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Reversal of thymic atrophy
Experimental Gerontology 39 (2004) 673–678 www.elsevier.com/locate/expgero
Reversal of thymic atrophy Sian M. Henson*, Jeffrey Pido-Lopez, Richard Aspinall Department of Immunology, Imperial College, Chelsea and Westminster Hospital, 369 Fulham Road, London SW10 9NH, UK Received 24 June 2003; received in revised form 25 September 2003; accepted 10 October 2003
Abstract Age-associated thymic atrophy is a key event preceding the inefficient functioning of the immune system, resulting in a diminished capacity to generate new T-cells. This thymic involution has been proposed to be due to changes in the thymic microenvironment resulting in its failure to support thymopoiesis. A key cytokine in the early stages of thymocyte development is IL-7 and expression levels are greatly reduced with age. The ability of IL-7 to restore the immune system by enhancing thymic output remains controversial. In this review, we highlight the advances in molecular approaches used to evaluate recent thymic emigrants and assess the success of these strategies in determining whether IL-7 can lead to immune reconstitution. q 2004 Elsevier Inc. All rights reserved. Keywords: Atrophy; Thymus; IL-7; TRECs; DECs
1. Age and the thymus The thymus is the primary organ for the production of ab T-cells and is thus essential for a functional immune system. However, removal of the thymus from children after six months of age (Brearley et al., 1987) does not result in overt immunodeficiency before the end of adolescence, a finding which lead to the assumption that the thymus is an organ that becomes redundant with time. This however, is not actually the case because peripheral T-cell numbers are maintained by a powerful homeostatic compensatory mechanism which causes the peripheral expansion of mature T-cells leading to the regeneration of the T-cell pool (Freitas and Rocha, 2000) Thymic function is at its most active during the foetal and perinatal periods but output declines with age resulting in a dramatic loss in the number of naı¨ve T-cells being produced. This decline in de novo T-cell production correlates with the atrophy of the thymic stroma, a condition known as thymic involution. Several changes to the thymic architecture can be observed, the perivascular space becomes enlarged leading to a decreased thymic epithelial space, an area that houses both the cortical and medullary components responsible for thymopoiesis (Steinmann, 1986). The perivascular space is enlarged by lymphocytic infiltration but as ageing * Corresponding author. Tel.: þ44-20-876-8250; fax: þ 44-20-87465997. E-mail address: [email protected] (S.M. Henson). 0531-5565/$ - see front matter q 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.exger.2003.10.030
progresses adipose tissue eventually replaces the lymphocytic perivascular space (Steinmann et al., 1985). However, it has been shown that the elderly still retain a limited thymic function, with thymocytes being produced by a small thymic rudiment composed of epithelium (Steinmann et al., 1985).
2. T-cell development and age changes Despite this loss in thymic output precursor T-cells continuously migrate from the bone marrow to the thymus throughout life. The earliest precursor cells that arrive at the murine thymus are CD32CD4loCD82 progenitors (Wu et al., 1991). These early precursors can be considered to be effectively triple negative (TN) because CD4 knock out mice showed normal progression through this early phase of development, suggesting that the expression of CD4 does not play a fundamental role at this stage of the pathway (Rahemtulla et al., 1991). This TN population can be further subdivided on the basis of expression of CD44 and CD25. The most immature stage being CD44þCD252, maturation through the T-cell developmental pathway proceeds through CD44þCD25þ then CD442CD25þ to a stage that is CD442CD252 and it is during the transition between these late stages that the majority of thymocyte expansion occurs. It is also during this period that T-cell receptor (TCR) b-chain rearrangement begins (Godfrey
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et al., 1993), the inducement of this rearrangement is thought to involve interleukin-7 (IL-7) and recombinationactivating genes 1 and 2 (RAG-1, RAG-2) (Muegge et al., 1993). Expression of the TCR b-chain at the thymocyte surface requires an a-chain equivalent to form the pre-TCR (pre-TCRa), this pre-TCRa induces expansion and differentiation of these TN cells into the double positive cortical CD4þCD8þ thymocytes (von Boehmer and Fehling, 1997). At this double positive stage the TCR a-chain undergoes rearrangement and a competent TCR ab receptor on these immature T-cells is the basis on which they undergo either positive or negative selection, processes which result in the loss of greater than 95% of developing thymocytes. The positively selected thymocytes finally form single positive T-cells expressing either CD4 or CD8 located in the thymic medulla. Age related involution has been described histologically as the loss of cortical areas of the thymus without much loss to medullary regions (Kraft et al., 1988) which would suggest a loss of the early stages of the T-cell developmental pathway without loss of stages found in the medulla. Indeed, it has been shown that there is a blockage in the T-cell developmental pathway occurring within the TN subset. The amount of CD44þCD25þ thymocytes and the progeny of these cells showed a marked reduction with age, however, no significant difference was seen in the CD44þCD252 population, leading to the suggestion that the lesion occurred after this subset (Aspinall, 1997). The expression of CD25 is associated with the rearrangement of TCR b-chain genes (Godfrey et al., 1994). The decreased capacity of aged cells to undergo the transition from CD44þCD252 to CD44þCD25þ led to the suggestion that thymic involution is the product of the failure of the cells to undergo differentiation through this particular stage of T-cell development. Indeed experiments using transgenic animals confirm this proposal that age-associated thymic atrophy is associated with problems with rearrangement and expression of the TCR b-chain genes. F5 transgenic mice that were carrying a complete TCR-ab transgene under the control of a CD2 minigene showed no age-associated thymic atrophy when compared to normal mice and a similar result was also observed when using the same transgenes on a RAG-RO background (Aspinall, 1997).
3. Age and the thymic microenvironment Many papers have been written about the possible mechanisms causing age-associated thymic atrophy, with theories ranging from having fewer or faulty progenitors to changes in the microenvironment of the thymus. However, it is now generally accepted that the loss in thymic function results from a failure of the thymic microenvironment to support thymopoiesis. This microenvironment is provided by the thymic stroma and cytokines and there is evidence that changes in both the functions of the thymic stroma and
in the levels of cytokines affect T-cell development (Mackall et al., 1998; Rodewald et al., 1997). One of the most important cytokines in the developmental pathway is IL-7, which is produced by MHC class IIþ thymic epithelial cells. IL-7’s role in thymocyte development has been linked to the survival and proliferation of thymocytes and is also thought to be important during the rearrangement of TCR b-chain (Moore et al., 1996; Oosterwegel et al., 1997). The level of IL-7 is reduced with age (Andrew and Aspinall, 2002; Ortman et al., 2002), however, it is not clear whether this reduction is due to loss of the IL-7-producing thymic epithelial cells or to a decline in epithelial cell functions. Connexin proteins form the gap junction channels between epithelial cells and proteins such as keratin-8, also expressed by cortical epithelium, can both be used as a measurement of the relative proportion of cortical epithelial cells in thymic stroma. Studies in aged mice show that both connexin 43 (Andrew and Aspinall, 2002) and keratin-8 (Ortman et al., 2002) decline with age, however, the decline of both these proteins was not as marked as the loss of IL-7 with age, suggesting that a reduction in IL-7 production is not matched by a similar loss of epithelial cells. It has been postulated that epithelial cells without the capacity to produce IL-7 are replacing cells that do have this capacity (Andrew and Aspinall, 2002). However, this may be an over simplification of the complexity of thymic involution because the transcription factor Foxn 1 (Whn) that is expressed by epithelial cells and is critical for thymic epithelial cell proliferation and differentiation is also significantly reduced in the aged thymus (Ortman et al., 2002). The importance of IL-7 is seen when it is added to TN thymocyte cultures isolated from old mice. IL-7 significantly reduced apoptosis and increased the percentage of live cells within CD44þCD25þ and CD442CD25þ subpopulations after 24 h and pro-survival effects remained after 5 days (Andrew and Aspinall, 2001). However, in order to test the effectiveness of IL-7 when administered in vivo an accurate measure of thymic function has to be found. Until recently thymic function could only be evaluated indirectly by phenotyping of naı¨ve T-cells in the peripheral pool or through the use of thymic scan. Thymic scans use computerised axial tomography to measure the volume of the thymus and correlates the measured volume with thymic output. Although this provides a useful estimate of thymic size, results have to be interpreted with care because any increase in thymic size may be due to the infiltration of T-cells into the thymus. The use of phenotypic markers to quantify the number of naı¨ve and memory T-cells has worked well in avian (Kong et al., 1998) and rodent models (Hosseinzadeh and Goldschneider, 1993) using young animals, however, such clarity of definition does not exist when applied to aged animals. For it has been shown that recent thymic emigrants can masquerade as phenotypic memory T-cells whilst the true, antigen specific clonally expanded memory T-cells can show phenotypic characteristics of naı¨ve T-cells (Wills et al., 1999). Indeed
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we have found the use of phenotypic analysis to reveal there to be no significant difference in the number of phenotypic naı¨ve (CD442CD45RBþ) or memory (CD44þCD45RB2) CD4þ or CD8þ cells isolated from mice aged 24 months after treatment with IL-7. A finding also shared by others, Fry and Mackall, using phenotypic analysis, showed that the administration of IL-7 to aged hosts did not lead to an increased thymic output, but a finding which was not corroborated by molecular analysis (Fry and Mackall, 2002).
4. Identification of recent thymic emigrants Researchers began to look for alternative, more sensitive methods of measuring thymic function. It was only with the development of a novel molecular method, TCR excision circle (TREC) analysis, that researchers have been able to gain a better idea about the number of recent thymic emigrants coming from the thymus. TREC analysis has been successfully used to study thymic output first in chickens (Kong et al., 1998) and then in humans (Douek et al., 1998) and it is only with the advent of this technique
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that scientists have had the power to distinguish naı¨ve from memory cells. TREC analysis quantitates the amount of episomal DNA circles that are produced during the rearrangement of ab TCR genes. A requirement for the productive rearrangement of the TCRA locus is the deletion of the TCRD locus contained within it. Rearrangements at the a/d locus produce two types of TREC that are found within about 70% of ab T-cells, a signal joint TREC and a coding joint TREC (Fig. 1). End-to-end ligation of the recombination signal sequences flanking the drec locus and the cJa locus removes the TCRD locus, forming the signal joint sequence. The recombined drec to cJa junction, the coding joint, is retained in the signal joint sequence until TCRAV to TCRAJ recombination occurs. A maximum of two signal joint TRECs and two coding joint TRECs can be present within one ab T-cell if the corresponding rearrangement occurs in both alleles. TRECs are not integrated into the genome, have been shown to be stable, are not duplicated during mitosis, and are therefore diluted out with each cellular division. Meaning, TREC levels are higher in thymocytes that have undergone b-rearrangement when compared to naı¨ve T-cells and likewise naı¨ve T-cells have higher TREC levels than memory T-cells. This method,
Fig. 1. Formation of T-cell receptor excision circles (TRECs). TRECs are formed when the TCR delta locus that is contained within the TCR alpha locus is deleted prior to the alpha gene rearrangement. The dRec and cJa are brought towards each other and cleaved. These cleaved portions are then ligated together to form an extrachromsomal circular DNA product containing the TCR delta locus known as the signal joint TREC. The recombined dRec to cJa junction forms the coding joint TREC and is removed from the signal joint when TCR Va to Ja recombination occurs.
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therefore, provides an accurate measure of the number of recent thymic emigrant and so thymic function. Indeed, many researchers have employed TREC methodology to determine changes in thymic function with age, high TREC levels were shown to be present during childhood but declined with increasing age (Douek et al., 1998). TRECs were also detectable in the elderly (Douek et al., 1998) confirming previous histological evidence for a continuance of thymic output with old age. Although TREC levels have been widely used as a measurement of thymic function the interpretation of TREC data need to be approached with care because TREC levels are determined not only by the output of the thymus but also by the extent of proliferation, cell death and intracellular degradation of these episomal circles. Currently, it is hard to determine the relative effects of thymic output and T-cell proliferation on TREC levels experimentally. Proliferation can be evaluated by the use of the intracellular stain Ki67, a nuclear antigen that is expressed in cells undergoing proliferation. Indeed, assessment of the levels of TRECs and Ki67 expression after IL-7 therapy in cynomolgus monkeys (Fry et al., 2003), showed that the number of TRECs after 11 days of treatment dramatically declined from the base line values and was accompanied by an increase in the expression of Ki67 leading the authors to conclude that IL-7 therapy induced T-cell turn over rather than thymopoiesis. However, the finding may not be a true representation of the effect of IL-7 therapy because the authors compared TREC values to a base-line figure measured before treatment had commenced. Thus, the drop in TREC values may be caused by the stress of the treatment regimen, as stress is known to induce apoptosis in the double positive thymocyte subset (Tarcic et al., 1998). An alternate way of determining whether measuring the TREC content provides a true representation of thymic function is through the use of mathematical modelling. An early simplistic model suggested that the decline in thymic function affects TREC numbers when accompanied by peripheral T-cell division (Hazenberg et al., 2000). The authors based their model on the assumption that
naı¨ve T-cell division and intracellular degradation of TRECs do not occur, however, this is unlikely to be the case for even though TRECs are stably maintained they have a finite lifespan. The half-life of TRECs in chickens has been shown to be 2 weeks (Kong et al., 1998), in rhesus macaques TRECs are still present 1 year after thymectomy (Sodora et al., 2000) and further more TRECs can still be detected in humans who have undergone thymectomy up to 40 years previously (Douek et al., 1998). Based on these observations more sophisticated models could be formed and when used predicted that the rate of TREC degradation was 0.002 per day, similar to the T-cell death rate (Ye and Kirschner, 2002). This mathematical model was also able to confirm previous reports that during ageing, the reduction in TRECs seen within CD4þ T-cells is caused by thymic impairment, whereas both increased peripheral T-cell division and decreased thymic output induce the decline in TREC levels within CD8þ T-cells, with T-cell death influencing TREC content in both CD4þ and CD8þ populations to a lesser extent (Douek et al., 1998; Steffens et al., 2000). One other confounding factor that complicates the interpretation of TREC data is the variety of different techniques used to measure TREC levels and the different units researchers choose to express TRECs, which together make it hard to compare between different studies. Another note of caution has to be made when transferring the TREC methodology from a human to a mouse model, for although the two systems are similar there are subtle differences that have the potential to greatly influence the TREC content. The mouse drec element is more promiscuous in its binding than that of the human, having the ability to bind other Ja gene segments as alternative acceptor sites to the cJa gene (Shutter et al., 1995), which results in a lower frequency of drec-cJa rearrangements. Also, in the mouse there are three drec elements and these drecs can use any of the other Jas and many do so in preference to cJa (Fig. 2). Therefore, the use of primers specific for the mouse produces PCR products with a range of sizes due to the promiscuity of the drec and its acceptor sites, leading to an underestimation of the number of TRECs present. Indeed,
Fig. 2. Increased promiscuity in excision points in the mouse compared with human. The mouse has three drec elements and any of these elements can use any of the Ja genes as 30 acceptor sites and many do so in preference to cJa, leading to a lower frequency of drec-cJa rearrangements.
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a recent report suggested that the administration of IL-7 to aged mice did not augment thymopoiesis, as demonstrated by a lack of difference in the number of TRECs per 100,000 thymocytes in saline-treated versus IL-7-treated young mice (Sempowski et al., 2002). However, the authors chose to measure TRECs and as mentioned above this can lead to an underestimation of TREC levels and subsequently a misinterpretation of the results. Indeed the standard errors of the mean for their TREC data are very large, in several cases being bigger than the measured TREC value, suggesting this to be the case. The standard error values are likely to represent the large error incurred by the promiscuity of the drec-cJa rearrangements. The products of the PCR formed using the primers designed by the investigators in question confirm this and Fig. 3 shows the multiple banding patterns produced by the PCR products, with the measured 93 base pair product not even being the major product formed. A more reliable molecular method for measuring thymic output in the mouse is through the use of TCR delta excision circle (dEC) analysis. This method is based on the fact that all murine episomal TREC DNA circles contain the TCRd constant gene (Pido-Lopez et al., 2002). Thus, when primers specific to the TCRd constant region are utilised they essentially detect all the TRECs present in murine T-cells with only one product generated allowing for a more accurate measure of thymic output (Fig. 4). When this dECs method was employed to measure thymic output following IL-7 treatment, IL-7 was shown to increase the level of dECs detected in IL-7-treated old mice compared to saline treated controls, suggesting that IL-7 could increase thymopoeisis (Pido-Lopez et al., 2002).
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Fig. 4. Single PCR product produced using dEC primers (Pido-Lopez et al., 2002). 100 ng of DNA was purified from ab T-cells from the spleens of 6 week old mice and used for PCR. A range of Mg2þ concentrations were used for the PCR reactions; 1.5, 3, and 4.5 mM, lanes 2–4, respectively, lane 1 contains the 100 bp ladder.
5. Summary We have discussed the importance of IL-7 in the T-cell developmental pathway, highlighting the reports that suggest the administration of IL-7 to aged animals causes de novo T-cell production by the thymus. However, we have also shown that the field remains dogged with controversy with many researchers failing to show any affect of IL-7 treatment on thymopoiesis. This controversy stems from the varying methods employed by researchers to identify recent thymic immigrants. To date the role played by IL-7 in vivo still remains unclear and what is needed is additional studies with carefully interpreted data to be undertaken in order to resolve the current discrepancies in the literature.
References
Fig. 3. Multiple PCR products produced using mouse TREC primers (Sempowski et al., 2002). 100 ng of DNA was isolated from ab T-cells from the spleens of 6-week-old mice and used for PCR. A range of Mg2þ concentrations were used for each reaction; 1.5, 3, and 4.5 mM, lanes 2–4, respectively, lane 1 contains the 100 bp ladder.
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Reversal of thymic atrophy Sian M. Henson*, Jeffrey Pido-Lopez, Richard Aspinall Department of Immunology, Imperial College, Chelsea and Westminster Hospital, 369 Fulham Road, London SW10 9NH, UK Received 24 June 2003; received in revised form 25 September 2003; accepted 10 October 2003
Abstract Age-associated thymic atrophy is a key event preceding the inefficient functioning of the immune system, resulting in a diminished capacity to generate new T-cells. This thymic involution has been proposed to be due to changes in the thymic microenvironment resulting in its failure to support thymopoiesis. A key cytokine in the early stages of thymocyte development is IL-7 and expression levels are greatly reduced with age. The ability of IL-7 to restore the immune system by enhancing thymic output remains controversial. In this review, we highlight the advances in molecular approaches used to evaluate recent thymic emigrants and assess the success of these strategies in determining whether IL-7 can lead to immune reconstitution. q 2004 Elsevier Inc. All rights reserved. Keywords: Atrophy; Thymus; IL-7; TRECs; DECs
1. Age and the thymus The thymus is the primary organ for the production of ab T-cells and is thus essential for a functional immune system. However, removal of the thymus from children after six months of age (Brearley et al., 1987) does not result in overt immunodeficiency before the end of adolescence, a finding which lead to the assumption that the thymus is an organ that becomes redundant with time. This however, is not actually the case because peripheral T-cell numbers are maintained by a powerful homeostatic compensatory mechanism which causes the peripheral expansion of mature T-cells leading to the regeneration of the T-cell pool (Freitas and Rocha, 2000) Thymic function is at its most active during the foetal and perinatal periods but output declines with age resulting in a dramatic loss in the number of naı¨ve T-cells being produced. This decline in de novo T-cell production correlates with the atrophy of the thymic stroma, a condition known as thymic involution. Several changes to the thymic architecture can be observed, the perivascular space becomes enlarged leading to a decreased thymic epithelial space, an area that houses both the cortical and medullary components responsible for thymopoiesis (Steinmann, 1986). The perivascular space is enlarged by lymphocytic infiltration but as ageing * Corresponding author. Tel.: þ44-20-876-8250; fax: þ 44-20-87465997. E-mail address: [email protected] (S.M. Henson). 0531-5565/$ - see front matter q 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.exger.2003.10.030
progresses adipose tissue eventually replaces the lymphocytic perivascular space (Steinmann et al., 1985). However, it has been shown that the elderly still retain a limited thymic function, with thymocytes being produced by a small thymic rudiment composed of epithelium (Steinmann et al., 1985).
2. T-cell development and age changes Despite this loss in thymic output precursor T-cells continuously migrate from the bone marrow to the thymus throughout life. The earliest precursor cells that arrive at the murine thymus are CD32CD4loCD82 progenitors (Wu et al., 1991). These early precursors can be considered to be effectively triple negative (TN) because CD4 knock out mice showed normal progression through this early phase of development, suggesting that the expression of CD4 does not play a fundamental role at this stage of the pathway (Rahemtulla et al., 1991). This TN population can be further subdivided on the basis of expression of CD44 and CD25. The most immature stage being CD44þCD252, maturation through the T-cell developmental pathway proceeds through CD44þCD25þ then CD442CD25þ to a stage that is CD442CD252 and it is during the transition between these late stages that the majority of thymocyte expansion occurs. It is also during this period that T-cell receptor (TCR) b-chain rearrangement begins (Godfrey
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et al., 1993), the inducement of this rearrangement is thought to involve interleukin-7 (IL-7) and recombinationactivating genes 1 and 2 (RAG-1, RAG-2) (Muegge et al., 1993). Expression of the TCR b-chain at the thymocyte surface requires an a-chain equivalent to form the pre-TCR (pre-TCRa), this pre-TCRa induces expansion and differentiation of these TN cells into the double positive cortical CD4þCD8þ thymocytes (von Boehmer and Fehling, 1997). At this double positive stage the TCR a-chain undergoes rearrangement and a competent TCR ab receptor on these immature T-cells is the basis on which they undergo either positive or negative selection, processes which result in the loss of greater than 95% of developing thymocytes. The positively selected thymocytes finally form single positive T-cells expressing either CD4 or CD8 located in the thymic medulla. Age related involution has been described histologically as the loss of cortical areas of the thymus without much loss to medullary regions (Kraft et al., 1988) which would suggest a loss of the early stages of the T-cell developmental pathway without loss of stages found in the medulla. Indeed, it has been shown that there is a blockage in the T-cell developmental pathway occurring within the TN subset. The amount of CD44þCD25þ thymocytes and the progeny of these cells showed a marked reduction with age, however, no significant difference was seen in the CD44þCD252 population, leading to the suggestion that the lesion occurred after this subset (Aspinall, 1997). The expression of CD25 is associated with the rearrangement of TCR b-chain genes (Godfrey et al., 1994). The decreased capacity of aged cells to undergo the transition from CD44þCD252 to CD44þCD25þ led to the suggestion that thymic involution is the product of the failure of the cells to undergo differentiation through this particular stage of T-cell development. Indeed experiments using transgenic animals confirm this proposal that age-associated thymic atrophy is associated with problems with rearrangement and expression of the TCR b-chain genes. F5 transgenic mice that were carrying a complete TCR-ab transgene under the control of a CD2 minigene showed no age-associated thymic atrophy when compared to normal mice and a similar result was also observed when using the same transgenes on a RAG-RO background (Aspinall, 1997).
3. Age and the thymic microenvironment Many papers have been written about the possible mechanisms causing age-associated thymic atrophy, with theories ranging from having fewer or faulty progenitors to changes in the microenvironment of the thymus. However, it is now generally accepted that the loss in thymic function results from a failure of the thymic microenvironment to support thymopoiesis. This microenvironment is provided by the thymic stroma and cytokines and there is evidence that changes in both the functions of the thymic stroma and
in the levels of cytokines affect T-cell development (Mackall et al., 1998; Rodewald et al., 1997). One of the most important cytokines in the developmental pathway is IL-7, which is produced by MHC class IIþ thymic epithelial cells. IL-7’s role in thymocyte development has been linked to the survival and proliferation of thymocytes and is also thought to be important during the rearrangement of TCR b-chain (Moore et al., 1996; Oosterwegel et al., 1997). The level of IL-7 is reduced with age (Andrew and Aspinall, 2002; Ortman et al., 2002), however, it is not clear whether this reduction is due to loss of the IL-7-producing thymic epithelial cells or to a decline in epithelial cell functions. Connexin proteins form the gap junction channels between epithelial cells and proteins such as keratin-8, also expressed by cortical epithelium, can both be used as a measurement of the relative proportion of cortical epithelial cells in thymic stroma. Studies in aged mice show that both connexin 43 (Andrew and Aspinall, 2002) and keratin-8 (Ortman et al., 2002) decline with age, however, the decline of both these proteins was not as marked as the loss of IL-7 with age, suggesting that a reduction in IL-7 production is not matched by a similar loss of epithelial cells. It has been postulated that epithelial cells without the capacity to produce IL-7 are replacing cells that do have this capacity (Andrew and Aspinall, 2002). However, this may be an over simplification of the complexity of thymic involution because the transcription factor Foxn 1 (Whn) that is expressed by epithelial cells and is critical for thymic epithelial cell proliferation and differentiation is also significantly reduced in the aged thymus (Ortman et al., 2002). The importance of IL-7 is seen when it is added to TN thymocyte cultures isolated from old mice. IL-7 significantly reduced apoptosis and increased the percentage of live cells within CD44þCD25þ and CD442CD25þ subpopulations after 24 h and pro-survival effects remained after 5 days (Andrew and Aspinall, 2001). However, in order to test the effectiveness of IL-7 when administered in vivo an accurate measure of thymic function has to be found. Until recently thymic function could only be evaluated indirectly by phenotyping of naı¨ve T-cells in the peripheral pool or through the use of thymic scan. Thymic scans use computerised axial tomography to measure the volume of the thymus and correlates the measured volume with thymic output. Although this provides a useful estimate of thymic size, results have to be interpreted with care because any increase in thymic size may be due to the infiltration of T-cells into the thymus. The use of phenotypic markers to quantify the number of naı¨ve and memory T-cells has worked well in avian (Kong et al., 1998) and rodent models (Hosseinzadeh and Goldschneider, 1993) using young animals, however, such clarity of definition does not exist when applied to aged animals. For it has been shown that recent thymic emigrants can masquerade as phenotypic memory T-cells whilst the true, antigen specific clonally expanded memory T-cells can show phenotypic characteristics of naı¨ve T-cells (Wills et al., 1999). Indeed
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we have found the use of phenotypic analysis to reveal there to be no significant difference in the number of phenotypic naı¨ve (CD442CD45RBþ) or memory (CD44þCD45RB2) CD4þ or CD8þ cells isolated from mice aged 24 months after treatment with IL-7. A finding also shared by others, Fry and Mackall, using phenotypic analysis, showed that the administration of IL-7 to aged hosts did not lead to an increased thymic output, but a finding which was not corroborated by molecular analysis (Fry and Mackall, 2002).
4. Identification of recent thymic emigrants Researchers began to look for alternative, more sensitive methods of measuring thymic function. It was only with the development of a novel molecular method, TCR excision circle (TREC) analysis, that researchers have been able to gain a better idea about the number of recent thymic emigrants coming from the thymus. TREC analysis has been successfully used to study thymic output first in chickens (Kong et al., 1998) and then in humans (Douek et al., 1998) and it is only with the advent of this technique
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that scientists have had the power to distinguish naı¨ve from memory cells. TREC analysis quantitates the amount of episomal DNA circles that are produced during the rearrangement of ab TCR genes. A requirement for the productive rearrangement of the TCRA locus is the deletion of the TCRD locus contained within it. Rearrangements at the a/d locus produce two types of TREC that are found within about 70% of ab T-cells, a signal joint TREC and a coding joint TREC (Fig. 1). End-to-end ligation of the recombination signal sequences flanking the drec locus and the cJa locus removes the TCRD locus, forming the signal joint sequence. The recombined drec to cJa junction, the coding joint, is retained in the signal joint sequence until TCRAV to TCRAJ recombination occurs. A maximum of two signal joint TRECs and two coding joint TRECs can be present within one ab T-cell if the corresponding rearrangement occurs in both alleles. TRECs are not integrated into the genome, have been shown to be stable, are not duplicated during mitosis, and are therefore diluted out with each cellular division. Meaning, TREC levels are higher in thymocytes that have undergone b-rearrangement when compared to naı¨ve T-cells and likewise naı¨ve T-cells have higher TREC levels than memory T-cells. This method,
Fig. 1. Formation of T-cell receptor excision circles (TRECs). TRECs are formed when the TCR delta locus that is contained within the TCR alpha locus is deleted prior to the alpha gene rearrangement. The dRec and cJa are brought towards each other and cleaved. These cleaved portions are then ligated together to form an extrachromsomal circular DNA product containing the TCR delta locus known as the signal joint TREC. The recombined dRec to cJa junction forms the coding joint TREC and is removed from the signal joint when TCR Va to Ja recombination occurs.
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therefore, provides an accurate measure of the number of recent thymic emigrant and so thymic function. Indeed, many researchers have employed TREC methodology to determine changes in thymic function with age, high TREC levels were shown to be present during childhood but declined with increasing age (Douek et al., 1998). TRECs were also detectable in the elderly (Douek et al., 1998) confirming previous histological evidence for a continuance of thymic output with old age. Although TREC levels have been widely used as a measurement of thymic function the interpretation of TREC data need to be approached with care because TREC levels are determined not only by the output of the thymus but also by the extent of proliferation, cell death and intracellular degradation of these episomal circles. Currently, it is hard to determine the relative effects of thymic output and T-cell proliferation on TREC levels experimentally. Proliferation can be evaluated by the use of the intracellular stain Ki67, a nuclear antigen that is expressed in cells undergoing proliferation. Indeed, assessment of the levels of TRECs and Ki67 expression after IL-7 therapy in cynomolgus monkeys (Fry et al., 2003), showed that the number of TRECs after 11 days of treatment dramatically declined from the base line values and was accompanied by an increase in the expression of Ki67 leading the authors to conclude that IL-7 therapy induced T-cell turn over rather than thymopoiesis. However, the finding may not be a true representation of the effect of IL-7 therapy because the authors compared TREC values to a base-line figure measured before treatment had commenced. Thus, the drop in TREC values may be caused by the stress of the treatment regimen, as stress is known to induce apoptosis in the double positive thymocyte subset (Tarcic et al., 1998). An alternate way of determining whether measuring the TREC content provides a true representation of thymic function is through the use of mathematical modelling. An early simplistic model suggested that the decline in thymic function affects TREC numbers when accompanied by peripheral T-cell division (Hazenberg et al., 2000). The authors based their model on the assumption that
naı¨ve T-cell division and intracellular degradation of TRECs do not occur, however, this is unlikely to be the case for even though TRECs are stably maintained they have a finite lifespan. The half-life of TRECs in chickens has been shown to be 2 weeks (Kong et al., 1998), in rhesus macaques TRECs are still present 1 year after thymectomy (Sodora et al., 2000) and further more TRECs can still be detected in humans who have undergone thymectomy up to 40 years previously (Douek et al., 1998). Based on these observations more sophisticated models could be formed and when used predicted that the rate of TREC degradation was 0.002 per day, similar to the T-cell death rate (Ye and Kirschner, 2002). This mathematical model was also able to confirm previous reports that during ageing, the reduction in TRECs seen within CD4þ T-cells is caused by thymic impairment, whereas both increased peripheral T-cell division and decreased thymic output induce the decline in TREC levels within CD8þ T-cells, with T-cell death influencing TREC content in both CD4þ and CD8þ populations to a lesser extent (Douek et al., 1998; Steffens et al., 2000). One other confounding factor that complicates the interpretation of TREC data is the variety of different techniques used to measure TREC levels and the different units researchers choose to express TRECs, which together make it hard to compare between different studies. Another note of caution has to be made when transferring the TREC methodology from a human to a mouse model, for although the two systems are similar there are subtle differences that have the potential to greatly influence the TREC content. The mouse drec element is more promiscuous in its binding than that of the human, having the ability to bind other Ja gene segments as alternative acceptor sites to the cJa gene (Shutter et al., 1995), which results in a lower frequency of drec-cJa rearrangements. Also, in the mouse there are three drec elements and these drecs can use any of the other Jas and many do so in preference to cJa (Fig. 2). Therefore, the use of primers specific for the mouse produces PCR products with a range of sizes due to the promiscuity of the drec and its acceptor sites, leading to an underestimation of the number of TRECs present. Indeed,
Fig. 2. Increased promiscuity in excision points in the mouse compared with human. The mouse has three drec elements and any of these elements can use any of the Ja genes as 30 acceptor sites and many do so in preference to cJa, leading to a lower frequency of drec-cJa rearrangements.
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a recent report suggested that the administration of IL-7 to aged mice did not augment thymopoiesis, as demonstrated by a lack of difference in the number of TRECs per 100,000 thymocytes in saline-treated versus IL-7-treated young mice (Sempowski et al., 2002). However, the authors chose to measure TRECs and as mentioned above this can lead to an underestimation of TREC levels and subsequently a misinterpretation of the results. Indeed the standard errors of the mean for their TREC data are very large, in several cases being bigger than the measured TREC value, suggesting this to be the case. The standard error values are likely to represent the large error incurred by the promiscuity of the drec-cJa rearrangements. The products of the PCR formed using the primers designed by the investigators in question confirm this and Fig. 3 shows the multiple banding patterns produced by the PCR products, with the measured 93 base pair product not even being the major product formed. A more reliable molecular method for measuring thymic output in the mouse is through the use of TCR delta excision circle (dEC) analysis. This method is based on the fact that all murine episomal TREC DNA circles contain the TCRd constant gene (Pido-Lopez et al., 2002). Thus, when primers specific to the TCRd constant region are utilised they essentially detect all the TRECs present in murine T-cells with only one product generated allowing for a more accurate measure of thymic output (Fig. 4). When this dECs method was employed to measure thymic output following IL-7 treatment, IL-7 was shown to increase the level of dECs detected in IL-7-treated old mice compared to saline treated controls, suggesting that IL-7 could increase thymopoeisis (Pido-Lopez et al., 2002).
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Fig. 4. Single PCR product produced using dEC primers (Pido-Lopez et al., 2002). 100 ng of DNA was purified from ab T-cells from the spleens of 6 week old mice and used for PCR. A range of Mg2þ concentrations were used for the PCR reactions; 1.5, 3, and 4.5 mM, lanes 2–4, respectively, lane 1 contains the 100 bp ladder.
5. Summary We have discussed the importance of IL-7 in the T-cell developmental pathway, highlighting the reports that suggest the administration of IL-7 to aged animals causes de novo T-cell production by the thymus. However, we have also shown that the field remains dogged with controversy with many researchers failing to show any affect of IL-7 treatment on thymopoiesis. This controversy stems from the varying methods employed by researchers to identify recent thymic immigrants. To date the role played by IL-7 in vivo still remains unclear and what is needed is additional studies with carefully interpreted data to be undertaken in order to resolve the current discrepancies in the literature.
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Fig. 3. Multiple PCR products produced using mouse TREC primers (Sempowski et al., 2002). 100 ng of DNA was isolated from ab T-cells from the spleens of 6-week-old mice and used for PCR. A range of Mg2þ concentrations were used for each reaction; 1.5, 3, and 4.5 mM, lanes 2–4, respectively, lane 1 contains the 100 bp ladder.
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