CD4⁺ T cells play an essential role in coordinating innate and adaptive immunity against multiple pathogens. Upon antigen encounter and exposure to inflammatory cytokines, CD4⁺ T cells display an inherent capacity to adapt diverse functional fates and facilitate immunity through their secretion of soluble meditators and direct cell interactions with other immune cell populations. In response to either an acute or persistent viral infection, naïve antigen-specific CD4⁺ T cells clonally expand and primarily differentiate into either T helper type 1 (Th1) or T follicular helper (Tfh) cells, which support cellular or humoral responses, respectively (Crotty, 2011; Sheikh and Groom, 2021).

Several recent studies have provided insight into how the fate commitment of CD4⁺ T cells is established early after CD4⁺ T cell priming. Notably, the strength of T cell receptor (TCR) and IL-2R signaling strongly influences this bifurcation decision, with increased TCR and IL-2R signaling generally favoring Th1 lineage commitment (Choi et al., 2011; Pepper et al., 2011; Johnston et al., 2012; Snook et al., 2018). Mechanistically, activation of the TCR and IL-2R signaling pathways results in the induction of Blimp-1, a transcription factor (TF) that is critical for Th1 differentiation and that also potently antagonizes Bcl-6, a transcriptional repressor that is required for Tfh differentiation (Johnston et al., 2009; Choi et al., 2011; Pepper et al., 2011). Additionally, early exposure of activated CD4⁺ T cells to IL-12 and type I IFN signaling can promote Th1 cell differentiation by inducing expression of T-bet (Hsieh et al., 1993; Manetti, 1993; Mullen et al., 2001; Ray et al., 2014), which is the lineage-defining TF that is required for Th1 development (Szabo et al., 2000). Th1 cells can then travel to sites of infection where they produce pro-inflammatory cytokines such as IL-2, IFN-γ, and TNF-α, which contribute to the activation of CD8⁺ T cells and macrophages (Sheikh and Groom, 2021). Thus, Th1 cells play a critical role in orchestrating immunity against intracellular pathogens. Conversely, dendritic cell presentation of antigen and secretion of IL-6 facilitates early Tfh differentiation by inducing Bcl-6 expression and the subsequent upregulation of CXCR5 (Crotty, 2011; Eto et al., 2011; Choi et al., 2013). Pre-Tfh cells then migrate from the T cell zone to the T cell-B cell border where they can interact with antigen-presenting B cells via ICOS-ICOSL and CD40-CD40L pathways, which are required for fully instilling the Tfh program (Han et al., 1995; Choi et al., 2011; Crotty, 2011). Mature Tfh cells then provide essential help signals to pathogen-specific B cells to facilitate germinal center (GC) reactions, B cell secretion of high-affinity antibodies, and the generation of memory B cells and long-lived plasma cells (Crotty, 2011).

After the initial T cell expansion phase, the pool of virus-specific CD4⁺ T cells begins to decline in both self-resolving and chronic infections, although the nature of the infection plays a major role in shaping the overall magnitude, subset distribution, and functional capacity of responding CD4⁺ T cells. In acute settings, following pathogen clearance, distinct populations of memory CD4⁺ T cells develop that are well equipped to confer long-lasting protection against re-infection. These memory cells include Th1- and Tfh-like subsets (which may represent T effector memory cells; Marshall et al., 2011; Pepper et al., 2011; Hale et al., 2013), tissue resident memory cells that provide rapid protection at frontline sites of infection (Iijima and Iwasaki, 2014; Schreiner and King, 2018), and a less differentiated T central memory (Tcm) cell population that displays an increased capacity to proliferate and produce IL-2 upon secondary challenge (Pepper and Jenkins, 2011). While several studies have provided evidence that differentiated effector Th1 and Tfh cells can give rise to long-lived memory cells, previous work further indicates that a memory precursor population develops among early responding CD4⁺ T cells and that this precursor population also significantly contributes to the establishment of durable and effective CD4⁺ T cell memory following resolution of the infection (Marshall et al., 2011; Pepper et al., 2011; Ciucci et al., 2019). Notably, similar to that of Tcm cells, this precursor subset displays high expression of CCR7, TCF-1, and Bcl2, and as such was referred to as Tcm precursor (Tcmp) cells (Ciucci et al., 2019). Importantly, these Tcmp cells display an augmented capacity to survive the contraction phase of acute infection and generate diverse effector cell populations during a secondary response (Marshall et al., 2011; Pepper et al., 2011). The development and functional fitness of Tcmp cells have recently been identified to be dependent on the TF Thpok (Ciucci et al., 2019), although previous work also indicates that Bcl-6, in addition to its well-established role in facilitating Tfh differentiation, is also important for the generation of Tcmp cells (Pepper et al., 2011). Thus, CD4⁺ T cell differentiation during acute viral infection is a well-synchronized process that results in appropriately balanced Th1, Tfh, and Tcmp cell formation at the peak of infection, and these subsets can then give rise to highly functional memory populations.

In contrast to acute settings, CD4⁺ T cells responding to persistent infection or cancer often acquire dysfunctional features, including a diminished capacity to produce pro-inflammatory cytokines that coincides with the upregulation of co-inhibitory receptors such as PD-1, Tim-3, and CTLA-4 (Fuller et al., 2004; Brooks et al., 2005; Crawford et al., 2014). Additionally, several studies have identified that Th1 formation and function is drastically reduced during chronic viral infection, and this decline in Th1 formation is accompanied by a relative increase in Tfh cell development (Fahey et al., 2011; Yamada et al., 2016). This shift in CD4⁺ T cell differentiation is thought to be important to limit Th1-mediated immunopathology (Penaloza-MacMaster et al., 2015), while at the same time promote Tfh-mediated antibody responses that are required for viral clearance from the periphery (Fahey et al., 2011; Harker et al., 2011; Cook et al., 2015; Greczmiel et al., 2017). Notably, recent work in the field has further demonstrated that in addition to the anticipated Th1 and Tfh populations that form during chronic viral infection, a previously unrecognized memory-like CD4⁺ T cell subset also develops, and this subset bears a transcriptional program similar to that of Tcmp cells from acute infection, as evident by its high expression of Tcf7, Ccr7, Il7r, and Bcl2 (Snell et al., 2021; Andreatta et al., 2022; Xia et al., 2022; Zander et al., 2022). Moreover, memory-like CD4⁺ T cells were found to display an augmented capacity to accumulate during persistent infection, and they also exhibited the propensity to give rise to either Th1 or Tfh cells, indicating that this subset displays some developmental plasticity (Xia et al., 2022; Zander et al., 2022). Thus, CD4⁺ T cells responding to chronic viral infection are also capable of exhibiting memory-like properties, even in the presence of ongoing viral replication. However, despite these recent advances, our understanding of how CD4⁺ T cells adapt under settings of persistent antigen exposure and inflammation remains incompletely understood. Moreover, although recent reports have demonstrated that several transcriptionally distinct CD4⁺ T cell subsets develop during acute viral infection (Ciucci et al., 2019; Khatun et al., 2021), whether analogous or additional heterogeneous populations of CD4⁺ T cells emerge during chronic viral infection, and how these subsets compare to those from acute infection have remained significant knowledge gaps in the field. Additionally, questions remain about how chronic viral infection perturbs the functional and transcriptional landscape of CD4⁺ T cell populations, and whether these alterations in the CD4⁺ T cell compartment are due to global differences in gene expression programs or stem from differences in the distribution pattern of T helper cell subsets that develop under these contexts.

While previous studies have demonstrated that most antigen-specific CD4⁺ T cell clones can give rise to multiple lineages following acute bacterial or viral infection (Tubo et al., 2013), recent evidence indicates that some clones may also display a biased cell fate decision (Khatun et al., 2021). Importantly, cell-intrinsic factors, such as TCR signal strength, TCR affinity for cognate peptide, and TCR chain usage have all recently been implicated in contributing to shaping CD4⁺ T cell fate decision during viral infection (Tubo et al., 2013; Tubo et al., 2016; Cho et al., 2017; Künzli et al., 2020; Khatun et al., 2021). Interestingly, TCR signal strength was found to exert opposite effects on the balance of Th1 to Tfh cells depending on whether the infection was acutely resolving or persistent (Künzli et al., 2020). However, we currently do not fully understand the extent by which TCR repertoire impacts cell fate decisions during chronic viral infection. Additionally, whether naïve T cells with particular TCR chains display the propensity to give rise to multiple independent lineages during chronic viral infection, or whether some clones preferentially acquire a particular differentiation program that may skew the T helper cell response in the setting of chronic infection, remains unclear.

In this study, we employed single-cell RNA sequencing (scRNA-seq) coupled with single-cell TCR sequencing (scTCR-seq) to investigate the transcriptional landscape of CD4⁺ T cells responding to chronic lymphocytic choriomeningitis virus (LCMV) infection. Notably, we uncovered previously unappreciated transcriptional heterogeneity among the virus-specific CD4⁺ T cell pool, including the identification of several transcriptionally distinct states within the Th1, Tfh, and T memory-like cell populations. Moreover, we found that, similar to findings from acute viral infection, the majority of CD4 T cell clones responding to chronic viral infection displayed remarkable developmental plasticity as evidenced by their membership across multiple distinct subsets, although a small fraction of clonotypes did appear to exhibit a preferred lineage choice. Lastly, using an integrated analysis to compare the transcriptional program and subset distribution of CD4⁺ T cells responding to either acute or chronic LCMV infection, we identified that persistent infection is not only associated with altered CD4⁺ T cell subset formation and gene expression profiles, but also changes in core gene expression modules that span multiple distinct T helper cell populations.

In this study, we demonstrate that CD4⁺ T cells responding to chronic LCMV infection are more heterogeneous than previously appreciated, with several transcriptionally distinct subsets being detected within Th1, Tfh, and T memory-like CD4⁺ T cell populations. We further demonstrate that while the progeny of most CD4⁺ T cell clones displayed immense developmental plasticity, suggesting a one cell-multiple fate model, a small fraction of clones appeared to exhibit a biased cell fate-decision, indicating that TCR usage may have an impact on CD4⁺ T cell differentiation. Additionally, we highlight some potential flow cytometric gating strategies that can be used to further interrogate these respective subsets and also provide evidence that the relative distribution of these populations differed substantially between acute and chronic viral infection. Lastly, our integrated analyses revealed that chronic viral infection results in vastly altered gene expression programs in responding CD4⁺ T cells, with evidence for not only lineage-specific gene expression profiles but also core gene expression programs that are upregulated and conserved across multiple distinct populations of T helper cells. Collectively, these data may serve as a useful resource to assess and compare the transcriptional landscape and differentiation trajectory of virus-specific CD4⁺ T cell clones during acute and chronic viral infection.

Several recent studies have identified that CD4⁺ T cells responding to acute viral infection are composed of multiple transcriptionally distinct CD4⁺ T cell subsets, including Th1, Tfh, and Tcmp cells (Ciucci et al., 2019; Khatun et al., 2021). While the differentiation and helper functions of Th1 and Tfh subsets in these acute settings are fairly well established, recent work has also yielded important insight into the transcriptional profile and developmental biology of Tcmp cells. Notably, Tcmp cells display high expression of memory cell markers CCR7, TCF-1, and Bcl-2 and have an intrinsic capacity to survive the contraction phase of acute infection and generate diverse effector cell populations during a secondary response (Ciucci et al., 2019; Marshall et al., 2011; Pepper and Jenkins, 2011). The development of Tcmp cells has been demonstrated to be dependent on both Thpok and Bcl6 (Ciucci et al., 2019; Pepper and Jenkins, 2011). Thus, our understanding of CD4⁺ T cell differentiation during acute infection is becoming increasingly more well defined. By comparison, our knowledge of how CD4⁺ T cells adapt in the face of persistent antigen exposure and inflammation remains incompletely understood. Moreover, whether diverse populations of CD4⁺ T cells also emerge during chronic infection or whether they can display memory-like properties have remained significant knowledge gaps in the field. Notably, recent studies have identified that a memory-like CD4⁺ T cell population develops alongside Th1 and Tfh cell subsets during chronic viral infection (Xia et al., 2022; Zander et al., 2022) and that this memory-like subset bears a strikingly similar transcriptional profile as Tcmp cells from acute LCMV infection (Zander et al., 2022). However, whether additional heterogeneity exists amongst the CD4⁺ T cell pool during chronic viral infection remains unclear. Moreover, an in-depth assessment into how the transcriptional landscape of CD4⁺ T cells differs between acute and chronic viral infection at the single-cell level has not previously been explored. Herein, we demonstrate that CD4 T cells responding to chronic viral infection are more heterogeneous than previously appreciated, with several transcriptionally distinct subsets arising during the early phase of chronic infection. These include two potentially unique Th1 cell subsets (Ly6c-hi and Lag3-hi cells), two Tfh-like subsets (GC Tfh2 and Tfh1), a memory-like population, and a pre-Tfh subset that exhibits both a memory-like signature and also expresses some Tfh-associated genes. Additionally, our scRNA-seq analyses identified a cluster of Slamf7-hi cells that may represent a cytolytic CD4 T cell subset (Cachot et al., 2021; Della-Torre et al., 2018), as well as a unique cluster that displayed increased expression of multiple ISGs, consistent with chronic infection driving a sustained type I IFN response (Teijaro et al., 2013; Wilson et al., 2013). Using spectral flow cytometry, we validate the formation of several of these subsets during chronic viral infection and also highlight a few potential gating strategies and markers that may be useful in interrogating these subsets further. However, future work will be important to elucidate the developmental relationship between these respective populations and to establish whether some of these newly identified subsets represent functionally distinct lineages or merely transitional states along the Th1, Tfh, or memory T cell developmental pathways.

Notably, while our TCR clonal trajectory analyses indicate that the majority of GP66-specific CD4⁺ T cell clones responding to chronic LCMV infection appear to be multipotent, a small fraction of T cell clones appeared to display a biased cell fate decision and favored a particular lineage. Overall, this finding is consistent with other recent studies from resolving infections (Tubo et al., 2013; Khatun et al., 2021) and suggests that the vast majority of naïve CD4⁺ T cells are capable of giving rise to multiple distinct lineages independent of TCR sequence, although TCR structure may still influence the differentiation trajectory of some select clones. As certain CDR3 motifs from the TCR alpha chain have previously been implicated as being a useful predictor of T cell fate during acute LCMV infection (Khatun et al., 2021), a deeper investigation into whether particular TCR alpha or beta chain sequences impact CD4⁺ T cell fate determination during chronic viral infection will be of future interest.

Consistent with chronic viral infection resulting in diminished Th1 differentiation and function (Fuller et al., 2004; Brooks et al., 2005; Fahey et al., 2011; Yamada et al., 2016), our integrated scRNA-seq analysis demonstrated that the proportions of both Ly6c-hi and Lag3-hi Th1 cells were dramatically reduced during chronic vs acute LCMV infection. Interestingly, module score analyses identified that loss of the Th1 gene expression program was not restricted to only the Th1 cell subsets but was conserved across multiple distinct subsets, indicating that loss of Th1-like pro-inflammatory activity may be a generalizable feature of T helper cell responses during chronic viral infection. This loss in Th1 cell subset formation coincided with increased frequencies of multiple subsets, including Slamf6-hi, pre-Tfh, ISG, and Slamf7-hi clusters, whereas the Tfh clusters remained similar between infections. Intriguingly, we further observed an enriched quiescence or memory-associated transcriptional profile across most CD4⁺ T cell subsets responding to chronic viral infection, despite viral load remaining high at this time point. Although the reasons for this remain unclear, it is possible that CD4⁺ T cells responding to chronic infection need to acquire a more quiescent or memory-like phenotype in order to mitigate Th1-mediated pathology (Penaloza-MacMaster et al., 2015). This enhanced acquisition of a memory-like phenotype is supported by the relative increased formation of Slamf6⁺ and pre-Tfh populations during persistent infection, which exhibit high expression of Tcf7, Il7r, Ccr7, and Bcl2. However, it is important to note that despite this enriched memory-associated program, most CD4⁺ T cell subsets responding to chronic LCMV infection also displayed increased TCR signaling and T cell dysfunction or exhaustion module scores, indicating that these cell subsets are not likely to be in a truly quiescent state nor do they fit the description of canonical memory populations. Consistent with this, we identified that Id3, a TF known to be critical for memory T cell formation following acute infection (Yang et al., 2011), was substantially diminished in most CD4⁺ T cell subsets from chronic infection compared to their acute infection counterparts. Moreover, in line with increased TCR signaling during chronic viral infection, we observed a conserved increase in the expression of AP-1 family members Jund and Junb, as well as in Cd69, Nr4a2, and Tox gene expression across all CD4⁺ T cell subsets during LCMV Cl13 infection. Similarly, exhaustion markers Lag3, Tigit, and Ctla4 were uniformly increased in all CD4⁺ T cell subsets responding to chronic viral infection. Intriguingly, while these data highlight a broad upregulation of dysfunctional programs spanning multiple distinct populations of CD4⁺ T cells during chronic viral infection, it is worthy to mention that Th1 cell subsets from chronic infection displayed markedly higher dysfunction and exhaustion module scores compared to all other subsets. This data, coupled with previous observations demonstrating a rapid loss of Th1 responses during chronic viral infection (which may possibly reflect clonal deletion) and that Th1 cells rapidly lose their capacity to produce pro-inflammatory cytokines as the infection progresses (Fuller et al., 2004; Brooks et al., 2005; Fahey et al., 2011; Osokine et al., 2014), indicate that Th1 cells do exhibit some cardinal features of the process of CD8⁺ T cell exhaustion. At the same time, however, Th1 cells (from both acute and chronic infection) displayed increased expression of effector CD8⁺ T cell genes (Figure 4—figure supplement 1E), and Th1 cells further exhibited the highest capacity to produce IFN-γ and TNF-α upon ex vivo peptide stimulation (Zander et al., 2022). Thus, while Th1 cells responding to chronic viral infection share some overlap in their transcriptional program with both effector and exhausted CD8 T cells, whether these cells (or other populations of CD4⁺ T cells) are truly ‘exhausted’ remains to be investigated further.

Collectively, our results highlight perturbed gene expression programs in CD4⁺ T cells during chronic viral infection, with apparent alterations in both lineage-specific T helper cell programs and also core expression modules that are differentially regulated across all CD4⁺ T cell subsets between acute and chronic viral infection. A future investigation into the transcriptional circuits underlying the altered CD4⁺ T cell differentiation that occurs during chronic infection may have important implications for strategies aimed at improving T cell-based immunotherapies during chronic infection.

The scRNA-seq and scTCR-seq data have been deposited in the GEO database (accession no GSE201730), and are available to the public.

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Author details

1. Ryan Zander

   Blood Research Institute, Versiti Wisconsin, Milwaukee, United States

   Contribution

   Conceptualization, Data curation, Software, Formal analysis, Validation, Investigation, Visualization, Writing - original draft, Writing - review and editing

   Contributed equally with

   Achia Khatun

   For correspondence

   Ryan-Zander@uiowa.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-7431-8535

2. Achia Khatun

   1. Blood Research Institute, Versiti Wisconsin, Milwaukee, United States
   2. Department of Microbiology and Immunology, Medical College of Wisconsin, Milwaukee, United States

   Contribution

   Conceptualization, Data curation, Software, Formal analysis, Visualization, Methodology, Writing - review and editing

   Contributed equally with

   Ryan Zander

   Competing interests

   No competing interests declared

3. Moujtaba Y Kasmani

   1. Blood Research Institute, Versiti Wisconsin, Milwaukee, United States
   2. Department of Microbiology and Immunology, Medical College of Wisconsin, Milwaukee, United States

   Contribution

   Software, Formal analysis, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-5753-5335

4. Yao Chen

   1. Blood Research Institute, Versiti Wisconsin, Milwaukee, United States
   2. Department of Microbiology and Immunology, Medical College of Wisconsin, Milwaukee, United States

   Contribution

   Data curation, Investigation

   Competing interests

   No competing interests declared

5. Weiguo Cui

   1. Blood Research Institute, Versiti Wisconsin, Milwaukee, United States
   2. Department of Microbiology and Immunology, Medical College of Wisconsin, Milwaukee, United States

   Contribution

   Conceptualization, Resources, Supervision, Funding acquisition, Writing - review and editing

   For correspondence

   weiguo.cui@bcw.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-1562-9218

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