Th1- and Th2-associated IgG subtypes in mice, humans, and cattle.
Helper CD4+ T cells are essential in shaping effective antibody response and cytotoxic T cell response against pathogen invasion. There are two subtypes of pathogen-specific helper T cells in mice and humans; type 1 (Th1) and type 2 (Th2), with Th1 producing interferon-gamma (IFNγ) and Th2 producing interleukin-4 (IL-4). While effective Th1 controls intracellular pathogens like viruses, efficient Th2 controls extracellular pathogens like most parasites. However, the most predominant CD4+ T cell subtype in cattle is Th0, which produces both IFNγ and IL-4, and only exists in small amounts in mice and humans. Moreover, in many bovine infections, both IFNγ and IL-4 were detected in the blood and both antigen-specific IgG2 (Th1 associated bovine antibody) and antigen-specific IgG1 (Th2 associated bovine antibody) were upregulated in the serum, suggesting bovine CD4+ T cell responses may vary from those in mice and humans. How bovine CD4+ T cell differentiation differs from that in mice and humans and how some critical bovine pathogens regulate immunity to establish chronic infections are largely unknown. This chapter summarizes current literature and identifies the knowledge gaps to provide insights into future research in the field.
- CD4+ T cell differentiation
- antigen-specific clones
- Th0 responses
- chronic infections
CD4+ T cells, also called helper T cells, are important regulators of adaptive immune responses, which are antigen-specific and critical in protecting animals from pathogen infections. The control of intracellular pathogens, such as viruses, primarily depends on antigen-specific CD8+ T cell response, whereas antibodies (produced by B cells) or humoral immune responses are mostly responsible for the control of extracellular pathogens such as most bacteria and parasites. CD4+ T cells are the lynchpin in shaping both CD8+ T cell and antibody responses [1, 2].
Common lymphoid progenitor cells migrate from the bone marrow into the thymus for further development and maturation into T cells. Inside the thymus, these progenitor cells proliferate into a large pool of T cells, with each expressing a unique T cell receptor (TCR) through a genetic recombination. After TCR recombination, T cells must go through two selection processes, and only a fraction of them pass through these selections and become either CD4+ or CD8+ T cells . Surviving CD4+ T cells then exit the thymus as naïve CD4+ T cells but without the ability to help CD8+ T cells and B cells. To become fully functional, naïve CD4+ T cells need to become activated and differentiated into specialized effector subtypes; helper type 1 (Th1) to facilitate CD8+ T cell responses, and helper type 2 (Th2) to facilitate antibody responses . Naïve CD4+ T cells constantly survey secondary lymphoid tissues to detect pathogens through their antigen-specific TCRs . As opposed to antibodies, which bind directly to pathogens or their derivatives, TCRs can only recognize short chains of amino acids (derived from pathogens) that are presented by major histocompatibility-II (MHC-II) expressed on antigen presenting cells (APCs) . This recognition process provides the 1st signal required to activate naïve CD4+ T cells. Along with the 1st signal, APCs also offer co-stimulation as the 2nd signal and cytokine signaling, as the 3rd signal, to the naïve CD4+ T cell. Combined, these three signals coordinate CD4+ T cell differentiation into distinct effector subtypes with different helper functions .
Studies in humans and mice have identified numerous helper subtypes, including: Th1, Th2, Th3, Th9, Th17, Treg, and Tr1 [2, 6]. Among these, Th1 and Th2 are considered to play major roles in defending the host from pathogen invasion [7, 8, 9]. Th1 cells help CD8+ T cells to gain killing functions, which leads to apoptosis of infected cells and induces Interferon gamma (IFNγ) mediated immunity [10, 11, 12, 13]. On the other hand, Th2 cells help B cells differentiate into plasma cells, which produce pathogen-specific antibodies . Antibodies or humoral immunity contribute to the control of extracellular pathogens by mechanisms like neutralizing toxins, preventing bacterial attachment to the host cell, and stimulating basophil and mast cells to release toxic chemicals that induce the expulsion of large gastrointestinal parasites [15, 16]. Although antibodies are mostly responsible for controlling extracellular pathogens, they can also play important roles in cell-mediated killing of intracellular pathogens . For instance, during intracellular infections in mice, Th1 cells help B cells become plasma cells that secrete antigen- specific immunoglobulin subtype G2a (IgG2a), which in turn can help killing infected cells through antibody dependent cytotoxicity (ADCC) [18, 19]. In short, Th1 is responsible for control of intracellular pathogens mostly through shaping CD8+ T cell responses and Th2 is for control of extracellular pathogens through antibody responses. In addition, antibodies can be involved in both Th1 and Th2 responses, but with unique subtypes, such as IgG2 for Th1, and IgG1 for Th2 in cattle. This will be discussed further in Section 2.
There are many similarities in the immune system across species. Therefore, knowledge generated from the research in mice and humans has been extensively applicable to study immune responses in cattle [20, 21, 22, 23]. In the past several decades, however, unique features have been discovered in the bovine immune system that are not shared with that of mice and humans, such as high prevalence of circulating γδ T cells , production of IL-10 by γδ T cells , regulation of CD4+ T cell activation by neutrophils , which are able to secrete IL-10, and high prevalence of hybrid helper T cells (
Cattle industry suffers billions of dollar’s losses annually due to infections, and many of the commercially available vaccines for cattle are not fully effective [29, 30, 31, 32]. Understanding the mechanisms underlying bovine CD4+ T cell differentiation, which seems to be partially different from that of mice and humans, is critical to identify novel strategies to achieve more effective immunity after vaccinations, such as through generating strong Th1 responses against intracellular pathogens and Th2 responses against extracellular pathogens. In this chapter, we will summarize the current knowledge and key findings on bovine CD4+ T cell responses, highlight the existing knowledge gaps, and provide some insights on future directions.
2. CD4+ T cells regulate adaptive immunity
Naive CD4+ T cells exit the thymus and search for pathogen-derived antigens presented by APCs in secondary lymphoid tissues (
2.1 Th1 cells coordinate CD8+ T cell response to intracellular pathogens
During the infection, the host responds to the intracellular pathogens by inducing cytokines such as IFNγ and IL-12 from APCs like macrophages and dendritic cells (DCs), which further leads to the polarization of CD4+ T cells into a Th1 subtype. IFNγ and IL-12 enhance the expression of transcription factor T-bet, which directs Th1 differentiation in the activated naïve CD4+ T cells (Figure 2a) [51, 52]. More specifically, when bound to their receptors on naïve CD4+ T cells, these cytokines induce the activation of transcription factor STAT-1 or STAT-4 respectively, which in turn causes T-bet upregulation . Subsequently, T-bet induces histone modification and binds to the promoter region of Th1-specific cytokine genes, which leads to enhanced expression of IFNγ [51, 52]. In addition, T-bet also inhibits Th2 differentiation by repressing the transcription of Th2 specific genes, such as
One key functions of differentiated Th1 cells is to facilitate the activation of CD8+ T cells by “conditioning” dendritic cells; a process that induces dendritic cell (DC) maturation by modifying their cytoskeletal structure, upregulating co-stimulatory molecules, and by enhancing their migration to secondary lymphoid tissues [55, 56, 57]. Once conditioned, these DCs can induce CD8+ T cell activation as shown in Figure 2(b). Although these two processes, conditioning of DCs and activation of CD8+ T cells, might occur simultaneously, some researchers argue that this process may occur in two sequential steps: conditioning DC first, followed by CD8+ T cell activation [56, 58, 59]. Activated CD8+ T cell secretes cytotoxicity-related proteins such as perforin and granzyme-B. While perforin forms pores at the cell membrane, granzyme enters through these pores and cause apoptosis of the infected cell . Additionally, antigen-specific CD8+ T cells can kill infected cells through caspase mediated pathway, when Fas molecules expressed on the infected cells interact with Fas Ligand expressed on the antigen-specific CD8+ T cells .
IFNγ is a critical cytokine performing multiple functions to assist Th1 response against intracellular pathogens in mice, humans and cattle . Although many types of immune cells can produce IFNγ including NK cells, DCs, macrophages and B cells, it is the signature cytokine of Th1 subtype . Th1 produced IFNγ plays a critical role in regulating the Th1 response. IFNγ can recruit immune cells to the site of infection and promote anti-microbial activities of neutrophils and macrophages by inducing oxidative burst and production of reactive oxygen species (ROS) [62, 63, 64, 65]. IFNγ is directly involved in blocking viral replication, as well as enhancing the cytotoxic activity of CD8+ T cells [66, 67]. Moreover, IFNγ can enhance the number, mobility, and cytotoxicity of CD8+ T cells [67, 68].
During infection caused by intracellular pathogens, Th1 produced IFNγ can induce IgG subtype switching in activated B cells. However, this subtype switching may differ among the species. For example, it induces production of IgG2a in mice and IgG2 in cattle but IgG1 and IgG3 in humans (Table 1) [18, 69, 73]. These IgG subtypes induced by IFNγ can facilitate multiple mechanisms such as ADCC to kill intracellular pathogens, such as
2.2 Differentiated Th2 cells coordinate humoral response against extracellular pathogens
During infections caused by extracellular pathogens, innate immune cells such as basophils, eosinophils, and innate lymphoid cells (ILCs) produce and secrete IL-4 [83, 84]. Together with 1st and 2nd signals, IL-4 signaling on naïve CD4+ T cell upregulates GATA-3 (GATA binding protein-3), a critical transcription factor for Th2 differentiation [85, 86]. GATA-3 knockout mice mounted impaired Th2 responses [87, 88]. When IL-4 binds to its corresponding receptor on the surface of naive CD4+ T cells, it activates STAT-6, which turns on pathways leading to GATA-3 expression (Figure 3a) [93, 94]. Consecutively, GATA-3 promotes Th2 differentiation by inducing histone acetylation and enhancing transcription of the IL4 gene [83, 95]. In addition, GATA-3 is capable of suppressing Th1 differentiation by downregulating transcription and expression of molecules such as the IL-12 receptor
Once differentiated, Th2 cells are capable of activating B cells to produce antibodies that defend the host against extracellular pathogens [97, 98]. During B cell activation, Th2 cells recognize peptide–MHC-II complexes expressed on B cells [99, 100] and provide co-stimulation via CD40L, which are both necessary for B cell activation  (Figure 3b). Importantly, IL-4 signaling induces isotype and subtype switching of B cells towards IgE and IgG1 production, which are key antibodies for controlling extracellular pathogens in mice and cattle .
Although antibodies can assist CD8+ T cell responses during intracellular infections, they play a major role in controlling infections caused by extracellular pathogens [13, 103, 104]. Antibodies can prevent the attachment of extracellular bacteria to the host cell, facilitate phagocytic killing, and neutralize toxins [13, 105, 106, 107, 108]. In addition, different antibody isotypes and subtypes can have different functions. For instance, IgE can bind to both low and high-affinity receptors (Fc
In addition to IL-4, other cytokines such as IL-5, IL-9 and IL-13 are also involved in the control of extracellular pathogens. For example, IL-9 promotes production of IgE and proliferation as well as maturation of mast cells, which rapidly infiltrate the site of infection [113, 114]. Similarly, IL-5 induces differentiation, maturation, and infiltration of eosinophils to the site of infection . Infiltrated mast cells and eosinophils, when cross-linked by antigen-specific IgE, degranulate (i.e., release histamine and leukotrienes) to kill or expel gastrointestinal parasites. IL-13 on the other hand, plays a significant role in the expulsion of parasites by inducing regeneration of the intestinal epithelium and contraction of smooth muscle cells in the intestine [98, 115]. Nevertheless, there are multiple cytokines involved in the differentiation of Th2 responses, but IL-4 is considered the most critical one.
2.3 Th1/Th2 cytokines induce immunoglobulin class switching during infection
Antibodies produced by activated B cells during infection are classified into five different classes (
2.4 Cytokines and transcription factors mediate Th1/Th2 cross-regulation
In humans and mice, multiple lines of evidence support that Th1 differentiation inhibits Th2 differentiation, and vice versa [120, 121]. For example,
2.5 Distinct Th1 and Th2 are the most dominant antigen-specific clones in mice and humans
In mice and humans, Mosmann et al. and Romagnani et al. stimulated single CD4+ T cells
2.6 Th0 is the most dominant antigen-specific clone in cattle
Just a few years after the discovery of the Th1/Th2 subtypes in humans and mice, Brown et al. successfully investigated bovine Th1/Th2 response through the establishment and analysis of antigen-specific CD4+ T cell clones. Peripheral blood mononuclear cells (PBMC) were purified from cattle challenged by experimental pathogens: either intracellular pathogens (
3. Many critical bovine pathogens induce Th0 responses
In cattle, mixed Th1/Th2 cytokines (both IFNγ and IL-4) have been detected in cultured PBMCs, or Draining Lymph Nodes (DLNs), or local tissues in large number of diseases. Most researchers commonly refer to this as the bovine Th0 response, which may include clones of all three types (Th1, Th2, and Th0) [128, 139, 140, 141]. It is important to note that while Th0 clones can produce both IFN γ and IL-4, Th1 and Th2 clones can only produce a single cytokine, either IFN γ or IL-4 (Figure 4). Therefore, a mixed population of Th1, Th2, and Th0 cells possibly contributes to the induction of Th0 responses in most of the bovine diseases as explained in Section 4.
4. Advancement of technology facilitates the progress in bovine immunology
Technology is a critical factor that drives the advancement of science, and bovine immunology is not an exception, particularly regarding bovine CD4+ T cell research. In the late 80s, the study of bovine Th1/Th2 responses depended heavily on the measurement of cytokines in the supernatant of cultured CD4+ T cells through simple biological assays such as ELISA, or detection of IgG subtypes in the serum of infected animals through ELISA or immunoblotting techniques. In this context, upregulation of supernatant IFNγ and serum IgG2 would represent a Th1 response, upregulation of IL-4 and detection of serum IgG1 would indicate a Th2 response [18, 80], and detection of both cytokines and both IgG subtypes (IgG1 and IgG2) would represent a Th0 response . In the late 90s, advancements in molecular biology enabled scientists to measure cytokines at the transcriptional level (mRNA). Thus, reverse transcription polymerase chain reaction (RT-PCR) was commonly used to detect the presence of mRNA of Th1/Th2 cytokines in PBMCs, DLNs, and tissues of infected cattle [143, 144, 145]. In the next decade, the advent of quantitative PCR (qPCR) improved the detection of Th1/Th2 transcripts from a qualitative to a quantitative level . Later, with the invention and use of flow cytometry, scientists were able to measure protein production of Th1/Th2 cytokines on a population level . More recently, some very exciting technological advancements have been developed, such as single-cell RNA sequencing, proteomics, metabolomics, confocal microscopy, which are considered excellent tools for a deeper understanding of immune mechanisms [148, 149, 150, 151, 152]. Therefore, the advancement of bovine immunology research is closely associated with the development of novel technology in science, especially in the context of understanding Th1/Th2 responses in cattle.
4.1 Most intracellular pathogens induce either a Th1 or Th0 response in cattle
During pathogen invasion, the host mounts a CD4+ T cell response that may or may not be effective enough to clear the infection. In humans, ineffective CD4+ T cell responses are associated with increased pathogenesis and progression towards chronic infections . Cattle mostly launch either Th1 or Th0 responses against intracellular pathogens [154, 155, 156, 157]. However, some bovine pathogens are able to establish chronic infections, which is possibly associated with ineffective CD4+ T cell responses [128, 158].
As observed in mice and humans, bovine Th1 responses are considered to be protective against diseases caused by intracellular pathogens such as
Bovine pathogens such as
During the early phases of Respiratory syncytial virus (RSV) infection in humans and mice, the host launches a Th1/Th2 mixed response (
The efficacy of Th0 responses in controlling infections caused by bovine intracellular pathogens is unclear. While Th0 responses seem ineffective against some bovine diseases such as tuberculosis, they can be protective against bovine babesiosis and non-cytopathic Bovine viral diarrhea virus (ncp- BVDV) infection [156, 157, 184]. In Babesiosis, both CD8+ T cell responses and humoral responses appear critical to clear infection. For instance, increased numbers of antigen-specific CD8+ T cells were detected in the peripheral blood of vaccinated animals . Similarly, transferring serum from an immune animal containing both IgG1 and IgG2 can clear infection of sick animals . In this regard,
Cattle might launch different immune responses against different biotypes of the same intracellular pathogen [145, 186, 187]. For instance, while Th0 response was induced against the non-cytopathic (ncp) biotype of Bovine viral diarrhea virus (BVDV), Th1 response was induced during infection caused by the cytopathic biotype (cp) . In experiments with T cells isolated from the ncp-BVDV infected cattle, IL-4 protein in the supernatant of CD4+ T cell culture and IFNγ protein in CD8+ T cell culture were detected, suggesting possible induction of Th0 response . More recently, Palomares et al. analyzed cytokine expression in tracheo-bronchial lymph nodes and found that both IFNγ and IL-4 were detected in ncp-BVDV-infected cattle, but IL-12 mRNA was only detected in cp-BVDV-infected cattle . Additionally, while only IgG2 was detected in the serum of cp-BVDV-infected cattle, both IgG1 and IgG2 were detected in ncp-BVDV infected cattle after day 35 of infection . These results collectively reveal that ncp-BVDV induces a Th0 response whereas cp-BVDV induces a Th1 response in infected cattle.
Thus, available literature supports the notion that cattle launch either Th1 or Th0 responses against most infectious diseases caused by intracellular pathogens (Table 2). Moreover, although further research is required to confirm these findings, the shift from an early Th1 or Th0 response towards a Th2 response is associated with progression of disease towards chronic condition.
|Disease||Detected cytokines||Serum antibodies||References|
|Theileriosis||IFNγ (RT-PCR)||—||[154, 163]|
|Anaplasmosis||IFNγ (ELISA)||IIgG2 /IgG1 + IIgG2||[155, 189]|
|Babesiosis||IFNγ + IL-4 (RT-PCR)||—||[22, 138, 156]|
|Respiratory syndrome||IFNγ+IL-4(flow cytometry)||IgE||[181, 182]|
|Bovine viral diarrhea||IFN γ / IFNγ + IL-4|
|IgG2/ IgG1 + IgG2||[145, 186, 187]|
|Tuberculosis||IFNγ to IL-4 shift (PCR)||IgG2 to IgG1 shift|||
|Paratuberculosis||IFNγ to IL-4 shift|
|IgG2 to IgG1 shift||[158, 191, 192]|
4.2 Most extracellular pathogens induce either a Th2 or Th0 response in cattle
In mice and humans, Th2 responses are typically effective in controlling extracellular pathogens. In this regard, Th2 cytokines can induce processes such as IgG subtype switching and migration of mast and eosinophils to the site of infection that are critical for defending the host against extracellular bacteria and parasites . In cattle, most of extracellular parasites induce either Th2 or Th0 responses [193, 194, 195]. However, some pathogens are capable of suppressing Th2 response, which is associated with the establishment of chronic infections .
Generally, Th2 responses are effective in controlling gastrointestinal nematodes such as
Interestingly, some extracellular parasites such as
Immune response against extracellular pathogens may vary at the systemic and local levels, such as in bovine trichomoniasis, where Th0 response is induced in the serum, and Th2 response in the mucosal secretion [220, 221]. More specifically,
Generally, Th2 response is effective in controlling extracellular bacteria . For instance, Th2 response controls
Collectively, the results obtained from multiple experiments indicates that extracellular pathogens typically trigger Th2 or Th0 responses in cattle as shown in Table 3, and some extracellular pathogens modulate initial Th2 or Th0 responses to ineffective Th1 responses that are associated with the development of chronic infection.
|Disease||Detected cytokines||Serum antibodies||References|
|Cooperiosis||IL-4 (q-PCR)||IgG1||[201, 202, 227]|
|Lung worm infection||IL-4 /IL-4+ IFNγ|
|IgG1, /IgG1 + IgG2||[228, 229]|
|Trichomoniasis||—||IgG1 + IgG2||[221, 222, 230]|
|IgG1||[211, 231, 232]|
|Ostertagiasis||IL-4 + IFNγ|
|IgG1 + IgG2||[146, 215, 218]|
4.3 Pathogens regulate the availability and the strength of three critical signals to suppress effective CD4+ T cell responses
Whenever a pathogen invades and starts multiplying, the host mounts a coordinated attack in order to clear the infection. To counteract the host attacks, some pathogens can interfere with helper T cell responses to establish chronic infections. This can be achieved through unique strategies that impair the availability or strength of the signals required for the activation and differentiation of CD4+ T cells (Figure 1). For example, pathogens such as
Bovine pathogens escape from effective CD4+ T cell responses in a very similar way to those of mice and humans. They can regulate the availability, type, and strength of three signals. Some pathogens such as Bovine herpes virus type-1 (BHV-1),
4.4 Pathogens regulate the CD4+ T cell differentiation process to establish chronic infections in cattle
In addition to regulating activation signals, during the course of infection, pathogens can also regulate CD4+ T cell differentiation to evade the effective immune response mounted by the host. As already explained, intracellular pathogens can shift effective Th1 response to an ineffective Th2 response; similarly, extracellular pathogens can shift an effective Th2 response to an ineffective Th1 response, in order to promote the chronic infection in the host. For example,
5. Conclusion and future directions
After receiving three stimulation signals from APCs, naïve CD4+ T cells differentiate into effector subtypes such as Th1, Th2, and Th0 cells. While clear-cut Th1 and Th2 are the common subtypes detected in mice and humans, hybrid Th0 is common in cattle infected by both intracellular and extracellular pathogens. In fact, Th0 responses induced in many bovine diseases might consist of a mixed population of Th1, Th2, and Th0 subtypes. Thus, despite similarities in general, bovine CD4+ T cell responses seem to be partially different from the Th1/Th2 responses classically defined in mice and humans. Therefore, understanding the mechanisms of bovine CD4+ T cell differentiation and its regulation by pathogens may facilitate the development of more effective vaccines and designing immune intervention strategies against important chronic bovine infectious diseases.
We gratefully acknowledge the Grant 2016-67015-24948 (to Z.X.) and Grant 2019-67015-29831 (to Z.X.), the Jorgensen Foundation (to Z.X.), and MAES program in University of Maryland (to Z.X.). The figures were created with BioRender.com.
Conflict of interest
The authors state no conflict of interests.