[enlarge]

Figure 1. T-cell development in the thymus. T cell progenitors develop in the bone marrow and migrate to the thymus. Early T cells are CD4-CD8- (double-negative thymocytes). After TCR rearrangement, In the thymus double-negative thymocytes differentiate into CD4⁺CD8⁺ (double-positive) thymocytes. Following interaction with self-peptide-MHC class I complexes, thymocytes become CD8⁺ T cells. Thymocytes, interacting with self-peptide-MHC class II complexes, become CD4⁺ T cells [1].

It is well established that CD4^- CD8^- T cell precursors migrate to the thymus where they undergo the following phenotypical stages: CD44⁺ CD25^- (DN1), CD44⁺ CD25⁺ (DN2), CD44^- CD25⁺ (DN3), and CD44^- CD25^- (DN4), followed by the progression of DN4 cells into the double-positive CD4⁺ CD8⁺ T cells [5] (Figure 1). Infection by various pathogens causes activation and proliferation of naïve T cells, which differentiate into lineages with effector and memory fates. Naive CD4⁺ T cells recognize antigens presented by major histocompatibility complex (MHC) class II on antigen-presenting cells. Depending on the specific stimuli, the CD4⁺ T cells can differentiate into various subtypes, including the helper T_H1, T_H2 and T_H17 cells and regulatory T cells (Tregs). A subset of T_H2 cells differentiate into allergic disease-related T_H2A cells, with a CD45RB^low CD27⁻ phenotype and coexpression of the chemoattractant receptor CRT_H2, the natural killer cell marker CD161, and the homing receptor CD49d [6]. Memory T cells vary in their surface receptor expression, effector and trafficking abilities. There are four major subsets of memory T cells: central memory, effector memory, tissue-resident memory and stem memory T cells. Multiple signals regulate the differentiation of CD4⁺ T cells into central and peripheral memory cells. CD4⁺ T central memory cells express CD62L and CCR7, which are important for their migration [7]. The peripheral T stem cell memory cells express CXCR3 and CD95 molecules. In addition, both naive and memory T-cell subsets express a variety of functional molecules (Table 1).

The second major group of T cells, CD8⁺ T cells, mediates direct killing of antigen-presenting target cells. Naive CD8⁺ T cells are activated upon recognition of antigens presented by MHC class I on dendritic cells in the spleen or lymph nodes. Activated CD8⁺ T cells expand and become effector CD8⁺ T cells. CD8⁺ T cells tend to be evaluated during the study for tumor-infiltrating T cells. For example, Vodnala SK et al. evaluated the effect of overabundance of potassium in the tumor microenvironment on CD8⁺ T cell stemness and dysfunction in tumors [9].

The majority of the T cells bear α and β chains in their T cell receptor (TCR). However, there is a population of T cells, which have TCR formed by γ and δ chains. These cells, gamma delta T cells, bind to BTN2A1 and BTN3A1 [10], are significantly enriched in epithelia [11, 12]. Gamma delta T cells regulate immune responses by various mechanisms, including suppression of effector T cell and T_H1 cell functions, blockage of neutrophil influx and regulation of antigen-presenting cell activity.

Naive T cells are considered as precursors of the majority of antigen-activated T cell subpopulations. Human naïve CD4⁺ T cells express CD45RA, CCR7, CD62L and CD27. Upon recognition of antigens presented by major histocompatibility complex (MHC) class II on antigen-presenting cells, naïve CD4⁺ T cells undergo proliferation and differentiation into functionally different T cell subsets including IFN-γ producing helper T cell-1 cells (T_H1), IL-4-producing T_H2, IL-17-producing T_H17 cells, and inducible regulatory T cells (iTregs) (Figure 2). Each T cell subset expresses specific transcription factors, such as T-bet (T_H1), GATA3 (T_H2), RORγt (T_H17), and Foxp3 (CD25⁺ Tregs) [13]. Grandclaudon M et al used IL-12Rb2 as a T_H1 cell marker [14]. With regard to T_H17 cells, their differentiation is under control TGF-β and IL-6-induced differentiation, IL-21-induced activation, and IL-23-regulated stabilization [15, 16]. As to iTregs, FOXP3 was found to an important marker of natural CD4⁺ CD25⁺ regulatory T cells. Moreover, transfection of CD4⁺ CD25^- T cells with Foxp3 stimulates their regulatory activity [17]. In addition, TGF-β was found to be crucial for the differentiation of naive CD4⁺ T cells into Foxp3⁺ Tregs [18]. Also, IL-2 is commonly required for TGF-β-regulated iTreg differentiation [19]. D Mathew et al examined the six CD4⁺ T cell subtypes in Covid-19 patient blood: naïve (CD45RA⁺ CD27⁺ CCR7⁺ CD95^-), central memory (CD45RA^- CD27⁺ CCR7⁺), effector memory 1 (CD45RA^- CD27⁺ CCR7^-), effector memory 2 (CD45RA^- CD27^- CCR7⁺), effector memory 3 (CD45RA^- CD27^- CCR7^-), and EMRA (CD45RA⁺CD27^- CCR7^-) in addition to circulating CD4⁺ T cells (CD45RA^- PD1⁺ CXCR5⁺) and activated circulating CD4⁺ T cells (CD38⁺ICOS⁺) [20].

[enlarge]

Figure 2. Differentiation of helper CD4⁺ T cell subsets. After activation by antigen-presenting cells, CD4⁺ T cells can differentiate into several subsets: T helper 1 (T_H1), T_H2, T_H17 and regulatory T cells. The differentiation of each T cell subset is regulated by different transcription factors [2].

With regard to cytotoxic T cells, there are several peripheral subsets of different subsets of CD8⁺ T cells based on the expression of CD45RA and CCR7: a CD45RA⁺ CCR7⁺ subset of naive cells, a CD45RA^- CCR7⁺ subset of antigen-experienced memory T cells, a CD45RA^- CCR7^- effector memory cell subset, and a CD45RA⁺ CCR7⁻ subset of differentiated, antigen-experienced effector cells. Also, there are effector memory CD8⁺ T cells expressing CD69 and CD103 and residing in non-lymphoid tissues [21, 22]. A subpopulation of CD8⁺ T cells shows a memory cell phenotype: CD62L^- /⁺ CCR7⁺ CD27^- /⁺ . Activated cytotoxic CD8⁺ T cells downregulate expression of L-selectin and CCR7 and upregulate surface expression of CD44, LFA-1 and/or α4β1 integrin. In addition, there are CD8^- cytotoxic T cells: CD4⁺ cytotoxic T cells and gamma delta T cells [23]. Hashimoto K et al shows that supercentenarians have much higher level of CD4⁺ cytotoxic T cells in blood circulation than young people [23]. D Mathew et al examined the six CD8⁺ T cell subtypes in Covid-19 patient blood with CD45RA, CD27, CCR7, and CD95 cell surface markers: naïve (CD45RA⁺ CD27⁺ CCR7⁺ CD95^-), central memory (CD45RA^- CD27⁺ CCR7⁺), effector memory 1 (CD45RA^- CD27⁺ CCR7^-), effector memory 2 (CD45RA^- CD27^- CCR7⁺), effector memory 3 (CD45RA^- CD27^- CCR7^-), and EMRA (CD45RA⁺CD27^- CCR7^-) [20].

There are several additional phenotypical markers expressed in both human and mouse Tregs. They include CTLA-4, CD103, GITR and OX40. In particular, CTLA-4 is important for both inhibitory functions and homeostasis of Tregs. Intracellular expression of CTLA-4 was observed in CD4⁺ CD25⁺ human Tregs [24]. Another marker, integrin α (CD103) is expressed by Tregs and CD4⁺ CD25⁺ CD103⁺ Tregs were demonstrated to produce IL-10 actively [25]. In addition, GITR (CD357) is expressed in CD4⁺ CD25⁺ human Tregs in peripheral blood [26]. Also, OX40 (CD134) was shown to stimulate the proliferation of CD4⁺ FoxP3⁺ Tregs [27]. Moreover, OX40 stimulates migration of Tregs into the peripheral lymphoid and other tissues during inflammation [28].

Several specific T cell subsets, including follicular B helper T cells (TFH), follicular regulatory T cells (TFR) and cytotoxic CD8⁺ T cells, reside in the germinal centers and regulate the B cell proliferation [29]. Among these T cell subpopulations, TFH cells belong to CD4⁺ T cells and assist follicular B cells located in secondary lymphoid tissues, such as lymph nodes, spleen, and tonsils. Concerning the specific markers, high expression of CXC-chemokine receptor 5 (CXCR5) characterizes TFH cells [30]. Its interaction with CXC-chemokine ligand 13 (CXCL13) produced by follicular stromal cells mediates the homing of TFH cells into lymphoid follicles [30]. The development of TFH cells is strongly dependent on IL-2 production, as naїve IL2-secreting CD4⁺ T cells are destined to differentiate into TFH cells, while other CD4⁺ T cells, which do not produce IL-2, develop into non-TFH cells [31]. In addition to a universal T cell marker Thy1 (CD90) and CXCR5, TFH cells express ICOS and PD-1 molecules. Upregulation of CXCR5 expression stimulates TFH cells to migrate into the germinal centers, where these cells stabilize their phenotype by contacts with local B cells via ICOS-ICOSL binding [32]. Concerning the regulation of TFH functions, γδ T cells (TCRγδ⁺ CXCR5⁺ T cells), which also reside in the lymph nodes, have recently been shown to present antigens to TFH cells and induce their activation [33].

[enlarge]

Figure 3. Stages of B cell development. B cell development starts in the bone marrow. Immature B cells migrate to the spleen. There are three subsets of mature B cells: follicular B2 cells, marginal zone B cells and B1 cells. Following exposure to antigens, B cells differentiate into antibody-producing plasma cells [3]. B2 cells may remain plastic and can differentiate to B1 cells upon the self-reactivity of B cell antigen receptor [4].

Cellular interactions with TFH cells regulate the proliferation and maturation of B cells in the germinal centers [34]. Besides, TFH cells secrete Il-4 and IL-21 cytokines, which are crucial for the functioning of the germinal centers [34]. Moreover, in the germinal centers, TFH cells are represented by at least two distinct subpopulations: IL-21⁺ T cells regulating the selection of high-affinity B cells and IL-4⁺ T cells promoting differentiation of plasmocytes [35]. A third type, secreting IL-4, IL-5, and IL-13, directs the class switching of B cells from IgG1 to high-affinity IgE during anaphylaxis, and coexpress transcription factors BCL6 and GATA3 [36]. Several studies have shown that chronic viral infection strongly induces differentiation of TFH, which leads to non-specific B cell activation [37].

In addition to TFH cells, researchers have identified TFR cells in the germinal centers. This subset of T cells expresses Foxp3 and also regulates the activity of germinal centers [38]. TFR cells suppress the proliferation of B cells and the production of IgM and IgG antibodies [38, 39] and diminish the secretion of IL-4 and IL-21 by TFH cells in the germinal centers [40].

The standard methods for measurement of T cell immune responses include Enzyme-Linked Immuno Spot assay (ELISpot), Intracellular Cytokine Staining assay (ICS), Tetramer assay and Flow Cytometry. The ELISpot and ICS assays apply in vitro stimulation to analyze the cytokine expression profiles of responding cells. The ELISpot method detects spots of cytokines secreted by individual cells, and ICS examines surface markers and produced cytokines. Multiple approaches can measure the proliferation of T cells in response to specific antigens, including thymidine incorporation assay, flow cytometric analysis of CD38 expression or ELISA detection of BrdU incorporation into DNA of proliferating T cells.

Many solid tumors are infiltrated by T cells such as CD8⁺ cells. Cytotoxic CD8⁺ T lymphocytes recognize an antigen-MHC complex on tumor cells, get activated and release perforin and granzymes causing apoptosis in target tumor cells [41]. Tumor antigen-specific T cells are used for the development of new immunotherapeutic methods. Cytotoxic T cells expressing chimeric antigen receptors (CAR T cells) have been effectively used for hematological malignancies [42]. In addition to CAR T lymphocytes, bispecific T cell engagers (BiTEs), represented by two scFvs, bind to both CD3 on T cells and a tumor cell antigen. One of the BiTEs, blinatumomab, was approved to treat B cell leukemia [43].

Tumor-infiltrating T cell subpopulations include CD4⁺ and CD8⁺ T cell subsets (Table 2). CD4⁺ T cells are represented by T helpers, such as T_H1, T_H2, T_H9, T_H1 7 and Tfh, and Tregs. The CD8⁺ T cell subsets consist of cytotoxic cells and mucosal-associated invariant T cells (MAIT). These T cell subpopulations are highly important for prognosis and prediction of treatment efficiency. CD8⁺ cytotoxic cells migrate into the tumor tissue and display cytotoxic activity against tumor cells [44].

γδ T cells, a T cell subset involved in both innate and adaptive immunity, may have both anti-tumor and pro-tumor functions [56]. Anti-tumor effects are mediated via their differentiation into cytotoxic T cells. Vδ1 and Vγ9Vδ2 T cells are two populations of γδ T cells, which are present in the tumor microenvironment. Both subsets develop cytotoxic capabilities [52]. The lysis of the target tumor cells by γδ T cells is mediated via different mechanisms involving granzyme B, perforin, Fas ligand. In addition, γδ T cells induce cytostatic effects by producing IFN-γ or TNF-α [53, 54]. In contrast to anti-tumor effects, IL-17⁺ γδ T cells are associated with the progression of ovarian tumors [57]. Based on their functional capabilities, γδ T_H1, γδ T_H2, γδ T_H17, γδ Tfh, and γδ Treg cells can be distinguished among the effector γδ T cells [56]. Notably, these cells can differentiate from one subset to another under the influence of different cytokines.

Among the T helpers, T_H9 cells are known to secrete IL-9 and IL-10 and inhibit tumor growth [45]. Low expression of IL-9R induces melanoma growth. Moreover, some T_H9 cells can secrete IFNg. Also, T_H9 cells, which infiltrate colorectal tumors, may be regulated through PD-1/PD-L1 pathway and may stimulate the proliferation of CD8⁺ cells. Expansion of T_H9 cells is usually accompanied by an increase of IL-9⁺ IL-4^- and IL-9⁺ IL-4⁺ cell subsets [58]. Surprisingly, other studies have shown that T_H9 cells may stimulate epithelial-mesenchymal transition and dissemination of lung tumor cells [59]. Furthermore, T_H9 cells infiltrate lung tumors and are associated with poor survival.

Tregs are known to infiltrate tumor sites and demonstrate immunosuppressive activity. Tumor-infiltrating Tregs were found to have high expression of CTLA-4, GITR and PD-1 [46]. Tregs express multiple chemokine receptors with corresponding ligands, such as CCR with CCL12, CCR4 with CCL17 and CXCR4 with CXCL1 [47]. With regard to phenotypical characteristics, tumor-infiltrating Tregs express immunosuppressive markers, such as iCTLA-4. In addition, these cells have upregulated expression of GITR, LAG3, OX40 and ICOS [48].

Mucosal-associated invariant T cells (MAIT) belong to innate T cells and are present mainly in mucosal tissues. They express a semi-invariant T cell receptor, which contains rearranged TCRβ chains with Vβ gene segments [49]. MAIT cells demonstrate strong cytotoxic activity in colon tumors. The functions of these cells include both production of T_H1 -mediated cytokines and direct lysis of neoplastic cells [50]. Circulation of MAIT cells was downregulated in patients with hepatocellular carcinoma and colorectal and lung tumors [60]. Notably, MAIT cells, which infiltrated tumor tissues in patients with colorectal carcinoma, secreted less IFNg than MAIT cells from intact livers.

With regard to memory T cells, Vahidi et al analyzed the presence of CD8⁺ memory T cell groups in draining lymph nodes from patients with breast tumors. Increased numbers of CD45^low central memory T cells were found in lymph nodes, indicating that the early differentiation of memory T cells is inhibited by tumor-produced regulatory factors [61]. The increase of tumor-infiltrating T cells usually correlates with positive outcomes in solid tumors. Tissue-resident memory T cells were associated with better prognosis and longer survival in patients with breast tumors [51].

There are three main subsets of naïve B lymphocytes: follicular B cells, marginal zone B cells and B1 B cells. Mature follicular B cells migrate through blood and lymph, reside in specific B cell areas of lymph nodes, Peyer’s patches, and the spleen and may present T-dependent antigens to T cells. Marginal zone CD19⁺ CD21⁺ CD23^- CD24⁺ IgM⁺ B cells reside in the marginal sinus of the spleen and mediate the transport of antigen in immune complexes. B1 cells are involved in the development of IgM responses to bacterial T cell-independent antigens. These cells can migrate from the peritoneum and reside in mesenteric lymph nodes. Memory B-cells are represented by three subsets: pre-switch IgD⁺ IgM⁺ CD27⁺ B cells, IgD^- IgM⁺ CD27⁺ B cells, post-switch IgA⁺ CD27⁺ and IgG⁺ CD27⁺ B cells and IgA⁺ CD27^- and IgG⁺ CD27^- memory B cells [62]. R Shi et al obtained memory B cells specific to SARS-CoV-2 RBD using the following markers: CD3⁻, CD16⁻, CD235a⁻, CD38⁻, CD19⁺, CD27⁺, IgG⁺ in addition to His⁺ (for His-tagged SARS-CoV-2 RBD) [63]. Circulating plasmablasts can be identified by the expression of CD38 and CD138 [64].

Expression of BCR expression is highly important for maintaining B cells in the peripheral immune system. However, only 30% of B cells in spleen develop into mature B cells. Moreover, mice, which have mutations in genes encoding BCR-related proteins, including BLNK, Btk, and Vav, show disruption of the maturation process [66, 67].

Lymphoid progenitors Lin^- KIT^low CSA1^low IL-7R⁺ are considered to be a lymphoid progenitor group and can differentiate into both B and T cells. Also, in vitro studies have demonstrated that B220^- CD19⁺ cells can differentiate into myeloid or B cells [68] and Lin^- KIT^low SCA1^low IL-7R⁺ FLT3⁺ CD34^- cells or B220^- KIT^low SCA1⁺ CD24⁺ CD43⁺ cells contain increased numbers of B cell precursors [69, 70].

Early B220⁺ precursors of B cells do not express cell surface immunoglobulin (Ig), reside in the bone marrow and include pre-pro-B cells, pro-B cells, and pre-B cells. Immature pre-B cells migrate to the spleen, where they differentiate into mature B cells and plasmocytes (Figure 3). Peripheral B cell subsets, including transitional, mature, memory and antibody-secreting cells, express different surface markers (Table 3).

In addition to IgG production, a subpopulation of splenic B cells can possess regulatory functions. Regulatory B cells (Bregs) affect various parts of the immune system with IL-10 playing a key role in these processes. The B10 subgroup of B cells was shown to act as regulatory cells in experimental models of lupus and autoimmune encephalomyelitis [71]. Moreover, IL-10 producing Bregs with the surface phenotype CD19⁺ CD24^hi CD38^hi were found in the peripheral blood in SLE patients [72]. In addition, regulatory phenotypes CD19⁺, CD24⁺ CD27⁺ and CD19⁺ IgD⁺ CD24^hi CD38^hi CD5^hi were shown to have suppressive functions in humans [73, 74].

Several subsets of Bregs were characterized in human peripheral blood. These subsets include B cells with different levels of maturity: transitional CD19⁺ CD24^hi CD38^hi Bregs [80, 81], CD19⁺ CD27^int CD38⁺, plasmablasts [82] and CD19⁺ CD25⁺ CD71⁺ B regulatory 1 cells [83]. Recent studies suggest that differentiation and stimulation of Bregs are likely to be induced by inflammation associated with either infection or autoimmune reactions. In particular, toll-like receptor agonists of bacterial origin were shown to activate Bregs in vitro [84, 85]. In addition, the proliferation of Bregs was reported in a murine model of autoimmune arthritis [86].

In addition to the subsets of Bregs mentioned above, Tim-1⁺ B cells were also shown to regulate immune reactions, since Tim-1 mucin domain-mutated mice develop autoimmune disorders [87]. Tim-1⁺ Bregs were identified within different B cell subpopulations, including CD19⁺ CD1d^hi CD5⁺, MZ and B1 cells [88]. Also, human CD73⁻ CD25⁺ CD71⁺ BR1 cells were demonstrated to be involved in the development of allergen tolerance [83]. Membrane regulatory molecules expressed by Bregs include CD25, CD71 and CD274 [72, 89, 90].

Activated B cells, regulatory B cells and memory B cells are among the most notable B cell subpopulations found within tumor tissues. The functions of tumor-infiltrating heterogenous B lymphocyte subsets include antigen presentation, secretion of antibodies and cytokines, and activation of T cells. In particular, B cells can promote T cell proliferation by functioning as antigen-presenting cells [97]. Tumor-specific antibodies, which recognize tumor antigens, may potentially be used to reduce tumor cells or activate cytotoxic T cells [98]. However, regulatory B cells may act as immune suppressors thus playing a protumor role in the tumor loci. These negative functions of regulatory B cells led to the promotion of therapeutic strategies based on B cell depletion [99].

The detection of B-lymphocytes in the tumor microenvironment has been associated with better prognosis in several tumors (Table 4). In particular, the increased numbers of CD20⁺ cells were associated with better survival in patients with pancreatic ductal adenocarcinoma [100]. A recent study analyzed different subsets of B cells present in tumor micronvironment [101]. Increased numbers of tumor-infiltrating naive B cells (CD20⁺ CD27^- IgM⁺ ), IgM⁺ memory B cells (CD20⁺ CD27⁺ IgM⁺ ), CD27^- isotype-switched memory B cells (CD20⁺ CD27^- IgM^- ), and plasma cells (CD20^- CD24^- CD27^hi CD38^hi ) were found to be associated with better survival in hepatocellular carcinoma [78].

With regard to regulatory subsets, Wu et al found elevated numbers of CD20⁺ CD27^- PD-L1⁺ regulatory B cells, which were positively associated with melanoma stage [75]. These PD-L1⁺ cells were suggested to act as T cell inhibitors. Furthermore, regulatory B cells suppress CD8⁺ cytotoxic activity by producing IL-10 [76]. In line with these data, CD19⁺ CD5⁺ CD43⁺ B1a Bregs were shown to suppress anti-melanoma immune response by secreting IL-10 and inhibiting T_H1 cytokine release by cytotoxic CD8⁺ T cells [77]. The selective inhibition of mitogen-activated protein kinase (MAPK) kinase (MEK), a crucial member of RAS pathway, suppresses regulatory B cells in lymph nodes without affecting humoral immunity [102].

In addition, IgD^- CD27⁺ memory, CD86⁺ CD21^- antigen-presenting and CD86⁺ activated B cells also infiltrated tumor tissues. Activated naïve B cells, which expressed elevated levels of CD80, CD86, CD44, CD69, and PD-L1, were found to suppress T_H1 7-cell expansion through the PD-1/PD-L1 pathway and stimulated differentiation of CD4⁺ T cells into T_H1 7 cells via the secretion of IL-27 and IL-6 [79]. Also, in a study of B cell subsets in hepatocellular carcinoma, the authors have shown that increased numbers infiltrating CD20⁺ B cells, IgM⁺ memory B cells, CD27^- isotype-switched memory B cells and plasma cells correlated with better survival [78].

One of the most important functions of B cells is antibody production. Enzyme-linked immunosorbent assay (ELISA) can analyze secreted antibodies, plaque-forming cell (PFC) assays can detect antibody-secreting B cells, and ELISPOT can indicate the number of antibody-producing B cells.

Labome surveys formal publications to develop Validated Antibody Database (VAD). Table 5 lists the most cited antibodies against T cell markers and B cell markers among the 60,000 articles Labome has surveyed.

CD45, also called leukocyte common antigen(LCA), regarded as a pan-immune marker, has also been found in rare epithelial cells in mouse intestine [103], more specifically in tuft-2 cells [104].