Collateral Damage: What Effect Does Anti-CD4 and Anti-CD8 α Antibody− Mediated Depletion Have on Leukocyte Populations? This information is current as of October 1, 2021. So Ri Jung, Tamara Suprunenko, Thomas M. Ashhurst, Nicholas J. C. King and Markus J. Hofer J Immunol published online 24 August 2018 http://www.jimmunol.org/content/early/2018/08/23/jimmun

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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 © 2018 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published August 24, 2018, doi:10.4049/jimmunol.1800339 The Journal of Immunology

Collateral Damage: What Effect Does Anti-CD4 and Anti-CD8a Antibody–Mediated Depletion Have on Leukocyte Populations?

So Ri Jung,*,†,‡,x Tamara Suprunenko,*,†,‡,x Thomas M. Ashhurst,†,‡,x,{,# Nicholas J. C. King,†,‡,x,{,# and Markus J. Hofer*,†,‡,x

Anti-CD4 or anti-CD8a Ab–mediated depletion strategies are widely used to determine the role of subsets. However, surface expression of CD4 and CD8a is not limited to T cells and occurs on other leukocyte populations as well. Using both unbiased t-distributed stochastic neighbor embedding of flow cytometry data and conventional gating strategies, we assessed the impact of anti-CD4 and anti-CD8a Ab–mediated depletion on non–T cell populations in mice. Our results show that anti-CD4 and anti- CD8a Ab injections not only resulted in depletion of T cells but also led to depletion of specific dendritic cell subsets in a dose- dependent manner. Importantly, the extent of this effect varied between mock- and virus-infected mice. We also demonstrate the Downloaded from importance of using a second, noncompeting Ab (clone CT-CD8a) to detect CD8a+ cells following depletion with anti-CD8a Ab clone 2.43. Our study provides a necessary caution to carefully consider the effects on nontarget cells when using Ab injections for leukocyte depletion in all experimental conditions. The Journal of Immunology, 2018, 201: 000–000.

ince 1986, injection of anti-CD4 or anti-CD8 Abs into mice CD8 surface complex differs between DCs (CD8a/CD8a homo- http://www.jimmunol.org/ has been a widely used technique to deplete CD4+ or CD8+ dimer) and T cells (CD8a/CD8b heterodimer) (12). However, S T cell subsets, respectively, in vivo (1–3). However, as the certain subsets of CD8+ T cells, such as intraepithelial lymphocytes, process of depletion relies on Ag–Ab interactions, concerns have express only CD8a and not CD8b on their surface and are thus not been raised about collateral effects on non–T cells with surface depleted by anti-CD8b Ab administration (13, 14). There is no expression of CD4 or CD8. The principal cell populations of equivalent strategy for specific CD4+ T cell depletion as cells ex- concern are dendritic cells (DCs), with specific DC subsets press CD4 as monomers or disulfide-linked homodimers (15, 16). expressing varying levels of CD4 or CD8a. DCs are key APCs Importantly, the precise effects of anti-CD4 or anti-CD8 Ab (reviewed in Ref. 4), and collateral depletion of these cells could injections on non–T cell populations are not well characterized

have far-reaching consequences for a host’s immune response, and, in many studies, not assessed or considered, despite their by guest on October 1, 2021 especially during viral infection. CD11b+ DCs express CD4, potential effect on experimental outcomes. In this study, we ana- whereas CD8a+ DCs are identified by the presence of CD8a lyzed the effects of anti-CD4 or anti-CD8a Ab injections on (5, 6). In addition, plasmacytoid DCs (pDCs) express a high level several leukocyte populations in the spleen and blood of naive and of CD4 and a varying level of CD8a (5, 6). Accordingly, signif- virus-infected mice. icant reductions in the number of CD4+ DCs or CD8a+ DCs are reported after anti-CD4 or anti-CD8a Ab injections, respectively Materials and Methods (7–9). To reduce the impact on DCs, Abs against CD8b, instead of Literature search the commonly used target CD8a,havebeenused(10,11),asthe Scientific literature was reviewed by searching for titles and abstracts using the following search strings: “(CD4 or L3T4) and (deplet* or ablat* or remov*)” or “(CD8 or Lyt-2) and (deplet* or ablat* or remov*)” in *School of Life and Environmental Sciences, The University of Sydney, Sydney, New South Wales 2006, Australia; †Marie Bashir Institute for Infectious Diseases PubMed with entries up until June 1, 2018, being considered in this study. and Biosecurity, The University of Sydney, Sydney, New South Wales 2006, Australia; ‡Charles Perkins Centre, The University of Sydney, Sydney, New South Mice Wales 2006, Australia; xBosch Institute, The University of Sydney, Sydney, New { Female C57BL/6 mice aged between 8 and 12 wk were obtained from South Wales 2006, Australia; Sydney Cytometry, Core Facility of The University of Sydney and Centenary Institute, Sydney, New South Wales 2006, Australia; Australian BioResources and housed in specific pathogen-free conditions and #Department of Pathology, School of Medical Sciences, Sydney Medical in the animal facility of the University of Sydney. Ethical approval for the School, The University of Sydney, Sydney, New South Wales 2006, Australia use of all mice was obtained from the University of Sydney Animal Care and Ethics Committee (AEC 1056/16). The lymphocytic choriomeningitis ORCIDs: 0000-0003-1611-7644 (S.R.J.); 0000-0001-7269-7773 (T.M.A.); 0000- 0002-3877-9772 (N.J.C.K.); 0000-0001-5111-3978 (M.J.H.). virus (LCMV) Armstrong (ARM) 53b stock was obtained originally from a triple plaque-purified clone that was subsequently passaged twice in Received for publication March 7, 2018. Accepted for publication August 1, 2018. BHK cells in Michael Oldstone’s (17) laboratory and kindly provided by This work was supported by a seeding grant from the Marie Bashir Institute for I. L. Campbell (18). For virus inoculation, mice were given i.p. injection of Infectious Diseases and Biosecurity at The University of Sydney (to M.J.H.). 500 PFU (low-dose) or 200,000 PFU (high-dose) LCMV-ARM 53b in Address correspondence and reprint requests to Dr. Markus J. Hofer, University 200 ml of PBS plus 2.5% FBS. Mock-infected mice received the same of Sydney, Maze Crescent, Building G08, Sydney, NSW 2006, Australia. E-mail volume of PBS plus 2.5% FBS without the virus. The anti-CD4 (clone address: [email protected] GK1.5; ATCC TIB-207) and anti-CD8a Abs (clone 2.43; ATCC TIB-210) Abbreviations used in this article: ARM, Armstrong; DC, dendritic cell; LCMV, were purchased from Bio X Cell (West Lebanon, NH) or produced in lymphocytic choriomeningitis virus; MFI, median fluorescence intensity; pDC, plas- house from hybridoma supernatant as described previously (18) macytoid DC; t-SNE, t-distributed stochastic neighbor embedding. and quantified by ELISA according to the manufacturer’s instructions (Invitrogen). Two different strategies were used to achieve sufficient de- Copyright Ó 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$35.00 pletion in mice infected with low-dose or high-dose LCMV: mice infected

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1800339 2 CD4 AND CD8a DEPLETION AFFECTS DIFFERENT T CELLS AND DCs with low-dose LCMV and corresponding mock-infected control mice were nine publications used an in vivo approach to deplete CD4+ cells injected i.p. with 10 mg in-house made anti-CD4 or anti-CD8a Abs in in a mouse model, of which 145 used anti-CD4 Ab injection 100 ml of PBS on the day of infection and on days 2, 4, and 6 postin- regimens. Of these, 137 manuscripts attempted to deplete CD4+ fection. By contrast, mice infected with high-dose LCMV and corre- sponding mock-infected control mice were injected i.p. with 200 mgof T cells only, whereas just seven manuscripts acknowledged that anti-CD4 or anti-CD8a Abs on the day of infection and on days 3 and 6 any CD4+ cells could have been affected by this methodology postinfection. Although the first is sufficient to achieve .95% reduction of (26–32). More than 80% of all published work performing anti- + + CD4 or CD8a leukocytes in peripheral blood from uninfected mice, the CD4 Ab–mediated depletion method used GK1.5. Similarly, of latter is more commonly used (e.g., Refs. 19–22). Control mice received an equal volume of PBS without Ab or 200 mg anti–keyhole limpet hemo- the 809 manuscripts found on CD8 depletion, 801 manuscripts cyanin (clone LTF-2; Bio X Cell; isotype control), respectively. had English full texts available, and 185 papers performed anti- CD8 Ab–mediated depletion of CD8+ cells in vivo. Six articles Flow cytometry stated that any CD8-expressing cells could have been affected On day 7 postinfection, mice were deeply anesthetized by halothane inhalation (27, 29, 30, 33–35), whereas 178 papers did not comment on cells and euthanized by exsanguination via cardiac puncture. In the case where liver other than T cells. Diverse clones of anti-CD8a and anti-CD8b leukocytes were isolated, the mice were perfused with sterile PBS. A one-tenth Abs were used, with anti-CD8a Ab clones 2.43 and 53-6.7 being volume of 0.5 M EDTA was added to collected blood to prevent coagulation, and RBCs were lysed using buffered ammonium chloride solution (170 mM the most common choices (Fig. 1B). These findings suggested NH4Cl, 20 mM HEPES [pH 7.4]). Single-cell suspensions of blood leuko- that although there is robust evidence for CD4 and/or CD8 sur- cytes, splenocytes, and liver leukocytes were stained for cell surface markers face expression on non–T cell populations, there is widespread using specific fluorophore-conjugated Abs optimized for flow cytometry. The uncertainty as to whether they are also affected by Ab injection reagents and Abs used in this study were as follows: LIVE/DEAD Fixable regimens.

Blue Dead Cell Stain (Thermo Fisher Scientific), anti-CD11b-BUV395 (clone Downloaded from M1/70; BD Biosciences), anti-B220-BUV737 (clone RA3-6B2; BD Biosci- Anti-CD8a Ab clone CT-CD8a is a suitable detection Ab ences), anti-MHCII(I-A/I-E)-BV510 (clone M5/114.15.2; BioLegend), anti- a Ly6C-BV605 (clone HK1.4; BioLegend), anti-Ly6G-BV650 (clone 1A8; postdepletion with anti-CD8 Ab clone 2.43 BioLegend), anti-CD8a-FITC (clone CT-CD8a; Thermo Fisher Scien- It was previously reported that anti-CD4 Ab clone RM4-4 is a tific), anti-Siglec-H-PE (clone 551; BioLegend), anti-CD3ε-PE/Cy5.5 (clone 145-2C11; Thermo Fisher Scientific), anti-NK1.1-PE/Cy5 (clone PK136; suitable detection Ab postdepletion with anti-CD4 Ab clone BioLegend), anti-CD8b-PE/Cy7 (clone H35-17.2; Thermo Fisher Scientific), GK1.5, being noncompetitive in its binding capacity (36). In the anti-CD4-allophycocyanin (clone RM4-4; BioLegend), anti-CD8a- case of anti-CD8a Ab–mediated depletion of CD8a+CD8b+ http://www.jimmunol.org/ allophycocyanin (clone 53-6.7; BioLegend; for in vitro blocking experiments), T cells, Abs targeting CD8b have previously been used for de- anti-CD45-Alexa Fluor 700 (clone 30-F11; BioLegend), and anti-CD11c- tection (37, 38). However, as CD8a+CD8b2 T cells and CD8a+ allophycocyanin/Cy7 (clone N418; BioLegend). For in vitro blocking experi- ments, two million splenocytes were incubated with serially diluted DCs do not have surface expression of CD8b, it was necessary to anti-CD8a Ab (clone 2.43) in 200 mlofPBSfor20minat4˚Cinthe identify an anti-CD8a Ab clone that does not compete with the dark prior to staining with fluorophore-labeled Abs. Stained and fixed cells commonly used anti-CD8a depleting Ab clone 2.43. were analyzed with a Becton Dickinson custom 10-laser LSR II flow We examined the binding capacity of anti-CD8a Ab clones 53-6.7 cytometer and FlowJo software (v.10.4.1). The staining index was calcu- lated using median fluorescence intensity (MFI) as follows: staining and CT-CD8a on splenocytes preincubated with anti-CD8a Ab index = (MFIpositive population 2 MFInegative population)/SDnegative population. clone 2.43. The anti-CD8b Ab clone H35-17.2 was included as a by guest on October 1, 2021 control, as it binds to a different Ag from the blocking Ab, and t-Distributed stochastic neighbor embedding of multicolor flow hence no competition was expected in CD8a+CD8b+ Tcells.The cytometry data via Barnes–Hut approximations binding capacity of each clone was assessed by calculating the As a dimensionality reduction technique for multicolor flow cytometry staining index, an estimate of the separation between the positive data, Barnes–Hut-implemented t-distributed stochastic neighbor em- and the negative populations, and the proportion of cells expressing bedding (t-SNE) was used (23, 24). Following color compensation, + the marker. Clone 53-6.7 showed a rapid decline in both the staining leukocytes were gated on live CD45 cells, followed by subgating for CD4+ and/or CD8a+ cells, of which 30,000 representative events were index and the percentage of positive cells to zero as the amount of randomly sampled using FlowJo. The t-SNE plugin in FlowJo was used clone 2.43 used in preincubation increased (Fig. 2A, 2B). Although to calculate t-SNE parameters. Subsequently, data were exported as staining with CT-CD8a showed a decline in the staining index as comma-separated values and visualized using a script in RStudio the amount of 2.43 Ab increased, it remained well above the basal (v1.1.383) (25). level even after preincubation with the maximal amount of 2.43 Ab. Results There was also a decline in the percentage of positive cells as the amount of blocking Ab increased. However, this decline in the Most scientific publications reporting the usage of anti-CD4 or percentage of positive cells with increasing amount of blocking Ab anti-CD8 Ab injection regimens focus on T cell ablations only was also observed to a similar degree in anti-CD8b Ab clone To survey the usage of anti-CD4 or anti-CD8 Ab injection regimens H35-17.2–stained cells (Fig. 2A, 2B). These results suggest that for depleting specific leukocyte subsets, we performed a literature anti-CD8a Ab clone CT-CD8a, but not 53-6.7, is a suitable, search on scientific publications in PubMed. A total of 13,080 and noncompeting detection Ab postdepletion with clone 2.43. 9335 entries were returned on searches for “(CD4 or L3T4) and (deplet* or ablat* or remov*)” or “(CD8 or Lyt-2) and (deplet* or Ab injection regimens target specific leukocyte populations a ablat* or remov*),” respectively (Fig. 1A). Although PubMed with surface expression of CD4 and/or CD8 allows for species restriction using medical subject headings key- To determine the effects of repeated anti-CD4 or anti-CD8a Ab word searches, species restriction to Mus musculus resulted in injections on multiple leukocyte populations, we analyzed spleno- removal of true-positive hits, and hence the search was not lim- cytes and blood leukocytes from naive and low-dose (500 PFU) ited to murine models. Instead, full-text analysis was performed LCMV–infected mice. LCMV infection was chosen as it is com- to remove false positives and to identify experimental details. For monly used to study immune responses, and Ab-mediated cell feasibility, the detailed full-text analysis was limited to articles depletion strategies have regularly been used in this model [e.g., published between January 1, 2016, and June 1, 2018 (Fig. 1B). (39, 40)]. Our results had shown that although the anti-CD8a de- Of the 1149 manuscripts that were found on CD4 depletion, 1138 tection Ab clone, CT-CD8a, is suitable to detect CD8a+ cells after manuscripts had English full texts available. Two hundred and depletion, this clone nevertheless showed a significant decline in the The Journal of Immunology 3 Downloaded from http://www.jimmunol.org/

FIGURE 1. Most of the scientific literature using anti-CD4 or anti-CD8 Ab–mediated depletion methods focuses on ablating T cell subsets. (A) Number by guest on October 1, 2021 of scientific manuscripts returned using the search strings “(CD4 or L3T4) and (deplet* or ablat* or remov*)” or “(CD8 or Lyt-2) and (deplet* or ablat* or remov*).” (B) Usage of anti-CD4 or anti-CD8 Ab-mediated depletion methods in the literature from January 1, 2016, to June 1, 2018, performing manual full-text searches. staining index (Fig. 2A, 2B). To account for this, we used the following anti-CD8a Ab injection regimen in both mock- and fluorescence minus one controls as the negative cutoffs to include LCMV-infected mice (Fig. 3C; arrowheads). Although there was a the CD4lo and CD8alo events when gating for CD4+ and/or CD8a+ reduction of CD4hi subset of pDCs following anti-CD4 Ab injection cells. regimen, CD4lo pDCs were retained (Fig. 3C; arrowheads). How- To examine the different leukocyte populations affected by the Ab ever, t-SNE analysis could not determine whether the disappearance injection regimens, t-SNE was performed as an unbiased categori- of CD4hi pDCs was because of specific ablation by anti-CD4 Ab zation and visualization method (Figs. 3, 4). t-SNE analysis on injections or the reduction in CD4 fluorescence intensity mediated mock-infected splenocytes pregated for live CD45+,CD4+, and/or by interactions between the depletion and detection Abs. Near- CD8a+ revealed that most leukocytes with surface expression of identical observations were made with t-SNE analysis of blood CD4 and/or CD8a were also CD3ε+ (CD4hi or CD8ahi)orB220+ samples (data not shown). (CD4lo or CD8alo) (Fig. 3B). As depicted by the insets, pDCs A recent report using anti-CD3ε Abs had shown off-target effects (Siglec-H+), CD11b+ DCs (CD11c+ CD11b+), and CD8a+ DCs on cells not expressing the CD3ε Ag (41). To assess any off-target (CD11c+ CD8a+) were also readily identified. When the effects of effects on CD42CD8a2 cells, we employed a manual gating low-dose Ab injection regimens and low-dose LCMV infection strategy to determine the changes in the numbers of CD42CD8a2 were examined, we found that Ab-mediated depletion was highly T cells, B cells, NK cells, neutrophils, and Ly6Chi monocytes effective and specific, with the most recognizable change being the (Fig. 3A). We found no significant changes in the numbers of these near-complete disappearance of CD3ε+ splenocytes with surface cells following Ab injection regimens (data not shown). Taken to- expression of CD4 in both mock- and LCMV-infected mice treated gether, these findings suggest that surface expression of CD4 and/or with anti-CD4 Ab (Fig. 3C). Similarly, there was a widespread loss CD8a is a necessary requirement for targeted depletion by the of CD3ε+ cells with surface expression of CD8a when mice were corresponding Ab injection regimens. Furthermore, these findings treated with anti-CD8a Ab (Fig. 3C). By contrast, B220+ cells show that the ablative efficacy of Ab injection regimens differs (CD4lo or CD8alo) were unaffected by Ab injection (Fig. 3C). between specific leukocyte subsets. There were variable effects on the DC populations, with CD11b+ To investigate Ab and virus dose-dependent effects on leukocyte DCs being largely unaffected following treatment with either anti- depletion efficacy, we performed t-SNE analysis on splenocytes, CD4 or anti-CD8a Ab, whereas CD8a+ DCs were greatly reduced blood leukocytes, and liver leukocytes from naive and high-dose 4 CD4 AND CD8a DEPLETION AFFECTS DIFFERENT T CELLS AND DCs

FIGURE 2. Anti-CD8a Ab clone CT-CD8a retains staining capacity on splenocytes preincubated with the anti-CD8a depletion Ab clone 2.43. (A) Splenocytes preincubated with various concentrations of anti-CD8a Ab clone 2.43 were stained with fluorophore-conjugated Downloaded from anti-CD8a Ab clone 53-6.7, clone CT-CD8a, or anti- CD8b Ab clone H35-17.2. (B) Staining index and percentage of positive cells as determined for each fluorophore-conjugated Ab. Dotted lines on the y-axis of staining index graphs indicate staining indices, as deter- mined from the fluorescence minus one control. Repre- sentative mean values from two independent experiments http://www.jimmunol.org/ are displayed from four experimental replicates with er- ror bars indicating SD. Some error bars were smaller than the symbols and are not visible in the figure. For statis- tical significance (one-way ANOVA with Dunnett post- test): *p , 0.05 compared with splenocyte preincubated with 1024 mg/ml of anti-CD8a Ab clone 2.43. by guest on October 1, 2021

(200,000 PFU) LCMV–infected mice that were treated with high- blood and liver samples from the same cohort of mice (data dose isotype anti-CD4 or anti-CD8a Abs (200 mg) (Fig. 4). As not shown). Moreover, CD42CD8a2 T cells, B cells, NK cells, above (Fig. 3B), t-SNE analysis on leukocytes pregated for live neutrophils, and Ly6Chi monocytes were not ablated following CD45+ CD4+ and/or CD8a+ identified CD3ε+ cells (CD4hi or high-dose Ab injections (data not shown). CD8ahi), B220+ cells (CD4lo or CD8alo), CD11b+ DCs (CD11c+ a CD11b+), CD8a+ DCs (CD11c+ CD8a+), and pDCs (Siglec-H+) Surface expression of CD4 and/or CD8 alone is not sufficient (Fig. 4A). Comparable to low-dose LCMV infection and low-dose condition for targeted ablation following Ab injection Ab injection regimens (Fig. 3C), mice that were given high-dose Our t-SNE analysis had shown that CD4+ and/or CD8a+ leuko- anti-CD4 or anti-CD8a Ab injections had a near-complete de- cytes were divided into three subsets, T cells (CD3ε+), B220+ pletion of CD3ε+ T cells (CD4hi or CD8ahi) but not B220+ cells cells, and DC subsets, and that the ablative efficacy of Ab injec- (CD4lo or CD8alo) (Fig. 4B). CD11b+ DCs were largely unaf- tion regimens varied between these groups. To quantitatively de- fected following high-dose anti-CD4 or anti-CD8a Ab injection termine the ablative effects of low-dose or high-dose anti-CD4 or regimens, whereas CD4hi pDCs were greatly reduced following anti-CD8a Ab injections on these cell populations separately, we high-dose anti-CD4 Ab injection in both mock- and LCMV- performed manual gating and quantitation. A reduction in number infected mice (Fig. 4B). Moreover, CD8a+ DCs were largely equivalent to more than 95% of CD4+ T cells or CD8a+CD8b+ ablated following high-dose anti-CD8a Ab injections (Fig. 4B). T cells was observed, independent of low-dose LCMV infection Near-identical observations were made with t-SNE analysis of in both the spleen and blood of mice treated with low doses of The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 3. Cell surface expression of CD4 and/or CD8a is a necessary but not sufficient requirement for ablation following low-dose Ab injection regimens in mock- or low-dose LCMV–infected mice. (A) Representative gating strategy shown for splenocytes. The same strategy was also employed for leukocytes from blood. (B) t-SNE analysis was performed on splenocytes (pregated for CD4+ and/or CD8a+ cells) from mock-infected mice, as described in Materials and Methods. Surface expression levels of CD4, CD8a, CD3ε, and B220 are shown. Insets depict surface expression levels of Siglec-H, CD11c, or CD11b as annotated. (C) t-SNE plots depicting surface expression levels of CD4 and CD8a on splenocytes from mock- or low-dose (500 PFU) LCMV– infected mice, treated with PBS, anti-CD4, or anti-CD8a Ab (10 mg) on the day of infection and days 2, 4, and 6 postinfection. Organs were harvested and analyzed on day 7 postinfection. Arrows indicate disappearance of T cell subsets, and arrowheads indicate disappearance of DC subsets. P1, neutrophils; P2, B cells; P3, NK cells; P4, Ly6Chi monocytes; P5, CD42CD8a2 T cells. 6 CD4 AND CD8a DEPLETION AFFECTS DIFFERENT T CELLS AND DCs Downloaded from http://www.jimmunol.org/

FIGURE 4. Cell surface expression of CD4 and/or CD8a is a necessary but not sufficient requirement for ablation following high-dose Ab injection regimens by guest on October 1, 2021 in mock- or high-dose LCMV–infected mice. (A) t-SNE analysis was performed on splenocytes (pregated for CD4+ and/or CD8a+ cells) from mock-infected mice as described in Materials and Methods. Surface expression levels of CD4, CD8a,CD3ε, and B220 are shown. Insets depict surface expression levels of Siglec-H, CD11c, or CD11b as annotated. (B) t-SNE plots depicting surface expression levels of CD4 and CD8a on splenocytes from mock- or high-dose (200,000 PFU) LCMV–infected mice treated with isotype, anti-CD4, or anti-CD8a Ab (200 mg) on the day of infection and days 3 and 6 postinfection. Organs were harvested and analyzed on day 7 postinfection. Arrows indicate disappearance of T cell subsets, and arrowheads indicate disappearance of DC subsets. anti-CD4 or anti-CD8a Abs, respectively (Fig. 5A–E). CD8a+ mice (40–50%) but not mock-infected mice, despite the absence CD8b2 T cells also showed a significant reduction, equaling ∼75% of the targeted Ag, CD8a (Fig. 5D). Contrary to the observations in both the spleen and blood of mock- and LCMV-infected mice made with t-SNE analysis (Fig. 3C), pDCs (CD4+ CD8avar) treated with anti-CD8a Ab (Fig. 5A–E). Furthermore, Ab treat- showed no ablation in either the spleen or blood of mock- and ments also reduced the number of CD4+CD8a+ double-positive LCMV-infected mice after being treated with low-dose anti-CD4 T cells in the spleen, with anti-CD4 treatment having greater effect or anti-CD8a Abs, suggesting that the apparent disappearance of (.97% reduction) than anti-CD8a treatment (80–85% reduction) in CD4hi pDCs in t-SNE analysis was due to decreased CD4 fluo- both mock- and LCMV-infected mice (Fig. 5D). Although double- rescence intensity from CD4hi to CD4lo, mediated by the inter- positive T cells were greatly reduced in the blood of mock-infected, actions between the depletion and detection Abs (Fig. 5C–E). anti-CD4-treated mice when compared with the control (∼90% Furthermore, there was a reduced efficacy of Ab-mediated de- reduction), anti-CD8a-treated mice did not show a significant re- pletion of cells in the blood of low-dose LCMV–infected mice duction in the number of these cells (Fig. 5E). Corroborating the (Fig. 5E) compared with the spleen of infected mice (Fig. 5D). findings in t-SNE analysis (Figs. 3, 4), B220+ cells (CD4lo or Most notably, 98% of CD4+CD8a+ double-positive T cells were CD8alo) were not changed in number (data not shown). depleted from the blood of mock-infected mice after anti-CD4 Ab CD11b+ DCs were not ablated by anti-CD4 Ab treatment in injections, but only 50% were ablated in mice infected with either the spleen or blood of mock- and low-dose LCMV–infected LCMV (Fig. 5E). CD8a+ DCs were depleted by 77% in mock- mice (Fig. 5A–E). By contrast, anti-CD8a Ab treatment signifi- infected mice but only by 50% in LCMV-infected mice after anti- cantly reduced (75–90%) numbers of CD8a+ DCs in the spleen, CD8a Ab treatment (Fig. 5E). independent of LCMV infection. Although a similar reduction of We confirmed these findings in mice that were either mock- or CD8a+ DCs was observed in the blood of uninfected mice, there high-dose LCMV–infected and treated with high-dose Ab injection was no statistically significant reduction in the blood of LCMV- regimens. High-dose LCMV infection mediated more widespread infected mice (Fig. 5E). There was also a significant reduction of changes in the number of leukocyte subsets, including a significant CD11b+ DCs in the spleen of anti-CD8a-treated, LCMV-infected expansion of CD8a+CD8b+ T cells when compared with low-dose The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 5. Ab injection regimens have Ab dose–dependent effects on distinct leukocyte populations. (A–C) Representative pseudocolor plots depicting changes in the proportions of leukocyte subsets in the spleen of mock- or low-dose LCMV–infected mice (500 PFU) treated with PBS, anti-CD4, or anti- CD8a Ab. Numbers indicate percentage of each subset as a proportion of the parent gating. Pregated on (A) live CD45+ CD3ε+,(B) live CD45+ CD11chi MHCIIhi,or(C) live CD45+ B220+.(D) Number of specific leukocyte populations in the spleen or (E) peripheral blood of (Figure legend continues) 8 CD4 AND CD8a DEPLETION AFFECTS DIFFERENT T CELLS AND DCs infection (Fig. 5D, 5E, 5I–K), indicating more robust immunolog- affected to the same degree. Importantly, only leukocytes with ical responses. Near-identical observations were made regarding the surface expression of CD4 and/or CD8a were depleted, indicating specific ablative effects of anti-CD4 or anti-CD8a Ab injection minimal off-target effects. regimens on leukocytes from the spleen and blood, with a few After anti-CD4 Ab injections, CD4+ T cells and CD4+CD8a+ exceptions (Fig. 5F–J). Notably, high-dose anti-CD8a Ab injections double-positive T cells were significantly ablated. However, resulted in a near-complete depletion of CD8a+ DCs (.96%) in the CD11b+ DCs were not affected by both low- and high-dose Ab spleen of mock- and high-dose LCMV–infected mice. Furthermore, injection regimens, despite expressing CD4. This finding differs contrary to low-dose Ab injection regimens, injections of high-dose from a previously published report that showed significant re- anti-CD4 or anti-CD8a Abs resulted in a significant reduction in the duction of CD4+CD8a2 DCs following anti-CD4 Ab injection number of pDCs in the spleen (∼50%) but not the blood. In the (8). This discrepancy may be the result of differences in the Ab liver, CD4+ T cells were significantly ablated following an anti-CD4 injection regimens. Martı´n et al. (8) injected 300 mg of anti-CD4 Ab injection regimen in both mock-infected (∼71%) and LCMV- clone GK1.5 daily for three consecutive days, whereas we used an infected mice (∼98%) (Fig. 5K). CD8a+CD8b+ T cells had a near- injection regimen of 10 mg every other day or 200 mg every third complete depletion (∼99% in both mock- and LCMV-infected day. Although both doses of Ab injections were sufficient to de- mice) following an anti-CD8a Ab injection regimen, and CD8a+ plete .95% of all CD4+ leukocytes in the spleen, peripheral CD8b2 TcellsandCD8a+ DCs were also significantly depleted in blood, and liver, our results indicate that depletion rates differ both mock- and high-dose LCMV–infected mice (Fig. 5K). The between specific subsets. The reduced susceptibility of DCs numbers of CD11b+ DCs and pDCs were changed following Ab compared with T cells may be due to the significantly lower level injections only in LCMV-infected mice (Fig. 5K). of CD4 on these cells. Notably, using t-SNE analysis, we observed Next, we assessed the relative surface expression levels of CD4 that the CD4hi, but not all of the CD4lo, subset of pDCs appeared Downloaded from and/or CD8a on T cells and DCs. In both the spleen and blood, to be reduced following low-dose anti-CD4 Ab treatment. By CD4+CD8a+ T cells had a relatively similar level of CD4 as CD4+ contrast, manual gating showed no effect on the overall number of T cells, whereas their CD8a level was significantly lower than those pDCs. The apparent depletion of pDCs seen in t-SNE analysis is of CD8a+CD8b+ T cells. Despite having detectable CD4 expres- thus likely a reduction in CD4 fluorescence intensity from CD4hi sion, CD11b+ DCs and pDCs had significantly lower CD4 surface to CD4lo, mediated by interactions between the depletion and expression when compared with CD4+ T cells in the spleen, blood, detection Abs. However, in contrast to low-dose Ab injections, http://www.jimmunol.org/ and liver (Fig. 6A, 6B). By contrast, CD8a+ DCs in the spleen but high-dose anti-CD4 and anti-CD8a Ab injections resulted in a not blood or liver had a similar level of CD8a when compared with significant reduction in the number of pDCs in the spleen but not CD8a+CD8b+ T cells, whereas the MFI of CD8a in pDCs was less blood or liver, suggesting a dose-dependent increase in collateral than one-tenth that of CD8a+CD8b+ T cells in the spleen, blood, ablative effects on non–T cell populations. and liver (Fig. 6A, 6B). No significant differences were observed in In contrast to anti-CD4 Ab injections, anti-CD8a Ab injection the levels of CD4 or CD8a when comparing leukocytes from the regimens ablated CD8a+ T cells and DCs in the spleen, blood, and spleen and blood of mock-versus low-dose LCMV–infected mice, liver but not CD4+CD8a+ double-positive T cells in the blood of with the exception of a significant decline in the surface level of either LCMV-infected or mock-infected mice. The collateral ef- by guest on October 1, 2021 CD8a on CD8a+CD8b+ T cells in the blood (Fig. 6A). By contrast, fects of anti-CD8a Ab injections on splenic CD8a+ DCs have the surface expression level of CD4 was significantly decreased in been reported previously by Zhan et al. (9) and are possibly due to CD4+ splenocytes (CD4+ Tcells,CD4+CD8a+ T cells, and pDCs), the equally high surface expression level of CD8a on DC and blood leukocytes (CD4+CD8a+ T cells and pDCs), and liver CD8a+ T cell subsets in the spleen. The comparatively higher leukocytes (pDCs) in mice infected with a high dose of LCMV level of CD4 versus CD8a on CD4+CD8a+ double-positive (Fig. 6B). The same was true for the surface expression of CD8a, T cells may also explain the greater efficacy of the anti-CD4 Ab which was significantly lower on CD8a+CD8b+ T cells in the than the anti-CD8a Ab in depleting these cells. However, despite spleen, blood, and liver of high-dose LCMV–infected mice com- having significantly lower surface expression of CD8a than pared with mock mice. CD8a was also significantly lower on CD8a+CD8b+ T cells, CD8a+CD8b2 T cells in the spleen, blood, CD8a+ DCs in the spleen and liver, but not blood, of high-dose and liver and CD8a+ DCs in the blood and liver were successfully LCMV–infected mice compared with mock-infected mice depleted. Furthermore, the similar level of CD8a on CD8a+ DCs (Fig. 6B). In summary, these findings suggest that CD4 and/or from the blood of mock- versus low-dose and high-dose LCMV– CD8a surface expression is not sufficient for targeted depletion infected mice indicates that the reduced depletion efficacy is not by corresponding Ab injection regimens. due to intrinsic reduction of the surface Ag level or increased numbers of immature CD8alo DCs emigrating from the bone Discussion marrow in response to infection. Interestingly, we observed an In this study, we demonstrated that the administration of anti-CD4 overall reduction in the efficacy of low-dose anti-CD4 and anti- or anti-CD8a Abs depletes cognate T cell and DC subsets. CD8a Ab injection regimens in the blood of LCMV-infected However, not all cell populations expressing the target Ags were mice, which was not seen in mice treated with high-dose Abs.

mock- or low-dose LCMV–infected mice treated with PBS, anti-CD4, or anti-CD8a Ab (gray, red, and blue symbols, respectively). Combined sample size of n = 10 per experimental group from two independent experiments. Bar represents median. (F–H) Representative pseudocolor plots depicting changes in the proportions of leukocyte subsets in the spleen of mock- or high-dose LCMV–infected mice (200,000 PFU) treated with isotype, anti-CD4, or anti-CD8a Ab. Numbers indicate percentage of each subset as a proportion of the parent gating. Pregated on (F) live CD45+ CD3ε+,(G) live CD45+ CD11chi MHCIIhi, or (H) live CD45+ B220+.(I) Number of specific leukocyte populations in the spleen, (J) peripheral blood, or (K) liver of mock- or high-dose LCMV– infected mice treated with isotype, anti-CD4, or anti-CD8a Ab (gray, red, and blue symbols, respectively). Sample size of n = 4. Bar represents median. For statistical significance (Mann–Whitney U test): ns, non-significant. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001 compared with PBS-injected control [for (D) and (E)] or isotype Ab–injected control [for (I)–(K)] within the respective infection group. #p , 0.05, ##p , 0.01, ###p , 0.001, ####p , 0.0001 compared with mock-infected control within the respective Ab injection group. The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 6. Specific leukocyte subsets have different surface expression levels of CD4 and/or CD8a.(A) MFI of CD4 (red columns) and CD8a (blue columns) on CD4+ T cells (live CD45+ CD3ε+ CD4+ CD8a2), CD8a+CD8b+ T cells (live CD45+ CD3ε+ CD42 CD8a+ CD8b+), CD8a+CD8b2 T cells (live CD45+ CD3ε+ CD42 CD8a+ CD8b2), CD4+CD8a+ T cells (live CD45+ CD3ε+ CD4+ CD8a+), CD11b+ DCs (live CD45+ CD11chi MHCIIhi CD11b+), CD8a+ DCs (live CD45+ CD11chi MHCIIhi CD8a+), and pDCs (live CD45+ B220+ CD11c+ Siglec-H+) in the spleen or blood of PBS-injected mock-infected mice (nonstriped columns) and PBS-injected low-dose LCMV–infected mice (500 PFU; striped columns). Combined sample size of n = 10 from two inde- pendent experiments. (B) MFI of CD4 (red columns) and CD8a (blue columns) on CD4+ Tcells,CD8a+CD8b+ Tcells,CD8a+CD8b2 Tcells,CD4+CD8a+ Tcells,CD11b+ DCs, CD8a+ DCs, and pDCs in the spleen, blood, or liver of isotype Ab-injected mock-infected mice (nonstriped columns) and isotype Ab-injected high-dose LCMV–infected mice (200,000 PFU; striped columns). Sample size of n = 4. Column represents median, and error bars indicate 95% confidence interval. For statistical significance (Mann–Whitney U test): *p , 0.05 compared with CD4+ Tcells(whencomparingMFIofCD4)andCD8a+CD8b+ T cells (when comparing MFI of CD8a) within the respective infection group. #p , 0.05 compared with respective cell type in the mock-infected group.

The finding in the low-dose depleted animals is consistent with a We observed no depletion of leukocytes that did not express the previous report that increased levels of immune complexes of viral target Ags following low- or high-dose Ab injection regimens. By Ags and host Abs in circulation impair FcgR-dependent Ab- contrast, a recent study looking at the effects of anti-CD3ε Ab mediated leukocyte depletion in the peripheral blood of mice injections reported off-target effects on non–CD3ε-expressing persistently infected with LCMV (42, 43) and suggests a general cells (41). These effects correlated with the dose of depleting Ab inhibitory effect of Ab-mediated depletion during infection. Im- used. It is unclear if a similar effect would occur at an even higher portantly, the absence of this effect in high-dose treated mice dose; nevertheless, off-target effects should be assessed for each emphasized the necessity to determine the effective dosage for Ab injection regimen anew. each experimental condition. Further, it has been proposed that Furthermore, our findings demonstrate the need for using detection collateral effects on DC subsets could be avoided by using Abs Abs that do not compete with depletion Abs to assess treatment targeting anti-CD8b instead of anti-CD8a to specifically ablate efficacy accurately. This is also evident from Zhan et al. (9), who used CD8a+CD8b+ T cells (10, 11). However, this strategy would not two different gating strategies in parallel to assess depletion of ablate the CD8a+CD8b2 T cells, which are mostly present in CD8a+ DCs; the first was based on the presence of CD8a,whereas intestinal epithelium (44) and hence may not be suitable for the second approach gated for CD205+CD11b2 DCs based on the studying intraepithelial immune interactions. observation that CD8a+ DCs are positive for CD205 and negative 10 CD4 AND CD8a DEPLETION AFFECTS DIFFERENT T CELLS AND DCs for CD11b (45). The first gating strategy indicated a near-complete is expressed by epidermal Langerhans’ cells predominantly as covalent dimers. ∼ Exp. Dermatol. 12: 700–711. depletion, whereas the second strategy showed a reduction of 70% 16. Matthias, L. J., P. T. Yam, X. M. Jiang, N. Vandegraaff, P. Li, P. Poumbourios, + 2 in the number of CD205 CD11b DCs (9). The difference between N. Donoghue, and P. J. Hogg. 2002. Disulfide exchange in domain 2 of CD4 is the two gating strategies is likely due to competitive binding be- required for entry of HIV-1. Nat. Immunol. 3: 727–732. 17. Dutko, F. J., and M. B. Oldstone. 1983. Genomic and biological variation among tween the depletion and detection Abs in the first approach and commonly used lymphocytic choriomeningitis virus strains. J. Gen. Virol. 64: further demonstrates the importance of using appropriate detection 1689–1698. strategies when assessing Ab-mediated depletion. 18. Hofer, M. J., W. Li, P. Manders, R. Terry, S. L. Lim, N. J. King, and I. L. Campbell. 2012. Mice deficient in STAT1 but not STAT2 or IRF9 develop a In summary, our results demonstrate that both anti-CD8a and lethal CD4+ T-cell-mediated disease following infection with lymphocytic anti-CD4 Ab injection regimens have no off-target effects on choriomeningitis virus. J. Virol. 86: 6932–6946. 19. Zhu, D., L. Liu, D. Yang, S. Fu, Y. Bian, Z. Sun, J. He, L. Su, L. Zhang, H. Peng, leukocytes that lack the cognate surface Ags. However, anti-CD4 and Y. X. Fu. 2016. Clearing persistent extracellular antigen of hepatitis B virus: and anti-CD8a Ab injections have dose-dependent collateral ef- an immunomodulatory strategy to reverse tolerance for an effective therapeutic fects on Ag-expressing, non–T cell populations. Furthermore, vaccination. J. Immunol. 196: 3079–3087. 20. Lv, J., Y. Xiong, W. Li, W. Yang, L. Zhao, and R. He. 2017. BLT1 mediates efficacy and collateral effects of Ab depletion varied between bleomycin-induced lung fibrosis independently of neutrophils and CD4+ T cells. uninfected and infected mice, emphasizing the need for multi- J. Immunol. 198: 1673–1684. variate assessment of treatment effects in all experimental groups. 21. Zhu, F., T. Liu, C. Zhao, X. Lu, J. Zhang, and W. Xu. 2017. Whole-killed blood- stage vaccine-induced immunity suppresses the development of malaria parasites in mosquitoes. J. Immunol. 198: 300–307. 22. Gilfillan, C. B., S. Kuhn, C. Baey, E. J. Hyde, J. Yang, C. Ruedl, and Acknowledgments F. Ronchese. 2018. Clec9A+ dendritic cells are not essential for antitumor CD8+ We thank Iain L. Campbell for LCMVARM 53b, Brendon Davis for technical T cell responses induced by poly I:C immunotherapy. J. Immunol. 200: 2978– assistance in generating neutralizing antibodies, and Claire L. Thompson for 2986. Downloaded from editorial help with the manuscript. We thank the Sydney Cytometry facility for 23. van der Maaten, L. 2014. Accelerating t-SNE using tree-based algorithms. J. Mach. Learn. Res. 15: 3221–3245. assistance with flow cytometry and data analysis. We thank Monica Cooper at 24. van der Maaten, L., and G. Hinton. 2008. Visualizing data using t-SNE. J. Mach. the University of Sydney Library for assistance with literature review. Learn. Res. 9: 2579–2605. 25. Ashhurst, T. M. 2017. tSNEplots v1.2.0. GitHub repository. DOI: http://doi.org/ 10.5281/zenodo.893859. Available at: https://github.com/sydneycytometry/ Disclosures tSNEplots. Accessed: December 4, 2017. The authors have no financial conflicts of interest. 26. Ruan, S., Y. Cai, A. J. Ramsay, D. A. Welsh, K. Norris, and J. E. Shellito. 2017.

B cell and antibody responses in mice induced by a putative cell surface pep- http://www.jimmunol.org/ tidase of Pneumocystis murina protect against experimental infection. Vaccine 35: 672–679. References 27. Moriguchi, T., S. Kaneumi, S. Takeda, K. Enomoto, S. K. Mishra, T. Miki, 1. Wofsy, D., and W. E. Seaman. 1985. Successful treatment of autoimmunity in U. Koshimizu, H. Kitamura, and T. Kondo. 2016. Ecrg4 contributes to the anti- NZB/NZW F1 mice with monoclonal antibody to L3T4. J. Exp. Med. 161: 378– glioma immunosurveillance through type-I interferon signaling. OncoImmunol- 391. ogy 5: e1242547. 2. Benjamin, R. J., and H. Waldmann. 1986. Induction of tolerance by monoclonal 28. Fujinami, N., T. Yoshikawa, Y. Sawada, M. Shimomura, T. Iwama, S. Sugai, antibody therapy. Nature 320: 449–451. S. Kitano, Y. Uemura, and T. Nakatsura. 2016. Enhancement of antitumor effect 3. Cobbold, S. P., G. Martin, S. Qin, and H. Waldmann. 1986. Monoclonal anti- by peptide vaccine therapy in combination with anti-CD4 antibody: study in a bodies to promote marrow engraftment and tissue graft tolerance. Nature 323: murine model. Biochem. Biophys. Rep. 5: 482–491. 164–166. 29. Indrova´, M., J. Rossowska, E. Pajtasz-Piasecka, R. Mikyskova´, J. Richter,

4. Belz, G. T. 2008. Getting together: dendritic cells, T cells, collaboration and J. Rosina, R. Sedlacek, and A. Fiserova´. 2018. The role of immune cell sub- by guest on October 1, 2021 fates. Immunol. Cell Biol. 86: 310–311. populations in the growth and rejection of TC-1/A9 tumors in novel mouse 5. Dong, M. B., M. J. Rahman, and K. V. Tarbell. 2016. Flow cytometric gating for strains differing in the H2-D haplotype and NKC domain. Oncol. Lett. 15: spleen monocyte and DC subsets: differences in autoimmune NOD mice and 3594–3601. with acute inflammation. J. Immunol. Methods 432: 4–12. 30. Kawakubo, N., S. Tanaka, Y. Kinoshita, T. Tajiri, Y. Yonemitsu, and T. Taguchi. 6. Vremec, D., and K. Shortman. 1997. Dendritic cell subtypes in mouse lym- 2018. Sequential actions of immune effector cells induced by viral activation of phoid organs: cross-correlation of surface markers, changes with incubation, dendritic cells to eliminate murine neuroblastoma. J. Pediatr. Surg. 53: 1615– and differences among thymus, spleen, and lymph nodes. J. Immunol. 159: 1620. 565–573. 31. Goff, P. H., T. Hayashi, W. He, S. Yao, H. B. Cottam, G. S. Tan, B. Crain, 7. Dunne, P. J., B. Moran, R. C. Cummins, and K. H. Mills. 2009. CD11c F. Krammer, K. Messer, M. Pu, et al. 2017. Synthetic Toll-like receptor 4 (TLR4) +CD8alpha+ dendritic cells promote protective immunity to respiratory infection and TLR7 ligands work additively via MyD88 to induce protective antiviral with Bordetella pertussis. J. Immunol. 183: 400–410. immunity in mice. J. Virol. 91: e01050-17. 8. Martı´n, P., G. M. del Hoyo, F. Anjue`re, S. R. Ruiz, C. F. Arias, A. R. Marı´n, and 32. Glanville, N., T. J. Peel, A. Schro¨der, J. Aniscenko, R. P. Walton, S. Finotto, and C. Ardavı´n. 2000. Concept of lymphoid versus myeloid dendritic cell lineages S. L. Johnston. 2016. Tbet deficiency causes T helper cell dependent airways revisited: both CD8alpha(-) and CD8alpha(+) dendritic cells are generated from eosinophilia and mucus hypersecretion in response to rhinovirus infection. PLoS CD4(low) lymphoid-committed precursors. Blood 96: 2511–2519. Pathog. 12: e1005913. 9. Zhan, Y., Y. Xu, S. Seah, J. L. Brady, E. M. Carrington, C. Cheers, B. A. Croker, 33. Poczobutt, J. M., T. T. Nguyen, D. Hanson, H. Li, T. R. Sippel, M. C. Weiser-Evans, L. Wu, J. A. Villadangos, and A. M. Lew. 2010. Resident and monocyte-derived M. Gijon, R. C. Murphy, and R. A. Nemenoff. 2016. Deletion of 5-lipoxygenase in dendritic cells become dominant IL-12 producers under different conditions and the tumor microenvironment promotes lung cancer progression and metastasis signaling pathways. J. Immunol. 185: 2125–2133. through regulating T cell recruitment. J. Immunol. 196: 891–901. 10. Pham, T. T., M. Verheijen, L. Vandermosten, K. Deroost, S. Knoops, K. Van den 34. Kolachala, V. L., S. Palle, M. Shen, A. Feng, D. Shayakhmetov, and N. A. Gupta. Eynde, L. Boon, C. J. Janse, G. Opdenakker, and P. E. Van den Steen. 2017. 2017. Loss of L--guided CD8+, but not CD4+, cells protects against is- Pathogenic CD8+ T cells cause increased levels of VEGF-A in experimental chemia reperfusion injury in a steatotic liver. Hepatology 66: 1258–1274. malaria-associated acute respiratory distress syndrome, but therapeutic VEGFR 35. Mathios, D., C. K. Park, W. D. Marcus, S. Alter, P. R. Rhode, E. K. Jeng, inhibition is not effective. Front. Cell. Infect. Microbiol. 7: 416. H. C. Wong, D. M. Pardoll, and M. Lim. 2016. Therapeutic administration of 11. Saunderson, S. C., and A. D. McLellan. 2017. Role of lymphocyte subsets in the IL-15 superagonist complex ALT-803 leads to long-term survival and durable immune response to primary B cell-derived exosomes. J. Immunol. 199: 2225– antitumor immune response in a murine glioblastoma model. Int. J. Cancer 138: 2235. 187–194. 12. Leggat, J. A., D. L. Gibbons, S. F. Haque, A. L. Smith, J. W. Wells, K. Choy, 36. Arora, S., R. A. McDonald, G. B. Toews, and G. B. Huffnagle. 2006. Effect of a C. M. Lloyd, A. C. Hayday, and A. Noble. 2008. Innate responsiveness of CD8 CD4-depleting antibody on the development of Cryptococcus neoformans-induced memory T-cell populations nonspecifically inhibits allergic sensitization. J. Al- allergic bronchopulmonary mycosis in mice. Infect. Immun. 74: 4339–4348. lergy Clin. Immunol. 122: 1014–1021.e4. 37. Burrack, K. S., S. A. Montgomery, D. Homann, and T. E. Morrison. 2015. CD8+ 13. Gapin, L., H. Cheroutre, and M. Kronenberg. 1999. Cutting edge: TCR alpha beta+ T cells control Ross River virus infection in musculoskeletal tissues of infected CD8 alpha alpha+ T cells are found in intestinal intraepithelial lymphocytes of mice. J. Immunol. 194: 678–689. mice that lack classical MHC class I molecules. J. Immunol. 163: 4100–4104. 38. Burrack, K. S., J. J. L. Tan, M. K. McCarthy, Z. Her, J. N. Berger, L. F. P. Ng, 14. Konno, A., K. Okada, K. Mizuno, M. Nishida, S. Nagaoki, T. Toma, T. Uehara, and T. E. Morrison. 2015. Myeloid cell arg1 inhibits control of arthritogenic K. Ohta, Y. Kasahara, H. Seki, et al. 2002. CD8alpha alpha memory effector alphavirus infection by suppressing antiviral T cells. PLoS Pathog. 11: T cells descend directly from clonally expanded CD8alpha +beta high TCRalpha e1005191. beta T cells in vivo. Blood 100: 4090–4097. 39. Matloubian, M., R. J. Concepcion, and R. Ahmed. 1994. CD4+ T cells are re- 15. Lynch, G. W., E. K. Slaytor, F. D. Elliott, A. Saurajen, S. G. Turville, quired to sustain CD8+ cytotoxic T-cell responses during chronic viral infection. A. J. Sloane, P. U. Cameron, A. L. Cunningham, and G. M. Halliday. 2003. CD4 J. Virol. 68: 8056–8063. The Journal of Immunology 11

40. Penaloza-MacMaster, P., N. M. Provine, E. Blass, and D. H. Barouch. 2015. CD4 43. Yamada, D. H., H. Elsaesser, A. Lux, J. M. Timmerman, S. L. Morrison, J. C. de T cell depletion substantially augments the rescue potential of PD- blockade la Torre, F. Nimmerjahn, and D. G. Brooks. 2015. Suppression of Fcg-receptor- for deeply exhausted CD8 T cells. J. Immunol. 195: 1054–1063. mediated antibody effector function during persistent viral infection. Immunity 41. Loubaki, L., T. Tremblay, and R. Bazin. 2013. In vivo depletion of leuko- 42: 379–390. cytes and platelets following injection of T cell-specific antibodies into mice. 44. Cheroutre, H., F. Lambolez, and D. Mucida. 2011. The and dark sides of J. Immunol. Methods 393: 38–44. intestinal intraepithelial lymphocytes. Nat. Rev. Immunol. 11: 445–456. 42. Wieland, A., R. Shashidharamurthy, A. O. Kamphorst, J. H. Han, R. D. Aubert, 45.Jiang,W.,W.J.Swiggard,C.Heufler, M. Peng, A. Mirza, R. M. Steinman, B. P. Choudhury, S. R. Stowell, J. Lee, G. A. Punkosdy, M. J. Shlomchik, et al. and M. C. Nussenzweig. 1995. The receptor DEC-205 expressed by den- 2015. Antibody effector functions mediated by Fcg-receptors are compromised dritic cells and thymic epithelial cells is involved in antigen processing. during persistent viral infection. Immunity 42: 367–378. Nature 375: 151–155. Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021