24 B-cell-T-cell co-opearation

Dr. M. N. Gupta

1. Objectives

• To learn about cell surface markers of APC, B cells and T-cells
• To understand how encounters of B cells and T-cells take place in primary form and germinal centres.
• To understand the role of surface markers in cell-cell cooperation
• To understand why innate immunity must precede acquired immunity

2. Concept Map

3. Description

In a way, the last few chapters can be thought of introducing the cast of drama. This chapter describes the drama and indicates the role played by each member of the cast.

Briefly B-cells need T-cell cooperation. Other APCs can also activate T-cells. This interaction leads to signalling events to result in specific gene transcription.

Signal transduction involves phosphorylation-dephosphorylation which is one of the common signal transduction mechanism in cell biology and cell metabolism in particular.

TcR recognition of the processed antigen is the first step in cell-cell cooperation. Once this interaction has taken place, accessory molecules firm up this binding between APC and T-cells.

The contacts between TcR, accessory and co-receptor molecules with antigen fragment:

MHC complex and ligands on APC are called “immunological synapse”.

Formation of this immunological synapse actually creates a signalling domain. The signalling to the genes initiates specific transcriptions. Phosphorylation and dephosphorylation is involved in the signal transduction.

The importance of B-cell- Th cell cooperation can be appreciated by recalling that these are thymus independent (T-independent) antigens. Consisting of repeating structures, these crosslink B-receptors.

These also result in secretion of IL-1, IL-6, and TNFα from macrophages. Most of the T-independent antigens are not degradable easily so cannot be processed for eliciting presentation to T-cells. Importance of this property can be gauzed by the fact that poly-L-amino acid (unlike poly-D-amino acids) are T-dependent antigens.

The non-involvement of T-cells means IL-2, IL-4 and IL-5 secreted by T-cells are not available. Normally T-independent antigens involve activation of CD5+ subsets of B-cells.

All these features result in poor primary and secondary responses. There is no class switching. There is no affinity maturation. The T-independent antigens induce a response which peaks earlier than the corresponding responses by T-dependent antigens.

As was mentioned earlier, many pathogens and their products are in fact T-independent antigens. To recollect that list, it includes endotoxins, dextrans, levans and polymerized flagellin in bacterial flagellae.

So, are there any advantages to the host in non-involvement of T-cells or absence of B-cell-T-cells cooperation in case of these T-independent antigen? To start with, as complex cell-cell interaction are not required, the response is rapid.

In any case, these are able to activate macrophages which produce cytokines (IL-1, IL-6 and TNF-α). The absence of T-cell secreted IL-2, IL-4 and IL-5 results in a short lived immune response and absence of IgG production class switching, T-independent antigens activate a B-cell subsets which has CD5 marker.

T-cells (Th cells) get clonally selected by any APC (which need not be B-cell). Hence, an antigenic peptide:MHC complex on any APC leads to clonal selection of T-cells specific to these antigen (Fragments).

These clonally selected T-cells there participates in T-cell-B cell cooperation. It may be emphasized that T-cells recognize some antigenic peptides and B-cells recognise different antigenic peptides arising from the same antigen.

Hence clonal selection of B-cells which is initiated by a soluble antigen and operated at the level of whole antigen is carried forward.

When B-cell-T-cell co-operation is initiated, B-cells also display antigenic peptide:MHC complex. The T-cell selection by APC operates via TcR. However, as has been mentioned earlier, APC also has co-stimulatory molecules which engage corresponding ligand on T-cells.

CD 86 is a common co-stimulator molecule on many APCs and it recognises CD28 on T-cells. These two interactions are the critical two signals which activate T-cell. T-cell-APC interacting in such a way are called “immunological synapse”

B-cells also similarly require two signals to become effector cells: BcR interacting with antigen and signals from Th cells for optimal differentiation and proliferation.

It is necessary to view activation of both B-cell and T-cell activation in the context of time and place. APC activation is often rapid and can be in response to antigen presence alone in the form of a pathogen. However, more complete APC response does require T-cell help in the form of cytokines secreted by T-cells.

This T-cell involvement produces more MHC class I or II molecules on APC, larger number of receptors for Fc and adhesion molecules. In turn, APC also secretes quite a few cytokines. Hence, T-cell cooperation makes APC function more effective as the immune cell component. This also indicates that adaptive immune responses evolved later and strengthened the innate immunity. So evolution segued in the adaptive immunity rather than creating a totally independent ab initio design. Discarding an existing design and creating a totally unrelated new design would have been perhaps wasteful approach.

Once B-cells become plasma cells and start secreting specific antibodies, they no longer express MHC class II molecule and no longer respond to any signals for differentiation and proliferation. At that stage, T-cell cooperation has ceased as it is no longer necessary.

At a specific point in time, T-cells have complete TcR. B-cells also have BcR containing surface IgM and IgD along with other surface molecules.

Sometime an antigen which is normally presented by class II pathway happens to be presented on MHC class I molecules. This mechanism called cross-presentation facilitates APCs to present viral antigens to CD8 Tc cells, even if APCs are not site of the infection.

Cell surface markers of cells involved as APC

As we have mentioned before, cell surfaces consist of diverse kind of molecules. These cell surface molecules becomes the best indentifying mark of individuality. Hence these are called cell markers.

mAbs are the best tools to recognise and catalogue the surface markers of different cells. CD (cluster of differentiation) numbers identify each marker systematically. As we have already seen, cells at different stages of development and differentiation often have different surface markers.

Thus, some are specific to the lineages (called lineage markers) while others appearing at different stages of development and differentiation often have different surface markers.

Those which appear only as a result of stimulus (upon activation) are called activation markers.

Thus, CD3 is a lineage marker for T-cells. CD1 is a maturation marker which appears in thymus. CD25 is the T-cell growth factor receptor and is thus activation marker of T-cells. Frequently, as happens with any nomenclature system, other names are also used which are more descriptive of the function of the molecule. For example, CD-25 is also called IL-2 receptor.

It happens in some cases that maturation marker for one lineage is activation marker for another type of cells. The MHC class II is there on B-cells during much of their existence, for human T-cells it is an activation marker. Also, activation markers may not appear but may increase in number upon stimulation. CD11a (LFA-1) on monocytes exemplifies this.

Many surface markers seem to have evolved from a common ancestral gene. Members of immunoglobulin superfamily shares many common structural features. This family includes CD2, CD3, CD4, CD8 etc. Another such family is integrin family. It has many sub families. Integrins are dimers of α type and β type chains. Each subfamily has common β chain but characteristic α chain.

Thus, we have β2-integrins with β-chains as CD18. The α-chains could be CD11a, CD11b, CD11c to form integrins LFA-1, Mac 1 and CR4 in leucocytes. Similarly β1- integrin subfamily shares CD29 and consists of very late activation (VLA) markers.

Uses of fluorescent antibodies and flow cytometry have further facilitated characterization of cells on the basis of their surface molecules.

Before we discuss cell interaction between APC, B-cells and T-cells, it will facilitate discussion if we at this point look at their surface markers. We have casually referred to them but we can especially focus on those which are involved in cell-cell cooperation.

Historically, the first marker discovered on T-cells was the one which enables T-cells to bind to sheep RBC, it was given the name CD2. However TcR is the lineage marker. Both TcR-1 (γ:δ) and TcR-2 (α:β) are associated with five polypeptide complex of CD3.

The role of CD3 is critical for signal transduction after T-cell activation. In humans, CD3 has membrane spanning γ, δ, ε, δ chains. γ, δ, ε are members of Ig superfamily. δ chains occur as dimers. In mice, instead of that δ occurs as heterodimer of δ-ε chains.

It is δ-δ dimer which is linked to the ITAM through which signal transduction occurs. Sometime Ti is used to refer to the part of TcR which is involved in binding of antigen:MHC complex. This in a way can be viewed as Fab (of TcR attached) to constant domains. We may recollect that this part of TcR also is formed by gene rearrangement similar to BcR.

The TcR-2 has two forms: CD4+ or CD8+. Expression of CD45RO/CD24 by CD4+ Th cells reflects the cell activation. The cytokines dictate expression of activation markers etc.

Some other markers also characterize all T-cells. These include CD2 which is also involved in cell activation. CD5 also is involved in the cell activation but is also present on a subset of B-cells.

As has been hinted, surface markers are species characteristics. Murine T-cells express thy-1 or ζ. Instead of CD5 of human T-cells, murine cells have Ly-1. The CD4 and CD8 equivalents are L3T4 and L-2.3 respectively.

The circulatory B-cells in peripheral blood express IgM and IgD with identical antigen binding specificities. The B-cells in intestinal mucosa has IgA instead. Again many other surface markers are present on human and murine B-cells. Some are homologoes in two, some are different.

The class II MHC antigens, of course, are most important. In mice, these are I-A or I-E and in humans these are HLA-DP, DQ and DR. Complement receptors notably for C3b (CR1, CD35) and C3d (CR2, CD21) are commonly found in both mice and human B-cells. FcγRII/CD32 are Fc receptors involved in negative signalling to B-cells.

Human B-cell lineage markers are CD19, CD20 and CD22, CD72-CD78. A subset of B-cells which shows specificity towards autoantigens including DNA, Fc of Ig, phospholipids and cytoskeletal component compounds express CD5 and a receptor for mouse erythrocytes!

Activation markers for T and B-cells

Both lymphocytes produce receptors for cytokines including IL-2. IL-2R in T-cells consists of a low affinity receptor of 55 kD (CD25) and a higher affinity receptor of 70kD. Activated T-cells sees expression of receptor for CDT1 (transferrin) and CD38. Interestingly, these two are present on T-cells prior to thymic entry, disappear during thymic stay and appear again on activation.

Memory cells carry the late activation marker CD29. These cells also express large number of receptors which are termed adhesion molecules. These prime these cells for again becoming effector cells on second encounter with the antigen.

B-cell‟s activation markers, apart from IL-2R, include receptor for molecules which are responsible for growth and differentiation such as IL-3, IL-4, IL-5 and IL-6. C8-71 and higher expression of MHC class II molecules is also seen. Again CD38 occurs during early stages, disappears during much of the later phases and only appears again when B-cells are terminally differentiated with plasma cells.

Another marker for terminally differentiated plasma cells is PCA-1. Memory cells in germinal centres (we will discuss these shortly in reasonable detail) in secondary lymphoid tissues do not express IgD or CD22.

Just to recollect, mononuclear phagocytes which arise from bone marrow stem cells and become “professional macrophages” are part of innate immunity and in that sense are not part of cell-cell cooperation which we are discussing largely in the context of adaptive immunity.

However no matter how innate immunity operated in early days of evolution, at present these macrophages do depend upon T-cell derived cytokines for efficient functioning in several cases.

These macrophages uptake particulate antigens: which pathogens are! While macrophages present at different locations even carry different names, these seem to be part of a network. This was learnt by observing that fine carbon particles injected intravenously ended up in organs which are known to contain these mononuclear phagocytes.

Location wise liver, kidney and brain, these are referred to as kupffer cells, intraglomerular mesangial cells and brain microglia. Aleveolar and serosal macrophages are not fixed and are “wandering macrophages”.

Human (as well as murine) macrophages have mannosyl-fucosyl receptors (MFR) responsible for binding to sugars on microbial surfaces. CD14 is a receptor for binding to a receptor for LBP (lipopolysaccharide binding protein). LBP in serum coats gram negative bacteria and thus facilitates their uptake by macrophages.

FcγR1 (CD64), FcγRII (CD32), FcγRIII (CD 16) are Fc receptors in human macrophages which have low, medium and high affinity for Igs via their Fc fragments. So, we can see again how innate immunity is connected to various arms of adaptive immunity.

Complement receptor CR1 (CD35, aC3b receptor) is also present along with CR3 (CD11b, C3bi receptor); the latter appearing as activated macrophage marker. LFA-1 (CD11a) and p150 (CD11c) are also similar markers.

Intracytoplasmic vesicles in macrophages also have typical activated markers such as CD11b and CD11c. MHC class II antigens are present on some macrophages which allows these to act as APCs for T-cells.

Macrophages have receptors for IL-4, IFN-γ and MIF (migration inhibition factor). That is how macrophages derive T-cell cooperation. In turn macrophages produce their own cytokines such as IFNs, IL-1 and TNF.

Human macrophages also express CD13, CD15, CD68 and VLA-4 (CD29/CD49d). None of these except possibly FcγR1 can serve as lineage marker for human macrophages.

Apart from B-cells and macrophages, dendritic cells are the third important APCs. How immature dendritic cells become dendritic cells in lymphoid tissue is discussed elsewhere.

Table 1: Dendritic Cells

There are various ways of classifying dendritic cells. Two major groups can be:

• One class expresses high levels of class II molecules. These act as APCs for T-cells.
• The second class resides in lymphoid tissue follicles. These donot express MHC class II molecules but have markers which enable these to interact with B-cells.

The first class can be further subdivided into two subclasses of myeloid derived dendritic cells and plasmacytoids which have longer life time than former kind and reside in bone marrow or peripheral organs.

CD44 is a marker on dendritic cells which acts as an adhesion molecule which participates in binding of dendritic cells to connective tissues.

Surface markers on dendritic cells include:

•  Mannosyl/fucosyl receptors

•  Toll like receptors (TLR1, 2, 3, 4, 5)

•  NOD-like receptors

•  R14-1-like receptors (for viral RNA)

•  Scavenger receptors (for carbohydrates/lipids)

Plasmacytrid dendritic cells have TLR10 (CD 290). A C-type lectin receptor called DC-SIGN was originally identified as adhesion molecule for ICAM 3 and hence was named Dendritic Cell Specific Intercellular adhesion molecule-3-Grabbing Non integrin. DC sign is also present on macrophages and specificity for carbohydrates on viruses, bacteria and fungi. DC-SIGN probably plays an important role between APC and T-cells.

Role of adhesion molecules

Interaction of T-cells to all other cells operates through a spectrum of adhesion molecules on T-cell surfaces recognising corresponding spectrum of adhesion molecules on the partner cell. These kinds of interaction are not antigen specific and in fact many adhesion molecules involved are not restricted to T-cell interactions.

The adhesion molecules include selectins, integrins, Ig superfamily members and some mucin like substances. L-selectin (CD 62L) present on naïve T-cells helps their migrating from the blood into peripheral lymphoid tissues. L-selectin binds to vascular addressin (mucin like molecules) and GlyCAM-1. In mucosal endothelium, L-selectin binds to MAdCAM-1, another addression.

All T-cells express a β2-integrin commonly known as LFA-1 (Lymphocyte function associated antigen-1). Also, present on macrophages and neutrophils, its common role is recruitment of cells to site of infection. T-cells also have CD2 and β1 integrins. β1 -integrins expression increases at late phase of activation and hence it is also called VLAs (very late activation antigens).

LFA-1 binds ICAMs and this is important for cell migration, T-cell-APC interaction or T-cell target cell interactions. ICAM-1 and 2 are present both on endothelium cells and APCs, ICAM-3 on APC. It interacts, for example strongly with DC-SIGN on dendritic cells.

The APC- T-cell encounter starts with LFA-2 (CD2) on T-cells recognising LFA-3 on APC. This is accompanied by LFA-1 on T-cells recognising ICAM-1 and -2 on APCs, ICAM-3 on T-cells recognising DC-SIGN on APCs.

T-cell- APC interaction generates a signal via TcR which in turn induces a conformational change in LFA-1 binding to ICAM-1 and -2 more strongly.

B-7 on APC recognising CD28 on T-cell generates the critical co-stimulatory signal which is required in addition to TcR-CD4 interacting with peptide:MHC class II on APC.

At this point, T-cell activation leads to its expressing higher level of CTLA-4 (CD152). CTLA-4 binds to B7 strongly by about 20x. This interaction displaces CD28-B7 interaction and this terminates T-cell proliferation.

B-cell-T-cell cooperation is based upon linked recognition. This means that both lymphocytes must recognise the epitopes on the same antigen. This necessitates that T-cell specific for antigen must have clonally selected to become activated before. So, B-cell-Tcell cooperation is preceded by an encounter of T-cell with an APC presenting the same antigen. Hence „antigen‟ may mean the pathogen. For example a virus coat will have several proteins. As far as T cell was clonally selected by one of those, it will cooperate with the B-cell provided peptide recognised by T-cell was a part of the antigen specific to the B-cell.

We have mentioned before that B-cells can also act as APC. In fact, this occurs during secondary response of humoral nature. The memory B-cells act as APC and are able to activate Th cells. As a follow up, these memory cells receive signals from activated Th cells and become effector cells again.

The CD40L appears only upon activation on T-cells and recognises CD40 on B-cells. Help from Th cells for B-cells proliferation, becoming either plasma cells or memory cells is critically dependent upon CD40-CD40L pair.

For both B-cells and T-cells to become activated, two signals are required. The second signal can come not only from a chemokine like interleukin but also from cell-cell interaction.

It is Th2 cells which secrete IL-4 and IL-5 which interact with B-cells and eosinophils. Th1 cells secrete IL-2 and γ-interferon and help Tc cells. Th1 cells also activate macrophages via IFN- γ and helps macrophages in destroying pathogen‟s responsible for infections like tuberculosis. Thus, unactivated macrophages would just offer innate immunity and are unable to fight such infections.

Th1 secreted IFN-γ induces expression of MHC class II molecules like endothelial cells enabling them to function as APC.

Local lymph nodes have T-cell zones through which naïve T-cells are passing continuously. Antigens processed and presented by APCs (such as dendritic cells) migrate to these T-cell zones and trap T-cells via T-cell-APC interaction already described. B-cells are also trapped by activation of adhesion molecules and chemokine receptors. This Th-B cell interaction establishes a primary focus of clonal selection. It is here that primary humoral immune response is initiated.

B-cells and T-cells which proliferate in primary focus can migrate into primary lymphoid follicle and form Germinal centres. These consist of proliferating B-cells, T-cells. It is at germinal centre that somatic hypermutation, affinity maturation and isotype switching takes place. Also, it is here that proliferating B-cells reduce IgD expression. Germinal centres are also the sites where B-cells differentiate either into plasma cells or memory cells.

What remains to be discussed is the detail of how peptides are presented by MHC molecules. While fine details of structures of MHC class II and class I molecule are different, then overall structure forms a similar peptide binding cleft.

This cleft in the case of MHC class II is open at both ends. Thus, ends of antigenic peptide binding to class I are somewhat buried in the latter, the corresponding parts of the peptide bound to MHC class I are not.

MHC molecule: peptide binding is very strong. So much so that isolated MHC molecules are invariably obtained in the bound peptide form. This is physiologically very relevant as antigen presentation by MHC is part of specificity aspect of adaptive immunity.

While MHC class I bind short peptides of 8-10 amino acids, peptides binding to MHC class II are not limited by this length. However, beyond 13-17 amino acid residues, the peptides bound to MHC class II are chopped away by peptidases.

Thus, APC, B-cells and T-cells interact to produce effective adaptive immunity. It is also clear from the preceding discussion that while innate immunity alone is many times not enough to fight infection. At the same time, it should also be realised that nonspecific innate immunity response which are initiated first are also responsible for adaptive immunity to take over and synergise with adaptive immunity.

In a bacterial infection, the first response is inflammation. Inflammation was recognized 200 yrs ago by its signs of tumor (swelling), rubor (reduces), calor (heat), dolor (pain). While inflammation can be also caused due to an injury like cutting, burning and exposure to a chemical, in the case of infection, it is followed by adaptive immune responses. Before that polymorphonuclear cells collect at the site within 30-60 min, in 5-6 hrs the monocytes start infiltrating the site. Macrophages set the stage for adaptive immune response if infection persists.

The immune response is fairly well orchestrated event with multiple elements. It is worth repeating that the seamless way the adaptive immunity synergised with innate immunity during evolution is an excellent example of evolution of biological design of living process.

Those who debated so acrimoniously in those days about relative importance of antibodies and cells in immune response never realized that:

•  Both were correct in a broad fashion to a varying degree.

•  The two arms of acquired immunity involves cooperation between humoral and cell mediated immunity.

Summary

• Cell surface marker of APC, B-cells and T-cells
• Lineage marker and activation marker for various cells
• Development of primary focus and germinal centres
• APC-T cells and B cell-T cell cooperation.