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Impact of secondary TCR engagement on the heterogeneity of pathogen-specific CD8+ T cell response during acute and chronic toxoplasmosis [1]

['Lindsey A. Shallberg', 'Department Of Pathobiology', 'School Of Veterinary Medicine', 'University Of Pennsylvania', 'Philadelphia', 'Pennsylvania', 'United States Of America', 'Anthony T. Phan', 'David A. Christian', 'Joseph A. Perry']

Date: 2022-08 

Abstract Initial TCR engagement (priming) of naive CD8+ T cells results in T cell expansion, and these early events influence the generation of diverse effector and memory populations. During infection, activated T cells can re-encounter cognate antigen, but how these events influence local effector responses or formation of memory populations is unclear. To address this issue, OT-I T cells which express the Nur77-GFP reporter of TCR activation were paired with the parasite Toxoplasma gondii that expresses OVA to assess how secondary encounter with antigen influences CD8+ T cell responses. During acute infection, TCR stimulation in affected tissues correlated with parasite burden and was associated with markers of effector cells while Nur77-GFP- OT-I showed signs of effector memory potential. However, both Nur77-GFP- and Nur77-GFP+ OT-I from acutely infected mice formed similar memory populations when transferred into naive mice. During the chronic stage of infection in the CNS, TCR activation was associated with large scale transcriptional changes and the acquisition of an effector T cell phenotype as well as the generation of a population of CD103+ CD69+ Trm like cells. While inhibition of parasite replication resulted in reduced effector responses it did not alter the Trm population. These data sets highlight that recent TCR activation contributes to the phenotypic heterogeneity of the CD8+ T cell response but suggest that this process has a limited impact on memory populations at acute and chronic stages of infection.

Author summary Following priming, the ability of effector CD8+ T cells to recognize class I restricted microbial antigens is important to control many acute and chronic infections. However the role that recent T cell receptor stimulation plays in the formation of ongoing T cell responses is unclear. Here, we utilize a genetic reporter of TCR stimulation, high parameter flow cytometry, and imaging to characterize TCR-driven phenotypes of pathogen specific T cell responses to the parasite Toxoplasma gondii during acute and chronic infection in the periphery and central nervous system. These data sets highlight the contribution of recent TCR activation on the phenotypic heterogeneity of the CD8+ T cell response but suggest that T cell memory formation and maintenance is independent of recent TCR activation.

Citation: Shallberg LA, Phan AT, Christian DA, Perry JA, Haskins BE, Beiting DP, et al. (2022) Impact of secondary TCR engagement on the heterogeneity of pathogen-specific CD8+ T cell response during acute and chronic toxoplasmosis. PLoS Pathog 18(6): e1010296. https://doi.org/10.1371/journal.ppat.1010296 Editor: Eric Y. Denkers, University of New Mexico, UNITED STATES Received: January 24, 2022; Accepted: May 6, 2022; Published: June 21, 2022 Copyright: © 2022 Shallberg et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All RNAseq files are available from the GEO database (accession number GSE188495.). Funding: This study was funded by the National Institute of Allergy and Infectious Diseases (5R01AI157247; 5T32AI00753223, R01NS112516; CAH, AAK, THH, https://www.niaid.nih.gov/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction CD8+ T cells play an important role in immunity to intracellular infections and multiple subsets including T effector, T memory, and T resident memory (Trm) cells have been associated with different anatomical locations and distinct functions [1–3]. During infection, dendritic cells in secondary lymphoid organs present microbially derived peptides via MHC Class I, activating T Cell Receptor (TCR) signaling of naive CD8+ T cells. This signal 1, along with co-stimulation (signal 2) and cytokines (signal 3) results in priming of naive CD8+ T cells and induces a proliferative burst. Consequently, an individual CD8+ T cell can produce 1,000 to 10,000 daughter cells within a week of stimulation, which in turn give rise to diverse effector and memory populations [4–10]. After priming, CD8+ T cells leave the secondary lymphoid organs and migrate to the periphery, where they can mediate TCR-dependent effector functions that include cytolysis of infected cells and secretion of cytokines (IFN-γ and TNF-α) required for resistance to infection. While early responses are dominated by the production of T effector populations it is clear that even during the early stage of infection that memory precursor populations are present [11,12]. As pathogen burden is controlled there is a contraction phase where the majority of effector CD8+ T cells undergo apoptosis, but a heterogeneous population of long-lived memory T cells remain in the circulation and reside in tissues [6,13,14]. During many chronic infections, a pool of effector and memory CD8+ T cells will persist that contribute to the long-term control of the pathogen [15–18]. Multiple studies have documented heterogeneity in the phenotype, function, longevity, and fate of these pathogen specific CD8+ T cells [8,12,19], but it is unclear whether re-encounter with cognate antigen at local sites of infection contributes to the observed diversity. In current models, antigen experienced T cells are less dependent on signal 2 and 3, and stimulation through the TCR is considered the critical signal that induces local effector activities [20,21]. However, previously activated T cells can respond to signals 2 and 3 to mediate effector functions that include the production of cytokines such as IFN-γ [22–24]. The ability to assess T cell function and activation status at sites of infection and inflammation has depended on changes in the expression of surface proteins such as CD69, CD44 and LFA-1, the use of genetically encoded reporters of cytokine production or the ability to secrete cytokines on re-stimulation, and even intravital imaging of their behavior [25–27]. These approaches have been complimented by protein and transcriptional profiling that have highlighted phenotypic and functional heterogeneity of CD8+ T cells associated with different pathogens [1,28,29]. Nevertheless, certain combinations of surface molecules, such as chemokine receptors as well as activating or inhibitory receptors, have helped define different T cell subsets. For example, in certain viral and bacterial infections, the use of graded expression of the chemokine receptor CX3CR1 along with KLRG1 has been used to distinguish effector and memory CD8+ T cell populations [8,9]. Likewise, even during acute toxoplasmosis, the co-expression of cell surface receptors KLRG1 and CXCR3 distinguishes CD8+ T cell subsets that have memory potential or are terminally differentiated, with continued presence of infection driving the KLRG1+CXCR3- terminally differentiated population [12,30]. As a consequence of T cell activation, changes in the levels of surface markers such as LFA-1, CD44, and CD69 have been useful to identify antigen experienced T cell populations, but it has been a challenge to distinguish those T cell populations that have undergone recent TCR mediated activation [31]. Thus, while TCR signals promote a marked increase in CD69, this transmembrane C type lectin associated with tissue retention is also upregulated by inflammatory signals, its downregulation is varied and following clearance of antigen CD69 is constitutively expressed on Trm populations [32–35]. Genetically encoded reporters of calcium flux have been useful for imaging of TCR signals in vivo [25], however these calcium fluxes are rapid and transient and this method does not permit long term tracking or isolation of antigen stimulated cells. Nur77 is an orphan nuclear hormone receptor that is upregulated quickly following stimulation of the TCR [36–39] and transgenic mice that express Nur77-GFP have been used to identify T cells that have experienced recent TCR engagement during priming and the effector phase of T cell responses [36,37,40]. T. gondii is a ubiquitous obligate intracellular parasite that naturally infects many intermediate hosts, including humans and mice [41]. Resistance to this intracellular parasite is dependent on cell mediated immunity and as a natural host for this pathogen, the mouse has provided a tractable model that helped establish the role of IFN-γ and CD8+ T cells in resistance to intracellular infection [12,16,42–44]. In mice, early infection is characterized by high levels of replication of the tachyzoite stage of the parasite and vascular dissemination to multiple tissues [45–47]. This phase of infection generates a robust T cell response that restricts parasite growth, but the ability of T. gondii to form latent tissue cysts in neurons allows infection to persist in the CNS [44,48]. Depending on the combination of mouse and parasite strains the chronic phase of infection can be asymptomatic or lead to an ongoing encephalitis characterized by the presence of parasite specific T cells that provide robust local protection but which never fully resolves [49]. In the latter setting, there is good evidence that antigen is processed and presented within the CNS [50–52] and long-term control of T. gondii is mediated by CD4+ and CD8+ T cell production of IFN-γ which activates a variety of anti-microbial mechanisms [53–55]. Likewise in humans, chronic toxoplasmosis is considered to be largely asymptomatic but in patients with acquired defects in T cell responses, the reactivation of the tissue cysts can lead to the development of toxoplasmic encephalitis (TE) [41,56]. Analysis of the CD8+ T cell responses in murine models of acute and chronic toxoplasmosis has highlighted mechanisms that generate and sustain CD8+ T cell responses in the periphery [30,57–59] as well as the presence of a sub-population of Trm-like cell populations in the CNS [12,34,57] that are mirrored in other infections [13,60]. The ability to modify T. gondii to express model antigens such as ovalbumin (OVA), combined with TCR transgenics has provided an important tool to understand T cell responses to this infection [51,52,61–63]. This approach allowed the study of early events that lead to parasite specific T cell responses, how these operate in the CNS, and the cells that present antigen in the brain [12,50–52,64,65]. Here, OT-I CD8+ T cells specific for the OVA peptide SIINFEKL which express Nur77-GFP were combined with T. gondii-OVA, which constitutively express OVA in the tachyzoite and bradyzoite life stages, to identify cells that had recently engaged their TCR and asses how this impacted function. Although TCR activity has been linked to memory formation [66], an adoptive transfer model indicated that during the acute phase recent TCR stimulation did not influence the ability to form long lived memory T cells. During the chronic stage of infection, tachyzoite replication in the CNS was associated with TCR activation, large scale transcriptional changes, and the acquisition of an effector T cell phenotype as well as the generation of a local population of Trm like cells. However, while inhibition of parasite replication resulted in reduced effector responses it did not alter the Trm population. These data sets highlight the contribution of recent TCR activation on the phenotypic heterogeneity of the CD8+ T cell response but suggest that T cell memory formation and maintenance is independent of recent TCR activation.

Discussion Our previous studies have shown that during TE, parasite-specific CD8+ T cells are highly motile and based on their patterns of migration a mathematical model was developed for the ability of these cells to find infected targets [72]. In that report it was unclear what proportion or how frequently the parasite specific CD8+ T cells in the periphery or CNS re-encounter antigen and how this might influence effector function or fate. Here we show that TCR engagement in the periphery and the CNS correlates with levels of infection in these compartments and was as high as ~40% but fell to 10–15% in the chronic stage of infection in the CNS. Given that this Nur77 reporter only provides a snapshot of how many T cells have seen antigen within a narrow time frame it appears that the frequency with which CD8+ T cells encounter cognate antigen is high. Similarly, studies utilizing calcium flux reporters in the CNS of mice with high VSV titers concluded that up to 30% of pathogen specific CD8+ T cells were being stimulated by antigen over a short time period [25]. Thus, despite the CNS being a tissue canonically considered immune privileged during TE there are robust levels of antigen presentation [50–52] and secondary T cell activation that is required for local pathogen control. While initial TCR stimulation is necessary for T cell priming, few studies have distinguished the role of subsequent antigen exposure on the function and fate of activated effector and memory CD8+ T cells in vivo. Many cytokines and transcription factors contribute to the development of T cell heterogeneity, and during toxoplasmosis these include the cytokines IL-7 and IL-12 [11,73], as well as transcription factors T-bet, c-Rel, BLIMP1, STAT1, and Bhlhe40 [74–79], yet it is unclear whether secondary antigen encounter and TCR activation alters CD8+ T cell fate decisions. The studies presented here show that TCR engagement of activated CD8+ T cells in a secondary site of infection correlated with profound transcriptional changes and expression of markers of effector T cells (for example co-expression of KLRG1 and CX3CR1 and cytokine production). It has been proposed that TCR signal strength and levels of inflammation during priming influence the balance between effector and memory CD8+ T cells [66,80]. However, in the transfer studies performed here recent TCR signals did not appear to impact the ability to form a memory CD8+ T cell population. Likewise, the presence of Trm CD8+ T cells in the CNS have been described for other infections [81] and TCR signal strength and duration influences the establishment and function of these Trm populations [82,83]. A Trm population has also been described during TE that produces IFN-γ and TNF-α [34] and the studies presented here revealed that the OT-I Trm-like cells received frequent TCR stimulation. However, while the absence of active parasite replication resulted in reduced TCR engagement there was no significant change in the frequency or number of these Trm cells in the CNS. Thus, both approaches that addressed the impact of secondary TCR activity on memory populations suggest that recent antigen encounter does not alter these fates and fit with the concept that Trm progenitors are generated early in effector responses [81,84]. Additionally, the use of sulfadiazine to restrict parasite replication, which may mimic chronic infection in humans, highlighted the persistence of the Trm population in the absence of replicating parasites. However, it is currently unclear whether parasite-specific Trm form in the CNS following T. gondii infection in humans, and whether these cells are critical in controlling chronic human disease. The CNS represents a unique site of inflammation during infection, and the ability of T. gondii to establish a persistent infection in this tissue provides the opportunity to study how TCR signals intersect with different phases of the T cell response in the brain. For example, it has been suggested that following challenge with viral or bacterial infections that pathogen replication in the brain is not required for resident CD8+ T cell populations to populate the CNS [85]. However, during toxoplasmosis, endothelial cells of the blood brain barrier are infected [45] and the data that associates CX3CR1 (a chemokine receptor responsible for leukocyte entry into the CNS [86]) with TCR activation in the vascular compartment of the CNS implies that antigen stimulation at the blood brain barrier may induce T cell extravasation into the parenchyma. The use of the Nur77-GFP reporter as a surrogate for TCR activation should provide a sensitive system to assess the host factors that contribute to compartment (vascular, endothelial, and parenchymal) specific T cell activity in the CNS required for T cell mediated protective immunity. In certain cancers and infection, one of the effects of repetitive antigen stimulation on CD8+ T cells is the induction of T cell exhaustion, denoted by an increase in inhibitory receptors and a decrease in cytokine secretion and cytolytic capacity [87–89]. During TE, parasite-specific T cells express high levels of inhibitory receptors such as PD-1 and TIGIT and it has been suggested that these T cell populations may be exhausted [88–91]. Indeed, there is a report that PD-L1 blockade leads to enhanced CD8+ T cell responses during TE [89], whereas in a similar setting the absence of TIGIT does not augment the T cell response [90]. The impact of inhibitory receptors during TE is complicated by evidence that PD-1 supports the generation of Trm in the CNS in viral infections [92–94]. Nonetheless, in our studies IFN-γ expression in the brain was closely correlated with expression of Nur77-GFP, indicating that TCR-independent bystander cytokine-mediated activation of CD8+ T cells [95–97] is not a major contributor to the production of IFN-γ. Moreover, the functional and transcriptional analysis did not reveal any early signs of exhaustion in Nur77-GFP+ OT-I. Indeed, the magnitude of differentially expressed genes associated with TCR activation in the CNS was similar in scope to the transcriptional changes observed during priming of OT-I T cells in other models [1]. Many of these upregulated genes were transcription factors and regulators (Irf8, Batf3, Bach2, Egr2) known to direct CD8+ T cell function, indicating that subsequent antigen encounter may restructure transcriptional programs. These results also revealed six clusters that were not segregated by Nur77-GFP expression and underscore the need for additional studies to distinguish how priming affects diversity versus the TCR-independent signals that affect the evolution of the T cell response during TE. There are several neurotropic infections that have distinct developmental states associated with latency and for T. gondii this is exemplified by the ability to form the tissue cyst. It has been proposed that the presence of this stage in neurons, a cell type known for low MHC-I expression [98], would be sequestered by the immune response. There is good evidence that T. gondii antigen is readily presented to T cells within the CNS [50,51,99], and the idea that neurons can act as a refuge has been challenged by evidence that MHC-I presentation of tachyzoite antigens by neurons contributes to parasite control [52]. Imaging of brains from mice chronically infected with T. gondii had higher numbers of Nur77-GFP+ OT-I surrounding tachyzoites but as the infection in the brain progresses the decline in levels of TCR engagement correlated with parasite control and the switch from tachyzoite to bradyzoite. However, there were examples where Nur77-GFP+ OT-I were in close proximity to intact cysts but whether this is due to presentation by infected neurons, other glial populations, or infiltrating DC or monocyte populations that interact with infected neurons [51] is currently unknown. Whether the cyst stage is under direct immune surveillance is also unclear but the ability of bradyzoites to actively block IFN-γ signaling [48] implies the need for the parasite to evade an active response against the cyst stage [44,100]. Currently, there is a paucity of host and parasite specific tools to dissect the ability of host cells to process and present antigens from the bradyzoite life-stage. Future experiments utilizing bradyzoite-specific secretion of model antigens combined with the Nur77-GFP reporter will be utilized to address this knowledge gap.

Materials and methods Ethics statement All mice were housed in a specific-pathogen free environment at the University of Pennsylvania School of Veterinary Medicine in accordance with federal guidelines and with approval of the Institutional Animal Care and Use Committee. Mice and infection Nur77-GFP reporter mice, C57BL/6 mice, CD45.1 C57BL/6 mice, and OT-I mice were purchased from Jackson Laboratories (for all materials and reagents, see Tables 1 and 2). DPE-GFP transgenic mice were originally obtained from Ulrich H. von Andrian (CBR, Harvard, Boston MA) and were crossed to OT-I mice. Group sizes for experiments were between 3–6 mice and are indicated in figure legends. The generation of CPS-OVA parasites and PRU-OVA parasites were described previously [29,62]. PPT PowerPoint slide

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TIFF original image Download: Table 1. Materials. https://doi.org/10.1371/journal.ppat.1010296.t001 PPT PowerPoint slide

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TIFF original image Download: Table 2. Reagents. https://doi.org/10.1371/journal.ppat.1010296.t002 Parasites were cultured and maintained by serial passage on human foreskin fibroblast cells in the presence of parasite culture media (DMEM, 20% medium M199, 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, 25 μg/ml gentamycin) which was supplemented with uracil for culturing CPS strain parasites (final concentration of 0.2 mM uracil). Tachyzoites of each strain were prepared for infection by serial 26g needle passage and filtered through a 5-μm-pore-size filter. 1 day prior to infection, 5,000 congenically distinct naive splenic Nur77-GFP OT-I were transferred i.v. into naive mice. Mice were infected i.p. with 104 live parasites. In indicated experiments, mice were treated with 250mg/L of sulfadiazine in drinking water for 4 weeks beginning at 3 weeks post infection. For adoptive transfers of OT-I from infected mice to naive mice, OT-I were double sorted on a BD FACS Aria. Each naive mice received an i.v. transfer of 150,000 Nur77-GFP- or Nur77-GFP+ pooled from 2 infected mice. Recipient mice were treated with sulfadiazine two days prior and 1 week following transfer to ensure T. gondii infection was not transferred. Parasite quantification by PCR Approximately 10mg of tissue was snap frozen and DNA was extracted with Qiagen DNeasy Blood and Tissue Kit according to manufacturer’s protocol. Real-time PCR was conducted with SYBR Green master mix according to manufacturer’s protocol, with 500ng of template DNA. T. gondii DNA was amplified with forward (5’-TCCCCTCTGCTGGCGAAAAGT-3’) and reverse (5’-AGCGTTCGTGGTCAACTATCGATTG-3’) primers to the B1 gene. PCR was performed on an Applied Biosystems ViiA7 with the following cycle conditions: Hold phase: 2min 50C, 10min 95C; PCR phase (occurs 50x): 15s at 95C, 1min @60C. Tissue processing Spleens were grinded through a 70uM filter and red blood cells lysed with ACK lysis buffer. Brains were chopped into 1mm sections and incubated at 37C for 1.5 hours with 250ug/mL Collagenase/Dispase and 10ug/mL DNase, passed through a 70uM filter, then leukocytes were isolated through 30% and 60% Percoll density centrifugation. Quantification of T cell cytokine production 1x106 brain mononuclear cells were cultured ex vivo at 37C for 4 hours in RPMI supplemented with 10% FBS. For restimulations, 1uM of peptide was added with Protein Transport Inhibitor Cocktail. For quantification of endogenous levels of cytokine production, only Protein Transport Inhibitor Cocktail was added. Bone marrow macrophage culture Bone marrow macrophages were isolated and cultured for 7 days with 30% L929 media in DMEM with 10% FBS, with media replenishing on day 4 after isolation. For peptide stimulations, 1uM of peptide was added to macrophages for 20 minutes and excess peptide was removed by washing 2 times. For parasite infection, a parasite to macrophage infection ratio of 2:1 was used, and excess parasites were removed by washing 2 times. CD8+ T cell were isolated by MACS bead enrichment. Macrophages and T cells were cultured at a 1:2 ratio. Flow cytometry Staining of extracellular antigens was performed following Fc receptor blocking with Rat IgG and anti-mouse CD16/CD32. Intracellular antigens were stained following fixation and permeabilization using the Foxp3 Transcription Factor Staining Buffer Set. All samples were run on a BD FACSymphony A5 or BD FACSCanto and analyzed on FlowJo v10 software (FlowJo, Ashland, OR). UMAP and Phenograph were utilized in FlowJo for dimensionality reduction and unsupervised clustering. Imaging Brains were fixed in 4% paraformaldehyde overnight, followed by 15% and 30% sucrose in PBS overnight. 12uM sections were stained with AF488 anti-GFP and AF647 anti-CD45.1 in 0.1% Triton-X100 and 0.05% Tween-20 in PBS. Slides were sealed with Prolong Diamond Antifade Mountant with DAPI. Slides were imaged at 60x magnification on a Leica SP5-II (Lecia, Wetzlar, Germany). Live imaging of brain explants was performed as previously described [72]. Briefly, mice were infected with PRU-OVA parasites expressing tdTomato. Four weeks following infection, mice received 2x106 activated DPE-GFP-OT-I i.v., and brains were imaged one week following transfer of DPE-GFP-OT-I. Imaging was performed on a Lecia SP5 multiphoton. RNA sequencing Nur77-GFP- and Nur77-GFP+ OT-I were double sorted as described above. Cells were collected in RPMI with 20% FBS and resuspended in Buffer RLT Plus. mRNA was isolated using RNeasy Micro Plus Kit and RNA quality was evaluated by High Sensitivity RNA Screen Tape on an Agilent 4200 TapeStation. cDNA library was prepared with Takara SMART-Seq HT kit and adapters ligated with Nextera XT DNA Library Preparation Kit and Index Kit v2 according to manufacturers’ protocols. AMPure XP beads were used for primer cleanup. 75-base pair reads were sequenced on a NextSeq 500 machine (Illumina, San Diego, CA)) according to manufacturer protocol. Kallisto was utilized to pseudo-align genes to version 75 of the mouse reference genome GRCm38. Transcripts that were represented in fewer than 3 of 4 samples in a group were excluded from analysis. The limma package in R was used to generate normalized counts per million, and differential genes (adjusted P-value>0.05) were identified with a log fold change cutoff of 0.3. The heatmaply package was used for heatmap generation. Gene set enrichment analysis was performed with GSEA software (v4.0.3, Broad Institute) with gene sets containing less than 10 genes excluded. Enrichment plots were generated in Cytoscape (v3.7.1) with an adjusted P-value cutoff of 0.05 and false discovery rate cutoff of 0.1 (pipeline adapted from [101]). Data is deposited in GEO (accession# GSE188495).

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