How Does The Body Achieve The Functional

Silencing Of Antigen Reactive Clones? Essay, Research Paper

How does the body achieve the functional silencing of antigen reactive clones?

The central tenet of the immune system is the ability to recognise and remove non-self components without affecting self components. The T cells and B cells of the immune system express a vast repertoire of antigen receptors. It is thought that the germline TCR repertoire is composed of in the region of at least 109 different specificities. Considering the potential number of antigens that may be bound by these receptors it is inevitable that a proportion of them will be targeted against self components.

Tolerance in the T cell repertoire.

Central tolerance.

three T cell mechanisms for self tolerance: clonal deletion, clonal anergy and antigen specific suppressor T cells.

Burnet proposed theory of clonal deletion in 1955.

Marrack (1987) showed that T cells bearing receptors containing a member of the Vb17 family were absent from mice expressing the MHC class-II molecule I-E, with which the Vb17 chain is reactive.

Further support has been provided by the absence of self-reactive clones in mice expressing a superantigen such as minor lymphocyte stimulating antigen – an antigen which binds to the TCR in a manner that is independent of the Va chain and thus binds a much larger proportion of T cells than conventional antigens.

in the thymus self reactive TCR present at the CD4+8+stage, not at the CD4+8- or CD4-8+ stage x deletion occurred during thymocyte development.

There is evidence that deletion occurs while thymocytes are in their double positive state -

CD8+ thymocytes bearing receptors specific for class II MHC molecules are deleted during thymocyte development – surprising since CD8+cells are usually MHC I restricted

If deletion was mediated via an interaction between TCR/CD8 and MHC/peptide – single positive cells would not be deleted because CD8 could not interact with the MHC II molecule

x deletion must take place at the CD4/8 double positive stage when CD4 is available to mediate the deletion of MHC II restricted CD8+ cells. Support for this notion has come from the use of anti-CD4 antibodies which block in vivo the clonal deletion of MHC II restricted CD8+ cells. This study does not preclude the possibility that deletion could occur at a stage later than the CD4+8- or CD4-8+ stage.

Clonal deletion does not occur in nude mice (lack the thymus, ha-ha but no thymus no T cells ) x that the involvement of the thymus in clonal deletion

Thymic stromal cells are either of bone marrow or epithelial origin

Most cells express both class I and class II molecules allowing them to participate in the deletion process

Since the epithelial components are resistant to X-irradiation, chimeras can be produced with thymuses composed of epithelial components from the irradiated host and bone marrow components, which are radiosensitive, which are transferred from another animal. The resulting chimera can then have thymus epithelial components expressing a different MHC haplotype from the donor bone marrow component.

In one series of experiments, mouse chimeras were constructed which had the class II molecule I-E localised to either the bone marrow or the thymus epithelium. These chimeras were compared with animals that had I-E totally absent from the body or present in all locations of the body.

In animals with no I-E 10% of immature thymocytes expressed the I-E specific TCR b chain Vb17, compared to 6% of mature thymocytes. This lower level of incidence in mature thymocytes is attributable to poor selection of the TCR, not deletion of these cells.

In animals with I-E on bone marrow alone deletion was observed i.e Vb17 was observed in immature thymocytes, not mature thymocytes. Having said this the immature thymocytes expressed less Vb17 than I-E negative control immature thymocytes, indicating that macrophages, B cells or dendritic cells could cause elimination of some immature thymocytes.

In animals with I-E expressed on the thymus epithelium no deletion of immature thymocytes was observed but mature thymocytes were deleted.

These experiments showed that thymocytes can be deleted by interaction with I-E & peptide on bone marrow derived cells in either the thymus medulla or cortex.

An interesting attempt to resolve the location of and the specific cell types involved in clonal deletion has come from the use of animals made transgenic with MHC genes under the control of tissue specific promoters. The value of these systems is dependant upon their ability to accurately produce a given tissue distribution of MHC gene expression.

Irradiation bone marrow chimeras in which Mls antigen expressed only by the radioresistant thymic epithelium of the host

Deletion of cells specific for the Mls antigen occurs by the transfer of the antigen from the cells of the host to the donor bone marrow-derived cells

Deletion will only occur if the donor bone marrow expresses a permissive MHC haplotype, e.g. one that can present Mls with high efficiency.

Clonal deletion is not observed if only the host thymic epithelium expresses the permissive MHC haplotype

In this case it is relevant to investigate whether the mice are tolerant to the antigen despite not having deleted the reactive T cells.

In mice in which only the thymic epithelium expresses the I-E and the reactive Vb17 T cells are not deleted, a tenfold reduction is observed in the MLR response to I-E (so some degree of tolerance)

In another example in chimeras that fail to delete Vb6 or Vb17 there is a drastic reduction in the proliferative response to the relevant antigens even though the response of these animals to control antigens is normal.

x tolerance can occur in the absence of deletion x clonal anergy: T cells are unresponsive to the antigen to which their TCR is targeted despite it being presented by an appropriate antigen presenting cell with all the costimulatory signals that are usually sufficient for T cell activation.

antigen transferred from host bone marrow to donor thymic epithelium, establish whether transfer could take place in the opposite direction.

Presentation restricted to permissive MHC on the thymic epithelium thus would cause tolerance by clonal anergy rather than deletion.

in chimeras formed from donor bone marrow expressing Mls and a non-permissive MHC haplotype and thymic epithelium with no Mls and a permissive haplotype, transfer of antigen did not occur.

Two possible conclusions to explain why deletion versus anergy would be obtained.

deletion by bone marrow derived elements, anergy thymic epithelium.

or

bone marrow responsible for producing both types of tolerance

deletion due to interaction of autoreactive T cells with Mls presented by permissive MHC haplotypes

anergy is produced it is by interaction with Mls presented by non-permissive haplotypes, which results in a lower level of TCR occupancy.

There is a basic problem in the current understanding of the mechanisms of positive selection, deletion, anergy and T cell activation. Current hypotheses state that they are all mediated through the same receptor, the T cell receptor. How can the ligation of the same molecule result in such a diverse array of consequences?

In order for a given thymocyte to survive to become a mature thymocyte it must be positively selected in the fetus by self antigen/MHC on the surface of thymus epithelial cells. If the animal is to be tolerant to self components then the thymocyte that has been positively selected on the basis of its reactivity to self must be deleted or anergised by the thymus. If this were the case for every developing T cell then no T cells would survive this process. At present there are two hypotheses to account for this seemingly counter-intuitive premise: the affinity hypothesis and the altered ligand hypothesis.

affinity hypothesis – thymocyte may express either a high affinity TCR or a low affinity TCR.

Both high and low affinity TCR s are positively selected, but only clones expressing a low affinity TCR will survive.

At present there is little knowledge about what the structural basis for this may be. Possibly thymus cortical cells express higher densities of accessory ligands and thus the process of positive selection is less particular about the affinity of the TCR involved.

altered ligand hypothesis – MHC molecules expressed in the cortical thymus epithelium bind to peptides that are not found elsewhere

If the bone marrow derived cells and not the thymus epithelial cells are responsible for deletion then the bone marrow component would not express the thymic self-peptides and thus not delete T cells reactive to these peptides

T cells would survive into the periphery

This model also has an advantage over the affinity hypothesis as it allows T cells to be reactive against foreign antigens without concurrently reacting against self as the self peptides that positively selected the T cells are sequestered in the thymus and are not present in the periphery. Still does not account for the T cells that are reactive against foreign antigens have been selected by self antigens and thus each foreign antigen encountered must have a self counterpart.

also there is evidence that thymus MHC is different than that elsewhere:

antibody developed which will bind to MHC in 15% of peripheral tissues but never thymus MHC

ome T-cell hybridomas targetted agianst MHC & peptide can react against thymus MHC alone

alternative hypothesis is that the developmental stage at which the T cell encounters the different thymic environments could be important.

features of the T cell that change during development: expression of accessory molecules, density of TCR, type of TCR associated signalling components, the inducibility of specific genes and the nature of the intracellular signalling pathway.

ligand binding of the TCR results in Ca2+ – cause immature thymocytes to apoptose, cause mature thymocytes to become activated

immature thymocytes exist at two stages: at one receptor engagement does not result in Ca2+ and so cell survives

at another stage Ca2+ and death occurs

difference between these two populations may be that the Ca2+ sensitive one does not have TCR coupled to CD3 and the resisitant one does, co anti-CD3 antibodies caused Ca2+ and death in both.

Why does the TCR – antigen/MHC interaction have two different consequences during positive and negative selection.

suggested signals that produce deletion are the same as those that produce activation of a T cell, i.e. antigen presented in association with MHC molecule with the necessary costimulatory signals such as IL-2

stage of development of the T cell may be important

Support for this theory has come from experiments in which splenic dendritic cells (which are capable of providing costimulatory signals to mature T cells) are added to fetal thymic cultures and subsequently induce tolerance..

what may cause an autoreactive T cell to become anergised rather than activated?

Jenkins proposes that CD4+ T cells are only activated for proliferation or deletion when the antigen is presented by an antigen presenting cell that is capable of providing the necessary costimulation.

antigen is presented in the absence of such costimulation x T cell becomes anergic

According to the experiments detailed above involving the radiation chimeras the bone marrow derived components of the thymus are responsible for clonal deletion whereas the thymic epithelium is responsible for clonal anergy. This situation is further complicated by the possibility that cells within the thymus are heterogenous. This may mean that only a subpopulation of these cells is capable of inducing anergy.

Peripheral tolerance.

Does thymus express all antigens that may be encountered in the periphery? -unlikely

thymus may not express all peripheral antigens, it may be capable of capturing antigens from the circulation, presenting them to APC s and tolerising clones reactive to them.

Evidence

radiation chimeras expressing Mls on the bone marrow components alone which implied that antigen could be taken up by cells in the thymus.

Myoglobin intravenously injected is presented by class II MHC expressing dendritic cells 15 minutes after injection.

If all peripheral antigens could be presented by this mechanism then this would be sufficient for peripheral tolerance.

CD8+ cytotoxic T cell antigens intracellular in origin and peptide fragments from them can not be picked up by APC s and carried to the thymus.

Tolerance may be maintained if only CD4+ T cells are tolerised to self antigens as the T helper cell is essential for the production of a CD8+ CTL response (so even if you did have self-reactive CD8 then still no CD8 response)

Tolerance to certain self antigens unnecessary if the cells in which they are expressed are incapable of presenting them to the relevant T cell.

e.g. if a cell that does not express class II MHC molecules on its surface. This is likely as it has long been known that class II MHC molecules have a restricted cellular distribution.

If a cell does not have the necessary class II molecule then it will be unable to activate a CD4+ T cell, and as mentioned before this cell is necessary for an efficient cell mediated or humoral response.

if a cell that normally does not express MHC II is induced to do so, for example by IFNg, it may become capable of antigen presentation to T helper cells. This is corroborated by data that suggests that autoimmunity can arise as a result of g-IFN production during infection.

clonal deletion in the periphery -

Intravenous injection of Mls-1a bearing splenocytes into adult mice does not cause the deletion of the reactive Vb6 and Vb8.1 bearing T cells

Neonatal thymectomy prevents clonal deletion of Vb11+ T cells in I-E expressing mice, indicating that the thymus is responsible for all clonal deletion of these cells.

exposure of thymocytes to calcium ionophores results in their deletion, not observed in T cells. This indicates that clonal deletion may be restricted to a stage in T cells’ lives that occurs in the thymus.

clonal anergy in the periphery -

In vivo mature cells bearing Mls-reactive TCRs have been rendered tolerant by the transfer of Mls+ cells.

these cells express IL-2 receptors indicating that they are responding to antigen, but they are unable to produce IL-2 in vitro.

These anergic T cells may act as antigen specific suppressors by competing with functional T cells for antigen but not responding to it. (strikes me as being rather wasteful, producing a complex cell just to function as a sink for antigen. And anyway, the antigen is present on cell surfaces so are we to beleive that these anergic cells cover self antigen in every possible place, – I don t think so!)

In order for potentially autoreactive T cells to be made anergic in the periphery while still maintaining a population of useful T cells it is useful to envisage a window of potential for tolerance for the T cell.

young T cell may emerge from the thymus and encounter self antigen

If the time at which it sees this antigen is during its window of tolerance then it becomes anergic.

If it is past the stage during which it may become anergic and the antigen is presented along with the necessary costimulation then the T cell may be activated.

These models do not, however, predict what the outcome may be if a CD4+8- thymocyte that has the potential to be anergised encounters both antigen and costimulation.

Tolerance in the B cell repertoire.

CD4+ T helper cell has a pivotal role in the production of an antibody response so no need for B cell tolerance?

It is possible that foreign antigen specific T cells may provide help for an autoreactive B cell if the foreign antigen and self antigen become noncovalently associated.

Reactive B cell binds self-antigen via sIg, takes it up, processes it and presents it to a T cell

If the self-antigen has a T-cell antigen that is recognised by the T-cell TCR then the B cell will act as an APC and present it to the T cell which will respond by providing help for the B cell

autoantibodies can be found in the serum of individuals with autoimmune disease

absence of high affinity antibodies indicates some form of tolerance in the B cell population.

evidence this tolerance is by no means absolute

Mice transgenic for the hen egg lysozyme antigen (HEL) produce autoantibodies when immunised with this protein conjugated to sheep red blood cells (SRBC) as a carrier. (Control transgenic animals just immunised with HEL do not show this response due to the tolerance of both T cell and B cell compartments.)

antibody levels low compared to non-transgenic controls, number of plasma cells produced by experimental animals can approach that of non-transgenic controls

affinities of the antibodies produced by the non-transgenic animals are of approximately one hundred times the affinity of those in the partially tolerised animals.

antibody titre and affinities of the antibodies affected by the tolerising mechanisms. (but surely more sensible to lower antibody titre rather than churn out useless low affinity antibodies.)

x high affinity antibody producing clones are those most susceptible to tolerisation. Mechanism of B cell tolerance may be dependent upon the binding of a critical number of antigen receptors. This is supported by the failure of tolerance in B cell clones producing high affinity antibodies in transgenic mice expressing low levels of HEL.

The first B cell clones produced following VDJ rearrangement express Igs with low affinity for antigens so may be less susceptible to tolerisaion but also less of a threat because they are low affinity.

antibodies produced after affinity maturation will be produced in the periphery and will be a threat if autoreactive so must be tolerised

Rearranged heavy and light chain transgenes encoding antibody specific for an antigen are inserted into a line of mice.

A separate line of mice is produced that are transgenic for the antigen that the antibody transgenes are specific for.

Cross the two lines x double transgenic mice that express both the transgenic self antigen and the transgenic antibody targeted against it.

Tolerance in these animals may be compared in mice that are only transgenic for the self antigen.

Two systems have been employed to this end: mice transgenic for HEL and its antibody and mice expressing various combinations of MHC haplotype and haplotype specific anti-MHC antibody.

Goodnow et al. produced transgenic mice carrying HEL under the control of the metallothionein or mouse albumen promoter.

For these mice HEL was self and consequently they were tolerant to it compared to non-transgenic littermates.

Mice were produced that were transgenic for an immunoglobulin that is specific for HEL.

To distinguish between transgenic Ig and endogenously rearranged Ig the heavy chain constant region Ig was derived from a different mouse strain so that cells could be fluorescently labelled for each allotype by monoclonal antibodies.

Fluorescence activated cell sorter analysis showed that 90% of peripheral B cells expressed only the transgenic Ig.

B cells that were transgenic for the Ig spontaneously differentiated into plasma cells and secreted IgM into the serum.

When the double transgenic mice were examined, however, they showed almost complete cessation of anti-HEL Ig secretion. FACS analysis of spleen and lymph node cells showed that anti-HEL B cells had not been deleted but had become anergic.

Nemazee and B rki introduced light and heavy chain genes encoding an antibody for H-2k MHC class I molecules into the germline of H-2d mice.

antibody not self reactive in the resulting animals and 50% of peripheral B cells expressed the transgene products with remainder expressing endogenous genes.

When these animals were crossed with non-transgenic H-2k/d heterozygous strain mice, anti H-2k antibody secretion was not observed in those progeny that expressed the H-2k MHC haplotype.

Unlike the case of the anti-HEL B cells mentioned above, no cells expressing the transgene could be detected, implying that in this case the autoreactive cells had been clonally deleted.

anergic cells observed in lysozyme mice expressed 90 to 98% less IgM on their surface – receptor down-regulation

may also have played a role in the clonal deletion in the H-2k transgenic mice since B cells in the bone marrow of these mice also showed reduced levels of surface IgM.

may be downregulation of IgM in the anti-MHC specific B cells prevented their homing to the peripheral lymphoid organs, accounting for their absence.

In the case of the anti-HEL B cells, they express IgD which could mediate this homing process. However despite IgD ligation being sufficient for activation, these cells are unresponsive and so a mechanism of tachyphylaxis whereby there are changes in the signal transduction across the membrane may be playing a part.- bit tenuous.