Chronic viral infection impairs immune memory to a different pathogen

Cheng Yang; Zhicui Liu; Ying Yang; Luis J. Cocka; Yongguo Li; Weihong Zeng; Hao Shen

doi:10.1371/journal.ppat.1012113

Abstract

Chronic viral infections cause T cell dysfunction in both animal models and human clinical settings, thereby affecting the ability of the host immune system to clear viral pathogens and develop proper virus-specific immune memory. However, the impact of chronic viral infections on the host’s immune memory to other pathogens has not been well described. In this study, we immunized mice with recombinant Listeria monocytogenes expressing OVA (Lm-OVA) to generate immunity to Lm and allow analysis of OVA-specific memory T (Tm) cells. We then infected these mice with lymphocytic choriomeningitis virus (LCMV) strain Cl-13 which establishes a chronic infection. We found that chronically infected mice were unable to protect against Listeria re-challenge. OVA-specific Tm cells showed a progressive loss in total numbers and in their ability to produce effector cytokines in the context of chronic LCMV infection. Unlike virus-specific T cells, OVA-specific Tm cells from chronically infected mice did not up-regulate the expression of inhibitory receptors, a hallmark feature of exhaustion in virus-specific T cells. Finally, OVA-specific Tm cells failed to mount a robust recall response after bacteria re-challenge both in the chronically infected and adoptively transferred naïve hosts. These results show that previously established bacteria-specific Tm cells become functionally impaired in the setting of an unrelated bystander chronic viral infection, which may contribute to poor immunity against other pathogens in the host with chronic viral infection.

Author summary

The impact of chronic viral infections on protective immunity to other pathogens remains largely unknown. Our data showed that mice infected with a chronic virus were incapable of conferring protection against bacterial reinfection. Mechanistically, infection by chronic virus not only reduced the number of bacterial-specific memory T cells, but also led to dysfunction of these cells as evident by diminished production of effector molecules and poor recall responses. Our results provide novel insights into immune mechanisms underlying the increased susceptibility to other pathogens in chronic virus infected hosts.

Citation: Yang C, Liu Z, Yang Y, Cocka LJ, Li Y, Zeng W, et al. (2024) Chronic viral infection impairs immune memory to a different pathogen. PLoS Pathog 20(3): e1012113. https://doi.org/10.1371/journal.ppat.1012113

Editor: Alec J. Hirsch, Oregon Health Science University, UNITED STATES

Received: October 23, 2023; Accepted: March 11, 2024; Published: March 28, 2024

Copyright: © 2024 Yang 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 relevant data are within the paper and its Supporting Information files.

Funding: This work was supported by National Institute of Health (R01AI130115 to HS), National Natural Science Foundation of China (32000664 to CY), Shanghai Pujiang Program (22PJD083 to WZ, and 22PJD057 to ZL), Natural Science Foundation of Shanghai (22ZR1467700 to WZ), Natural Science Foundation of Chongqing (cstc2021jcyj-msxmX0306 to YL), and Dr. Song Li donation to HS. 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 are an important component of the immune system in controlling intracellular pathogens. After acute infection, naïve CD8⁺ T cells become activated, proliferate and differentiate into a large population of cytotoxic and effector T cells, which contribute to the clearance of pathogens [1]. Following antigen clearance, the expanded effector CD8⁺ T cell population contracts by apoptosis, with a minor subset, termed memory precursors, surviving and further differentiating into mature memory T (Tm) cells [2–4]. Tm cells acquire stem cell-like property of self-renewal in the absence of antigenic stimulation [5]. When re-encounter with antigen, these memory subsets are able to immediately produce a large amount of effector cytokines and rapidly proliferate into secondary effectors to provide strong protective immunity [6–9]. Therefore, proper maintenance of Tm cell populations is crucial to sustain long-lasting protection against re-infection by previously encountered pathogens.

In contrast to acute infections, antigen-specific T cells fail to differentiate into memory subsets during many chronic infections. Activated virus-specific T cells gradually lose the capacity to produce cytokines, such as IL-2, TNF-α and IFN-γ, and exhibit reduced cytotoxicity, poor memory recall responses [10]. They also show progressive loss in numbers due to impaired homeostatic proliferation [11]. This state of “exhaustion” is due to prolonged antigenic and inflammatory stimulation [12,13]. T cell exhaustion was first described in mice infected with the clone 13 (Cl-13) strain of LCMV [12]. Compromised T cell function has now been observed in other chronic viral infections both in animal models and humans, such as human immunodeficiency virus (HIV), hepatitis B virus (HBV) and hepatitis C virus (HCV) [14].

Chronic viral infections are common in humans, with each individual carrying 8~12 chronic infections on average [15]. Over the past two decades, tremendous efforts have been undertaken to uncover the molecular mechanisms underlying the exhaustion of T cells specific to the ongoing chronic viral infections, including the identification of transcriptional, epigenetic and metabolic alterations involved in driving this process [16]. However, little is known about how chronic viral infections may affect pre-existing bystander Tm cells and protective immunity to other pathogens that were induced by prior infections and/or vaccinations. This question is of great clinical relevance, since the functionality of the existing memory pool is critical to hosts’ immune competence, and any dysregulation to this memory repertoire may lead to increased susceptibility to re-infections from previously encountered pathogens [17].

In the current study, we infected mice with Lm-OVA to establish immunological memory to the bacteria, and subsequently infected these animals with LCMV Armstrong (Arm) or Cl-13 strain to establish acute or chronic infections, respectively. Our data showed that chronically infected hosts had significantly higher bacterial burdens and mortality rate following a bacteria challenge, and bacteria-specific Tm cells were functionally impaired. Specifically, we observed a large decrease in total numbers of bacteria-specific Tm cells, as well as their capacity to produce effector cytokines, leading to a failure in mounting a robust recall response by Tm cells both in the chronically infected and adoptively transferred naïve hosts. Unlike virus-specific T cells, which had sustained expression of inhibitory receptors during chronic viral infection, bacteria-specific Tm cells did not express high levels of inhibitory molecules (PD-1, LAG-3 and TIM-3). Our results provide direct evidence that competent adoptive immunity to previously encountered pathogens can be dysregulated by a bystander chronic viral infection, and uncover novel immunological mechanisms leading to impaired protective immunity to other pathogens in chronically infected hosts.

Results

Chronic viral infection impairs protective immunity to bacteria re-infection

To examine the effect of a chronic viral infection on protective immunity against an unrelated pathogen, we first immunized mice with recombinant Lm (Lm-OVA), which is a widely used model to study T cell mediated protective immunity [18]. More than 30 days after Lm-OVA immunization, we infected the immunized mice with either Arm or Cl-13 strain of LCMV that causes acute and chronic infection, respectively. As expected, LCMV-specific (D^b/GP₃₃⁺) CD8⁺ T cells from Cl-13 infected mice showed exhaustion phenotypes including: 1) higher levels of the inhibitory receptors (PD-1, TIM-3 and LAG-3), 2) downregulated expression of memory-associated molecules (CD44 and CD127), 3) less production of effector cytokines (IFN-γ, TNF-α and IL-2), and 4) reduced cytotoxicity as measured by expression of CD107a (a marker expressed on the cell surface of CD8⁺ T cells following stimulation with cognate peptides that correlates with cytolytic activity) [19] (S1 Fig). At day 100 post LCMV infection, we challenged these mice with a lethal dose of Lm-OVA (Fig 1A). All Lm-OVA-immune mice recovered from an acute Arm infection (Lm-OVA/Arm) were resistant to bacterial challenge, while more than half of Lm-OVA-immune mice with Cl-13 infection (Lm-OVA/Cl-13) died by day 5 post Lm-OVA challenge (Fig 1B). On day 3 after Lm-OVA challenge, the bacterial loads in the spleen and liver of Lm-OVA/Cl-13 mice were 4–6 logs higher than in those of Lm-OVA/Arm mice (Fig 1C). These results show that protective immunity against Lm re-infection is impaired by a chronic but not an acute LCMV infection.

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Fig 1. Chronic virus infection compromises protective immunity to Lm-OVA.

(A) Experimental design to study the impact of chronic virus infection on protective immunity against Lm-OVA re-infection. Mice were immunized with ΔactA Lm-OVA (2×10⁶ CFU), and then infected with LCMV Arm (Lm-OVA/Arm) or Cl-13 (Lm-OVA/Cl-13) 30 days later. All mice were challenged with wild type (WT) Lm-OVA (1×10⁶ CFU) at day 100 after LCMV infection. (B) Survival of Lm-OVA/Arm and Lm-OVA/Cl-13 mice after Lm-OVA challenge. (C) Bacteria load in the spleens and livers of Lm-OVA/Arm or Lm-OVA/Cl-13 mice at day 3 after Lm-OVA challenge. * p<0.05; *** p<0.001.

https://doi.org/10.1371/journal.ppat.1012113.g001

Chronic viral infection causes progressive loss of pre-existing bystander Tm cells specific to a different pathogen

Chronic LCMV Cl-13 infection is known to induce functional exhaustion of LCMV-specific T cells [10]. To investigate possible mechanisms for the loss of protective immunity to Lm-OVA (Fig 1), we asked if pre-existing bystander Tm cells specific to Lm-OVA are also driven to exhaustion by chronic Cl-13 infection. Mice were immunized and boosted with Lm-OVA, and more than 30 days later were infected with LCMV Arm or Cl-13. The number, phenotype and functionality of both Lm-OVA-specific (K^b/OVA₂₅₇⁺) and LCMV-specific (D^b/GP₃₃⁺) CD8⁺ T cells were examined at different time points by serially bleeding and assayed by tetramers staining and intracellular cytokine staining (ICS). In Lm-OVA/Arm infected mice, while the frequency of K^b/OVA₂₅₇⁺ T cells decreased on day 8 following LCMV Arm infection due to extensive expansion of LCMV-specific T cells at this time point (Fig 2A), their numbers remained stable thereafter (Fig 2B). In contrast, the frequency and number of K^b/OVA₂₅₇⁺ Tm cells dropped progressively in Lm-OVA/Cl-13 infected mice, and by 60 days post Cl-13 infection, more than 90% of K^b/OVA₂₅₇⁺ Tm cells were depleted in the peripheral blood, resulting in a significantly lower number of K^b/OVA₂₅₇⁺ Tm cells in Lm-OVA/Cl-13 than Lm-OVA/Arm mice (Fig 2A and 2B). Notably, D^b/GP₃₃-specific T cells became almost undetectable in the blood of Lm-OVA/Cl-13 mice as early as 20 days after Cl-13 infection, which was much more rapid compared to the depletion of K^b/OVA₂₅₇-specific T cells (Fig 2A and 2B). The progressive loss of OVA₂₅₇-specific CD8⁺ Tm cells was also evident when assayed by ICS for IFN-γ production following cognate peptide stimulation (S2A and S2B Fig). Similar results were observed in Lm-specific CD4⁺ Tm (LLO₁₉₀⁺) cells, which were stably maintained in Lm-OVA/Arm mice but progressively lost in Lm-OVA/Cl-13 mice (S2C and S2D Fig). The profound loss of Tm cells was observed not only in the blood but also in the spleen and liver of Lm-OVA/Cl-13 mice, there were significantly lower numbers of K^b/OVA₂₅₇⁺ Tm cells in the organs of Lm-OVA/Cl-13 than in those of Lm-OVA/Arm mice on day 90 after LCMV infection (Fig 2C). Together, these data show that chronic viral infection leads to the progressive loss of pre-existing Tm cells specific to other unrelated pathogens.

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Fig 2. Chronic virus infection depletes Lm-OVA-specific Tm cells.

Mice were immunized with Lm-OVA, and infected with LCMV as described in Fig 1. (A) Lm-OVA immunized mice were left uninfected (Ctrl) or infected with LCMV Arm or Cl-13, and representative figures of virus (D^b/GP₃₃)- or bacteria (K^b/OVA₂₅₇)-specific CD8⁺ T cells in the blood of these mice at different time points after LCMV infection were shown. (B) Quantification of K^b/OVA₂₅₇⁺ (left) and D^b/GP₃₃⁺ (right) CD8⁺ T cells in the blood of Ctrl, Arm and Cl-13 mice at different time points after LCMV infection. In order to better track dynamic changes of Lm-OVA specific T cells, mice with higher numbers of K^b/OVA₂₅₇-specific memory T cells were selected for infection with either Arm or Cl-13, while those with lower K^bOVA₂₅₇-specific memory T cells remained uninfected as Ctrl. Asterisks indicate the statistical significance between Arm and Cl-13 mice. (C) Representative dot plot (left) and numeric summary (right) of K^b/OVA₂₅₇-specific CD8⁺ T cells in the spleen and liver of Lm-OVA/Arm or Lm-OVA/Cl-13 mice at day 100 after LCMV infection. * p<0.05; ** p<0.01; ***p<0.001.

https://doi.org/10.1371/journal.ppat.1012113.g002

Chronic viral infection induces functional impairment of pre-existing bystander Tm cells

In addition to profound loss of bystander Tm cells, the decrease of IFN-γ production indicated that functionality of bystander Tm cells might also be impaired by chronic Cl-13 infection. To further investigate this possibility, we determined the expression of CD107a and multiple cytokines (IFN-γ, TNF-α and IL-2) following OVA₂₅₇ peptide stimulation of samples from blood, and spleens harvested at more than 90 days after LCMV infection. Compared with Lm-OVA/Arm mice, samples from Lm-OVA/Cl-13 group had much lower percentages of CD8⁺ T cells producing cytokines (IFN-γ, TNF-α and IL-2) or expressing CD107a (Fig 3A and 3B), indicating a reduction in cytokine production and cytotoxicity. Furthermore, OVA₂₅₇-specific T cells from Lm-OVA/Cl-13 mice produced significantly lower amounts of IFN-γ and TNF-α on per cell basis as measured by geometric mean florescent intensity (gMFI) (Fig 3C and 3D). Since T cells producing more than one cytokine confer superior protection against invaded pathogens [20], we asked whether OVA₂₅₇-specific T cells from Lm-OVA/Cl-13 mice were less polyfunctional in comparison with their counterparts from Lm-OVA/Arm mice. To this end, CD107a⁺ OVA₂₅₇-specific CD8⁺ Tm cells were further analyzed for cytokines production at day 90 post LCMV infection. Most CD107a⁺ OVA₂₅₇-specific CD8⁺ Tm cells co-produced at least two of the three cytokines (IFN-γ, TNF-α and IL-2) in Lm-OVA/Arm mice. By contrast, approximately half of them from Lm-OVA/Cl-13 mice produced none of the three aforementioned cytokines in the blood and spleen (Fig 3E and 3F). When analyzing the polyfunctionality of OVA₂₅₇-specific CD8⁺ Tm cells at different time points after LCMV infection, we found that polyfunctional OVA₂₅₇-specific CD8⁺ Tm cells remained stable in Lm-OVA/Arm mice until the end of our experiments. In Lm-OVA/Cl-13 mice, however, OVA₂₅₇-specific CD8⁺ Tm cells lost the capacity to produce IL-2 as early as day 40 post Cl-13 infection. The proportion of multi-functional T cells decreased while monofunctional T cells (CD107a⁺/IFN-γ^-/TNF-α^-/IL-2^-) gradually increased from day 60 after Cl-13 infection (Fig 3G). Similar to OVA₂₅₇-specific CD8⁺ Tm cells, LLO₁₉₀-specific CD4⁺ Tm cells also showed reduced polyfunctionality in Lm-OVA/Cl-13 mice (S3 Fig). Together, these data indicate that chronic viral infection not only causes progressive loss of pre-existing bystander Tm cells, but also impairs their effector functions.

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Fig 3. Chronic virus infection progressively impairs the function of Lm-OVA-specific Tm cells.

Infection of mice was the same as described in Fig 2. (A and B) Percentages of CD107a, IFN-γ, TNF-α and IL-2 expression in CD8⁺ T cells after stimulation with OVA₂₅₇ peptide in the blood (A) and spleen (B) of Lm-OVA/Arm and Lm-OVA/Cl-13 mice at day 90 post LCMV infection. (C and D) Per-cell levels of IFN-γ and TNF-α production in CD8⁺ T cells after stimulation with OVA₂₅₇ peptide in the blood (C) and spleen (D). (E and F) Co-production of IFN-γ, TNF-α and IL-2 within CD107a⁺CD8⁺ T cells in the blood (E) and spleen (F) of Lm-OVA/Arm and Lm-OVA/Cl-13 mice at day 90 after LCMV infection, in the presence of OVA₂₅₇ peptide stimulation. (G) Dynamic change of CD107a⁺CD8⁺ T cells that co-produce IFN-γ, TNF-α and IL-2 in the blood of Lm-OVA/Arm and Lm-OVA/Cl-13 mice, following OVA₂₅₇ peptide stimulation. gMFI, geometric mean florescent intensity. Functions: 1, CD8⁺ T cells expressing CD107a alone; 2–4, CD8⁺ CD107a⁺ T cells co-producing one, two or all of the three cytokines (IFN-γ, TNF-α and IL-2).

https://doi.org/10.1371/journal.ppat.1012113.g003

The majority of T cell population become partially dysfunctional after chronic viral infection

Since we have observed that both virus- and bacteria-specific T cells were functionally impaired after a chronic viral infection, we hypothesized that chronic LCMV infection may have broad impacts on the functionality of T cells. To test our hypothesis, we isolated mononuclear cells from the spleens and livers of Lm-OVA/Arm and Lm-OVA/Cl-13 mice at more than 90 days after LCMV infection, as well as Lm-OVA immune mice (no LCMV infection) as the control (Ctrl). We then performed ICS for multiple cytokines (IFN-γ, TNF-α and IL-2) following short stimulation with phorbol 12-myristate 13-acetate and ionomycin (PMA/I), which bypasses proximal TCR signaling and induce most effector and memory (but not naïve) T cells to produce cytokines [21]. The results showed that Ctrl and Lm-OVA/Arm group had similar numbers of CD8⁺ T cells producing IFN-γ, TNF-α and IL-2. In contrast, frequencies of CD8⁺ T cells producing IFN-γ, TNF-α or IL-2 were much lower in Lm-OVA/Cl-13 mice (Fig 4A). More importantly, a striking decrease was observed in the percentages of multi-functional CD8⁺ T cells co-producing IFN-γ/IL-2 or IFN-γ/TNF-α in Lm-OVA/Cl-13 mice, in comparison with Ctrl and Lm-OVA/Arm mice (Fig 4A and 4B). Similar results were observed in CD4⁺ T cells (Fig 4C and 4D). These data suggest that a large fraction of T cells, both virus-specific and non-virus-specific, are functionally impaired in the context of a chronic viral infection.

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Fig 4. Chronic virus infection induces functional defects to the majority of T cell population.

Infection of mice was mentioned in Fig 2. More than 90 days after LCMV infection, mononuclear cells isolated from the spleen and liver of Ctrl, Lm-OVA/Arm (Arm) or Lm-OVA/Cl-13 (Cl-13) mice were stimulated with PMA and ionomycin, followed by ICS for IFN-γ, TNF-α and IL-2. (A) Representative figures showing the production of cytokines by CD8⁺ T cells. (B) Statistical presentation of CD8⁺ T cells co-producing IFN-γ/IL-2 or IFN-γ/TNF-α. (C) Representative images for the production of cytokines by CD4⁺ T cells. (D) Summary graphs for the frequency of CD4⁺ T cells that co-produce IFN-γ/IL-2 or IFN-γ/TNF-α.

https://doi.org/10.1371/journal.ppat.1012113.g004

Effects of chronic LCMV infection on bystander Tm cells differ from exhaustion of LCMV-specific T cells

Loss of bystander K^b/OVA₂₅₇⁺ Tm cells was a slow process, becoming profound only after 60 days post Cl-13 infection. This was in contrast to the depletion of LCMV-specific D^b/GP₃₃⁺ T cells, which was evident as early as day 20 after Cl-13 infection (Fig 2). High and sustained expression of multiple inhibitory receptors is one of the major hallmarks of exhausted T cells [22,23]. To investigate whether bystander K^b/OVA₂₅₇⁺ Tm cells have the properties of exhausted T cells, expression of inhibitory receptors PD-1, TIM-3 and LAG-3 was monitored in Lm-OVA/Arm and Lm-OVA/Cl-13 mice. Consistent with previous reports [22,24], virus-specific T cells primed by Arm infection rapidly down-regulated the expression of PD-1. In contrast, expression of PD-1 was maintained at a high level for weeks post Cl-13 infection (Fig 5A). Interestingly, PD-1 was not up-regulated on bacteria-specific T cells at various time points post LCMV infection in the peripheral blood, regardless of Arm or Cl-13 infection (Fig 5A). A similar trend was also observed for the inhibitory marker TIM-3 (Fig 5B). At the end point of our experiments (> day 90 post LCMV infection), we measured the expression of inhibitory receptors on virus- and bacteria-specific T cells both in spleens and livers. We found that virus-specific T cells in Cl-13 infected mice expressed high levels of PD-1, TIM-3 and LAG-3 as expected. Unlike virus-specific T cells, increased expression of the three aforementioned inhibitory receptors were not observed on K^b/OVA₂₅₇⁺ Tm cells from Cl-13 infected mice (Fig 5C and 5D). Therefore, dysfunctional bacteria-specific Tm cells do not appear to display the major characteristics of T-cell exhaustion during a chronic viral infection.

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Fig 5. Lm-OVA-specific Tm cells do not up-regulate the expression of inhibitory molecules during chronic virus infection.

Infection of mice was described in Fig 2. (A and B) Expression of PD-1 (A) and TIM-3 (B) on K^b/OVA₂₅₇- and D^b/GP₃₃-specific CD8⁺ T cells in the blood of Lm-OVA/Arm and Lm-OVA/Cl-13 mice at different time points after LCMV infection. (C and D) Expression of PD-1, TIM-3 and LAG-3 on K^b/OVA₂₅₇- and D^b/GP₃₃-specific CD8⁺ T cells in the spleens (C) and livers (D) of Lm-OVA/Arm and Lm-OVA/Cl-13 mice at day 90 after LCMV infection.

https://doi.org/10.1371/journal.ppat.1012113.g005

Bystander Tm cells derived from chronic virus infected hosts fail to recall upon bacteria re-challenge

Protective immunity against Lm re-infection is mediated by CD4⁺ and CD8⁺ Tm cells, with CD8⁺ T cells playing a predominant role [25]. To examine if loss of protective immunity against Lm reinfection in Lm-OVA/Cl-13 mice (Fig 1) is due to impaired memory recall of bacteria-specific Tm cells, we examined recall responses of OVA₂₅₇-specific CD8⁺ and LLO₁₉₀-specific CD4⁺ Tm cells on day 5 following Lm-OVA challenge. In comparison with Lm-OVA/Arm mice, Lm-OVA/Cl-13 mice had much lower percentages of OVA₂₅₇-specific CD8⁺ T cells in their spleens and livers after Lm challenge, as determined by tetramer staining or ICS following stimulation with cognate peptide (Fig 6A and 6B). Similarly, we observed much lower proportions of LLO₁₉₀-specific CD4⁺ T cells in Lm-OVA/Cl-13 than in Lm-OVA/Arm mice by day 5 after Lm challenge (S4 Fig). Together, these data suggest that the recall capacity of both CD4⁺ and CD8⁺ Tm cells specific to Lm was impaired by chronic Cl-13 infection.

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Fig 6. Chronic virus infection negatively impacts the memory recall of Lm-OVA-specific Tm cells.

(A and B) Infection of mice was described in Fig 2. Mice were challenged with WT Lm-OVA on more than 100 days after LCMV infection, recall responses of K^b/OVA₂₅₇-specific Tm cells in the spleen and liver of Lm-OVA/Arm and Lm-OVA/Cl-13 mice were determined by MHC Class I tetramer staining (A) or ICS for IFN-γ after cognate peptide stimulation (B) on day 5 post Lm-OVA challenge. (C) Design for adoptive transfer experiment. CD8⁺ T cells were purified from the spleens of Lm-OVA/Arm and Lm-OVA/Cl-13 mice (CD45.2) on more than 90 days after LCMV infection, and then purified cells containing the same number of K^b/OVA₂₅₇-specific T cells were transferred into congenic naïve recipients (CD45.1), which were challenged with WT Lm-OVA 1 day later. (D) Survival of mice that were non-transferred with cells (non-transfer) or receiving donor CD8⁺ T cells isolated from Lm-OVA/Arm (Arm) or Lm-OVA/Cl-13 (Cl-13) mice, following Lm-OVA challenge. (E and F) Percentage of donor-derived CD8⁺ T cells (CD45.2) (E), and fraction (F, left) or absolute number (F, right) of donor-derived K^b/OVA₂₅₇-specific T cells in the spleens and livers of adoptively transferred hosts on day 5 after Lm-OVA challenge.

https://doi.org/10.1371/journal.ppat.1012113.g006

To further test whether bystander Tm cells were capable of recalling after removal from the chronically infected hosts, we adoptively transferred an equal number of K^b/OVA₂₅₇-specific CD8⁺ T cells, which were purified from Lm-OVA/Arm or Lm-OVA/Cl-13 mice (CD45.2) on more than 90 days after LCMV infection, into congenic recipients (CD45.1). One day after transfer, recipient mice were challenged with a lethal dose of Lm-OVA (Fig 6C). The majority of mice (8/9) receiving donor cells from Lm-OVA/Arm mice survived from bacterial challenge. In contrast, most mice receiving donor cells from Lm-OVA/Cl-13 mice succumbed to bacterial challenge on day 5, which were comparable to non-transfer control mice (Fig 6D). Donor T cells and OVA₂₅₇-specific CD8⁺ Tm cells from Lm-OVA/Arm mice expanded and constituted a much higher proportion of total CD8⁺ T cell population in recipients, compared to those from Cl-13 infected mice (Fig 6E and 6F). Together, these data show that the functionality of pre-existing bystander Tm cells is intrinsically impaired by chronic virus infection, resulting in a diminished capacity to mount a strong recall response and confer protective immunity against bacterial reinfection. This impairment persists even after the bystander cells are removed from the chronic viral infection milieu.

Discussion

The formation of competent immunological memory is critical for rapid control of infections by previously encountered pathogens [4,9]. After maturation, Tm cell maintenance is dependent on environmental signals. During chronic viral infections, persistent antigen exposure can impact the memory repertoire [15]. Numerous data have shown that chronic virus infections negatively affect the development program of virus-specific T cells, which leads to a failure in the formation of functional Tm cells and impaired control of viral infections [10,12,13,26]. However, little is known about how chronic viral infections alter immune responses from the memory pool to unrelated pathogens. Herein, by employing two murine model pathogens, we showed that Lm-OVA immunized mice failed to mount an effective recall response to efficiently eradicate bacteria from a secondary challenge if the host suffered from a chronic virus infection (Fig 1). We found that chronic virus infection not only diminished the pool of pre-existing Lm-OVA specific CD4⁺ and CD8⁺ Tm cells, but also impaired their capacity to produce effector cytokines as well as mount recall responses (Figs 2, 3, 6, and S2–S4). Consistent with our data, Barnstorf et al. observed loss of adoptively transferred transgenic OT-I cells and impairment of their functions by chronic infection with a different LCMV strain, docile [17]. Instead of using monoclonal OT-I cells, our experimental system allowed us to examine a natural, polyclonal population of OVA-specific memory T cells induced by a bacterial infection. In contrast to the results from Barnstorf et al., we discovered clear phenotypic differences between virus-specific and non-virus-specific memory T cells (see below). More importantly, our study showed for the first time that a chronic infection impaired immunity to another pathogen, resulting in loss of protection against re-infection. These results have important clinical implications, and it would be critical to determine experimentally if our findings are applicable to other chronic infections, particularly in human diseases.

Although both virus- and non-virus-specific T cells become functionally impaired following chronic virus infection, our results showed clear phenotypic differences between these two populations (Figs 2 and 5). Most importantly, non-virus-specific T cells did not show upregulation of inhibitory receptors (Fig 5), a hallmark feature of exhaustion in virus-specific T cells. In addition, we observed differences in kinetics and tissue distribution between the two populations. While virus-specific T cells were rapidly expanding within the first week, we observed substantial loss of non-virus-specific memory T cells during this period in both Arm and Cl-13 infected mice, though the loss is more profound in Cl-13 than Arm-infected mice (Fig 2B). The initial loss of non-virus-specific memory T cells correlated with the timing of active viral replication, and higher viral replication likely contributed to more loss in Cl-13 infected mice (Fig 2B). After the clearance of an acute Arm infection, both virus-specific and non-virus-specific memory T cells remained stable and functional. In chronic Cl-13 infected mice, there was continuous loss in the number and function of non-virus-specific T cells, though much delayed in kinetics compared to virus-specific T cells. Virus-specific T cells were mostly depleted from the peripheral blood and became functionally impaired after 21 days. In contrast, profound loss of non-virus-specific T cells occurred much later (> 60 days, Fig 2), and these cells were still able to produce substantial amounts of cytokines at 40 days after chronic viral infection (Figs 3 and S2). Although virus-specific T cells were undetectable in peripheral blood at late time-points (Fig 2), they were preserved in the spleen and liver (Figs 5 and S1). However, we did not identify an enrichment site for non-virus-specific T cells (Figs 2 and 5).

The mechanisms of how non-virus-specific memory T cells are affected by a chronic viral infection are not known. The phenotypic and kinetic differences we observed between virus-specific and non-virus-specific T cells suggest that there are likely differences in the underlying mechanisms. In the canonical model proposed from existing data, T cell exhaustion is generally triggered by three signals: 1) persistent antigenic stimulation, 2) sustained co-inhibitory signals transduced by immune receptors, and 3) profound inflammation within the chronic infection environment [16,27]. Virus-specific T cells receive cumulative signals from TCR, inhibitory receptors (like PD-1), and inflammatory cytokines. In contrast, non-virus-specific T cells likely do not receive all three signals. First, antigenic stimulation is unlikely involved in functional defects of non-virus-specific memory T cells, since LCMV does not have epitopes that are cross-reactive with OVA-specific T cells. Second, sustained inhibitory signals may only play a minor role in functional defect of non-virus-specific T cells. Barnstorf et al. reported less than 1/3 of memory OT-I CD8⁺ T cells in chronic virus infected host express high levels of PD-1 [17]. We did not observe elevated expression of PD-1, TIM-3 and LAG-3 on OVA-specific T cells at any time-points during chronic virus infection (Fig 5). Consistent with the “Signal Strength” model [16], lack of antigenic stimulation and weak inhibitory signals may explain delayed onset of functional defects in non-virus-specific T cells, compared to virus-specific T cells.

Non-virus-specific T cells may experience the same profound inflammatory environment as virus-specific T cells in chronically infected hosts [16,27]. In support of a role for inflammatory signals, loss of non-virus-specific memory T cells and their functions correlates with timing and levels of active viral replication that drives inflammatory responses. Inflammatory cytokines such as type I IFNs (IFN-α/β) and IL-12 are the major sources of signal 3 required for T cell responses [28]. Type I IFNs and IL-12 are known to promote the generation of terminally differentiated T cells (KLRG1⁺CD127^lo) [29,30]. Type I IFNs have also been reported to induce apoptosis of memory phenotype T cells (CD44^hi) [31]. In addition, Barnstorf et al. observed sustained IL-6 production during chronic LCMV infection; IL-6 induces terminal differentiation on bystander memory OT-I cells [17]. These inflammatory cytokines together may play critical roles in the numeric loss and phenotypical alterations of non-virus-specific memory T cells. Interestingly, neither blockade of IFNAR nor IL-6R is sufficient to fully restore the number and function of bystander memory CD8⁺ T cells [16,17], suggesting the involvement of additional factors in driving bystander memory T cell dysfunction. These factors could include: 1) dysregulated expression of critical survival factors (e.g. IL-7, IL-15 for homeostatic proliferation/maintenance) [11], and 2) skewed differentiation of immunosuppressive dendritic cells (DCs) by sustained inflammatory signals [32]. Further studies are needed to investigate the role of these factors and fully understand the underlying mechanisms driving functional defects of non-virus-specific T cells during chronic viral infection.

Non-virus-specific Tm cells from chronic virus infected hosts were not only defective in effector functions, but also incapable of mounting an effective recall response (Figs 3, 6, and S2–S4). Similarly, Barnstorf et al. showed that memory OT-I CD8⁺ T cells cannot mount a robust secondary expansion in chronic LCMV infected mice [17]. The reasons for impaired secondary expansion are not known and could be due to limited expression of IL-2Ra following re-encounter of antigen [33], lack of DCs for efficient antigen presentation [32,34], or loss of response to inflammatory cytokines [35]. Furthermore, our results showed that non-virus-specific T cells from chronical Cl-13 infected mice were defective in recall responses even after transferred into new naïve recipients and were unresponsive to even PMA/I stimulation which bypasses proximal TCR signaling. (Figs 4 and 6). These results indicate that a chronic viral infection causes intrinsic defects in pre-existing bystander Tm cells specific to other pathogens (Fig 6). We and others have previously shown that epigenetic remodeling plays a critical role in transcriptional activation of the key effector genes and functional impairment during T cell exhaustion [36–43]. It remains to be defined how a chronic viral infection alters the transcriptional circuits and epigenetic landscapes of bystander Tm cells. Investigation into these questions will likely identify druggable targets to restore the function of compromised non-virus-specific T cells.

In summary, our study shows that bystander chronic viral infections are detrimental to the maintenance of long-lasting protective immunity against previously encountered pathogens. The results call for further investigations into broad impact that chronic exposure to inflammatory signals has on the immune system. Understanding the underlying mechanisms will help develop novel therapies that prevent the exhaustion of virus-specific T cells, while retaining the immune memory and competence to control other pathogens.

Materials and methods

Mice and ethics statement

Six-week-old female C57BL/6 (CD45.2⁺ or CD45.1⁺) mice were purchased from the National Cancer Institute (Bethesda, MD). Mice were maintained under specific pathogen-free conditions at animal facilities of the University of Pennsylvania, and were used in accordance with the Animal Care Guidelines of the University of Pennsylvania. Animal experiments were approved by the University of Pennsylvania Institutional Animal Care and Use Committee (IACUC, protocol # 806299).

Infections

Mice were immunized intravenously (i.v.) with Lm-OVA, as described previously [29]. More than 30 days after Lm-OVA infection, mice were infected with either LCMV Arm or Cl-13 strain, as described [23,44]. CD4⁺ T cells were depleted by intraperitoneal injection of 200 μg anti-CD4 antibody (GK1.5, BioXcell) at 1 day prior and post LCMV infection [44]. For rechallenge experiments, mice were infected i.v. with 1×10⁶ CFUs of WT Lm-OVA.

Flow cytometry

Surface staining and intracellular cytokine staining (ICS) were described previously [42]. Briefly, single-cell suspensions from peripheral blood, spleen or liver were stained with LIVE/DEAD Fixable Aqua Dye (Invitrogen) to exclude dead cells, then incubated with surface antibodies (eBiosciences) in combination with tetramers (NIH Tetramer Core Facility, Atlanta, GA). For ICS, cells were first cultured at 37°C for 5 hours in the complete medium (RPMI 1640 containing 10% FBS, 1% HEPES, 1% non-essential amino acid, 2 mM L-glutamine, 50 μM β-mercaptoethanol, 100 U/ mL penicillin and 100 μg/mL streptomycin; Gibco), in the presence of LLO_190-201, GP_33-41 or OVA_257-264 peptides (1 μg/mL), or PMA (50 ng/mL) + Ionomycin (0.5 μg/mL) (Sigma Aldrich). Brefeldin A and monensin were added to the culture for the last 4 hours. Cells were stained for viability and surface antigens as described above, followed by permeabilization (BD Cytofix/Perm) for intracellular staining. Samples were acquired on LSRII flow cytometer (BD Biosciences) and data were analyzed with FlowJo software (Treestar). Details about the reagents and softwares we used are shown in S1 Table.

Adoptive cell transfer

CD8⁺ T cells were purified from splenocytes by magnetic cell sorting (Miltenyi Biotec.), stained with K^b/OVA₂₅₇ tetramer to determine the number of OVA-specific cells. 5000 OVA-specific CD8⁺ T cells were transferred i.v. to recipient mice, which were challenged with Lm-OVA 1 day later as described previously [29].

Statistical analysis

Statistical analyses were described previously [45]. Briefly, the data were first evaluated for normality by the Shapiro-Wilk normality test. If the data passed normality test, we performed statistical analysis with unpaired Student’ s t-test, or one-way ANOVA with subsequent Tukey’s post-tests; if not, data were analyzed by the Mann-Whitney U test, or Kruskal-Wallis test followed by Dunns multiple-comparison test. Statistical significance was defined as follows: * p < 0.05, **p < 0.01, and ***p < 0.001.

Supporting information

S1 Data. Excel spreadsheets contain the underlying numerical data for Figs 1B, 1C, 2B, 2C, 3A, 3B, 3C, 3D, 3E, 3F, 3G, 4B, 4D, 6A, 6B, 6D, 6E, 6F, S2B, S2D, S3, S4A and S4B.

https://doi.org/10.1371/journal.ppat.1012113.s001

(XLSX)

S1 Table. Details about the reagents and software we used.

https://doi.org/10.1371/journal.ppat.1012113.s002

(XLSX)

S1 Fig. Virus-specific T cells become exhausted following chronic virus infection in Lm-OVA immunized mice.

(A) Experimental design. Mice were infected with ΔactA Lm-OVA, and more than 30 days after Lm-OVA infection, mice were infected with LCMV Arm or Cl-13. Virus-specific T cells were detected at day 45 after LCMV infection. (B) Expression of CD44, CD127, PD-1, TIM-3 and LAG-3 on D^b/GP₃₃⁺ CD8⁺ T cells in the spleens of Lm-OVA/Arm or Lm-OVA/Cl-13 mice. (C) Expression of CD107a and production of IFN-γ, TNF-α and IL-2 by CD8⁺ T cells from the spleens of Lm-OVA/Arm or Lm-OVA/Cl-13 mice after GP₃₃ peptide stimulation.

https://doi.org/10.1371/journal.ppat.1012113.s003

(TIF)

S2 Fig. Chronic virus infection leads to a progressive loss of Lm-OVA-specific Tm cells.

Infection of mice was the same as described in Fig 2. (A-D) Peripheral mononuclear cells isolated from Lm-OVA/Arm and Lm-OVA/Cl-13 mice at various time points post LCMV infection were stimulated with OVA₂₅₇ (A & B) or LLO₁₉₀ (C & D) peptides, respectively. Dynamic changes in the percentages of CD8⁺ (A & B) and CD4⁺ T cells (C & D) producing IFN-γ after peptide stimulation were shown. Asterisks indicate the statistical significance between Arm and Cl-13 mice (*, p<0.05; **, p<0.01; ***, p<0.001).

https://doi.org/10.1371/journal.ppat.1012113.s004

(TIF)

S3 Fig. Lm-OVA-specific CD4⁺ Tm cells lose polyfunctionality after chronic virus infection.

Infection of mice was described in Fig 2. More than 90 days after LCMV infection, CD4⁺ T cells in the spleen of Lm-OVA/Arm or Lm-OVA/Cl-13 mice were analyzed for their co-production of IFN-γ, TNF-α and IL-2, in the presence of LLO₁₉₀ peptide stimulation. Cytokines: 1, IFN-γ⁺TNF-α^-IL-2^-; 2, IFN-γ⁺TNF-α⁺IL-2^- and IFN-γ⁺TNF-α^-IL-2⁺; 3, IFN-γ⁺TNF-α⁺IL-2⁺.

https://doi.org/10.1371/journal.ppat.1012113.s005

(TIF)

S4 Fig. Lm-OVA-specific CD4⁺ Tm cells display impaired recall in chronic virus infected host.

Infection and challenge of mice were the same as described in Fig 1. (A & B) Recall responses of I-A^b/LLO₁₉₀-specific CD4⁺ T cells in the spleen and liver of Lm-OVA/Arm and Lm-OVA/Cl-13 mice were analyzed by MHC Class II tetramer staining (A) or ICS for IFN-γ after LLO₁₉₀ peptide stimulation (B).

https://doi.org/10.1371/journal.ppat.1012113.s006

(TIF)

Acknowledgments

We thank members of Shen lab for their assistance in conducting this research, and NIH Tetramer Core Facility for providing tetramers.

References

1. Harty JT, Tvinnereim AR, White DW. CD8+ T cell effector mechanisms in resistance to infection. Annu Rev Immunol. 2000;18:275–308. pmid:10837060.
- View Article
- PubMed/NCBI
- Google Scholar
2. Wherry EJ, Ahmed R. Memory CD8 T-cell differentiation during viral infection. J Virol. 2004;78(11):5535–45. pmid:15140950; PubMed Central PMCID: PMC415833.
- View Article
- PubMed/NCBI
- Google Scholar
3. Kaech SM, Ahmed R. Memory CD8+ T cell differentiation: initial antigen encounter triggers a developmental program in naive cells. Nat Immunol. 2001;2(5):415–22. pmid:11323695; PubMed Central PMCID: PMC3760150.
- View Article
- PubMed/NCBI
- Google Scholar
4. Williams MA, Bevan MJ. Effector and memory CTL differentiation. Annu Rev Immunol. 2007;25:171–92. pmid:17129182.
- View Article
- PubMed/NCBI
- Google Scholar
5. Murali-Krishna K, Lau LL, Sambhara S, Lemonnier F, Altman J, Ahmed R. Persistence of memory CD8 T cells in MHC class I-deficient mice. Science. 1999;286(5443):1377–81. pmid:10558996.
- View Article
- PubMed/NCBI
- Google Scholar
6. Kaech SM, Wherry EJ, Ahmed R. Effector and memory T-cell differentiation: implications for vaccine development. Nat Rev Immunol. 2002;2(4):251–62. pmid:12001996.
- View Article
- PubMed/NCBI
- Google Scholar
7. Gourley TS, Wherry EJ, Masopust D, Ahmed R. Generation and maintenance of immunological memory. Semin Immunol. 2004;16(5):323–33. pmid:15528077.
- View Article
- PubMed/NCBI
- Google Scholar
8. Masopust D, Ahmed R. Reflections on CD8 T-cell activation and memory. Immunol Res. 2004;29(1–3):151–60. pmid:15181278.
- View Article
- PubMed/NCBI
- Google Scholar
9. Rocha B, Tanchot C. CD8 T cell memory. Semin Immunol. 2004;16(5):305–14. pmid:15528075.
- View Article
- PubMed/NCBI
- Google Scholar
10. Wherry EJ, Blattman JN, Murali-Krishna K, van der Most R, Ahmed R. Viral persistence alters CD8 T-cell immunodominance and tissue distribution and results in distinct stages of functional impairment. J Virol. 2003;77(8):4911–27. pmid:12663797; PubMed Central PMCID: PMC152117.
- View Article
- PubMed/NCBI
- Google Scholar
11. Wherry EJ, Barber DL, Kaech SM, Blattman JN, Ahmed R. Antigen-independent memory CD8 T cells do not develop during chronic viral infection. Proc Natl Acad Sci U S A. 2004;101(45):16004–9. Epub 2004/10/27. pmid:15505208; PubMed Central PMCID: PMC524220.
- View Article
- PubMed/NCBI
- Google Scholar
12. Zajac AJ, Blattman JN, Murali-Krishna K, Sourdive DJ, Suresh M, Altman JD, et al. Viral immune evasion due to persistence of activated T cells without effector function. J Exp Med. 1998;188(12):2205–13. pmid:9858507; PubMed Central PMCID: PMC2212420.
- View Article
- PubMed/NCBI
- Google Scholar
13. Fuller MJ, Khanolkar A, Tebo AE, Zajac AJ. Maintenance, loss, and resurgence of T cell responses during acute, protracted, and chronic viral infections. J Immunol. 2004;172(7):4204–14. pmid:15034033.
- View Article
- PubMed/NCBI
- Google Scholar
14. Shin H, Wherry EJ. CD8 T cell dysfunction during chronic viral infection. Curr Opin Immunol. 2007;19(4):408–15. pmid:17656078.
- View Article
- PubMed/NCBI
- Google Scholar
15. Virgin HW, Wherry EJ, Ahmed R. Redefining chronic viral infection. Cell. 2009;138(1):30–50. pmid:19596234.
- View Article
- PubMed/NCBI
- Google Scholar
16. McLane LM, Abdel-Hakeem MS, Wherry EJ. CD8 T Cell Exhaustion During Chronic Viral Infection and Cancer. Annu Rev Immunol. 2019. pmid:30676822.
- View Article
- PubMed/NCBI
- Google Scholar
17. Barnstorf I, Borsa M, Baumann N, Pallmer K, Yermanos A, Joller N, et al. Chronic virus infection compromises memory bystander T cell function in an IL-6/STAT1-dependent manner. J Exp Med. 2019;216(3):571–86. pmid:30745322; PubMed Central PMCID: PMC6400541.
- View Article
- PubMed/NCBI
- Google Scholar
18. Shen H, Miller JF, Fan X, Kolwyck D, Ahmed R, Harty JT. Compartmentalization of bacterial antigens: differential effects on priming of CD8 T cells and protective immunity. Cell. 1998;92(4):535–45. pmid:9491894.
- View Article
- PubMed/NCBI
- Google Scholar
19. Betts MR, Brenchley JM, Price DA, De Rosa SC, Douek DC, Roederer M, et al. Sensitive and viable identification of antigen-specific CD8+ T cells by a flow cytometric assay for degranulation. J Immunol Methods. 2003;281(1–2):65–78. pmid:14580882.
- View Article
- PubMed/NCBI
- Google Scholar
20. Almeida JR, Price DA, Papagno L, Arkoub ZA, Sauce D, Bornstein E, et al. Superior control of HIV-1 replication by CD8+ T cells is reflected by their avidity, polyfunctionality, and clonal turnover. J Exp Med. 2007;204(10):2473–85. pmid:17893201; PubMed Central PMCID: PMC2118466.
- View Article
- PubMed/NCBI
- Google Scholar
21. Oh-hora M, Rao A. Calcium signaling in lymphocytes. Curr Opin Immunol. 2008;20(3):250–8. Epub 2008/06/03. pmid:18515054; PubMed Central PMCID: PMC2574011.
- View Article
- PubMed/NCBI
- Google Scholar
22. Barber DL, Wherry EJ, Masopust D, Zhu B, Allison JP, Sharpe AH, et al. Restoring function in exhausted CD8 T cells during chronic viral infection. Nature. 2006;439(7077):682–7. pmid:16382236.
- View Article
- PubMed/NCBI
- Google Scholar
23. Im SJ, Hashimoto M, Gerner MY, Lee J, Kissick HT, Burger MC, et al. Defining CD8+ T cells that provide the proliferative burst after PD-1 therapy. Nature. 2016;537(7620):417–21. pmid:27501248; PubMed Central PMCID: PMC5297183.
- View Article
- PubMed/NCBI
- Google Scholar
24. Wherry EJ, Ha SJ, Kaech SM, Haining WN, Sarkar S, Kalia V, et al. Molecular signature of CD8+ T cell exhaustion during chronic viral infection. Immunity. 2007;27(4):670–84. pmid:17950003.
- View Article
- PubMed/NCBI
- Google Scholar
25. Foulds KE, Zenewicz LA, Shedlock DJ, Jiang J, Troy AE, Shen H. Cutting edge: CD4 and CD8 T cells are intrinsically different in their proliferative responses. J Immunol. 2002;168(4):1528–32. pmid:11823476.
- View Article
- PubMed/NCBI
- Google Scholar
26. Wherry EJ. T cell exhaustion. Nat Immunol. 2011;12(6):492–9. pmid:21739672.
- View Article
- PubMed/NCBI
- Google Scholar
27. Mescher MF, Curtsinger JM, Agarwal P, Casey KA, Gerner M, Hammerbeck CD, et al. Signals required for programming effector and memory development by CD8+ T cells. Immunol Rev. 2006;211:81–92. pmid:16824119.
- View Article
- PubMed/NCBI
- Google Scholar
28. Curtsinger JM, Mescher MF. Inflammatory cytokines as a third signal for T cell activation. Curr Opin Immunol. 2010;22(3):333–40. Epub 2010/04/07. pmid:20363604; PubMed Central PMCID: PMC2891062.
- View Article
- PubMed/NCBI
- Google Scholar
29. Pearce EL, Shen H. Generation of CD8 T cell memory is regulated by IL-12. J Immunol. 2007;179(4):2074–81. pmid:17675465.
- View Article
- PubMed/NCBI
- Google Scholar
30. Stelekati E, Shin H, Doering TA, Dolfi DV, Ziegler CG, Beiting DP, et al. Bystander chronic infection negatively impacts development of CD8(+) T cell memory. Immunity. 2014;40(5):801–13. pmid:24837104; PubMed Central PMCID: PMC4114317.
- View Article
- PubMed/NCBI
- Google Scholar
31. Bahl K, Kim SK, Calcagno C, Ghersi D, Puzone R, Celada F, et al. IFN-induced attrition of CD8 T cells in the presence or absence of cognate antigen during the early stages of viral infections. J Immunol. 2006;176(7):4284–95. Epub 2006/03/21. pmid:16547266.
- View Article
- PubMed/NCBI
- Google Scholar
32. Cunningham CR, Champhekar A, Tullius MV, Dillon BJ, Zhen A, de la Fuente JR, et al. Type I and Type II Interferon Coordinately Regulate Suppressive Dendritic Cell Fate and Function during Viral Persistence. PLoS Pathog. 2016;12(1):e1005356. Epub 2016/01/26. pmid:26808628; PubMed Central PMCID: PMC4726812.
- View Article
- PubMed/NCBI
- Google Scholar
33. Williams MA, Tyznik AJ, Bevan MJ. Interleukin-2 signals during priming are required for secondary expansion of CD8+ memory T cells. Nature. 2006;441(7095):890–3. Epub 2006/06/17. pmid:16778891; PubMed Central PMCID: PMC2776073.
- View Article
- PubMed/NCBI
- Google Scholar
34. Sevilla N, McGavern DB, Teng C, Kunz S, Oldstone MB. Viral targeting of hematopoietic progenitors and inhibition of DC maturation as a dual strategy for immune subversion. J Clin Invest. 2004;113(5):737–45. Epub 2004/03/03. pmid:14991072; PubMed Central PMCID: PMC351322.
- View Article
- PubMed/NCBI
- Google Scholar
35. Keppler SJ, Aichele P. Signal 3 requirement for memory CD8+ T-cell activation is determined by the infectious pathogen. Eur J Immunol. 2011;41(11):3176–86. Epub 2011/08/11. pmid:21830209.
- View Article
- PubMed/NCBI
- Google Scholar
36. Fann M, Godlove JM, Catalfamo M, Wood WH 3rd, Chrest FJ, Chun N, et al. Histone acetylation is associated with differential gene expression in the rapid and robust memory CD8(+) T-cell response. Blood. 2006;108(10):3363–70. pmid:16868257; PubMed Central PMCID: PMC1895425.
- View Article
- PubMed/NCBI
- Google Scholar
37. Northrop JK, Thomas RM, Wells AD, Shen H. Epigenetic remodeling of the IL-2 and IFN-gamma loci in memory CD8 T cells is influenced by CD4 T cells. J Immunol. 2006;177(2):1062–9. pmid:16818762.
- View Article
- PubMed/NCBI
- Google Scholar
38. Araki Y, Fann M, Wersto R, Weng NP. Histone acetylation facilitates rapid and robust memory CD8 T cell response through differential expression of effector molecules (eomesodermin and its targets: perforin and granzyme B). J Immunol. 2008;180(12):8102–8. pmid:18523274; PubMed Central PMCID: PMC2493419.
- View Article
- PubMed/NCBI
- Google Scholar
39. Northrop JK, Wells AD, Shen H. Cutting edge: chromatin remodeling as a molecular basis for the enhanced functionality of memory CD8 T cells. J Immunol. 2008;181(2):865–8. pmid:18606637.
- View Article
- PubMed/NCBI
- Google Scholar
40. Weng NP, Araki Y, Subedi K. The molecular basis of the memory T cell response: differential gene expression and its epigenetic regulation. Nat Rev Immunol. 2012;12(4):306–15. pmid:22421787; PubMed Central PMCID: PMC4686144.
- View Article
- PubMed/NCBI
- Google Scholar
41. Yang C, Khanniche A, DiSpirito JR, Ji P, Wang S, Wang Y, et al. Transcriptome Signatures Reveal Rapid Induction of Immune-Responsive Genes in Human Memory CD8(+) T Cells. Sci Rep. 2016;6:27005. pmid:27243788; PubMed Central PMCID: PMC4886650.
- View Article
- PubMed/NCBI
- Google Scholar
42. Zhang F, Zhou X, DiSpirito JR, Wang C, Wang Y, Shen H. Epigenetic manipulation restores functions of defective CD8(+) T cells from chronic viral infection. Mol Ther. 2014;22(9):1698–706. pmid:24861055; PubMed Central PMCID: PMC4435497.
- View Article
- PubMed/NCBI
- Google Scholar
43. Dispirito JR, Shen H. Histone acetylation at the single-cell level: a marker of memory CD8+ T cell differentiation and functionality. J Immunol. 2010;184(9):4631–6. pmid:20308634.
- View Article
- PubMed/NCBI
- Google Scholar
44. He R, Hou S, Liu C, Zhang A, Bai Q, Han M, et al. Follicular CXCR5- expressing CD8(+) T cells curtail chronic viral infection. Nature. 2016;537(7620):412–28. pmid:27501245.
- View Article
- PubMed/NCBI
- Google Scholar
45. Zeng W, Qin S, Wang R, Zhang Y, Ma X, Tian F, et al. PDL1 blockage increases fetal resorption and Tfr cells but does not affect Tfh/Tfr ratio and B-cell maturation during allogeneic pregnancy. Cell Death Dis. 2020;11(2):119. Epub 2020/02/14. pmid:32051396; PubMed Central PMCID: PMC7016117.
- View Article
- PubMed/NCBI
- Google Scholar

[ref1] 1. Harty JT, Tvinnereim AR, White DW. CD8+ T cell effector mechanisms in resistance to infection. Annu Rev Immunol. 2000;18:275–308. pmid:10837060.
View Article
PubMed/NCBI
Google Scholar

[2] View Article

[3] PubMed/NCBI

[4] Google Scholar

[ref2] 2. Wherry EJ, Ahmed R. Memory CD8 T-cell differentiation during viral infection. J Virol. 2004;78(11):5535–45. pmid:15140950; PubMed Central PMCID: PMC415833.
View Article
PubMed/NCBI
Google Scholar

[6] View Article

[7] PubMed/NCBI

[8] Google Scholar

[ref3] 3. Kaech SM, Ahmed R. Memory CD8+ T cell differentiation: initial antigen encounter triggers a developmental program in naive cells. Nat Immunol. 2001;2(5):415–22. pmid:11323695; PubMed Central PMCID: PMC3760150.
View Article
PubMed/NCBI
Google Scholar

[10] View Article

[11] PubMed/NCBI

[12] Google Scholar

[ref4] 4. Williams MA, Bevan MJ. Effector and memory CTL differentiation. Annu Rev Immunol. 2007;25:171–92. pmid:17129182.
View Article
PubMed/NCBI
Google Scholar

[14] View Article

[15] PubMed/NCBI

[16] Google Scholar

[ref5] 5. Murali-Krishna K, Lau LL, Sambhara S, Lemonnier F, Altman J, Ahmed R. Persistence of memory CD8 T cells in MHC class I-deficient mice. Science. 1999;286(5443):1377–81. pmid:10558996.
View Article
PubMed/NCBI
Google Scholar

[18] View Article

[19] PubMed/NCBI

[20] Google Scholar

[ref6] 6. Kaech SM, Wherry EJ, Ahmed R. Effector and memory T-cell differentiation: implications for vaccine development. Nat Rev Immunol. 2002;2(4):251–62. pmid:12001996.
View Article
PubMed/NCBI
Google Scholar

[22] View Article

[23] PubMed/NCBI

[24] Google Scholar

[ref7] 7. Gourley TS, Wherry EJ, Masopust D, Ahmed R. Generation and maintenance of immunological memory. Semin Immunol. 2004;16(5):323–33. pmid:15528077.
View Article
PubMed/NCBI
Google Scholar

[26] View Article

[27] PubMed/NCBI

[28] Google Scholar

[ref8] 8. Masopust D, Ahmed R. Reflections on CD8 T-cell activation and memory. Immunol Res. 2004;29(1–3):151–60. pmid:15181278.
View Article
PubMed/NCBI
Google Scholar

[30] View Article

[31] PubMed/NCBI

[32] Google Scholar

[ref9] 9. Rocha B, Tanchot C. CD8 T cell memory. Semin Immunol. 2004;16(5):305–14. pmid:15528075.
View Article
PubMed/NCBI
Google Scholar

[34] View Article

[35] PubMed/NCBI

[36] Google Scholar

[ref10] 10. Wherry EJ, Blattman JN, Murali-Krishna K, van der Most R, Ahmed R. Viral persistence alters CD8 T-cell immunodominance and tissue distribution and results in distinct stages of functional impairment. J Virol. 2003;77(8):4911–27. pmid:12663797; PubMed Central PMCID: PMC152117.
View Article
PubMed/NCBI
Google Scholar

[38] View Article

[39] PubMed/NCBI

[40] Google Scholar

[ref11] 11. Wherry EJ, Barber DL, Kaech SM, Blattman JN, Ahmed R. Antigen-independent memory CD8 T cells do not develop during chronic viral infection. Proc Natl Acad Sci U S A. 2004;101(45):16004–9. Epub 2004/10/27. pmid:15505208; PubMed Central PMCID: PMC524220.
View Article
PubMed/NCBI
Google Scholar

[42] View Article

[43] PubMed/NCBI

[44] Google Scholar

[ref12] 12. Zajac AJ, Blattman JN, Murali-Krishna K, Sourdive DJ, Suresh M, Altman JD, et al. Viral immune evasion due to persistence of activated T cells without effector function. J Exp Med. 1998;188(12):2205–13. pmid:9858507; PubMed Central PMCID: PMC2212420.
View Article
PubMed/NCBI
Google Scholar

[46] View Article

[47] PubMed/NCBI

[48] Google Scholar

[ref13] 13. Fuller MJ, Khanolkar A, Tebo AE, Zajac AJ. Maintenance, loss, and resurgence of T cell responses during acute, protracted, and chronic viral infections. J Immunol. 2004;172(7):4204–14. pmid:15034033.
View Article
PubMed/NCBI
Google Scholar

[50] View Article

[51] PubMed/NCBI

[52] Google Scholar

[ref14] 14. Shin H, Wherry EJ. CD8 T cell dysfunction during chronic viral infection. Curr Opin Immunol. 2007;19(4):408–15. pmid:17656078.
View Article
PubMed/NCBI
Google Scholar

[54] View Article

[55] PubMed/NCBI

[56] Google Scholar

[ref15] 15. Virgin HW, Wherry EJ, Ahmed R. Redefining chronic viral infection. Cell. 2009;138(1):30–50. pmid:19596234.
View Article
PubMed/NCBI
Google Scholar

[58] View Article

[59] PubMed/NCBI

[60] Google Scholar

[ref16] 16. McLane LM, Abdel-Hakeem MS, Wherry EJ. CD8 T Cell Exhaustion During Chronic Viral Infection and Cancer. Annu Rev Immunol. 2019. pmid:30676822.
View Article
PubMed/NCBI
Google Scholar

[62] View Article

[63] PubMed/NCBI

[64] Google Scholar

[ref17] 17. Barnstorf I, Borsa M, Baumann N, Pallmer K, Yermanos A, Joller N, et al. Chronic virus infection compromises memory bystander T cell function in an IL-6/STAT1-dependent manner. J Exp Med. 2019;216(3):571–86. pmid:30745322; PubMed Central PMCID: PMC6400541.
View Article
PubMed/NCBI
Google Scholar

[66] View Article

[67] PubMed/NCBI

[68] Google Scholar

[ref18] 18. Shen H, Miller JF, Fan X, Kolwyck D, Ahmed R, Harty JT. Compartmentalization of bacterial antigens: differential effects on priming of CD8 T cells and protective immunity. Cell. 1998;92(4):535–45. pmid:9491894.
View Article
PubMed/NCBI
Google Scholar

[70] View Article

[71] PubMed/NCBI

[72] Google Scholar

[ref19] 19. Betts MR, Brenchley JM, Price DA, De Rosa SC, Douek DC, Roederer M, et al. Sensitive and viable identification of antigen-specific CD8+ T cells by a flow cytometric assay for degranulation. J Immunol Methods. 2003;281(1–2):65–78. pmid:14580882.
View Article
PubMed/NCBI
Google Scholar

[74] View Article

[75] PubMed/NCBI

[76] Google Scholar

[ref20] 20. Almeida JR, Price DA, Papagno L, Arkoub ZA, Sauce D, Bornstein E, et al. Superior control of HIV-1 replication by CD8+ T cells is reflected by their avidity, polyfunctionality, and clonal turnover. J Exp Med. 2007;204(10):2473–85. pmid:17893201; PubMed Central PMCID: PMC2118466.
View Article
PubMed/NCBI
Google Scholar

[78] View Article

[79] PubMed/NCBI

[80] Google Scholar

[ref21] 21. Oh-hora M, Rao A. Calcium signaling in lymphocytes. Curr Opin Immunol. 2008;20(3):250–8. Epub 2008/06/03. pmid:18515054; PubMed Central PMCID: PMC2574011.
View Article
PubMed/NCBI
Google Scholar

[82] View Article

[83] PubMed/NCBI

[84] Google Scholar

[ref22] 22. Barber DL, Wherry EJ, Masopust D, Zhu B, Allison JP, Sharpe AH, et al. Restoring function in exhausted CD8 T cells during chronic viral infection. Nature. 2006;439(7077):682–7. pmid:16382236.
View Article
PubMed/NCBI
Google Scholar

[86] View Article

[87] PubMed/NCBI

[88] Google Scholar

[ref23] 23. Im SJ, Hashimoto M, Gerner MY, Lee J, Kissick HT, Burger MC, et al. Defining CD8+ T cells that provide the proliferative burst after PD-1 therapy. Nature. 2016;537(7620):417–21. pmid:27501248; PubMed Central PMCID: PMC5297183.
View Article
PubMed/NCBI
Google Scholar

[90] View Article

[91] PubMed/NCBI

[92] Google Scholar

[ref24] 24. Wherry EJ, Ha SJ, Kaech SM, Haining WN, Sarkar S, Kalia V, et al. Molecular signature of CD8+ T cell exhaustion during chronic viral infection. Immunity. 2007;27(4):670–84. pmid:17950003.
View Article
PubMed/NCBI
Google Scholar

[94] View Article

[95] PubMed/NCBI

[96] Google Scholar

[ref25] 25. Foulds KE, Zenewicz LA, Shedlock DJ, Jiang J, Troy AE, Shen H. Cutting edge: CD4 and CD8 T cells are intrinsically different in their proliferative responses. J Immunol. 2002;168(4):1528–32. pmid:11823476.
View Article
PubMed/NCBI
Google Scholar

[98] View Article

[99] PubMed/NCBI

[100] Google Scholar

[ref26] 26. Wherry EJ. T cell exhaustion. Nat Immunol. 2011;12(6):492–9. pmid:21739672.
View Article
PubMed/NCBI
Google Scholar

[102] View Article

[103] PubMed/NCBI

[104] Google Scholar

[ref27] 27. Mescher MF, Curtsinger JM, Agarwal P, Casey KA, Gerner M, Hammerbeck CD, et al. Signals required for programming effector and memory development by CD8+ T cells. Immunol Rev. 2006;211:81–92. pmid:16824119.
View Article
PubMed/NCBI
Google Scholar

[106] View Article

[107] PubMed/NCBI

[108] Google Scholar

[ref28] 28. Curtsinger JM, Mescher MF. Inflammatory cytokines as a third signal for T cell activation. Curr Opin Immunol. 2010;22(3):333–40. Epub 2010/04/07. pmid:20363604; PubMed Central PMCID: PMC2891062.
View Article
PubMed/NCBI
Google Scholar

[110] View Article

[111] PubMed/NCBI

[112] Google Scholar

[ref29] 29. Pearce EL, Shen H. Generation of CD8 T cell memory is regulated by IL-12. J Immunol. 2007;179(4):2074–81. pmid:17675465.
View Article
PubMed/NCBI
Google Scholar

[114] View Article

[115] PubMed/NCBI

[116] Google Scholar

[ref30] 30. Stelekati E, Shin H, Doering TA, Dolfi DV, Ziegler CG, Beiting DP, et al. Bystander chronic infection negatively impacts development of CD8(+) T cell memory. Immunity. 2014;40(5):801–13. pmid:24837104; PubMed Central PMCID: PMC4114317.
View Article
PubMed/NCBI
Google Scholar

[118] View Article

[119] PubMed/NCBI

[120] Google Scholar

[ref31] 31. Bahl K, Kim SK, Calcagno C, Ghersi D, Puzone R, Celada F, et al. IFN-induced attrition of CD8 T cells in the presence or absence of cognate antigen during the early stages of viral infections. J Immunol. 2006;176(7):4284–95. Epub 2006/03/21. pmid:16547266.
View Article
PubMed/NCBI
Google Scholar

[122] View Article

[123] PubMed/NCBI

[124] Google Scholar

[ref32] 32. Cunningham CR, Champhekar A, Tullius MV, Dillon BJ, Zhen A, de la Fuente JR, et al. Type I and Type II Interferon Coordinately Regulate Suppressive Dendritic Cell Fate and Function during Viral Persistence. PLoS Pathog. 2016;12(1):e1005356. Epub 2016/01/26. pmid:26808628; PubMed Central PMCID: PMC4726812.
View Article
PubMed/NCBI
Google Scholar

[126] View Article

[127] PubMed/NCBI

[128] Google Scholar

[ref33] 33. Williams MA, Tyznik AJ, Bevan MJ. Interleukin-2 signals during priming are required for secondary expansion of CD8+ memory T cells. Nature. 2006;441(7095):890–3. Epub 2006/06/17. pmid:16778891; PubMed Central PMCID: PMC2776073.
View Article
PubMed/NCBI
Google Scholar

[130] View Article

[131] PubMed/NCBI

[132] Google Scholar

[ref34] 34. Sevilla N, McGavern DB, Teng C, Kunz S, Oldstone MB. Viral targeting of hematopoietic progenitors and inhibition of DC maturation as a dual strategy for immune subversion. J Clin Invest. 2004;113(5):737–45. Epub 2004/03/03. pmid:14991072; PubMed Central PMCID: PMC351322.
View Article
PubMed/NCBI
Google Scholar

[134] View Article

[135] PubMed/NCBI

[136] Google Scholar

[ref35] 35. Keppler SJ, Aichele P. Signal 3 requirement for memory CD8+ T-cell activation is determined by the infectious pathogen. Eur J Immunol. 2011;41(11):3176–86. Epub 2011/08/11. pmid:21830209.
View Article
PubMed/NCBI
Google Scholar

[138] View Article

[139] PubMed/NCBI

[140] Google Scholar

[ref36] 36. Fann M, Godlove JM, Catalfamo M, Wood WH 3rd, Chrest FJ, Chun N, et al. Histone acetylation is associated with differential gene expression in the rapid and robust memory CD8(+) T-cell response. Blood. 2006;108(10):3363–70. pmid:16868257; PubMed Central PMCID: PMC1895425.
View Article
PubMed/NCBI
Google Scholar

[142] View Article

[143] PubMed/NCBI

[144] Google Scholar

[ref37] 37. Northrop JK, Thomas RM, Wells AD, Shen H. Epigenetic remodeling of the IL-2 and IFN-gamma loci in memory CD8 T cells is influenced by CD4 T cells. J Immunol. 2006;177(2):1062–9. pmid:16818762.
View Article
PubMed/NCBI
Google Scholar

[146] View Article

[147] PubMed/NCBI

[148] Google Scholar

[ref38] 38. Araki Y, Fann M, Wersto R, Weng NP. Histone acetylation facilitates rapid and robust memory CD8 T cell response through differential expression of effector molecules (eomesodermin and its targets: perforin and granzyme B). J Immunol. 2008;180(12):8102–8. pmid:18523274; PubMed Central PMCID: PMC2493419.
View Article
PubMed/NCBI
Google Scholar

[150] View Article

[151] PubMed/NCBI

[152] Google Scholar

[ref39] 39. Northrop JK, Wells AD, Shen H. Cutting edge: chromatin remodeling as a molecular basis for the enhanced functionality of memory CD8 T cells. J Immunol. 2008;181(2):865–8. pmid:18606637.
View Article
PubMed/NCBI
Google Scholar

[154] View Article

[155] PubMed/NCBI

[156] Google Scholar

[ref40] 40. Weng NP, Araki Y, Subedi K. The molecular basis of the memory T cell response: differential gene expression and its epigenetic regulation. Nat Rev Immunol. 2012;12(4):306–15. pmid:22421787; PubMed Central PMCID: PMC4686144.
View Article
PubMed/NCBI
Google Scholar

[158] View Article

[159] PubMed/NCBI

[160] Google Scholar

[ref41] 41. Yang C, Khanniche A, DiSpirito JR, Ji P, Wang S, Wang Y, et al. Transcriptome Signatures Reveal Rapid Induction of Immune-Responsive Genes in Human Memory CD8(+) T Cells. Sci Rep. 2016;6:27005. pmid:27243788; PubMed Central PMCID: PMC4886650.
View Article
PubMed/NCBI
Google Scholar

[162] View Article

[163] PubMed/NCBI

[164] Google Scholar

[ref42] 42. Zhang F, Zhou X, DiSpirito JR, Wang C, Wang Y, Shen H. Epigenetic manipulation restores functions of defective CD8(+) T cells from chronic viral infection. Mol Ther. 2014;22(9):1698–706. pmid:24861055; PubMed Central PMCID: PMC4435497.
View Article
PubMed/NCBI
Google Scholar

[166] View Article

[167] PubMed/NCBI

[168] Google Scholar

[ref43] 43. Dispirito JR, Shen H. Histone acetylation at the single-cell level: a marker of memory CD8+ T cell differentiation and functionality. J Immunol. 2010;184(9):4631–6. pmid:20308634.
View Article
PubMed/NCBI
Google Scholar

[170] View Article

[171] PubMed/NCBI

[172] Google Scholar

[ref44] 44. He R, Hou S, Liu C, Zhang A, Bai Q, Han M, et al. Follicular CXCR5- expressing CD8(+) T cells curtail chronic viral infection. Nature. 2016;537(7620):412–28. pmid:27501245.
View Article
PubMed/NCBI
Google Scholar

[174] View Article

[175] PubMed/NCBI

[176] Google Scholar

[ref45] 45. Zeng W, Qin S, Wang R, Zhang Y, Ma X, Tian F, et al. PDL1 blockage increases fetal resorption and Tfr cells but does not affect Tfh/Tfr ratio and B-cell maturation during allogeneic pregnancy. Cell Death Dis. 2020;11(2):119. Epub 2020/02/14. pmid:32051396; PubMed Central PMCID: PMC7016117.
View Article
PubMed/NCBI
Google Scholar

[178] View Article

[179] PubMed/NCBI

[180] Google Scholar

Figures

Abstract

Author summary

Introduction

Results

Chronic viral infection impairs protective immunity to bacteria re-infection

Chronic viral infection causes progressive loss of pre-existing bystander Tm cells specific to a different pathogen

Chronic viral infection induces functional impairment of pre-existing bystander Tm cells

The majority of T cell population become partially dysfunctional after chronic viral infection

Effects of chronic LCMV infection on bystander Tm cells differ from exhaustion of LCMV-specific T cells

Bystander Tm cells derived from chronic virus infected hosts fail to recall upon bacteria re-challenge

Discussion

Materials and methods

Mice and ethics statement

Infections

Flow cytometry

Adoptive cell transfer

Statistical analysis

Supporting information

S1 Data. Excel spreadsheets contain the underlying numerical data for Figs 1B, 1C, 2B, 2C, 3A, 3B, 3C, 3D, 3E, 3F, 3G, 4B, 4D, 6A, 6B, 6D, 6E, 6F, S2B, S2D, S3, S4A and S4B.

S1 Table. Details about the reagents and software we used.

S1 Fig. Virus-specific T cells become exhausted following chronic virus infection in Lm-OVA immunized mice.

S2 Fig. Chronic virus infection leads to a progressive loss of Lm-OVA-specific Tm cells.

S3 Fig. Lm-OVA-specific CD4+ Tm cells lose polyfunctionality after chronic virus infection.

S4 Fig. Lm-OVA-specific CD4+ Tm cells display impaired recall in chronic virus infected host.

Acknowledgments

References

S3 Fig. Lm-OVA-specific CD4⁺ Tm cells lose polyfunctionality after chronic virus infection.

S4 Fig. Lm-OVA-specific CD4⁺ Tm cells display impaired recall in chronic virus infected host.