E, studies characterizing MTV used pooled viral stocks harvested 2 2 7F). By 3 wk p.i., we observed that CD8 / mice had a 4-log from the thymus, spleen, kidney, and pancreas from day-7 p.i. increase in MRV genome copies in the thymus and $5-log in- Webster Swiss neonates. In contrast, we used viral stocks isolated crease in the spleen (Fig. 7G, 7H). Interestingly, when we infected from BALB/c thymi alone, because our analyses showed that viral 2 2 TCRb / mice, which already have severe reduction of CD4+ copy numbers are much higher in infected thymi compared with cells before infection, we found that infected mice died at similar other peripheral organs. Although these differences could be due 2 2 kinetics to infection of CD8 / mice, strongly suggesting that the to viral titer differences or genetic drift of the viral genome, these 2 2 loss of CD4+ T cells during infection of CD8 / mice did not ﬁndings raise the possibility that MRV and MTV are not identical. enhance lethal pathology (Fig. 7I). These data argue that other cell Nonetheless, we showed that MRV-infected neonatal B6 mice can populations are infected and extend pathology associated with control viral copy number and robustly recover CD4+ cells in the MRV infection. thymus and periphery p.i. The major phenotypes followed in studies on MTV were thymic Discussion necrosis and speciﬁc loss of CD4+ T cells. Several studies sug- In this study, we established neonatal MRV infection in the B6 gested that these phenotypes were consistent with MTV having mouse strain. Previous reports on MTV suggested that neonatal B6 preferential tropism for CD4+ cells. In this study, we showed that 3218 CD8 CONTROL OF MRV Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 5. Mature CD8+ T cells with effector phenotype infiltrate MRV-infected thymi. P0 B6 mice were infected i.p. with MRV. Thymi were analyzed by flow cytometry. Thymocytes were pregated on live lymphocytes. (A and B) Representative flow cytometry and absolute number and frequency of CD8 ISP (CD8+CD42CD24+TCRblow) cells or mature CD8+ T cells (CD8+CD42CD242TCRbhigh) in infected or control thymi. (C) Representative flow cytometry for surface CD8b on CD8+ T cells. (D) Geometric mean fluorescence intensity of TCRb from infected or control thymi. Representative flow cytometry (E) and absolute number (F) of CD62L, CD44, CD69, and KLRG1 expression on CD8+ T cells. Open circles represent uninfected mice; filled circles represent infected mice. Data are representative of two or more independent experiments with n = 3–5 mice per experiment. *p , 0.05, ***p , 0.001, ****p , 0.0001, unpaired two-tailed t test. The Journal of Immunology 3219 Downloaded from http://www.jimmunol.org/

FIGURE 6. Thymic CD8+ T cells with a restricted TCR Vb repertoire and intracellular effector molecules accumulate in MRV-infected mice. Representative flow cytometry (A) and frequency (B)ofTCRVb3,6,8.3,and13onCD8+ T cells. Representative flow cytometry (C) and frequencies (D) from intracellular staining of unstimulated thymocytes for IFN-g and granzyme B. Open circles represent uninfected mice; filled circles represent infected mice. Data are representative of two or more independent experiments with n = 3–5 mice per experiment. **p , 0.01, ***p , 0.001, ****p , 0.0001, unpaired two-tailed t test. by guest on September 26, 2021

MRV indeed can directly infect CD4+ developing thymocytes but This hypothesis would suggest that virus-specific CD8+ T cells can be found at higher frequencies in thymocytes not expressing would need to expand from the small pool of mature CD8+ T cells CD4. Infection of the DN subset could indicate that MRV has tro- already present in neonatal mice. Thus, the myriad potential pism for cells recently entering the thymus from the bone marrow or mechanisms for selective loss of CD4-expressing cells will require perhaps even more immature hematopoietic precursors. HHV6 was additional experimental analysis. initially identified as human B cell lymphotropic virus, but in our We also determined that activated CD8+ T cells infiltrated the studies we did not identify B cell tropism for MRV (S.J. Patel and thymus of infected animals. These cells expressed effector mol- W.M. Yokoyama, unpublished observations). Moreover, our studies ecules and possessed restricted TCR repertoires. Infection of mice of Rag12/2 animals strongly suggest that MRV infects cells other lacking CD8+ T cells prevented recovery of peripheral CD4+ than mature T and B cells, especially in the spleen where viral titers T cells and caused lethality, which was associated with elevated were very high. Future identification of infected cells will be critical viral titers in the thymi and spleens of CD82/2 animals compared to more comprehensively characterize MRV tropism. with B6 controls. We also found that the lethality observed was An important outstanding question is why CD4-expressing cells not dependent on acute CD4+ T cell loss, because TCRb2/2 mice, appear to be selectively susceptible to MRV-mediated depletion lacking CD4+ cells before infection, were similarly susceptible to with increased apoptosis. We hypothesize that MRV may have MRV as CD82/2 mice. Taken together, these experiments indicate evolved to temporarily hamper the immune system during acute that MRV infection is controlled by CD8+ T cells. infection to more efficiently disseminate and/or establish latency. During acute MRV infection, neonatal mice have severely impaired For example, MRV may express proteins that interact with mol- immune responses. The near-complete loss of CD4+ cells eliminates ecules specifically expressed in CD4+ T cells, ultimately affecting important Th subsets from enhancing the antiviral immune response. susceptibility to apoptosis following viral infection. Another po- Specifically, these deficient mice need to properly polarize, activate, tential mechanism of CD4+ thymocyte depletion includes the in- and prime the CD8+ T cells without Th cell–derived cytokines. fection of parenchymal cells or APCs, which interact with CD4- Perhaps not surprisingly, we found that mature B cells, and pre- expressing thymocytes. Specifically, infection of thymic stromal sumably Abs, were not required for control of neonatal MRV in- cells could alter presentation of peptides to CD4+ thymocytes, fection. In the absence of CD4+ T cells in the periphery, B cells may leading to their deletion, but this would not totally explain loss of not garner appropriate T cell help for class switching and somatic mature CD4+ T cells already present in the spleen. Finally, it is hypermutation. Thus, during neonatal thymus infection that causes possible that CD8+ T cells may be uniquely resistant to lytic in- severe CD4+ T cell loss, CD8+ T cells appear to be especially im- fection by MRV, even though we showed that they can be infected. portant for clearing MRV and preventing death. 3220 CD8 CONTROL OF MRV

FIGURE 7. CD8+ T cells, but not mature B cells, are required for recovery after MRV infection. P0 B6 mice were infected i.p. with MRV. (A) Time course from ﬂow cytometry on peripheral blood from CD82/2 mice. (B) Kaplan–Meier curve Downloaded from comparing infected with uninfected CD82/2 animals. (C) Time course from ﬂow cytometry on peripheral blood from mMT mice. (D) Kaplan– Meier curve comparing infected with uninfected mMT animals. MRV genome copy number from thymus (E) or spleen (F) of P0 infected mice har- 2/2

vested on day 7 p.i. from B6 mice or CD8 mice. http://www.jimmunol.org/ MRV genome copy number from thymus (G)or spleen (H) of P0 infected mice harvested on day 21 p.i. from B6 mice or CD82/2 mice. (I) Kaplan– Meier curve comparing infected with uninfected TCRb2/2 animals. Gray points and lines represent uninfected mice; black points and lines represent infected mice. Data in (A) and (C) are representative of two independent experiments with n = 3–6 mice in each group. Data in (B) and (D) are pooled from two independent experiments with n = 12 and by guest on September 26, 2021 n = 10 mice, respectively. Data in (E)–(H) are pooled from two independent experiments (n =4– 7). Data in (I) are pooled from two independent experiments (n = 6–9). Viral titers were compared using an unpaired two-tailed t test on log10 relative genome copies. Survival analyses were conducted using the log-rank Mantel–Cox test. **p , 0.01, ***p , 0.001, ****p , 0.0001.

Although MRV infection of adult mice does not lead to similar CD8+ T cells may also be important in the control of MRV in adult reductions in CD4+ T cells, we can detect viral genome copies in mice, CD8+ T cell control may not be as dominant in adult mice as the thymus of adult animals infected with MRV (S.J. Patel and it is in neonatal mice. Further study on MRV infection of adult W.M. Yokoyama, unpublished observations). Many aspects of the mice will clarify these issues. innate and adaptive immune systems mature as mice age, and a Only a small fraction of humans with acute primary HHV6 combination of these factors likely protects adult mice from CD4+ infection present with symptoms of exanthem subitum. The mech- T cell depletion (25, 26). However, although we hypothesize that anisms explaining why some patients present with disease and The Journal of Immunology 3221 others appear to acquire HHV6 via subclinical infection are un- 10. Challoner, P. B., K. T. Smith, J. D. Parker, D. L. MacLeod, S. N. Coulter, known. Our studies suggest that roseoloviruses are controlled by T. M. Rose, E. R. Schultz, J. L. Bennett, R. L. Garber, M. Chang, et al. 1995. + + Plaque-associated expression of human herpesvirus 6 in multiple sclerosis. Proc. CD8 T cells and that impairment of the responsiveness of CD8 Natl. Acad. Sci. USA 92: 7440–7444. T cells can enhance viral pathogenesis. Perhaps patients with 11. Chapenko, S., A. Millers, Z. Nora, I. Logina, R. Kukaine, and M. Murovska. exanthem subitum have impaired CD8+ T cell responses, and this 2003. Correlation between HHV-6 reactivation and multiple sclerosis disease activity. J. Med. Virol. 69: 111–117. accounts for their disease presentation. A subset of children with 12. Goodman, A. D., D. J. Mock, J. M. Powers, J. V. Baker, and B. M. Blumberg. primary HHV6 have persistent viral DNA in their peripheral 2003. Human herpesvirus 6 genome and antigen in acute multiple sclerosis le- blood, and this too may be due to impaired CD8+ T cell function sions. J. Infect. Dis. 187: 1365–1376. 13. Knipe, D. M., and P. M. Howley, eds. 2013. Fields Virology. Wolters Kluwer (27). Human roseolovirus reactivation occurs during immune Health/Lippincott Williams & Wilkins, Philadelphia. suppression, another scenario in which CD8+ T cell surveillance is 14. Becerra, A., L. Gibson, L. J. Stern, and J. M. Calvo-Calle. 2014. Immune re- curtailed. Autologous T cell therapy for prophylaxis against sponse to HHV-6 and implications for immunotherapy. Curr. Opin. Virol. 9: 154– + 161. HHV6 reactivation has focused on activation and transfer of CD4 15. Leibovitch, E. C., G. S. Brunetto, B. Caruso, K. Fenton, J. Ohayon, D. S. Reich, T cells (28). Our studies suggest that CD8+ T cells may provide and S. Jacobson. 2014. Coinfection of human herpesviruses 6A (HHV-6A) and enhanced protection. HHV-6B as demonstrated by novel digital droplet PCR assay. PLoS One 9: e92328. 16. Nastke, M. D., A. Becerra, L. Yin, O. Dominguez-Amorocho, L. Gibson, Acknowledgments L. J. Stern, and J. M. Calvo-Calle. 2012. Human CD4+ T cell response to human herpesvirus 6. J. Virol. 86: 4776–4792. We thank Wandy Beatty and Jeff Elsner (Washington University Molecular 17. Patel, S. J., G. Zhao, V. R. Penna, E. Park, E. J. Lauron, I. B. Harvey, Microbiology Imaging Facility) for assistance with TEM imaging and mem- W. L. Beatty, B. Plougastel-Douglas, J. Poursine-Laurent, D. H. Fremont, et al. bers of the Yokoyama Laboratory for excellent discussions. 2017. A murine herpesvirus closely related to ubiquitous human herpesviruses Downloaded from causes T-cell depletion. J. Virol. 91: e02463–16. 18. Gobbi, A., C. A. Stoddart, M. S. Malnati, G. Locatelli, F. Santoro, N. W. Abbey, Disclosures C. Bare, V. Linquist-Stepps, M. B. Moreno, B. G. Herndier, et al. 1999. Human The authors have no ﬁnancial conﬂicts of interest. herpesvirus 6 (HHV-6) causes severe thymocyte depletion in SCID-hu Thy/Liv mice. J. Exp. Med. 189: 1953–1960. 19. Tanner, A., S. A. Carlson, M. Nukui, E. A. Murphy, and B. K. Berges. 2013. Human herpesvirus 6A infection and immunopathogenesis in humanized Rag22/2 References 2 2 gc / mice. J. Virol. 87: 12020–12028. http://www.jimmunol.org/ 1. Braun, D. K., G. Dominguez, and P. E. Pellett. 1997. Human herpesvirus 6. Clin. 20. Horvat, B., B. K. Berges, and P. Lusso. 2014. Recent developments in animal Microbiol. Rev. 10: 521–567. models for human herpesvirus 6A and 6B. Curr. Opin. Virol. 9: 97–103. 2. Blazsek, A., P. Sillo, N. Ishii, P. Gergely, Jr., G. Poor, K. Preisz, T. Hashimoto, 21. Morse, S. S., N. Sakaguchi, and S. Sakaguchi. 1999. Virus and autoimmunity: M. Medvecz, and S. Ka´rpa´ti. 2008. Searching for foreign antigens as possible induction of autoimmune disease in mice by mouse T lymphotropic virus triggering factors of autoimmunity: Torque teno virus DNA prevalence is ele- (MTLV) destroying CD4+ T cells. J. Immunol. 162: 5309–5316. vated in sera of patients with bullous pemphigoid. Exp. Dermatol. 17: 446–454. 22. Rowe, W. P., and W. I. Capps. 1961. A new mouse virus causing necrosis of the 3. Shimazu, Y., T. Kondo, T. Ishikawa, K. Yamashita, and A. Takaori-Kondo. 2013. thymus in newborn mice. J. Exp. Med. 113: 831–844. Human herpesvirus-6 encephalitis during hematopoietic stem cell transplanta- 23. Morse, S. S., and J. E. Valinsky. 1989. Mouse thymic virus (MTLV). A mam- tion leads to poor prognosis. Transpl. Infect. Dis. 15: 195–201. malian herpesvirus cytolytic for CD4+ (L3T4+) T lymphocytes. J. Exp. Med. 4. Yamashita, N., and T. Morishima. 2005. HHV-6 and seizures. Herpes 12: 46–49. 169: 591–596. 5. Kondo, K., H. Nagafuji, A. Hata, C. Tomomori, and K. Yamanishi. 1993. As- 24. Lauritsen, J. P., M. D. Christensen, J. Dietrich, J. Kastrup, N. Odum, and

sociation of human herpesvirus 6 infection of the central nervous system with by guest on September 26, 2021 C. Geisler. 1998. Two distinct pathways exist for down-regulation of the TCR. recurrence of febrile convulsions. J. Infect. Dis. 167: 1197–1200. J. Immunol. 161: 260–267. 6. Zerr, D. M., A. S. Meier, S. S. Selke, L. M. Frenkel, M. L. Huang, A. Wald, 25. Maro´di, L. 2006. Neonatal innate immunity to infectious agents. Infect. Immun. M. P. Rhoads, L. Nguy, R. Bornemann, R. A. Morrow, and L. Corey. 2005. A population-based study of primary human herpesvirus 6 infection. N. Engl. J. 74: 1999–2006. Med. 352: 768–776. 26. Levy, O. 2007. Innate immunity of the newborn: basic mechanisms and clinical 7. Yamanishi, K., T. Okuno, K. Shiraki, M. Takahashi, T. Kondo, Y. Asano, and correlates. Nat. Rev. Immunol. 7: 379–390. T. Kurata. 1988. Identiﬁcation of human herpesvirus-6 as a causal agent for 27. Caserta, M. T., M. P. McDermott, S. Dewhurst, K. Schnabel, J. A. Carnahan, exanthem subitum. Lancet 1: 1065–1067. L. Gilbert, G. Lathan, G. K. Lofthus, and C. B. Hall. 2004. Human herpesvirus 6 8. Ablashi, D. V., H. B. Eastman, C. B. Owen, M. M. Roman, J. Friedman, (HHV6) DNA persistence and reactivation in healthy children. J. Pediatr. 145: J. B. Zabriskie, D. L. Peterson, G. R. Pearson, and J. E. Whitman. 2000. Frequent 478–484. HHV-6 reactivation in multiple sclerosis (MS) and chronic fatigue syndrome 28. Gerdemann, U., L. Keukens, J. M. Keirnan, U. L. Katari, C. T. Nguyen, (CFS) patients. J. Clin. Virol. 16: 179–191. A. P. de Pagter, C. A. Ramos, A. Kennedy-Nasser, S. M. Gottschalk, 9. Ablashi, D. V., W. Lapps, M. Kaplan, J. E. Whitman, J. R. Richert, and H. E. Heslop, et al. 2013. Immunotherapeutic strategies to prevent and treat G. R. Pearson. 1998. Human herpesvirus-6 (HHV-6) infection in multiple scle- human herpesvirus 6 reactivation after allogeneic stem cell transplantation. rosis: a preliminary report. Mult. Scler. 4: 490–496. Blood 121: 207–218.