Unlike the cells of the innate immune system, T and B lymphocytes possess one unique and profoundly important property. On their surfaces, these cells also display a receptor complex capable of recognizing foreign (nonself) structures—with exquisite specificity. This receptor complex is both randomly generated (by somatic recombination of genetic elements ultimately encoding the mature receptors) and clonally distributed among progeny lymphocytes during their differentiation from progenitor cells. Therefore, of the approximate 10⁹ naive T and B lymphocytes in the human adaptive immune system, each cell may, in principle, display a unique receptor directed to a specific foreign antigenic structure. This common property of the antigen receptor on T and B lymphocytes (i.e., random generation by somatic DNA recombination and clonal receptor display) immediately raises two important questions. First, if these antigen receptors are stochastically generated, how is the generation of lymphocytes with receptors to self-structures (e.g., tissue proteins) avoided/prevented? Second, if the frequency of lymphocytes with a receptor directed to a specific foreign antigen (e.g., the coat proteins of an invading virus) is extremely low (1 in 10⁷ lymphocytes), how can those rare lymphocytes locate the antigen quickly enough to respond before the infection is beyond control? As is discussed below, it is the unique structure of the primary (central) lymphoid organs where lymphocyte progenitors develop into mature T and B lymphocytes, and of the secondary (peripheral) lymphoid organs where mature T and B lymphocytes respond to invading microorganisms that provides the answers to these questions.

Mature T and B lymphocytes are derived from lymphoid progenitors present in the bone marrow, as well as in sites of hematopoiesis in developing fetus (e.g., the liver) through a process of differentiation and selection linked to, and driven by, the antigen receptors displayed by these cells.²²¹,²³³ As described in Table 9.2, B-lymphocyte lineage development occurs in the bone marrow and follows a well-defined, step-wise program of gene
activation (e.g., expression of the recombination activating gene 1 and 2 [RAG1 and 2], lymphoid DNA-specific recombinases), and sequential rearrangement of immunoglobulin (Ig) V_H, D_H, J_H, and C_H heavy (H) chain genetic elements (by random somatic recombination), followed by rearrangement of the Ig K/λ V_L-J_L-C_LK/l light (L) chain locus elements. Then B-lymphocyte progenitors transit from the pro-B, through the pre-B, and to the surface immunoglobulin-positive immature B-lymphocyte stage. It is during this development/differentiation program that the B-lymphocyte receptor (B-cell receptor [BCR]) diversification occurs through random association of one of the 40 V_H gene segments with one of the 25 D_H and 6 J_H and 70 V_L with 9 J_L to yield (after nucleotide addition and/or excision at the site of gene segment recombination) 10⁷ or more different potential BCR H and L chain combinations. During this development process in the bone marrow, T-Lymphocytes expressing a BCR with reactivity to self-molecules (i.e., proteins, lipids, carbohydrates, nucleic acids) will, as a result of BCR engagement, undergo a process of negative selection. These self-reactive B cells are largely eliminated/deleted through interaction of the BCR on the developing cells, with self-constituents resulting in apoptosis or in activation (“anergy”) of self-reactive B lymphocytes. The immature B lymphocytes that escape negative selection undergo final maturation, migrate from the bone marrow to join the pool of mature B lymphocytes that circulate through the blood, and populate the secondary lymphoid organs. B lymphocytes ultimately express their effector activity by releasing the BCR of the mature B lymphocyte in a secreted form as immunoglobulin (antibody) molecules. This fact has the following two important implications for the recognition of viruses by B lymphocytes: (a) the B-lymphocyte response to viruses will be particularly effective when interacting with free/extracellular virus constituents (e.g., virions); and (b) the BCR and its antibody product will be sensitive to the conformation of viral constituent recognized.

T-Lymphocyte lineage development follows a program similar to that of B lymphocytes. The T-cell antigen receptor (TCR) is also a heterodimer consisting of two polypeptide chains, each of which is formed by somatic recombination of variable (V), diversity (D), and joining (J) segments encoding the variable portion of one chain (or V and J joining for the other chain) to a constant (C) gene segment. As in B lymphocytes, the randomly recombined gene segments encoding the V region gene segment joined to the respective C region gene segment for the two TCR chains encodes the TCR on the developing T lymphocyte. Unlike the BCR, however, the TCR always remains cell associated. Therefore, the TCR is normally directed to foreign antigens (or self-structures) that are cell associated. Consequently, T lymphocytes are uniquely suited to recognize cells that have been invaded by microorganisms (e.g., viruses and intracellular bacteria). Unlike the BCR, the TCR does not recognize intact microbial products, but rather recognizes small fragments of molecules—typically, short peptide fragments of microbial or self-proteins bound to protein products of the mammalian MHC genetic locus (e.g., the classically defined major human transplantation antigens). These MHC gene products expressed on cell surfaces serve as molecular platforms, both selecting and displaying the self-peptides involved in TCR selection during T-lymphocyte precursor development, and presenting microbial peptides to mature T lymphocytes responding to uptake/infection of cells by the microbes. The steps in the processing (fragmenting) and presentation of both self and microbial gene products by the MHC locus protein, and the structure of the major classes of MHC locus proteins (i.e., MHC class I and II proteins) are discussed below.

The generation of mature T lymphocyte from committed lymphocyte progenitors (arising in the bone marrow) occurs primarily in the thymus (Fig. 9.1). Here, immature thymocytes undergo the developmental program resulting in the formation of mature T lymphocytes, which then leave the thymus, enter the circulation, and ultimately populate secondary lymphoid organs. Two classes of mature T lymphocytes are produced in the thymus. The major population (∼80% of the peripheral T lymphocyte pool) are the α⁺β⁺ T cells (because their heterodimeric TCR express two chains encoded by TCR α and β loci). The second minor population of mature T cells are the γ⁺δ⁺ T cells (because they express a related two-chain TCR derived from the TCR γ and δ loci). Much of our current information on T-lymphocyte development comes from analysis of α⁺β⁺ T-cell development. These T lymphocytes represent the T-cell subset that responds primarily to viral infection, and the selection process of these T cells in the thymus will be emphasized here. The γ⁺δ⁺ T-cell subset likely plays a secondary role in the adaptive response to most virus infections, and the properties/function of this T-cell subset is summarized below.

T-Lymphocyte development in the thymus proceeds in a stepwise fashion with the sequential generation and expression of one (i.e., TCR-β or TCR-δ), and then the other (i.e., TCR-α or TCR-γ) TCR chain by random somatic recombination as thymocytes mature. A complex of four cell-surface molecules called CD3 (CD3 γ, δ, ε, and ξ), which are noncovalently associated with the TCR and are critical for signaling through the TCR, are also expressed during T-lymphocyte development. Specific antigen recognition by the TCR-α:β (or γ:δ) heterodimer is transduced into the cells by the CD3 complex. Concomitant with the expression of the rearranged TCR chains and the signal transducting CD3 complex, the developing thymocytes simultaneously express two co-receptor molecules, CD4 and CD8. Early in the developmental program, thymocytes do not express CD4 or CD8, and they are classified as immature, double-negative thymocytes. As thymic development/differentiation proceeds, immature thymocytes that express both CD4 and CD8 co-receptors (as well as CD3 complex and the TCR-α and β chains) and, at this point, are classified as are CD4⁺/CD8⁺ double-positive thymocytes. CD4 and CD8 molecules recognize structurally conserved domains on the MHC class II and I molecules, respectively. As a result of binding to MHC molecules displaying self-peptides on the surface of thymic stromal cells, CD4 or CD8 also delivers signals to the developing immature thymocytes. The combined signaling by TCR/CD3 complex and the CD4 or CD8 co-receptor directs the development of the immature thymocytes into single-positive CD4⁺ or CD8⁺ mature thymocytes (expressing TCR, CD3, and either CD4 or CD8).

Because the TCR does not recognize foreign molecules directly (but rather small fragments of molecules bound to MHC molecules), the TCR generated and expressed on developing thymocytes must recognize both MHC molecules and the peptide fragments bound to these molecules. The source of these peptides bound by MHC class I and class II molecules are self-proteins expressed by several different cell types in the thymus. The selection of the thymocytes that ultimately become mature co-receptor single-positive T lymphocytes (CD4⁺ or CD8⁺) and populate the secondary lymphoid organs is a process in the thymus of positive and negative selection.

At a specific development stage, immature double-positive thymocytes (expressing the randomly generated α : β TCR, CD3, and both CD4/CD8) encounter, within the thymic cortex, thymic cortical epithelial cells displaying MHC class I and II molecules complexed with self-peptides. Most of these thymocytes (∼80%) have randomly generated TCR that are unable to bind to the self-peptide/MHC molecular complexes. In the absence of a signal delivered through the TCR, these thymocytes will die by apoptosis. Those double-positive thymocytes whose TCR can bind the self-peptide/MHC complexes above a threshold avidity augmented by either a CD4 or a CD8 co-receptor binding to the MHC molecule will undergo positive selection and survive. If the thymocyte TCR can bind peptides bound to MHC class II molecules in association with the CD4 co-receptor, this interaction will result in the silencing of the CD8 co-receptor gene. This signaling event results in the development of a CD4⁺ single-positive thymocyte whose TCR recognizes (is restricted to) peptides bound to MHC class II molecules. Similarly, the double-positive thymocytes whose TCR and associated CD8 molecule can recognize MHC class I/self-peptide complexes with sufficient avidity to survive (i.e., those that are positively-selected) will give rise to MHC class I–restricted CD8⁺ single-positive thymocytes by a similar silencing mechanism of the CD4 gene.

After undergoing positive selection, the surviving single-positive thymocytes are all auto-reactive (i.e., directed to self-peptide/MHC complexes). Therefore, a process of negative selection must then ensue to eliminate/reduce self-reactive, mature peripheral T cells. Although less well understood, negative selection of the α : β TCR-expressing single-positive (CD4⁺ or CD8⁺) thymocytes probably occurs in both the thymus cortex and medulla. Negative selection is most efficiently mediated by mononuclear cells of hematopoietic origin (e.g., dendritic cells and macrophages) that simultaneously display MHC class I and II molecules expressing self-peptides. The 10% to 20% of thymocytes that survive positive selection, by definition, are auto-reactive. It is likely (but not certain) that the intracellular signal transduction cascade associated with TCR engagement is altered in the cortical and medullary single-positive thymocytes following positive selection. As a result these cortical and medullary thymocytes acquire the TCR-signaling profile of mature T lymphocytes. Consequently, when the thymocytes encounter the self-peptide/MHC complexes displayed by hematopoietic origin cells like DC in the cortex and medulla, those thymocytes whose TCR/CD4 or CD8 co-receptor complex recognizes self-peptide/MHC complexes with a sufficient avidity to activate will be eliminated (i.e., undergo negative selection—death by apoptosis). Thymocytes directed to self- “tissue specific” molecules (e.g., insulin) are deleted through the action of the autoimmune regulator (AIRE) gene. This gene is expressed in thymic medullary stromal cells and triggers the transient low level expression of many extrathymic tissue-specific self-proteins in these cells resulting in negative selection of tissue-reactive single-positive thymocytes. Therefore, only about 2% to 5% of immature thymocytes will survive the positive and negative selection processes. These remaining thymocytes will have the properties of mature CD4⁺ or CD8⁺ T lymphocytes (i.e., they have TCR that recognize peptide/MHC complexes, but will only interact with the MHC molecules with sufficient avidity to activate when the appropriate foreign [microbial] peptides are bound to the MHC molecule). Following positive and negative selection, these single-positive α : β T lymphocytes (CD4⁺ or CD8⁺) will leave the thymus to populate the secondary lymphoid organs as peripheral T cells (Fig. 9.1). The CD4⁺ or CD8⁺ α : β T cells represent 70% to 80% of the circulating extrathymic T-cell population—the primary T-cell population that responds to virus infection.

The second (minor) population of thymus-derived cells that populates peripheral sites is that of the γ : δ T cells, so named because they employ a heterodimeric TCR whose variable (and constant) region is encoded by a distinct set of genetic loci (the γ locus and the δ locus) homologous to the α and β TCR loci¹⁰³ (Table 9.3). These TCR genes rearrange in the thymus in a manner analogous to that of the
dominant α : β TCR T-cell homolog. The generation of γ : δ T cells in the thymus precedes α : β T-cell development. The γ : δ T cells are generated in two waves during T-cell development in the fetus. The first wave of maturing thymocytes consists of cells with limited TCR V gene diversity (i.e., utilize one or few Vγ and Vδ rearrangement in their TCR). After selection, these γ : δ T cells leave the thymus, traffick through the circulation, and preferentially localize to epithelial surfaces, particularly the skin, gut, and urogenital tract. The second wave of γ : δ T cells, as with α : β T cells, displays a highly diverse TCR array, and these cells localize to the secondary lymphoid tissues. Unlike the α : β TCR, however, the γ : δ TCR does not recognize peptides bound to the MHC class I or II locus products. Rather, γ : δ T cells either directly recognize structures such as heat shock proteins displayed on the surface of the infected cells, or recognize lipid or glyco/phospholipids moieties bound to MHC-like molecules (e.g., nonpolymorphic MHC Ib molecules) on the surface of stressed (infected) cells. The subset of intraepithelial γ : δ T cells, in particular, differs from the α : β T cells, and appears to function like cells of the innate immune system, recognizing not a specific pathogen, but alterations in epithelial cells induced by infection with microbes such as bacteria. At present, no direct role for γ : δ T cells in the control of virus infection has been firmly established although this T-cell subset has been implicated in the control of HIV and herpesvirus infection in humans.⁵

Among the other T-lymphocyte subsets (Table 9.3) generated during development are NK T cells, so-called because these α : β TCR-expressing T cells display cell-surface markers shared with NK cells, most notably the C-type lectin NK 1.1. NK T cells are divided into two subsets CD4⁺ CD8− or CD4−CD8− based on co-receptor expression. One subset of NK T cells employs a range of α : β V gene combinations in their expressed TCR. The other NK T-cell subset is similar to CD4− and CD8− γ : δ T cells, in that the cells utilize a limited number of TCR α : β V genes in receptor generation and are classified as invariant NK T cells (iNK T cells). The iNK T cells recognize foreign and self-lipid/glycolipid bound to MHC-like cell-surface CD1 molecules. The NK T cells with diverse TCR usage by contrast, directly recognize self- or microbial gene products rather than peptide/MHC complexes. Although not as yet certain, the CD1/lipid complexes and the self-moieties (e.g., heat shock proteins) recognized by NK T cells are likely produced or upregulated by cells in response to virus infection.

One T-lymphocyte subset has recently been recognized as a critical regulator of the adaptive immune responses to both infectious agents and self-molecules: the regulator T lymphocyte (T_reg) subset. The TCR on T_reg cells utilize rearranged α : β TCR gene products, and appear (at least in experimental animals) to arise late in thymic development (i.e., after the major wave of both γ : δ αvδα : β T-cell generation). As mature thymocytes, T_reg cells appear able to recognize self-peptides bound to MHC molecules and directly activate without undergoing negative selection. Therefore, T_reg cells that display primarily the CD4 co-receptor exist both in the thymus (as mature thymocytes) and in the periphery (i.e., in secondary lymphoid organs) as activated CD4⁺ T cells. The encounter with self and the resulting activation of T_reg cells, however, does not result in autoimmunity. Rather, recent evidence suggests that these T cells suppress/downregulate the activity of conventional CD4⁺ and CD8⁺ T cells responding to viral infection. Although both the role of T_reg cells in adaptive immune responses and their mechanism of action are not yet fully understood, these cells may prevent the development of excessive injury during T-cell responses to infection, and suppress the development of autoimmune responses during viral infection. Recent emerging information on the role of T_reg cells in virus infection is discussed in a separate section below.

The mature naïve (resting) B and T lymphocytes that egress from their development site (bone marrow and thymus, respectively) enter the blood, and then migrate to secondary (peripheral) lymphoid organs (SLOs). The SLOs play a critical role in the initiation of the adaptive (T- and B-cell) immune response to viruses and other microbes. The major SLOs are the lymph nodes (LNs), the spleen, and the so-called mucosal-associated lymphoid tissue (MALT). LNs are highly organized structures located at vessel convergence points in the lymphatic system, a system that collects fluids and cells from the tissues of most internal organs (e.g., liver, stomach) and body surfaces (e.g., skin, mucosa of the gastrointestinal [GI], genitourinary [GU], and respiratory tracts). The capacity of lymphatic vessels to collect antigen (e.g., virus, viral constituents) from the body surfaces/tissues, and deliver them to the LNs (where antigen concentrates) is essential for the efficient triggering of the adaptive immune response to microbes. The spleen plays a role similar to that of the LN network; however, the spleen is uniquely suited to collect antigen (virus) directly from the bloodstream, because of its unique architecture and extensive blood circulation. The third class of SLOs, the MALT, is composed of specialized lymphoid aggregates associated with major mucosal surfaces and sites of microbial invasions (e.g., the GI, GU, and respiratory tracts). The gut-associated MALT (also called GALT) includes tonsils, adenoids, appendix, and Peyer’s patches in the small intestine. In the respiratory tract are similar, but more diffuse, aggregates of lymphoid tissues associated with the epithelium of small/medium airways (i.e., bronchial-associated lymphoid tissue [BALT]). As with the other SLOs, the MALTs are sites of antigen localization where the initiation/induction of the adaptive immune response can occur.

The common feature of SLOs is the localization of adaptive immune cells (i.e., T and B cells) to distinct regions of the SLOs. For example, most B cells are localized in distinct follicles within the LN cortex. Activation of B cells specific for a pathogen/antigen leads to their proliferation and formation of a germinal center within the LN cortex. T cells within the LN localize to, and are diffusely distributed within, the LN paracortical areas. Other cell types are also distributed within the outer cortex and central medulla of the normal LN. These include stromal cells and cells of macrophage and DC lineage. These cells (particularly DC) play a crucial role in the induction of the adaptive immune response, and serve to direct T-cell/B-cell localization to distinct LN regions through the constitutive release of small (low molecular weight) chemotactic proteins—chemokines. A similar segregation of B cells/T cells to distinct regions occurs within the white pulp of the spleen and the MALT (likewise, under control of chemokines elaborated by SLO-resident cells).

The body’s complement of B and T cells consists of more than 10⁹ cells, each of which may display a unique antigen receptor directed to a specific epitope. Therefore, the frequency of B or T cells directed to a specific pathogen is extremely low (e.g., a frequency of 1 of 10⁴ to 10⁶ naïve B or T cells). The problem faced by the adaptive immune systems is that these rare, antigen-specific cells are distributed randomly throughout the body, and must be rapidly mobilized to respond to invading organism. These rare antigen-specific cells must first contact antigen, then activate and generate effector cells capable of eliminating the pathogen. The solution to this problem is achieved through the process of lymphocyte recirculation through the SLO.⁵⁷,²⁷³

Naïve (primary) T cells and most naïve B cells do not permanently reside in the SLOs. Rather, the T and B cells are constantly migrating from the blood through the SLOs and back to the bloodstream. Lymphocyte recirculation is controlled by the expression of distinct homing receptors (adhesion molecules) whose ligands are expressed on cells within the SLO. Naïve CD4⁺ and CD8⁺ T cells within the bloodstream express on their surface the adhesion/homing receptor, CD62L (L-selectin). T cells entering the blood vessels encounter the CD62L ligands—the mucin-like moieties CD34 and GlyCam-1 that are constitutively displayed in the endothelium of specialized high endothelial venules (HEVs) within the LN cortex. The interaction of CD62L (on the T cell) with its endothelial cell ligands arrests the T cell’s migration through the blood vessel, which results in its binding to the HEV endothelial cell. Other adhesion receptors displayed on endothelial cells that recognize carbohydrate ligands displayed on the surface of lymphocytes (as well as the α_Lβ₂ integrin, lymphocyte function-associated antigen 1 [LFA-1] displayed on lymphocytes) also contribute to the arrest of T-cell migration. The naïve circulating T cells also constitutively express a chemokine receptor (CCR7) that recognizes specific chemokines (CCL19, CCL21) displayed on the surface of the HEV endothelial cells. These chemokines are produced by the HEV cells and by cell types within the LN (e.g., DC and stromal cells). The chemokines produced by the latter cell types diffuse into the HEV and are bound to/displayed on, the surface of the HEV endothelial cell. The interaction between the CCR7 receptor on the arrested T cells and its chemokine ligand triggers the migration of the naïve T cells through the HEV and into the LN cortex (parafollicular T-cell region). If the T cells do not encounter antigen, they traffic through the LN cortex to the LN medulla, and then exit the LN though the efferent lymphatic vessels, and ultimately return to the bloodstream where the process of recirculation though the SLO begins again. The egress of naïve T cells from the LN is as noted above, controlled by a chemotactic receptor S1P1R for the lipid sphingosine-1-P (S1P). S1P is present at higher concentrations in lymph and blood than in the LN. S1P1R engagement attracts naïve T cells away from the LN and back into the circulation. Antigen recognition by T cells transiently downregulates S1P1R retaining the antigen detecting T cells in the LN.

Naïve B cells traffic through the LN by similar mechanisms and transiently localize to the lymphoid follicles along a chemokine gradient mediated by a specific chemokine receptor, CXCR5, displayed on the surface of circulating naïve B cells. Lymphocyte trafficking through the spleen and MALT are likewise regulated by the interplay of adhesion receptor/ligand interactions and chemokines constitutively expressed in these SLOs. In the case of MALT, a set of unique receptor/ligand interactions (restricted to these sites) guides the trafficking of adaptive immune cells to these specialized mucosal sites.

Afferent lymphatic vessels are localized in all body surfaces/tissues, and they drain into the LN. Virus infection of a body surface/tissue triggers the innate immune system—the initial defense against infection. Innate immune cells located at the body surfaces/tissues (specifically, DC and monocyte/macrophages) encounter pathogen, take up virus/viral antigen, and then activate/migrate through the afferent lymphatics to the draining LN. In the draining LN, these migrating cells (along with DC and, to a lesser extent, monocyte/macrophages present in the LN cortex) serve as antigen-bearing presenting cells (APCs) to the naïve B and T lymphocytes trafficking through the LN. As a result of encounter with antigen displayed on the APCs, the lymphocytes with antigen receptors specific for the antigen or pathogen will no longer traffic through the LN, but rather will be retained in the LN (due to S1P1R downregulation) and initiate the process of activation and differentiation, leading to the generation of specific effector B and T cells directed to the pathogen. Therefore, the SLO concentrate antigen in a specific site where the rare antigen-specific, naïve B and T cells circulating through the body can encounter and respond to the pathogen. Afferent lymphatics can also carry large particulates (e.g., viruses as well as “soluble” viral proteins) directly to the LN where the particulates can be captured by macrophages lining subcapsular lymph node sinuses and subsequently delivered to DC or presented to naïve T cells, as well as directed along with soluble antigens through fibroreticular conduits to B cells and a lymphoid follicles.⁹⁵,¹²⁵

If infection by a virus is localized to the body surface (e.g., papillomavirus infection of the skin or influenza virus infection of the respiratory tract), then the adaptive immune response to that pathogen will be induced primarily in the LN draining these sites. Virus infections that result in systemic spread (i.e., viremia) will also induce adaptive immune responses in the spleen. Viruses that infect sites where MALT is prominent (e.g., rotavirus infection in the Peyer’s patches of the small intestine) will trigger the local induction of the adaptive immune responses at the site of MALT localization.¹⁹⁶

The precise localization of T cells, B cells, DCs, and macrophages within the SLOs is controlled by chemokines, which are largely produced by stromal cells of the SLOs, and by bone marrow–derived cells (e.g., SLO-resident DCs). In draining LN, naïve (primary) T cells that are present in the circulation will enter the LN through the specialized HEV, where they encounter mature DCs (and macrophages) displaying processed antigen bound to MHC molecules, which are localized to sites in the parafollicular cortex of the LN facilitate interaction with T cells. Most naïve CD8⁺ T cell and CD4⁺ T cells entering the LN will not recognize the antigen (i.e., they have TCRs that fail to recognize the processed antigen with sufficient avidity to activate the cells). These T cells will ignore the DCs and migrate from the LN cortex to the medulla, then enter the efferent lymphatics, and ultimately return to the bloodstream to continue their perpetual search for relevant antigen.²⁷³ A similar process of lymphocyte egress through the HEV occurs in the MALT; for example, Peyer’s patches of the small intestine where antigen uptake by mucosal DCs and direct viral antigen transfer to the gut MALT through specialized intestinal epithelial cells, called M cells, result in antigen delivery to these mucosal lymphoid aggregates.¹⁵⁰ Viruses that enter the bloodstream (i.e., produce viremia) as well as any antigen-bearing tissue DCs that, on maturation, bypass the LN and gain access to the circulation will localize to the splenic white pulp. Here, circulating T and B lymphocytes enter the splenic white pulp by transiting through splenic arterioles where viral antigen-bearing DCs can encounter lymphocytes. The lymphocytes that do not recognize antigen will exit the splenic white pulp by entering trabecular veins, and re-enter the circulation.

The rare naïve CD8⁺ T cells and CD4⁺ T cells that recognize processed antigenic peptides displayed on the APC surface in the SLO do so through TCR engagement. The activation of naïve T cells leading to the induction of the adaptive immune response is a multistep process (at both the molecular and cellular level).¹¹² For induction of antigen-specific (virus-specific) CD8⁺ T-cell responses, the activation process requires both interaction of the antigen-specific TCR α : β chain complex with processed antigen fragments bound to MHC class I molecules on the APC surface, with sufficient avidity and interaction of the CD8 molecule on the T cell with a region of the MHC class I molecule distant from the site of peptide binding. To fully activate the naïve CD8⁺ (or CD4⁺) T cells, additional signaling events must occur, including binding of the co-stimulatory receptor, the CD28 molecule, displayed on naïve T lymphocytes with its ligands, CD80/86, which are displayed at high levels on mature (activated), antigen-bearing DCs. The sequence of events resulting in the activation of antigen-specific naïve CD4⁺ T cells parallels the steps in CD8⁺ T-cell activation, except that the antigen-recognizing TCR α : β chain complex must bind (with sufficient avidity) the antigenic peptides bound to MHC class II molecules displayed by APC; and the CD4 co-receptor on CD4⁺ T cells, in turn, must interact with a distinct site (outside of the region of peptide binding) on the MHC class II molecule. One important early consequence of CD4⁺ T-cell activation by antigen is the upregulation of the expression of the CD154 molecule on activated CD4⁺ T cells. CD154 (also called CD40 ligand or CD40L) is a member of the TNF gene family, and, as its name implies, is a ligand for CD40 (a TNF receptor gene family member). CD40 is expressed on both activated/mature DCs and antigen-activated B cells. Engagement of CD40 on DCs and B cells by CD154 (CD40L) on activated CD4⁺ T cells has important consequences for the function of these cells. Engagement of CD40 on DCs results in increased synthesis of certain cytokines (notably IL-12) by the antigen-bearing DCs in the SLO, which promotes CD4⁺ and CD8⁺
T-cell differentiation into effectors.¹⁴² For B cells, the interaction between CD40-activated B cells and CD40L expressed on activated CD4⁺ T cells is critical for B-cell differentiation into antibody-secreting effector B cells (plasma cells).¹⁸² Strategies to modulate the CD40/CD40L interaction in vivo may have important consequences in vaccine development.

The engagement of the TCR/co-receptor complex, and the CD28 co-stimulatory receptor on T cells in the SLO are the first two critical steps in the induction of a T-cell response to a foreign antigen. This initial antigen encounter by the TCR on specific T cells is called signal 1 in the lexicon of lymphocyte activation/differentiation. The engagement of the CD28 co-stimulatory receptor is designated as signal 2. As a consequence of their interaction with antigen and co-stimulatory ligand displaying DC APC, the naïve T cells halt their transit through the SLO (i.e., downregulation of S1P1R, and are retained there). The APC/T cell encounter leading to signal 1 and 2 transmission in the T cells appears to be of short duration (from 6 to 24 hours), after which the initial programming for T-cell activation/differentiation is independent of additional interaction with antigen.¹⁷⁴,²⁹³ Delivery of signals 1 and 2, although necessary, is not sufficient in itself, however, to ensure full T-cell activation/differentiation and the generation of effector and memory T cells to the antigen. A third signal, signal 3, is required to complete the T-cell activation/differentiation program in the SLO. Signal 3 is delivered by soluble mediators (i.e., cytokines and chemokines) produced primarily by cells of the innate immune system (e.g., pDCs mature/activated in cDCs, NK cells, NK T cells), which enter the SLO in response to infection/antigen deposition in the SLO. IL-12 produced by mature (activated) DC in the SLO is a particularly critical type of signal 2 in T-cell responses to virus, because engagement of the IL-12 receptor on activated CD8⁺ T cell and CD4⁺ T cells promotes their differentiation into effector T cells with potent antiviral activity (e.g., the generation of cytolytic T cells).¹⁹⁹

Other cytokines produced during T-cell activation (e.g., type 1 and type II/III IFNs [IFN-α, β, IFN-γ, IFN-l, respectively], IL-1, IL-4, IL-6, IL-10 and so on) by innate immune cells responding in the SLOs to virus infection will dramatically modulate the magnitude and type of both the effector and memory T-cell responses. These and other soluble mediators modify the course of T-cell activation/differentiation during the T cell’s residence in the SLO.¹⁴²

Similarly, in addition to CD28, other co-stimulatory receptors belonging (like CD28) to the Ig receptor gene family (e.g., Cytolytic T-Lymphocyte antigen-4 [CTL A-4]; inducible T-cell co-stimulator [ICOS]; programmed death-1 [PD-1]) and the TNF receptor gene family (e.g., CD27, OX40, 4-1BB, CD40L) whose expression on activated T cells is temporarily regulated following T-cell activation in the SLO, can either positively (ICOS, CD27) or negatively (PD-1, CTLA-4) regulate the tempo and magnitude of T-cell activation and differentiation into effector cells in the SLO or in peripheral sites of virus infection following interaction with their perspective ligands on APC.²⁴⁸,²⁵²

The temporal sequence of signal 1, 2, and 3 delivery to T lymphocytes in the SLO is reflected in the morphologic change of the naïve (primary) T cells from small, resting cells to activated lymphoblasts. This is followed by proliferative expansion of the antigen-specific T-cell clones in the SLO, and their differentiation into both effector T cells and an expanded population of memory CD8⁺ T cell and CD4⁺ T cells. T-cell proliferation is extremely rapid (cell cycle time of ≤6 hours and in one report ≤2 hours,³¹⁷ with estimates of individual naïve T-cell precursors undergoing 8 to 20 divisions during this programmed proliferation.¹³⁷ During their proliferative expansion and differentiation in the SLO, the responding T cells lose cell-surface adhesion molecules (e.g., CD62L) and receptors (e.g., CCR7) necessary for naïve T-cell circulation, and they upregulate the expression or express de novo adhesion molecules (e.g., the β2 integrin, LFA-1, and β1 integrin, very late antigen 4 [VLA-4], respectively), and chemokine receptors (e.g., CCR5, CXCR3) essential for effector T cells to localize to sites of inflammation.¹⁶⁵ At the end of the proliferation/differentiation sequence, most activated antigen-specific effector T cells will upregulate S1P1R and exit the SLO (through the efferent lymphatics in the case of T cells responding in the draining LN), enter the circulation, and traffic to the site of infection/inflammation. The homing of activated T cells is controlled by chemokines released by innate immune cells (as well as by epithelial and endothelial cells and fibroblasts) at the site of inflammation/infection (e.g., CXCL9 [Mig/CXCL9 (Monocline Induced by Gamma interferon)], CXCL10 [IP-10 (Interferon gamma induced Protein-10)], and CXCL11 [I-TAC (Interferon-inducible T cell Alpha Chemoattractant)] for CXCR3; and CCL3 [macrophage inflammatory protein-1α; MIP-1α] and CCL4 [MIP-1β] for CCR5). This homing is also controlled by the inflammation-induced upregulation of integrin ligands (e.g., intercellular adhesion molecule-1 [ICAM-1] for LFA-1, and vascular adhesion molecule [VCAM] for VLA-4) and adhesion receptors (P-selectin, E-selectin) on endothelial/epithelial cells at these sites.

The generation of effector T cells in the SLOs responding to infection can occur in as few as 5 to 7 days in both experimental models and human viral infection.⁶⁸ Given the rapid replication of most viruses, it is obviously essential to get specific T cells activated and mobilized as soon as possible through the concentration of antigen and the accumulation of the rare antigen-specific T cells in the SLO, followed by rapid proliferative expansion/differentiation of these cells into effectors in the SLO. The rapid induction of an adaptive immune response to virus infection is not a foregone conclusion, however. The process of T- and B-cell activation can be delayed for weeks to months in the case of infection with viruses such as HIV and HCV, which induce chronic infection.¹¹¹,¹⁸⁷ In these instances, the virus has the capacity to suppress/delay the induction of the adaptive immune response.

The process of naïve B-cell activation in the SLOs is similar to that of naïve T-cell activation. In the case of B-cell responses to viruses and other T cell–dependent antigens, however, antigen-specific CD4⁺ T cells play a critical role in driving B-cell activation/differentiation. As with naïve T cells, naïve B cells enter the LN though the HEV; unlike T cells, however, the B cells migrate from the paracortical (T-cell rich) area of the LN and into the B cell–enriched LN cortical follicle. This migration is controlled by the chemokine CXCL13, produced by a stromal cell type resident in the follicle (called a follicular DC because of its dendritic morphology, but not of hematopoietic origin). CXCL13 is the ligand for CXCR5 chemokine receptor that is constitutively expressed by naïve B cells. If the B cell does not encounter antigen in the follicle, it will ultimately migrate out of the cortical follicle to the medulla, then enter the efferent lymphatics, and rejoin the circulating pool of B cells in the bloodstream.

The B cells that display a BCR antigen receptor (i.e., the cell-surface membrane-bound form of the immunoglobulin molecule that recognizes antigen) for an antigen present in the LN would encounter this antigen immediately after migrating out of the HEV in the parafollicular (T-cell rich area) of the LN. In the case of viral antigen, the relative contribution of free virus, soluble viral proteins, and viral proteins transported by migrant DC/macrophages from the infection site to the reservoir of antigen present in the SLO and available to specific B cells is not certain. However, it has been recently appreciated that structures (i.e., conduits) within the SLO (in the case of LN) direct particulates like viruses and soluble proteins directly to regions of naïve B-cell accumulation in the SLOs.¹²⁵ Binding of antigen to the BCR (i.e., cross-linking cell surface immunoglobulin) provides the initial stimulus (signal 1) for specific B-cell activation. Signal 1 delivery halts the constitutive migration of the B cell from the paracortical T-cell zone to the LN follicle, and activates the chemokine-dependent migration of the B cells to the junction between the T-cell zone and the follicle. The strength of signal 1 can be greatly enhanced if the BCR co-receptor complex²⁰¹ is ligated simultaneously with the BCR by interaction with complement components bound to antigen or antigen–antibody complexes. In addition, cross-linking of the immunoglobulin receptor induces internalization of the antigen–immunoglobulin complex into an endosomal compartment where the antigen is fragmented and associates with MHC class II molecules that are constitutively expressed by B cells. The antigen/MHC class II complexes are then cycled to the B-cell surface, where they are available for recognition by antigen-specific CD4⁺ T cells. Another consequence of signal 1 delivery (by antigen to B cells) is CD40 upregulation. The CD4⁺ T cells, which interact with antigen-stimulated B cells, are themselves activated by prior encounter with antigen/MHC class II complexes on APC (i.e., mature, antigen-bearing DC), and have upregulated CD154 (CD40L) expression. The activated antigen-specific CD4⁺ T cells recognize the antigen/MHC complexes on the antigen-stimulated B cells, and deliver signal 2 to the B cells in two forms: interaction of CD40L (on T cells) with CD40 (on B cells), and the release of T-cell–derived cytokines (notably, IL-4, IL-21, and/or IFN-γ) whose synthesis is triggered in the CD4⁺ T-cell TCR recognition of antigenic peptides displayed by the B cells. Antigen-activated CD4⁺ T cells in LN may support B cell responses by differentiating into so-called T follicular helper (T_FH) cells. These activated CD4⁺ T cells appear to represent a distinct CXCR 5⁺/PD-1⁺ CD4⁺ T-cell subset, the generation of which is in part regulated by IL-6 produced within the responding LN. The cells secrete IL-21 in response to TCR engagement and also express CD154 (CD40L). The interaction of the T_FH with the naïve parafollicular B cells through CD40 engagement (on B cells) by CD40L on the T cells, and IL-21 signaling through its receptor on naïve T cells helps support the formation of B-cell germinal centers.

The fully activated (signals 1⁺2⁺) B-cell lymphoblasts undergo programmed proliferation after migration to the follicles to produce germinal center, which are follicular structures consisting primarily of responding/proliferating B-cell lymphoblasts. The proliferation/differentiation of the germinal center B-cell lymphoblasts results in the formation of antibody-secreting plasma cells, which migrate from the LN to the sites of infection/inflammation, and more importantly to the bone marrow where the plasma cells serve as a depot of long-lived antibody-secreting cells.¹⁶⁷ B cells that encounter antigen without CD4⁺ T-cell help (i.e., receive signal 1, but not signal 2) are capable of producing specific secreted antibodies; however, these antibodies will be of the immunoglobulin M (IgM) isotype and of low affinity. The isotype switch of antibody production from IgM to the IgG, IgA, and so on isotypes, as well as, the process of antibody affinity maturation requires T-cell help (signal 2).¹⁶⁷