18 B-cell surface molecules

Dr. M. N. Gupta

1. Objectives

• To understand the early phase of development of B-cell and role of bone marrow stromal cells.

• To understand the role of gene segment recombination at these various stages.

• To understand the relative role of bone marrow and peripheral tissue in this development.

2. Concept Map

3. Description

Lymphocytes are of two kinds B-cells and T-cells. Both evolve from the stem cells. For both cells there are two broad phases of cell development. Antigen dependent and antigen-independent. Today we discuss details of antigen independent phase of the B-cell development.

While discussing clonal selection theory, we had discussed some features like how clones are selected from pre existing ones. In this discussion we will look at the creation of these clones of B-cells from the stem cell present in the bone marrow.

The essential initial step in the adaptive immune response is the interaction of B-lymphocyte cell surface with an antigen.

The exact nature of this contact, though may be different when macrophages and T-cells are present.

Nevertheless the contact changes the nature of gene expression in B-lymphocytes.

A chain of events starts which results in multiplication and differentiation of B-lymphocytes.

Before this B-lymphocytes were already specialized cells in the sense that these had the capacity to respond to the presence of antigen.

Their differentiation finally results in their becoming the plasma cells secreting antibody specific to antigens or becoming memory cells.

B–cells differentiation in fetal liver and then in bone marrow

By about 8-9 weeks of pregnancy in women (about 14 days in mouse), B-cell precursors can be identified in fetal liver.

Later, production of these gradually stops and is taken over by bone marrow.

Our insight into the stages of B-cell differentiation has come from studies on various animals in embryonic stages. There are some similarities in these steps in mice, chickens and humans.

In the chicken, the site of B-cell lymphopoiesis is a lympho-epithelial organ called the Bursa of Fabricius. In the early days many studies were devoted to identifying Bursa or its equivalent in mammals. Ultimately it was established that mammals do not have bursa or an equivalent organ. In mammals, these cells develop from the lymphoid stem cell in the fetal liver.

In chicken/quails, stem cells appear into bursa by about 10-14 days of life of embryo.

In other systems, bone marrow takes over differentiation of B-cells. This is true of other cells as well-erythrocytes, granulocytes, monocytes and platlets.

Stromal cells of bone marrow play crucial role in the development of B-cells. The word stroma is derived from a Greek word which means mattress. B-cells taken out of bone marrow fail to grow in vitro. Hence support of stromal cells is necessary.

These stromal cells are mixture of many phenotypes and resemble fibroblasts, endothelial cells and smooth muscle cells. These stromal cells produce collagen, laminin and actin.

Stromal cells are believed to support B-cell development by not only providing specific contacts but also via growth factors which are responsible for multiplication as well as differentiation.

Stem cell factor (SCF) is one such growth factor which is membrane bound cytokine on stromal cells. It interacts with tyrosin kinase present on the surface of precursor of B-cell. The stromal cell derived factor or pre-B cell growth stimulating factor (SDF-1/PBSF) is also required. Mice which lack the gene for this factor tail to produce developed B-cells.

The role of SDF-I is believed to be in retention of precursor B-cells in the marrow. At some stage, Interleukin (IL-7) is also required for B-cell development.

During development, B-cells migrate within bone marrow becoming more independent of the stromal cells.

From inner bone surface, pre B-cells more to the marrow cavity and are exported from sinuses.

The immature B-cells develop into mature B-cells in peripheral lymphoid organ such as spleen.

More than 75% of B-cells which mature in bone marrow undergo apoptosis and undergo phagocytosis by macrophages present in the bone marrow.

For example murine B-cells are produced at the rate of 5×107 per day. However murine spleen at any time contains around 7×107 B-cells only.

Apoptosis or development into mature plasma cell are not the only two fate of the precursor B-cells. B-cells which produce receptors on their surface which react with self antigens either die or are inactivated or undergo receptor rearrangement.

If the self antigen is multivalent, apoptosis is the dominating outcome. This is called clonal deletion.

In other cases, receptor editing leads to a new receptor. Some other cells may become unresponsive to the antigen. Such ignorant cells are not able to sense self antigen either because it is in low concentration or inaccessible due to sequestration or is unable to crosslink the B- cell receptors.

B-cell development is characterized by appearance of cell surface proteins

Pro B-cells are progenitor cells with very little capacity for self renewal. These are first identified stage next to pluripotent stem cells. Early pro-B cells are identified by joining of DH to JH. Late pro B cells contain VH joining DJH. The large pre-B cells have complete µ chain expressed intracellularly. Some µ chain may start appearing on the cell surface. In such cases, the µ chain appears in combination with a surrogate light chain. This together is called pre- B cell receptor.

At small pre B-cell stage, heavy chain segment rearrangement is stopped. Expression of surrogate light chain also stops. The µ chain occurs only intracellularly.

The next stage in the B-cell development is the immature B-cell. This contains complete IgM on the surface. All these stages of B-cell development are antigen independent stages.

In mammalian fetus, all these stages occur in liver and spleen. After birth, all these stage of development takes place in bone marrow.

Pre-B cells are seen in human fetal liver by 7th or 8th week of gestation. By 9th week of gestation, B-cells in fetal liver have surface receptors for the complement and IgM. By 10th – 12th lymphocyte expressing other Ig class are present. By 15th week, distribution of B-cells in the blood, spleen, lymph nodes and those expressing different Ig isotypes is similar to an adult.

While IgM synthesis can be seen by 10th-12th week of gestation, IgG synthesis, takes place later. Serum and secretory IgA is detectable only by 13th week. The neonatal serum contains no IgA, small amount of IgM and maternal IgG In neonatats, the IgM response is longer lasting than adults, IgG and IgA responses are weak. This is presumably due to lack of T-cell cooperation. Neonates also lack IgG2 subisotype and thus are unable to respond to carbohydrate antigens.

In the adult, the immature B-cells are subjected to a selection process and those whose receptors have affinity towards self-antigens undergo either of the three processes we mentioned.

Cells now move over to secondary lymphoid tissues. The ones which survive there further develop into mature B-cells. These express both IgD and IgM. These are also called naïve B cells and recirculate through peripheral lymphoid tissues. Only when clonally selected after they encounter the specific antigen, they become plasma cells which actively secrete IgM initially and then IgG.

These various stages of development of B-cells in the bone marrow are nurtured by stromal cells and various cytokines and growth factors as already discussed.

Apart from appearance of these immunoglobulin or their chains, these stages of development are also accompanied by display of various other cells surface proteins.

Kit, the receptor for SCF-1 stimulates both lymphoid and myeloid developments in the bone marrow. It is turned off at the late pro B stage. At large pre B cell stage CD25, a low affinity IL-7 receptor is expressed. Kit and IL-7 are involved in pro-B and pre B cell proliferation.

The role of rearrangement of gene segments for Ig

The assembly of V gene segment by recombination is an imprecise process as random addition of nucleotides around the joining points is involved. We may recollect that this is one built in strategy for creating diversity of antibody specificities.

It is estimated that about 2 out of the 3 events generate out of frame sequence. These non productive joins have no use. B-cells which end up making such non productive joins are eliminated.

Allelic exclusion:

A B-cell either uses the gene from the paternal chromosome or from maternal chromosome. Once, an Ig DNA segments are successfully rearranged, the cell is committed to express that particular V regions on both L and H chains, other V gene segment rearrangements are not allowed.

If the right gene rearrangement does not take place, the B-cell tries it again till it gets it right. If the cell runs out of option, it stops. Similarly, once it gets it right it stops and no further rearrangement takes place.

If VH rearrangement is right, no further VH rearrangement takes place. Similarly, if the right VL rearrangement takes place, no further VL rearrangement takes place.

This cell and all its progeny are committed to produce Ig with the V region and hence with same corresponding specificity.

Cell surface markers are designated by acronym CD (cluster determinant) followed by a number. These numbers have no physiological significance but merely indicate their order of discovery. Anti CD-4 implies an antibody for CD4 cell surface antigen.

One of the earliest such marker is CD45R. This is a variant of CD45 which has been also called common leucocyte antigen. This is because variants of CD45 also appears on the surface of T-cells, monocytes, neutrophils CD45R is seen in pro-B and all subsequent B-cell stages right upto plasma cells. It is a tyrosin phosphatase in mouse, the nomenclature is different and equivalent of CD45R is called B22O.

CD19 is another protein which is present at all stages of B-cell development. Both CD45R and CD19 participate in B-cell receptor signalling.

B-cell receptor signalling is important for the development of B-cells. So all B-cell precursors start assembly of CD45R and CD19. Similarly receptor for IL-7 (essential facts for the growth of both B-cells and T-cells) is expressed on the B-cell precursors.

Use of anti-IL-7 antibody or mutants in which IL-7 receptors is inactivated stop B-cell development which unequivocally establish the role of these cell surface markers.

CD43, also known as mucin leukosialin appear at pro-B cell stage but is not their on immature B-cells. CD-43 is an adhesion molecule participating in interaction of B-cell precursors with other cells. One such important interaction is with stromal cells.

Figure 8: Four different productive rearrangements of a Vk and Jk gene segment. Only one strand of the DNA is shown for simplicity. The four different in phase joining of the two gene segments produce three different amino acid sequences at the seam between the segments

Figure 9: Examples of junctional diversity arising from the joining of the JH and D gene segments to form VH genes. (a) The joining of D gene segments to the JH1 gene segment. (b) The joining of homologous D gene segments to different VH segments and the same JH gene segment

Successful or productive rearrangement implies expression of a functional polypeptide.

Non-productive rearrangement implies an incorrect reading frame or creation of a termination codon at downstream to the joining parts.

Non-productive rearrangement seldom lead to formation of mRNA but polypeptide expression is very rare.

During B-cell development, H-chain rearrangement produce pre B-cells. After that L chains rearrangement leads to Bµ cells.

First K-chain is rearranged and only if k-chain rearrangement is unsuccessful, cell attempts λ chain rearrangement. This is inferred from the observation that B-cells generally do not have λ-chain rearrangement. On the other hand cells which ultimately express λ-chain do have rearrangement of k-chains.

The signal for the termination of H-chain rearrangement takes place before L-chain rearrangement is initiated.

The termination of H-chain rearrangement is seen by insertion of H-chain in the endoplasmic reticulum. Appearance of IgM on the cell surface indicate that L-chain rearrangement is terminated.

This well orchestrated regulation ensures that any B-cell produces Ig with λ as the light chain or K as the L-chain. The result is immature B-cell but which is committed to produce ab only of one specificity.

H-chain gene rearrangement is initiated at early pro B cell stage with D gene segment joining JH gene segment. This occurs at both alleles of the H-chain. This results in late pro-B cells.

Most D-JH joining in humans are productive because of the sequence of human D gene segments. Presumably, having two successful D-JH rearrangement is set against large failure rate for V to DJH joining.

It is V to DJH rearrangement takes place only on one chromosome to attempt µ H-chain formation. Successful formation of µ H-chain leads to pre-B cell stage.

In case of an unsuccessful µ chain gene formation, cell tries rearrangement on another chromosome. This is the way allelic exclusion is ensured.

The large pre B-cell with the µ-chain is now ready to divide and proliferate. Pro B-cells with non-productive rearrangement for producing µ chain gene are eliminated. The transient pre- B cells receptor with µ chain as its part provides the signal to proliferate. It also ensures that inability to receive the signal leads to elimination of the cells.

The pre B-cell receptor, as discussed before is made up of µ-chain along with a surrogate light chain. This surrogate L-chain consist of two fragments joined noncovalently. One is λ5 which is quite similar to C-domain of the λ-chain. Another is V pre B similar to V domain of the light chain but has an extra amino terminal region. The µ chain, surrogate light chain are also associated with two accessory chain Igα and Igβ to form pre B-cell receptor complex.

This pre B-cell receptor complex

• Resembles B-cell receptor

• Is expressed at low level

• Is formed transiently

The successful/productive rearrangement of a heavy chains and formation of pre B receptor complex stops division of large pre B-cells and they become small pre B cells.

Small pre-B cells initiates rearrangement of light chain in immature B-cell which leads to IgM display on the surface along with Igα and Igβ. This sends stop signal to L-chain rearrangement.

Thus, we finally have a mature B-cell with a defined specificity.

These B-cells with the functional B-cell receptor undergo a check for capacity to distinguish between self and nonself antigens. If the B-cell binds with a significant having constant to a self antigen, it will not mature any further, this is the negative selection process.

The positive selection for further maturation presumably come from Igα, In mice, B-cells which lack cytoplasmic domain in their Igα show 4-folds reduction in narrow and 100 fold reduction in number of peripheral B-cells.

Then Igα possibly plays a role in B-cells migrating to periphery and their survival there.

When B-cells leave bone marrow, these express sIgM (surface IgM) and small amount of sIgD (surface IgD)

Fully mature B-cells have low expression of sIgM and large levels of sIgD.

From the steady state levels of the B-cells in peripheral tissue in the unimmunized animals, arrival of new B-cells entering is probably balanced by death of a similar number of B-cells.

The access to the follicles in the peripheral lymphoid tissues which provide survival and maturation signals seem to be the main parameter which decides the fate of B-cells, those cannot compete are eliminated.

Those which survive seem to have receptors CXCR5 for chemoattractant BLC expressed by follicular dendritic cells. They also have higher level of expression of CD21 which is involved in signalling by B-cells.

It is believed that a signal from Tyrosinase kinase Syk is also necessary for survival and maturation of B-cells.

We have already discussed earlier how mature B-cell once clonally selected becomes plasma cells when we talked about clonal selection theory. So, this discussion has been retrospective in nature to look at the earlier stages of development of B-cells. It also covered how other feature of clonal selection theory operate. These include one B-cell producing ab of the same specificity and B-cells not secreting ab against self antigen.