I. Lymphocyte homeostasis. B cells. (Fabien Agenes & Manuela Rosado).
In the adult mouse the number of B cells remains constant in spite of the continuous production of new cells in the bone marrow (BM). The mechanisms that determine the number of lymphocytes in an adult mouse are poorly understood. It is not known why in normal physiological conditions the number of lymphocytes is what it is. We used B-cell deficient mice, with a developmental arrest of B cell production at the pro-B to pre-B stage of B cell differentiation to study the ability of a limited number of normal B cell precursors to populate peripheral B cell pools. We established mouse chimeras containing different numbers of competent precursor pre-B cells. In the chimeras reconstituted with mixtures of BM cells from normal and B-cell deficient donors, we showed that the physiological number of peripheral B cells was not determined by the number of B cell precursors. Mice containing less than 25% of the normal number of pre-B cells had reduced peripheral B cell numbers. A normal sized peripheral B cell pool, however, was generated in mice containing only 30% of the normal number of pre-B cells. These results demonstrate that about 1/3 of the normal number of BM B cell precursors suffices to maintain the peripheral B cell pool size. A similar conclusion was obtained after parabiosis between one normal and two or three B cell deficient mice. In these circumstances B cell production was restricted to the BM of the normal mouse since no chimerism was detectable in the BM of the different partners. In mice triads it was found that each individual mouse had physiological B cell numbers i.e. the B cell production of one mouse was sufficient to populate the peripheral pools of three mice. These results demonstrate that peripheral B cells number is not determined by the rates of BM B cell production, but it is limited at the periphery.
In the same set of chimeras we also showed that the compartment of activated IgM-secreting B cells was controlled by autonomous homeostatic mechanisms since the number of cells it comprises was regulated independently of the size of the mature B cell pool. These results support a model of the immune system in which the size of the different B cell compartments, i.e. pre-B, resting B and IgM-secreting, is autonomously regulated. They suggest a hierarchical organization in which the first priority is the maintenance of normal serum IgM levels to ensure both a first natural barrier of protection while keeping a maximum of repertoire diversity among the resting B cell compartment.
In an immune system where there is a continuous excess of B cell production and the total number of B cells is kept constant each newly produced B cell can only establish itself upon loss of other cells. The persistence of B cells at the periphery could, however, be modified by the continuous arrival of the newly formed BM migrants while the fate of the latter at the periphery could also be altered by the presence or absence of resident B cell populations. We investigated the possible mutual influences between established resident populations of peripheral B cells and newly formed B cell migrants. The questions addressed by these studies were: What is the role of B cell production in peripheral B cell renewal, are there feedback mechanisms that control new B cell entry into the peripheral B cell pools? To answer these questions we compared: 1) the persistence of peripheral B cells in the presence or absence of new BM B cell production. For this purpose we studied the fate of mature resting B cells transferred alone or simultaneously with a pool of precursor cells into immune-deficient mice (Rag2-/-); 2). We compared the fate of a population of B cells (recent BM migrants or mature B cells) in the presence of a previously established peripheral B cell population. In this case we followed the seeding and the persistence of a second population of B cells in B cell deficient mice previously injected with a cohort of mature B cells. Our results support the notion that the homeostatic regulation of the resting and activated B cell compartments is autonomous indicating that the two B cell populations belong to different immunological niches.
Can an established B cell population alter the fate of recent new B cell migrants at the periphery? The answer is yes. We demonstrated that in the presence of an established population of activated B cells there is a diminution in IgM production by a subsequently introduced B cell population. These findings indicate that there are mechanisms of feedback regulation controlling terminal B cell differentiation and the total number of activated B cells.
In conclusion, we showed that B cells remaining after two weeks of transfer in immune-deficient hosts represent a stable B cell population which persists for prolonged periods of time by self-renewing, resists replacement and can modify the fate of newly coming B cells. These findings suggest that the priority of the immune system is the production of circulating IgM levels to ensure a first barrier of protection against infection and that once a particular B cell is selected it can be kept by the system.
T cells. (Afonso Almeida, Jose Borghans & Nicolas Legrand).
We developed a novel strategy that allows a quantitative assessment of the contribution of the thymus T cell production and export to the establishment and maintenance of the peripheral T cell pools. We studied the ability of a limited number of T cell precursors to populate the peripheral T cell pools. We used TCRa-/-, CD3e-/- and Rag2-/- T-cell deficient mice, with a developmental arrest of T cell production, to establish mouse chimeras containing different numbers of competent precursor cells. Lethally irradiated alymphopenic B6.Rag2-/- mice were reconstituted with T cell depleted BM cells from normal B6.Ly5a donors alone or from normal B6.Ly5a and T-cell deficient B6.Ly5b mice mixed at several ratios. In the resulting chimeras, since normal Ly5a competent precursor cells are diluted among Ly5b incompetent mutant precursors, the rate of T cell production in the thymus should vary according to the fraction of competent Ly5a cells present in the initial BM inoculum.
In the mixed B6.Ly5bTCRa-/-/B6.Ly5a chimeras the total numbers of DP thymocytes, composed of both competent and incompetent cells, remained identical in all chimeras studied. The number of competent DP Ly5a cells, however, increased with the number of competent DN cells present in the thymus. The number of competent DP cells was linearly proportional to the number of competent DN cells i.e. a two-fold lower number of competent DN cells resulted in a two-fold lower number of competent DP cells. In conclusion our results indicate that the size of the DP compartment is determined by the influx of cells from the DN compartment, suggesting that in a normal mouse the number of DN precursor cells strictly determine the number of DP cells. In the thymus of all T cell deficient/normal mixed BM chimeras the number of TCRhighSPCD4 or TCRhighSPCD8 cells was proportional to the number of competent DP cells. A two-fold lower number of competent DP cells gave rise to a two-fold lower number of TCRhigh mature SP cells recovered from the thymus.
We studied the size of the CD8 and CD4 peripheral T cell pools in mice with reduced thymus T cell production and export. In contrast to what we found in the thymus, the total number of CD4 and CD8 cells in the periphery was not proportional to the number of cells in the previous compartment, i.e. thymus SPCD4 and SPCD8 cells. In most chimeras with reduced numbers of thymus SP cells the sizes of the peripheral T cell compartments were similar to those in the chimeras with normal numbers of thymus SP cells. A 100-fold reduction in the number of SP CD4 cells and SP CD8 cells reduced the peripheral CD4 and CD8 compartments only 4-fold and 8-fold, respectively. This suggests that in the presence of low T cell numbers a compensatory homeostatic mechanism favors T cell activation and proliferation, as to attain normal peripheral T cell numbers. In concordance, we found that the lower was the number of peripheral CD8+ or CD4+ T cells the higher was the fraction of activated CD4+CD45RBlow and CD8+CD44+ T cells. These findings demonstrate that the numbers of peripheral CD4 and CD8 T cells are only partly determined by the rates of thymus cell production and export. In conclusion these results show that in young adult mice there is an excess of thymus cell production since most chimeras with low numbers of SP thymocytes have normal peripheral T cell numbers. The establishment of the physiological sized peripheral T cell pool, however, requires a minimal thymus output since chimeras with very low numbers of SP thymus cells were not able to fully reconstitute the CD4 and the CD8 peripheral compartments.
Peripheral CD4+ T cells consists of functionally distinct subsets. A population of CD45RBhighCD25-CD4+ T cells enriched in naive cells, which represents about 2/3 of the peripheral CD4 T cells and populations of activated CD45RBlowCD25-CD4+ and CD45RBlowCD25+CD4+ T cells. The activated CD4+ T cells have been shown to exert important regulatory functions.
We investigated the mechanisms controlling the expansion of CD45RBhighCD25-CD4+ and CD45RBlowCD25+CD4+ T cells after transfer into T cell deficient CD3e-/- hosts. In particular we investigated if CD45RBlowCD25+CD4+ T cells, which have been described to exert regulatory function, could also govern peripheral CD4+ T cell homeostasis. In the hosts both CD4+ T cell populations proliferated extensively, but while the CD45RBhighCD25-CD4+ T cells reached equilibrium at 1-2x107 cells, the CD45RBlowCD25+CD4+ T cells did only attain ten fold lower numbers. Sequential cell transfers into the same hosts authenticate that there is a homeostatic control of expansion of the naive CD4 T cells and corroborate the role of cellular competition in the process.
When the two T cell populations were co-injected, the presence of CD45RBlowCD25+CD4+ T cells limited the expansion of the CD45RBhighCD25-CD4+ T cells. These effects were dependent on the relative number of the two cell populations. Suppression of CD45RBhighCD25-CD4+ T cell growth was obvious when the number of CD45RBlowCD25+CD4+ T cells exceed by a factor of ten the number of the CD45RBhighCD25-CD4+ T cells, less noticeable and variable when the two populations were present at similar numbers and absent at lower CD25+/CD25- cell ratios. In sequential cell transfer experiments we found that the transfer of a limited number (5x104) of CD45RBlowCD25+CD4+ T arrests the growth of an expanding population of 5x106 resident CD45RBhighCD25-CD4+ T cells. Adoptive cell transfers into secondary recipients of CD45RBlowCD25+CD4+ T cells shows that only the cells, which remain CD25+ display suppressor capacity. By which mechanisms do the CD45RBlowCD25+CD4+ T cells limit the expansion of CD45RBhighCD25-CD4+ T cells? We showed that the CD25+ cells do not completely block division of CFSE labeled CD4+ T cells, but either decrease the rate of cell division or the accumulation (survival) of the dividing cells. The control of peripheral T cell expansion by the CD45RBlowCD25+CD4+ T cells is not mediated by IL-10, since cells from IL-10-deficient mice are as effective as cells from normal donors in suppressing the expansion of the CD45RBhighCD25-CD4+ T cells. In conclusion these studies make evident the role of T cell interactions in the homeostatic control of the size of the peripheral CD4+ T cell pool. CD45RBlowCD25+CD4+ T cells act by limiting the accumulation of dividing CD45RBhighCD25-CD4+ naive T cells, i.e. they control differentiation of naive CD4 T cells into the activated cellular compartment.
II. Lymphocyte competition.(Fabien Agenes, Angela McLean & Manuela Rosado).
In an immune system where new lymphocytes are continuously produced in excess but their total numbers are kept constant, newly generated cells have to compete with other newly produced or resident cells to survive. Competition can be defined as "an interaction between two populations, in which, for each, the birth rates are depressed or the death rates increased by the presence of the other population". There are two main established criteria accepted as evidence of competition among populations: 1. The presence of competitors should modify the equilibrium size of a population and 2. The presence of competitors should alter the dynamics, e.g. the life span of a population. The question of whether competition arises between B and T cells was addressed by comparing the development and the fate of BCR-Tg, TCR-Tg and non-Tg populations in several different lines of mouse BM chimeras. It was found that: a) when injected alone, Tg and non-Tg cell populations show an identical behavior and generate peripheral pools of similar size. b) When Tg and non-Tg cells are mixed in the same host they initially accumulate at the same rate. However, after reaching steady state numbers there is a preferential selection of the non-Tg cells at the periphery. These observations fulfil the first criteria for competition since they demonstrate that the presence of non-Tg populations modifies the number of the Tg cells. In these experiments it was also found that the life expectancy of the Tg B and Tg T cells varied according to the presence and the type of other competing cells. These latter findings fulfil the second main criteria required for the definition of competition as they prove that the presence of competitors alters the life span of a population.
Competition may arise through different processes. In interference competition populations may interact directly with each other or one population can prevent a second population from occupying a habitat and from exploiting the resources in it. Thus, although interference competition may occur for a resource it is "only loosely related to the resource level". In exploitation competition different populations have a common need for resources present in limited supply. In this case competition is directly related to level of resources available. We may define "resource" as any factor, which can lead to increased cell survival or growth through at least some range of their availability. On a broad sense a resource is any factor, which is "used" by a cell and for that reason is no longer available to other cells. In the immune system many molecules can function as resources, e.g. antigen, MHC molecules, ligands for co-stimulatory and adhesion molecules, mitogens, interleukins, chemokines, hormones and other growth factors, etc. Resources can be external to the immune system or be produced by the lymphocytes themselves. By producing their own resources lymphocytes also contribute to generate their own ecological "space". It is now critical to establish a hierarchy of resources to define possible mechanisms of immune intervention.
We have compared the reconstitution capacity of B cells from Igk-/- mice and found that they are out-competed from the peripheral B cell pools by B cells from control mice, suggesting that BCR diversity plays a major role in B cell competition. We have also shown that the over expression of the bcl2 oncogene confers a competitive advantage in peripheral reconstitution by B cells from bcl2 Tg donors, confirming that cell survival is an important factor in B cell competition. We also addressed the role of surface IgD in B cell competitive capacity, by comparing the peripheral reconstitution capacity and the survival of B cells from IgD-/- and control IgD+ mice with no conclusive differences. Other factors are currently under scrutiny.
III. Lymphocyte survival.In an immune system where the total number of cells is limited, cell survival can no longer be a passive phenomenon, but rather a continuous active process where each lymphocyte must compete with other lymphocytes. It can be said that lymphocytes follow the Red Queen Hypothesis postulate "it takes all the running you can do to keep in the same place".
T cells. (Nicolas Legrand in collaboration with Corinne Tanchot & Benedita Rocha).
We have investigated the conditions necessary in vivo for the survival and expansion of naive and memory antigen-specific CD8+ T cells. To characterize the functional properties and the requirements for persistence of memory T cells, we used monoclonal T cell populations. Transgenic (Tg) mice bearing a Tg ab TCR specific for the HY male antigen restricted to MHC class I H-2Db and deficient in the recombinase gene RAG2 (TgRAG2-), were used to obtain monoclonal populations of CD8+ T cells.
To study the TCR interactions required for the survival or division of naive CD8 T cells we compared their fate after transfer into irradiated hosts, that differed in MHC class I and HY antigen expression. These hosts were: C57BL/6 CD8-/- deficient male (HY+H-2b+) and female (HY-H-2b+) mice; female H-2Db-deficient mice, that lack the MHC class I restriction element of this TgTCR but express other MHC class I molecules including H-2Kb (HY-H-2Db-class I+) and female class I- mice deficient in both H-2Db and b2-microglobulin (HY-Db-b2m-). These were used rather than b2m-deficient mice, because the latter mice express some H-2Db enough to induce deletion of male-specific Tg T cells in male mice. To correlate cell survival with interactions with MHC class I restricting element, we also used female mice expressing H-2Db but lacking H-2Kb (HY-Db+Kb-). These host mice were irradiated and injected two days latter with naive T cells. Recovery of Tg cells was evaluated at days 1, 2, 7 and 13 after transfer. Tg T cell division was studied one week after transfer by following BrdU incorporation.
One day after cell transfer the fraction of donor cells homing to the pool of lymphoid organs studied was the same in all groups of host mice (about half of the donor cell population). Naive T cells could survive in a resting state in female CD8-deficient mice: they did not incorporate BrdU and the number recovered was constant from day one up to two weeks after injection. Expansion of naive cells required stimulation with male antigen because they divided only after transfer into male CD8-deficient hosts. Naive T cells survival required the right MHC restricting element. In mice lacking H-2Db, or expressing no class I, (H2-Db-b2m-) naive cells did not survive but decayed to an average of 3% of the injected cohort at one week, 1% at 13 days and were undetectable at 2 weeks. This decay correlated with the absence of interactions with the MHC restriction element, because naive H-2Db restricted Tg cells persisted after transfer into H-2Kb-deficient mice expressing H-2Db. Thus, as described during thymus positive selection a minimal state of cell activation may allow survival, in the absence of cell division.
More recently, we have derived lines of double CD3e-/-H-2Db-/- and CD3e-/-H-2Kb-/- mice. We also derived 3 different lines of monoclonal TCR Tg mice: aHYRag2-/-, P14Rag2-/- and OT-1Rag2-/- mice. The HY and P14 TCR are restricted to MHC class I H-2Db. The OT-1 receptor is restricted to MHC class I H-2Kb. We follow the fate of the naive monoclonal CD8 T cells from these donor mice in non-irradiated CD3e-/-H-2Db-/- and CD3e-/-H-2Kb-/- mice and confirm that the survival of the naive CD8 T cells is strictly dependent on the presence of the correct MHC class I restricting element. The use of T cell deficient hosts formally excludes any possible involvement of the hosts T cells in the disappearance of the transferred T cells and allows the use of non-irradiated mice as recipients. In these non-irradiated CD3e-/- MHC class I deficient hosts, however, the decay of the transferred naive CD8 T cells was faster and detectable as early as 24 hours after inoculation.
We next studied the TCR interactions required to maintain CD8+ T cell memory. To obtain memory cells, female naive Tg T cells were stimulated with relatively low doses of male antigen in vivo in male' female bone marrow (BM) chimeras. We produced male/female B mice by injecting a mixture of 90% female and 10% male BM cells from CD3e-deficient mice into Rag-deficient female hosts. These hosts did not have endogenous T cells and 10% of BM-derived cells were of male origin. Naive T cells transferred into these chimeras expanded.
To identify the TCR interactions required for the survival or division of memory CD8 T cells, purified (>97%) memory populations (depleted of B cells and other class II positive antigen-presenting cells) were injected into irradiated hosts that differed in MHC class I or antigen presentation. Transgenic T cell recovery was determined at days 1, 2, 7, and 11 after transfer, while T cell division evaluated by BrdU incorporation at day 7. At 24 hours after transfer homing of memory Tg cells was the same in all groups of host mice and similar to that of naive Tg cells. When memory Tg cells were stimulated after transfer into male CD8- mice their rate of division (>90% BrdU+ cells) was higher than that of naive cells. In contrast to naive cells, memory cells transferred into female CD8- hosts also divided extensively (70% where BrdU+) and survived and divided in mice lacking the H-2Db restricting element (42% BrdU+). In mice lacking class I (H2-Db-b2m-mice), about 30% of memory cells still incorporated BrdU, indicating a response to autocrine or environment growth factors, even in the absence of T cell stimulation. However, this response was not sufficient to maintain memory T cells; they disappeared progressively. Two weeks after T cell transfer, donor cells in class I deficient (H2-Db-b2m-) host mice were barely detectable.
IV. Lymphocyte selection.B cells. (Emmanuelle Gaudin & Manuela Rosado).
Lymphocyte repertoires are shaped in primary lymphoid organs at the early stages of B and T cell development by episodes of positive and negative selection. To have a positive selection event it may suffice that one lymphocyte meet its specific antigen. In contrast, complete negative selection requires that all lymphocytes from the same clone meet the antigen. Whatever the selection mechanism involved it is evident that the dose of antigen must play a major role in the establishment of both B and T cell repertoires. We studied the effects of varying quantities of self-antigen in B cell development. We revisited a mouse model in which egg hen lysozyme (HEL) specific transgenic B cells develop in the presence of the neo self-antigen HEL. We derived mice transgenic (Tg) for either the anti-HEL BCR (MoMD4), a soluble form of HEL (ML5) or a membrane form of HEL (KLK3) in a Rag2-deficient background. Non-Tg Rag2-/- host mice were lethally irradiated and reconstituted with a mixture of 50% BM cells from MoMD4 aHEL donors and 50% of BM cells from Rag2-/- and HEL Tg (ML5.Rag2-/- or KLK3.Rag2-/-) donors mixed at different ratios. By using host and donor Rag2-/- mice we made sure that the only lymphocytes present in the chimeras were Tg B cells specific for HEL. By fixing at 50% the fraction of MoMD4 cells in the injected BM cohort we made sure that the rate of B cell production was the same in all chimeras studied. By modifying the proportions of non-Tg and HEL Tg cells in the remaining 50% BM inoculum we changed the levels of self-antigen production. As HEL Tg precursor cells are diluted among non-Tg precursors the production of HEL should vary according to the fraction of HEL Tg cells present in the initial BM inoculum. Indeed we found that this was the case. By ELISA we quantified the amount of HEL present in the serum of the different mouse chimeras after reconstitution. We found that indeed the levels of HEL diminished with decreasing numbers of injected Tg precursor cells. Six to eight weeks after reconstitution the chimeras were sacrificed and the number and phenotype of BM pre-B and B cells, spleen B cells and the serum IgM levels evaluated.
Development of HEL specific MoMD4 B cells in the absence or in the presence of variable quantities of soluble and membrane HEL was studied. In ML5.Rag2-/- hosts reconstituted with MoMD4 BM the total pre-B and B cell numbers recovered were identical to that obtained in non-Tg Rag2-/- recipients. The mature B cells expressed low levels of surface IgM, while IgD levels remained unchanged. The serum IgM levels were low as compared to mice where MoMD4 B cells nurtured in absence of HEL. We conclude that the presence of concentrations of about 20ng/ml of soluble HEL induce changes in the functional state of the HEL-specific B cells, while lower levels of HEL do not affect the developing B cells.
In the KLK3.Rag2-/- mice reconstituted with MoMD4 BM, B cells were rare and the serum concentrations of IgM were very low. By decreasing the fraction of HELm Tg cells injected in the chimeras we recovered increasing numbers of mature B cells and serum IgM levels. These results demonstrate that negative selection of self-reactive B cells is complete only when the antigen is present in sufficient amounts. High avidity B cells can persist in the presence of self-antigen.
The BCR Tg used by the MoMD4 B cells codes for a high affinity anti-lysozyme antibody. In normal development B cell interactions with self-antigens are likely to involve both high and low avidity interactions. We recently crossed the MD4 (aHEL) and SP6 (aTNP) Tg lines and obtained mice bearing two IgH and two IgL Tgs in a Rag2-/- background. In these MoMD4.MoSP6 DIgTg mice most (>98%) B cells bind HEL and express the 20.5 SP6 Tg Ig heavy chain. The level of HEL binding by the DIgTg B cells is reduced to 30% of that of the MoMD4 single BCR B cells, indicating that the overall avidity of these cells for HEL is lower than that of MoMD4 B cells. We studied the development of the low avidity HEL specific DIgTg B cells in the absence or in the presence of variable quantities of HEL. In ML5.Rag2-/- hosts reconstituted with BM cells from DIgTg mice or in chimeras injected with 50% BM cells from ML5.Rag2-/- donors the number of mature BM and spleen B cells was higher than in HEL- chimeras. The increase in B cell numbers was due to the augmentation of HEL specific single expressing cells. These results indicate that small local concentrations of HEL are sufficient to promote selection and survival of the HEL specific B cells. In the KLK3.Rag2-/- hosts dual BCR B cells are deleted in spite of their lower avidity for HEL. By decreasing the amounts of HEL in the mixed KLK3.Rag2-/- BM chimeras we recover ten times more B cells than in the control HEL- chimeras. Over 99% of the B cells were HEL specific single expressing cells. In the same chimeras the IgM levels were also increased to a similar extent. In mice reconstituted with fewer HELm Tg BM cells the numbers of B cells were as in HEL hosts, but the IgM levels were 20-30 times higher. These results show that low levels of self-antigen positively select for low avidity self-reactive B cells. The finding that B cell numbers are increased both in the BM and spleen and that the number of BM pre-B cells remains unchanged suggest that positive selection occur at the pre-B to B cell transition in the BM. We provided the first clear demonstration for positive selection of conventional B cells. Moreover, the presence of low quantities of self-antigens induces the activation of the low avidity dual receptor B cells and increased levels of serum IgMs. Most of these IgMs are self-reactive and these mice are full of IgM-HEL immune complexes. These results represent an important contribution to the origin of most physiological self-reactive Igs: they suggest that these Igs are produced upon the activation of low avidity self-reactive B cells by self-antigens present in weak quantities.
T cells.(Nicolas Legrand).
Antigen receptors of B and T lymphocytes are subjected to allelic exclusion. In general, for T cells the T cell receptor (TCR) b chain gene segments start to rearrange on one chromosome and continue on the second chromosome only when the first attempt resulted in a non-productive gene. Allelic exclusion of the TCR, however, is never failing proof. Different studies indicate that about 1% of the mature ab T cells contain two productive TCR b alleles. Moreover, in contrast to the TCR b locus, TCR a chain rearrangements proceed simultaneously in both chromosomes and about 30% of the ab T cells express two TCR a chains. In normal mice, the likelihood of generation of T cells with dual specificity is therefore elevated. Potentially the allelically included T cells could play an important role in auto-immunity. In dual receptor cells a second self reactive TCR could bypass thymic negative selection in virtue of its lower expression. If a non-self antigen stimulates the naive dual receptor T cells, these T cells once activated would acquire a lower threshold of activation respond to self-peptide/MHC and cause disease. The role of dual receptor T cells in auto-immune diseases is, however, strictly dependent in their ability to be first, positively selected in the thymus and second, to survive in the peripheral pools. Considering that both thymus positive selection and peripheral T cell survival require receptor engagement by MHC molecules, it is likely that the presence of two receptors bearing different specificities may impose some constraints to the selection, survival and functional abilities of dual receptor T cells. To examine these questions we studied T cell selection in the thymus and periphery of Rag2-deficient animals bearing two complete rearranged ab TCR transgenes. One receptor is specific for the HY male antigen (Vb8.2VaT3.70) and the second is specific for the gp33-41 peptide of LCMV (Vb8.2Va2), and both are restricted to the same MHC H-2Db class I molecule.
In the MoaHY.MoP14 DTg mice thymus T cell development only gives rise to CD8 T cells, indicating that the association between the different ab chains of the two TCR Tgs was not able to generate MHC class II restricted CD4 T cells. The lack of significant T cell development in MHC class I H-2Db-/- chimeras further demonstrates that all TCR specificities generated are restricted to the H-2Db molecule.
In female DTg mice ~80% of the peripheral CD8 T cells express two TCR a-chains, but single VaT3.70+ and single Va2+ cells were also present. Several findings suggest that while 99% of the CD8 T cells from DTg mice are Vb8.2+, most cells either do not express or express poorly the Vb8.1 chain of the P14 TCR. First, internalization of the Vb8.2 chain of the aHY TCR Tg leads to complete co-internalization of both Va chains. Secondly, in vitro stimulation of CD8 T cells from DTg mice with the LCMV gp33-41 peptide failed to induce cell proliferation. These DTg mice provide therefore a unique model to study the functional behavior of the dual TCRa CD8 T cells.
Exposure of mature DTg cells from female mice to the male HY antigen in vivo i.e. after transfer into male hosts, results in the selective expansion of both HY-specific single Va2-T3.70+ and dual Va2+T3.70+ cells. The in vivo immune response of single Va2-T3.70+ was, however, more efficient as indicated by the faster rate of expansion. The single aHY cells were susceptible to the tolerogenic effects of excess antigen since after day 7 after transfer into male hosts these cells became unable to proliferate after in vitro stimulation and were replaced by dual TCR cells and by Va2+T3.70- non-HY specific cells. The dual receptor exposed in vivo to the male antigen also expanded, but at a slower rate and did not disappear at latter times after transfer. They remained susceptible to the induction of peripheral tolerance. The fact that the dual TCRa cells proliferate poorly to the specific peptide but persist and are not replaced allows them to persist to exert important in vivo regulatory functions.
Using a competitive re-population strategy we directly accessed the accumulation of dual and single receptor CD8 T cells in the peripheral pools of different BM chimeras. When alone T cells from DTg and single Tg donors show similar behavior and generate peripheral pools of similar size. When mixed in the same host, single receptor expressing CD8 T cells out-competed dual receptor T cells from the peripheral pools following an hierarchy where MoP14 > MoaHY * DTg cells.
What happens to T cell development in the presence of the male HY self-antigen? In male DTg mice the presence of a second TCR Tg reduces the expression of the aHY TCR allowing the appearance of significant numbers of DP thymocytes and TCRhighCD8+ SP T cells. During the DP to SP transition only the cells expressing lower levels of CD8 and of the aHY TCR are positively selected. Our results also show that, even if the presence of a second TCR permits the development and emigration of mature CD8 T cells expressing a self-reactive TCR, these peripheral dual receptor T cells are functionally tolerant. Dual receptor CD8 cells from male mice in spite of the increased expression of the P14 TCR b chain are still unable to proliferate to the LCMV gp33-41 peptide. Like the CD8 T cells from conventional Rag+ aHY Tg and MoaHY male mice these DTg CD8 T cells show reduced surface expression of CD8 and of the aHY specific TCR and they lack responsiveness to cognate stimuli. Eighty percent of the MoaHY cells and about 40% of the dual receptor cells from male mice express the activation/memory CD44 marker and produce g-INF mRNA and IL-10 mRNA, suggesting that these tolerant cells may have important regulatory functions.
Our results indicate that an optimal level of signal may be required to trigger peripheral T cell survival: low TCR and CD8 levels compensate for excess of ligand, high TCR expression the lack of CD8 co-receptors, etc. Successful survival implies therefore a process of adaptation of lymphocyte populations to the host environment. Thus, in contrast to MoaHY males where TCR+CD8- (DN) cells represent 60% of the peripheral TCR+ cell pool, in DTg male mice most peripheral TCR+ cells are CD8+. It has been claimed that in aHY Tg mice the peripheral DN cells belong to gd T cell lineage, and that expression of the gd TCR receptor could ensure maturation and survival of the cells. Our current observations in Rag2-/- MoaHY mice demonstrate that the TCR+ DN cells do not require expression of gd TCRs to survive. Our findings suggest that in male MoaHY in the presence of an excess of self-antigen peripheral T cells can survive in absence of the CD8 and CD4 co-receptors if expressing higher levels of TCR. In fact in DTg male mice the few peripheral TCR+ DN cells show higher levels of aHY TCR expression.
In normal mice TCRa inclusion occurs in 30% of T cells which may represent an auto-immune hazard. In DTg mice the presence of a second TCR allows a significant number of dual receptor CD8 T cells expressing a self-reactive receptor to escape central deletion by self-antigen. These non-deleted cells migrate to the peripheral pools but are not fully competent to respond to cognate stimuli. Dual receptor cells, which develop in absence of self-antigen show efficient responses to immunization, remain sensitive to induction of peripheral tolerance, but are not replaced after exposure to high levels of self-antigen. They may, therefore, put forth, important regulatory functions in vivo. In overall dual TCR CD8 T cells have a poor competitive fitness when compared to single TCR cells and are counter-selected in the peripheral T cell pools. The ensemble of these characteristics may contribute to decrease the auto-immune hazard that dual receptor cells could represent in normal physiological conditions.