The multifaceted role of CD4⁺ T cells in the regulation of CD8⁺ T cell memory maturation

Abstract

Following infection, T cells differentiate into a heterogeneous population of effector T cells that can mediate pathogen clearance. A subset of these effector T cells possesses the ability to survive long-term and mature into memory T cells capable of providing long-term immunity. Understanding the signals that regulate the development of memory T cells is crucial to efforts to design vaccines capable of eliciting T cell-based immunity. CD4⁺ T cells are essential for the formation of protective memory CD8⁺ T cells following infection or immunization. However, until recently, the mechanisms by which CD4⁺ T cells act to support memory CD8⁺ T cell development following infection were unclear. Here, we discuss recent studies that provide insight into the multifaceted role of CD4⁺ T cells in the regulation of memory CD8⁺ T cell differentiation.

Memory CD8⁺ T cells are a principal component of immunity against intracellular pathogens such as viruses. They are distinguished by their capacity to survive long-term and undergo rapid and robust proliferation and acquisition of effector function upon antigen re-exposure ¹. Memory CD8⁺ T cells can vary in their phenotype, localization, and function allowing them to protect the host against a broad array of potential insults. CD8⁺ T cells can mediate the killing of infected cells through multiple mechanisms including expression of granzymes and perforin, as well as the secretion of cytokines such as interferon γ (IFN γ) and tumor necrosis factor α (TNFα). Unlike neutralizing antibodies that largely recognize proteins expressed on pathogen surfaces, CD8⁺ T cells respond to peptide sequences presented on the surface of antigen presenting cells. This allows them to recognize internal proteins of the pathogen that are less subject to evolutionary pressure and thus tend to be more highly conserved among different pathogen variants. Therefore, CD8⁺ T cells possess the potential to provide broadly reactive protection against viruses such as influenza or HIV that rapidly mutate their surface proteins ².

Despite the utility of memory CD8⁺ T cells in protection against pathogens that rapidly mutate to elude neutralizing antibodies, the development of T cell based vaccines has proven problematic ³. This failure is largely due to an incomplete understanding of the signals and cell types that operate at different stages of the immune response to influence the quantity and quality of developing memory CD8⁺ T cells. Additionally, knowledge of how CD8⁺ T cells are able to enter and maintain residence in mucosal tissues is of critical importance as many of the infections to which effective vaccines have not been developed initially infect mucosal sites such as the lungs, reproductive tract, and skin. In order to generate a protective memory CD8⁺ T cell response it is important to understand the signals needed to position them at the initial site of infection, as well as to induce a circulating memory pool capable of preventing outgrowth of the pathogen.

The T cell response to acute infection can typically be divided into three phases —priming and expansion, resolution and contraction, and memory. During the first phase, naïve CD8⁺ T cells divide and differentiate into effector cells that acquire the ability to produce IFNγ, TNFα and cytotoxic proteins such as granzymes and perforin ⁴. Following viral clearance, contraction and resolution ensues in which the majority of the effector CD8⁺ T cells die with ~5–10% surviving. These enter the third stage — the ‘memory’ phase — and are maintained long-term by signals such as IL-7 and IL-15 ⁵. While considerable heterogeneity exists among long-lived CD8⁺ T cells, they are typically divided into resident (T_RM), effector (T_EM), and central (T_CM) memory cells. Differences in their localization, recall ability, and effector functions allow them to provide overlapping layers of protection against potential reinfection ⁶. T_RM cells are located in mucosal sites such as the lungs, skin, and reproductive tract and are unique from other memory populations in that they do not reenter the circulation. They are characterized by high expression of CD103 and CD69 and act as sentinels to provide immediate protection upon local secondary infection through direct effector functions and the recruitment and reactivation of immune cells ^7–12. The developmental pathway and function of T_RM cells are reviewed elsewhere in this issue. [Cite Mackay, Mueller review] T_EM cells can migrate between tissues and secondary lymphoid organs and provide immune surveillance. T_EM cells have constitutive expression of some effector functions and lack expression of L-selectin (CD62L) and CCR7 ⁵. T_CM cells reside in secondary lymphoid organs and are typically characterized by expression of these molecules needed for entry therein. They possess the greatest proliferative potential among the memory T cell subsets and can rapidly expand and differentiate following rechallenge.

Considerable work over the past decade has sought to elucidate the signaling and transcriptional networks governing CD8⁺ T cell fate decisions, with these studies reviewed recently ⁵. This Review will focus on the essential role of CD4⁺ T cells in the development of memory CD8⁺ T cells ^13–19. They regulate CD8⁺ T cell memory development through multiple mechanisms, depending on the type of infection or immunization ²⁰. Here we will discuss recent studies that provide mechanistic insight into the process underlying the necessity of CD4⁺ T cells in memory CD8⁺ T cell maturation and function in different settings.

CD4⁺ T cell help following immunization

Following immunization, CD4⁺ T cells are necessary for the induction of a robust primary CD8⁺ T cell response (Fig. 1a)^21–28. Additionally, CD8⁺ T cells formed in the absence of CD4⁺ T cell help are impaired in their long-term maintenance and display poor proliferative ability upon secondary challenge ^13,16,29,30. Considerable work has sought to understand the processes underlying these defects.

Figure 1. CD4⁺ T cell help to CD8⁺ T cells during immunization.

a. Following immunization, naïve CD4⁺ T cells interact with and license cognate dendritic cells (DCs) through a CD40-dependent process. Licensed DCs express higher levels of MHC and costimulatory molecules and can recruit naïve CD8⁺ T to their cognate DC through secretion of the chemokines CCL3, 4, and 5. Licensed DCs also secrete IL-12 and IL-15, which increase expression of IL-2R (CD25) on CD8⁺ T cells and promote cell survival, respectively. Enhanced expression of CD25 facilitates the responsive of CD8⁺ T cells to IL-2 and promotes CD8⁺ T cell survival and their ability to proliferate upon secondary antigen encounter. Both CD4⁺ and CD8⁺ T cells act as sources of the IL-2, with CD4⁺ and CD8⁺ T cells in some settings also directly interacting through CD40:CD40L. b. Regulatory T (T_REG) cells can modulate the CD8⁺ T cell response following immunization by suppressing the maturation state of DCs, thereby limiting their ability to stimulate CD8⁺ T cells. By limiting chemokine secretion by DCs, T_REG cells can destabilize CD8⁺ T cell: DC interactions and accordingly promote the induction of high affinity effector and memory CD8⁺ T cells. T_REG cells also limit the sensing of IL-2 by CD8⁺ T cells by competing for available IL-2 and limiting the expression of CD25 by CD8⁺ T cells through control of DC secretion of IL-12.

A primary mechanism by which CD4⁺ T cells support the CD8⁺ T cell response following immunization is through the “licensing” of dendritic cells (DCs). CD4⁺ T cell help is required for DCs to increase their antigen-presentation and co-stimulatory capacity to levels sufficient to induce a robust effector CD8⁺ T cell response, with both the CD4⁺ and CD8⁺ T cells recognizing antigen presented by the same DC ²². This licensing is mediated through CD40: CD40L interactions between the DC and cognate CD4⁺ T cell that allow for the functional maturation of DCs ^21,24. The licensed DCs can subsequently interact with CD8⁺ T cells and induce a strong primary response ²³. In some settings, CD40: CD40L interactions can also occur directly between CD8⁺ T cells and CD4⁺ T cells ¹⁶.

CD4⁺ T cells also can support the primary CD8⁺ T cell response through facilitation of their interaction with DCs. Prior to antigen encounter, CD8⁺ T cells in the draining lymph node (dLN) upregulate the chemokine receptor CCR5. These cells can then migrate towards the site of antigen-specific DC: CD4⁺ T cell interactions due to the production of chemokines such as CCL3 and CCL4 by the licensed DCs ³¹. This guidance of naïve CD8⁺ T cells provides an explanation for how they can rapidly find their cognate antigen-presenting cell following immunization, despite the low frequency of both of these populations. CD4⁺ T cells licensing of DCs also can facilitate entry of naïve CD8⁺ T cells into the dLN through expansion of the arteriole feeding the dLN, as well as enlargement of the dLN itself, although the precise mechanism by which CD4⁺ T cells regulate this process remains to be determined ³².

Further insight was provided by two recent studies evaluating the spatiotemporal interactions of DCs and cognate T cells ^33,34. One found that CD4⁺ T cell activation precedes that of CD8⁺ T cells and occurs through interactions with migratory DCs ³⁴. Activated CD4⁺ T cells upregulate CD40L and subsequently license cross-presenting XCR1⁺ DCs that can engage and activate antigen-specific CD8⁺ T cells ³⁴. A separate study found that both CD4⁺ and CD8⁺ T cells are activated by distinct DC subsets (non-infected and infected DCs, respectively) at separate anatomical locations ³³. These activated T cells then interact with cognate XCR1⁺ DCs in clusters, which act as the platform by which CD4⁺ T cell help is provided to the CD8⁺ T cells ³³. Going forward it will be important to clarify differences between these and previous works regarding whether CD8⁺ T cells are activated by distinct DCs prior to engagement with XCR1⁺ DCs, and if initial engagement of CD8⁺ T cells with XCR1⁺ DCs precedes licensing of these DCs by CD40L-expressing CD4⁺ T cells or whether theses interactions occur simultaneously.

In addition to their role in the primary response, CD4⁺ T cells are necessary for robust secondary expansion of memory CD8⁺ T cells ^{13,16,27,29,30,35,36}. One suggested mechanism for this requirement is through regulation of tumor-necrosis factor-related apoptosis-induced ligand (TRAIL) ^37,38. “Helpless” CD8⁺ T cells rapidly induce TRAIL expression upon restimulation and undergo activation-induced cell death, although TRAIL deficiency does not seem to rescue secondary responsiveness of “helpless” cells in all settings ³⁰. The ability of CD8⁺ T cells to respond upon secondary challenge appears to be imprinted during their initial priming phase ^13,14, but interestingly, the provision of IL-15 during vaccination is sufficient to rescue the secondary cytotoxic T lymphocyte response in the absence of CD4⁺ T cells, likely due to IL-15 mediated suppression of the proapoptotic molecule Bax and induction of the anti-apoptotic molecule Bcl-xl ^28,37. IL-15 production by antigen presenting DCs is necessary even in the presence of CD4⁺ T cells to allow for the secondary responsiveness of CD8⁺ T cells. These studies suggest a model in which DCs gain the ability to secrete IL-15 following CD4⁺ T cell-mediated licensing.

IL-2 is also important during priming to imprint the secondary responsiveness of CD8⁺ T cells ^39,40. CD4⁺ T cells have been suggested as a critical source of IL-2, although other studies have found that CD8⁺ T cells produce the IL-2 that is necessary for their own development. ^{26,27,41–43}, with licensed DCs enabling such autocrine secretion. Autocrine IL-2 allows for the induction of NGFI-A binding protein 2 (Nab2) in CD8⁺ T cells, which mediates suppression of TRAIL expression and allows for secondary expansion of these cells ⁴³. Licensed DCs also produce IL-12p70 that acts directly on antigen-specific CD8⁺ T cells to increase their expression of CD25, rendering these cells more responsive to IL-2 ^41,44.

Regulatory CD4⁺ T (T_REG) cells can act to modulate IL-2 exposure of effector CD8⁺ T cells during the priming phase and are necessary for the generation of functional memory CD8⁺ T cells following immunization (Fig. 1b) ^45–48. For instance, competitive consumption of IL-2 by T_REG cells can limit the availability of IL-2 to early activated CD8⁺ T cells, which suppressed CD8⁺ T cell expansion, but enhanced the functional responsiveness of memory CD8⁺ T cells following secondary challenge ⁴⁵. While autocrine IL-2 expression is necessary for the generation of functional memory CD8⁺ T cells, prolonged exposure to IL-2 early after immunization promotes their terminal differentiation ^49,50. T_REG cells also modulate the maturation state of DCs through leukocyte-associated antigen 1 (LFA-1) and cytotoxic T lymophocyte-associated antigen 4 (CTLA4)-mediated regulation of CD80/86 expression, which may influence the ability of DCs to stimulate CD8⁺ T cells during priming ^51,52. T_REG cells in addition regulate the stability of interactions between DCs and CD8⁺ T cells through suppression of chemokine production by DCs, and are critical for the development of high-avidity effector and memory CD8⁺ T cells ^53–57. Additional mechanisms by which T_REG cells can regulate memory formation following infection are discussed in the following section.

CD4⁺ T cell help following systemic infection

Despite it being appreciated that CD4⁺ T cells are necessary for CD8⁺ T cell memory maturation following acute pathogen infection, the mechanism(s) by which this help is mediated, and which CD4 T cells are involved is still being understood ^{14,19,58–61}. One study showed that CD4⁺ T cells are required after the priming and expansion phase (i.e., during the contraction phase) and that this help can be antigen non-specific⁶⁰. CD4⁺ T cell help following LCMV infection is independent of CD40-mediated licensing of DCs or CD8⁺ T cells, with passive transfer of LCMV-specific antibodies sufficient to restore a protective memory CD8⁺ T cell response in the absence of CD40-CD40L interactions through the facilitation of viral clearance ^62,63. “Helpless” CD8⁺ T cells displayed increased expression of T-bet, a transcription factor involved in fine-tuning CD8⁺ effector and memory T cell differentiation, with reduction of T-bet expression restoring the functionality of these cells ^64,65. Additionally, CD4⁺ T cell help influences the epigenetic state of memory CD8⁺ T cells with “helpless” CD8⁺ T cells displaying reduced acetylation at the IFNγ locus and increased methylation at the IL-2 promoter resulting in reduced responsiveness upon restimulation ⁶⁶. Interestingly, treatment with a histone deacetylase inhibitor is sufficient to restore the responsiveness of “helpless” CD8⁺ T cells ⁶⁷.

Effector CD4⁺ T cells induced following pathogen infection display considerable phenotypic and functional heterogeneity that can influence their ability to provide help (Box 1). Insight into the specific mechanism(s) underlying CD4⁺ T cell help was provided by the finding that IL-10, which is normally thought of as an immunosuppressive cytokine, is important for memory CD8⁺ T cell development. Although normal numbers of memory CD8 T cells form in the absence of IL-10, the maturation of these cells was impaired, particularly the development of T_CM cells ^68–70. The cellular source of IL-10 needed to promote the phenotypic and functional qualities of the memory CD8⁺ T cells was unclear from these studies. Multiple cell types including myeloid cells, DCs, and T cells can secrete IL-10 following LCMV infection ^71,72. Interestingly, and somewhat counterintuitively, we found that T_REG cells act as the relevant source of IL-10 required for memory CD8⁺ T cell maturation following LCMV infection. In line with the kinetics of CD4 T cell help identified earlier ⁶⁰, T_reg cell-derived IL-10 primarily acted during the resolution phase to promote memory maturation through suppression of proinflammatory cytokine production by DCs (Fig. 2a, b) ⁴⁷. Conceptually similar, a separate study found that T_REG cells influence CD8⁺ T cell memory maturation through CTLA-4 mediated suppression of effector and proliferation programs in effector CD8⁺ T cells ⁴⁸. Thus, T_REG cells through multiple mechanisms can promote functional memory CD8⁺ T cell development.

Box 1. Effector CD4⁺ T cell differentiation.

Following antigen recognition, naïve CD4⁺ T cell can differentiate into distinct effector T helper cell lineages capable of differentially regulating the immune response. The inflammatory environment in which recently activated CD4⁺ T cells develop in drives their polarization and allows T cells to match their effector function to the pathogen encountered. Viral infection induces high levels of pro-inflammatory cytokines such as IL-12, IFNγ, and type I IFNs that promote the induction of Th1 cells. Conversely, IL-4 produced following helminth infection or allergic responses drives Th2 cell differentiation, while fungal infection induces TGFβ and IL-6 secretion that promote Th17 cell differentiation ¹⁰⁸. T follicular helper (Tfh) cell differentiation is regulated by STAT3 signaling cytokines such as IL-6 and IL-21 and is dependent on iterative interactions with cognate DCs and B cells ^111–115. CD4⁺ T cell subsets can be identified through 1) expression of cell surface markers such as Ly6C, PSGL1, CXCR5, and PD1; 2) expression of canonical transcriptions factors T-bet, Gata3, Rorγt, and Bcl6; and 3) secretion of cytokines such as IFNγ, TNFα, IL-4, IL-5, IL-13, IL-17, and IL-21. Considerable plasticity exists among effector CD4⁺ T cell subsets in vivo which helps facilitate the generation of a diverse immune response capable of mediating pathogen clearance, promoting the humoral response, and restraining immunopathology ¹⁰⁸.

Figure 2. T_REG cells promote memory CD8⁺ T cell maturation during viral infection.

a. Following acute viral infection, T_REG cells expansion is initially suppressed due to the presence of high levels of type I IFNs. As type I IFNs wane, T_REG cell numbers increase with these cells adopting a more effector like phenotype marked by increased expression of IL-10 and CTLA-4. During the resolution phase of infection, T_REG cell-derived IL-10 acts to suppress the maturation state of DCs and limit their production of proinflammatory cytokines. T_REG cell expression of CTLA-4 acts in a similar manner through modulation of the CD28-CD80/86 signaling axis. Low levels of proinflammatory signals allow for the continued maturation of effector CD8⁺ T cells into functional memory CD8⁺ T cells. b. In the absence of T_REG cell-derived IL-10 and/or CTLA-4, DCs adopt a more mature phenotype and secrete higher levels of proinflammatory cytokines. The enhanced levels of proinflammatory cytokines are sensed by effector CD8⁺ T cells and drive these cells to adopt a more terminally differentiated phenotype, limiting their ability to proliferate and mediate protective immunity upon reencounter with the pathogen.

In addition to this function, T_REG cells can act during the priming phase to suppress the magnitude of the CD8⁺ T cell response thereby restricting the number of cells that survive into the memory phase ^73–75. However, type I IFNs generated early after infection can directly inhibit T_REG cell activation and proliferation facilitating the generation of an optimal effector T cell response ⁷⁶. As type I IFNs wane, T_reg cell expansion occurs with the newly populating T_REG cells displaying an activated phenotype with more robust IL-10 expression relative to T_REG cells present at steady state (Box 2) ^47,77. IL-10-competent T_REG cells are primarily located in the white pulp of the spleen, near to DCs as well as memory precursor CD8⁺ T cells, positioning them optimally to suppress the activation state of DCs thus insulating CD8⁺ T cells from excess bystander inflammation and preserving their memory precursor-state ^{47,65,78–80}.

Box 2. Regulatory CD4⁺ T cell differentiation.

Regulatory CD4⁺ T (T_REG) cells express the transcription factor FoxP3 and are critical in the prevention of excess immunopathology or autoimmunity through multiple mechanisms¹¹⁶. T_REG cells possess considerable functional and phenotypic heterogeneity. Central or naïve T_REG cells express CD62L and are predominantly found in circulation and secondary lymphoid tissues. Following exposure to antigen and/or IL-2, T_REG cells adopt a more effector-like state and downregulate CD62L and progressively upregulate CD69 and KLRG1. Acquisition of a more effector like phenotype is accompanied by enhanced expression of suppressive molecules such as IL-10 and CTLA4 with KLRG1⁺ T_REG cells representing a terminally differentiated population ^47,77.

Effector T_REG cells appear to co-opt the transcriptional network of effector CD4⁺ T cells in order to match their suppressive function to their present environment. T-bet⁺ T_REG cells express the chemokine receptors CXCR3 and CCR4 and regulate skin and lung inflammation. STAT3-expressing T_REG cells upregulate CCR6 and regulate gut homeostasis, while Bcl6-expressing T_REG cells display high levels of CXCR5 and regulate the germinal center response. T_REG cells also accumulate in adipose tissue, with these cells distinguished by expression of PPARγ and are important in regulating adipose metabolism ¹¹⁷. T_REG cells have recently been identified in the muscle where they are promote muscle repair through secretion of the growth factor amphiregulin ¹¹⁸.

CD4⁺ T cell help following mucosal infection

CD8⁺ T_RM cells are critical in guarding mucosal surfaces against pathogen challenge ⁸¹. While CD4⁺ T cells are not needed to initiate a primary virus-specific response following mucosal infection by pathogens such as influenza, they are necessary for the optimal development of a memory CD8⁺ T cell population capable of mediating protective immunity ^18,75. However, until recently the role of CD4⁺ T cells in mediating tissue-specific CD8⁺ T cell memory was unclear. Insight into this question was provided by work showing that CD4⁺ T cell help is needed for entry into the female reproductive tract following mucosal viral infection ⁸². CD4⁺ T cells indirectly mediated the entry of CD8⁺ T cells into the tissue through IFNγ-dependent induction of chemokines by locally infected cells (Fig. 3a) ⁸². While CD4⁺ T cells do not appear necessary for CD8⁺ T cell entry into the skin following viral infection, CD4⁺ skin T_RM cells can facilitate recruitment of circulating CD8⁺ T cells into the skin in a CXCR3-dependent manner following challenge with Leishmania major ^83,84.

Figure 3. CD4⁺ T cell help to CD8⁺ T cell during mucosal infection.

a. Following influenza virus infection, CD4⁺ T cells rapidly migrate from the draining lymph node (dLN) to the lung airways where they mediate the release of chemokines from epithelial cells via secretion of IFNγ. As CD8⁺ T cells move from the dLN to the lung parenchyma they upregulate CD69, likely due to exposure to inflammatory cytokines and T cell receptor signaling. These cells can then migrate towards the chemokine gradient surrounding the airway where they encounter TGFβ, which subsequently induces the expression of CD103 and suppression of the transcription factor T-bet, thereby promoting the establishment of a lung T_RM cell population. b. Following herpes simplex virus infection, CD4⁺ T cells migrate from the draining lymph node (dLN) to the female reproductive tract (FRT) where they mediate the release of chemokines from the infected tissue via secretion of IFNγ. CD4⁺ T cell-derived IFNγ is necessary for CD8⁺ T cells to migrate into the virally infected FRT. c. Following West Nile virus infection, CD8⁺ T cells primed in the dLN migrate to the central nervous system (CNS) parenchyma. T_REG cells also traffic to the CNS where they modulate the levels of TGFβ. T_REG cell-dependent control of TGFβ may modulate the expression of CD103 expression on CD8⁺ T cells and accordingly influence their ability to reside long-term in the brain.

We extended these studies to find that CD4⁺ T cells are important for the development of airway-homing CD8⁺ T_RM cells following influenza virus infection ⁸⁵. While CD8⁺ T cells entered the lung in the absence of CD4⁺ T cell help, they failed to properly localize to the lung airways and had a reduced ability to recruit CD8⁺ T cells from circulation and mediate protective immunity upon heterosubtypic challenge. “Helpless” CD8⁺ T cells also displayed enhanced expression of T-bet, which rendered these cells less responsive to TGFb-mediated induction of CD103, an integrin essential for T_RM cell maintenance in the tissue ⁸⁵. Together, these studies provided a model in which IFNγ-producing CD4⁺ T cells directed effector CD8⁺ T cell migration into particular areas of certain mucosal tissues that then facilitated their exposure to signals, such as TGFβ, necessary for their continued maturation into CD103⁺ T_RM cells (Fig. 3b).

T_REG cells also may play a role in the formation of CD8⁺ T_RM cells. In the absence of T_REG cells, reduced numbers of CD8⁺ T_RM cells were retained in the central nervous system (CNS) following West Nile virus infection. The reduction in T_RM cell numbers is associated with decreased amounts of TGFβ in the CNS suggesting that T_reg-dependent modulation of TGFβ levels may be important in driving CD8⁺ T_RM cell formation and retention in mucosal tissues (Fig. 3c)⁸⁶. Together these findings highlight a new, perhaps ironic, role for T_REG cells in providing “helper” as opposed to suppressive functions for the generation of long-term T cell immunity.

CD4⁺ T cell help following chronic infection

CD4⁺ T cells play critical roles in the long-term maintenance of CD8⁺ T cell responses in multiple models of chronic viral infection ^87–91. A central component of CD4⁺ T cell help in this setting is the secretion of IL-21 ^92,93. IL-21 sensing by CD8⁺ T cells is necessary for the avoidance of clonal deletion and maintenance of effector activity even in the presence of CD4⁺ T cells ⁹⁴. IL-21-production by CD4⁺ T cells in individuals infected with the human immunodeficiency virus type 1 (HIV-1) correlates with CD8⁺ T cell functionality and viral control, suggesting that this pathway might be an important therapeutic target ^95–97.

Persistence of high antigen levels is a key driver of CD8⁺ T cell functional exhaustion during chronic viral infection ⁹⁸. Therefore, an additional indirect role of CD4⁺ T cell-derived IL-21 in bolstering the CD8⁺ T cell response during chronic infection may be through promoting viral control. Multiple effector CD4⁺ T cell subsets are capable of secreting IL-21 following viral infection including follicular helper T (Tfh) and Th1 cells ⁹⁹. Viral persistence promotes the differentiation of Tfh cells, which are necessary for the maintenance of the germinal center (GC) response and the continued production of virus-specific antibodies in the face of prolonged high levels of virus replication and immunosuppression ^100,101. Tfh cell-derived IL-21 is also critical for the GC response with a deficiency in IL-21 resulting in impaired maintenance of the GC along with reduced affinity maturation and isotype class switching ^102–104. Together, these studies suggest that Tfh-cell derived IL-21 is needed to promote the virus-specific humoral response thus allowing for the continued control of viremia and the prevention of terminal exhaustion.

IL-2 treatment also can enhance CD8⁺ T cell responses during chronic infection and allows for enhanced control of viral burden ^40,105. The beneficial effect of such treatment is limited in the absence of CD4⁺ T cells, which can serve as a source of IL-2 during chronic infection ¹⁰⁶. IL-2 therapy also results in an increase in the number of T_REG cells ¹⁰⁶. T_REG cells adopt an activated phenotype during chronic viral infection and have enhanced expression of molecules related to their suppressive activity including CTLA4, CD39, and IL-10 ^71,107. Depletion of T_REG cells during chronic infection resulted in a marked expansion of functional CD8⁺ T cells through a process dependent on CD4⁺ T cells and the expression of costimulatory molecules on DCs ¹⁰⁷. T_REG depletion alone was not sufficient to reduce viral burden, but did lead to significant reduction in viral titers when combined with PD-L1 blockade ¹⁰⁷. Therefore, in contrast to acute infection, suppression of the maturation state of DCs by T_REG cells during chronic infection serves to dampen long-term effector CD8+ T cell responses.

Concluding remarks and perspective

A temporal model for the function of CD4⁺ T cells in regulating CD8⁺ T cell maturation into distinct subsets capable of mediating protective immunity following viral infection has begun to emerge (Fig. 4).

Figure 4. Temporal model of CD4⁺ T cell help during viral infection.

a. Following infection, antigen-specific T cells rapidly proliferate during priming and differentiate into cytotoxic T lymphocytes (CTLs) that mediate viral clearance. Most of these cells die over the next several weeks during the resolution phase of the response. Only a small percentage of effector T cells (5–10%) survive and further develop into functional mature memory CD8⁺ T cells. b. CD4⁺ T cells play distinct roles during these phases to regulate the development of CD8⁺ T cell memory. During the priming phase, CD4⁺ T cells license the entry of CD8⁺ T cell into mucosal tissues and promote viral clearance through the induction of a virus-specific effector CD4⁺ T cell and humoral response. Later in the resolution phase, T_REG cell-derived IL-10 facilitates the maturation of a mature memory CD8⁺ T cell population and can promote functional quiescence of memory cell through expression of the inhibitory receptor CTLA-4. T_REG cells may also modulate TGFβ levels in mucosal sites to promote CD103 expression and accordingly regulate T_RM cell development. During the memory phase, the CD4⁺ T cell-dependent immune response allows for continued suppression of viral outgrowth.

During the priming phase CD4⁺ T cells, through IFNγ-mediated chemokine induction, license CD8⁺ T cell entry into mucosal tissues and facilitate their migration into a tissue microenvironment where they can sense signals necessary for their long-term residence ^82,85. Effector CD4⁺ T cells also help facilitate viral clearance through the induction of an anti-viral humoral response, as well as directly through the secretion of effector molecules and cytokines ¹⁰⁸. Rapid viral clearance is important in restricting the degree of exposure of CD8⁺ T cells to antigen and inflammation thus preventing functional exhaustion. High levels of type I IFN present during this stage of infection also restrict T_REG cell expansion and allow for a robust effector T cell response ⁷⁶.

During the resolution phase, as type I IFN levels wane, there is an expansion of activated T_REG cells. IL-10 secretion by activated T_REG cells can suppress the maturation state of DCs and limit their secretion of pro-inflammatory cytokines thus allowing for the preservation of less differentiated effector CD8⁺ T cells ⁴⁷. T_REG cell expression of CTLA-4 acts in a similar manner by limiting CD80/86 stimulation on DCs by CD28 ⁴⁸. Effector CD8⁺ T cells are then able to continue to mature and develop into memory CD8⁺ T cells capable of rapidly responding upon pathogen reencounter. T_REG cells also act during the resolution phase to modulate TGFβ levels within mucosal tissues thereby bolstering the induction of CD8⁺ T_RM cells ⁸⁶. Following the resolution phase, CD4⁺ T cells may contribute to the maintenance of memory CD8⁺ T cells through suppression of the outgrowth of viral reservoirs that could drive terminal exhaustion of CD8⁺ T cells.

This Review has focused on recent advances providing insight into the role of CD4⁺ T cell help in promoting the maturation of memory CD8⁺ T cell following immunization and infection. As our understanding of the mechanisms underlying CD4⁺ T cell help grows it will be important to harness these findings therapeutically to bolster vaccine efficacy. Considering the difficulties surrounding previous attempts at developing effective T cell based vaccines it may be necessary to modify existing vaccination approaches to more closely mirror the natural response to infection. This may be particularly relevant for vaccines to mucosal viruses such as influenza and HIV to which it will likely be necessary to induce T_CM, T_EM, and T_RM populations in order to optimally protect the host. Work is underway to develop vaccines capable of driving CD8⁺ T cells into mucosal sites and exposing them to the signals necessary for their continued maintenance in the tissue, similar to the function of CD4⁺ T cells during mucosal infection ^9,109. Therapeutic interventions designed to suppress inflammatory levels in individuals following vaccination through expansion of T_REG cells or provision of IL-10 may also be a novel approach to bolstering systemic T cell immunity. Individuals suffering from chronic infection have impaired development of protective immunity following vaccination, potentially due to high levels of bystander inflammation, and would be an attractive target for this type of intervention ¹¹⁰. Continued study of the mechanisms underlying CD4⁺ T cell help should provide clearer understanding into how we can harness the utility of CD4⁺ T cells to improve vaccine efficacy.