**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 *β2,* IFNγ, STAT-4, and possibly T-bet [96].

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 [101] (**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 [102].

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

### **Figure 3.**

*Th2 help to the activation of B cell. A) IL-4 binds to its receptor on naïve CD4+ T cells during activation, which induces GATA-3 activation and Th2 differentiation. This figure was adapted from previous reviews [46, 89, 90]. B) Once differentiated, Th2 cells secrete IL-4 and provide antigen stimulation and co-stimulation to a B cell. This figure was adapted from previous reviews [91, 92].*

bacteria to the host cell, facilitate phagocytic killing, and neutralize toxins [13, 105–108]. In addition, different antibody isotypes and subtypes can have different functions. For instance, IgE can bind to both low and high-affinity receptors (Fc*ε*RI and Fc*ε*RII) on mast cells and basophils, which results in the degranulation and release of chemicals (*e.g.,* histamine, leukotrienes) that either kill parasites directly, or induce hyper-contraction of intestinal smooth muscle to promote their expulsion [109–112].

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 [114]. 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 (*i.e.,* IgM, IgG, IgA, IgD and IgE) based on their structure [116]. Among them, IgG is the most abundant in serum, and it has four different subtypes, namely: IgG1, IgG2, IgG3 and IgG4 [116]. Each antibody has two structural segments (heavy and light chains) and two functional segments (Fab and Fc portions). While association of heavy chain with the light chain at the Fab portion forms antigen-binding sites, only the constant portion of the heavy chain constitutes the Fc segment that regulates the effector function of the antibody. During infection, activated B cells undergo isotype or subtype switching, a process that involves switching of Fc segment but not of the Fab segment. Briefly, DNA in B cells contains multiple heavy chain constant genes (or CH genes) that encode various types of Fc segments [117]. During infections, Th1 and Th2 cytokines provide signals to the activated B cells to select a specific CH gene for the heavy chain, thus producing a specific isotype or subtype of immunoglobulins with the same antigen specificity [118]. For example, IFNγ can induce subtype switching to IgG2a to enhance the killing of infected cells in mice; similarly, IL-4 can induce switching to IgG1 to promote humoral immunity (**Table 1**) [70–72, 119]. Historically, characterizing serum IgG subtypes was a common practice to define the immune response in clinically ill cattle; the greater concentration of serum IgG2 typically indicated a Th1 response, whereas greater IgG1 indicated a Th2 response. Interestingly, the Th1 induced IgG subtypes may vary among the mice, humans and cattle species as shown in **Table 1**.


**Table 1.**

*Th1- and Th2-associated IgG subtypes in mice, humans, and cattle.*
