Neonatal thymectomy identifies two major pools of sessile and recirculating peripheral T cells which appear to be under separate homeostatic control

Craig P. Cunningham, Wayne G. Kimpton, Anita Fernando and Ross N. P. Cahill

Laboratory for Foetal and Neonatal Immunology, School of Veterinary Science, University of Melbourne, Parkville, Victoria 3052, Australia

Correspondence to: R. N. P. Cahill


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In this study the role of the thymus in the development of sessile T cell populations resident in spleen and lymph nodes (LN) was contrasted with the development of recirculating T cell populations trafficking between blood and lymph. Extensive analysis of the composition and the rate of growth of the secondary lymphoid tissues and recirculating lymphocyte pool coupled with neonatal thymectomy revealed that the sessile and recirculating T cell populations showed different degrees of thymic dependency and increased in size at different rates, suggesting these two populations might be under separate homeostatic control. Neonatal thymectomy also resulted in a much greater depletion of CD8+ and {gamma}{delta} TCR+ T cell subsets compared with CD4+ T cells in the sessile and recirculating T cell pools, and greatly reduced the number of T cells homing to peripheral lymph nodes compared with those homing to the gut.

Keywords: neonatal thymectomy, recirculating T cell pool, T cell homeostasis


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The peripheral T cell pool can only grow by thymic export or self-renewal. The relative contribution of thymic export and peripheral expansion to the growth and maintenance of the peripheral T cell pool during young life remains relatively unexplored (1–3). In mice, thymic export begins around the time of birth (3) and early neonatal thymectomy in mice prevents the development of the peripheral T cell pool (4). In contrast to mice, thymic export in sheep and humans (both of which have a long gestation period) begins early during gestation, and the peripheral T cell pool of fetal lambs and humans is well developed at birth (5,6). In sheep, thymic export begins in the first third of gestation (gestation = 150 days) and the thymus reaches its maximum size (as a percentage of body weight) by ~120 days gestation (7). Early (60–100 days) fetal thymectomy largely prevents the normal growth and development of the peripheral T cell pool in fetal lambs (8,9), and in this regard resembles the effects of neonatal thymectomy of mice. The sheep thymus continues to grow after birth, reaching its maximum size at ~2 months of age (7), although the rate of thymic export from the thymus is the same in both fetal and postnatal lambs (10). These observations suggest that thymic export may continue to play a significant role in the growth of the peripheral T cell pool during postnatal life in young lambs. However, most work on thymic export has been conducted in adult mice. It has been suggested that in adults recent thymic emigrants are preferentially incorporated in the peripheral T cell pool causing displacement of pre-existing T cells (11). Other studies in mice suggested incorporation of recent thymic emigrants was random (12). More recently it was shown that multiple thymus grafting under the kidney capsule increased the size of the T cell pool and provided further evidence that emigrants are preferentially incorporated in the periphery (13).

In mice, the effects of neonatal thymectomy on the size of the peripheral T cell pool are only observed when thymectomy is performed in the first few days of life, after which thymectomy has little impact (14). Similarly, adult thymectomy in mice has little immediate impact on the size of the peripheral T cell pool (15), although the repertoire of peripheral T cells is considerably reduced due to homeostatic expansion of pre-existing peripheral T cells (11–13,16,17). Based on the effects of thymectomy in mice during neonatal life we asked what would be the effect(s) of neonatal thymectomy in lambs, which are both immunologically competent and have a well-developed peripheral T cell pool at birth (5,18,19)? It might be predicted that neonatal thymectomy in sheep should have little observable effect on the size of the peripheral T cell pool, given the maturity of the lymphoid system in the newborn lamb.

The relationship between the thymus and the growth and development of the peripheral T cell pool in postnatal lambs was examined by thymectomizing lambs shortly after birth. Neonatal thymectomy was found to have a profound impact on both the size and composition of the peripheral T cell pool in postnatal lambs. The results showed that the presence of the thymus was an absolute requirement for the normal growth of the peripheral T cell pool during early postnatal life in lambs.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Animals and surgery
All animals were obtained from I. and L. Grant (Geelong, Australia) from a controlled breeding program. Merino lambs were born on site in our animal house facility and housed with their mothers in pens. Anaesthesia was induced using thiopentone sodium (Pentothal, 50 mg/ml; Rhône Mérieux, Pinkenba, Australia) and maintained on halothane 1.5% (Fluothane; Zeneca, Macclesfield, UK) and oxygen in a semi-closed circuit system (Midget 3; CIG Medishield, North Ryde, Australia). For intrathoracic surgical procedures, positive pressure ventilation was performed prior to opening of the thorax and continued till after the chest had been adequately sealed.

Thymectomy
Thymectomy was performed according to a previously described method (20) which allows complete visualization of the entire thymus and its complete removal. Controls were unoperated animals of the same age.

Lymphatic cannulation
The efferent lymph ducts draining the prescapular and mesenteric lymph nodes (MLN) were cannulated using modifications of previously described techniques (21–23). For the MLN a large lymphatic duct draining 90% of the intestines was cannulated.

Blood collection and isolation of blood lymphocytes
Blood was collected from the jugular vein in EDTA (6.2 mg/ml blood; Ajax Chemicals, Melbourne, Australia) and lymphocytes were isolated from red blood cells using Tris-buffered ammonium chloride to lyse red cells. Cell concentrations were determined using a model FN Coulter counter (Coulter, Dunstable, UK). Blood lymphocyte counts were determined using a hemocytometer and differential counts performed on Geimsa-stained whole-blood smears. Blood volume was calculated using the weight of the animals on the basis of 74 ml/kg in lambs of this age (24). Total blood lymphocyte and subset counts were then determined by the formula lymphocyte concentration/mlxblood volume.

Lymph collection
Lymph was collected in sterile polypropylene bottles containing heparin at a final concentration of 100 U/ml (preservative free; DBL, Mulgrave, Australia). Lymph collection bottles were changed at regular intervals and the cell concentration in each collection was determined as above.

Cell suspensions
Animals were killed by injecting an overdose of barbiturate anaesthetic and tissues collected for analysis. LN (prescapular, prefemoral, popliteal, mesenteric and caudal mediastinal) and thymus were weighed and placed in PBS containing BSA (2%), sodium azide (0.1%) and EDTA (0.4%) (FACS buffer). Organs were gently teased through a stainless steel mesh into FACS buffer, washed 3 times and resuspended in FACS buffer as previously described (10). The total number of cells/g tissue was calculated after allowing for cells lost during preparation and cell concentrations were determined as above using a Coulter counter. Spleen cell suspensions were made in the same way except after the first wash; cells were placed in Tris-buffered ammonium chloride to lyse red blood cells and then washed 3 times in FACS buffer.

mAb
The mAb directed against sheep lymphocyte surface antigens CD5 (25-91), CD4 (44-38/44-97), CD8 (38-65) and {gamma}{delta} TCR (86D) were obtained as tissue culture supernatant or as ascites fluid from Dr M. Brandon (University of Melbourne), and have been described previously (25–27).

Immunofluorescent staining and flow cytometry
Cells were washed 3 times in FACS buffer before immunofluorescent staining and between reaction steps. All incubations were for 30 min and cells were kept at 4°C at all times. Cells (3x106) were first incubated with 50 µl mAb, followed by phycoerythrin-conjugated anti-mouse IgG (Fab')2 (Silenus Laboratories, Hawthorn, Australia). All samples were analyzed as fresh preparations on FACScan/FACSCalibur flow cytometers (Becton Dickinson, Sunnyvale, CA). The machines were calibrated with beads (CaliBRITE; Becton Dickinson) using AutoCOMP software (Becton Dickinson). Additional compensation was set by eye daily. Acquisition and analysis of date was performed using CellQuest software (CellQuest version 3.1f; Becton Dickinson). Dead cells and granulocytes were gated out on the basis of forward and side angle scatter and autofluorescence.

Statistical analysis
A Student's unpaired t-test was used to compare between two groups of normally distributed data. Statistical analysis was performed using Minitab 10.5 Xtra software (Minitab, State College, PA).


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Neonatal thymectomy largely prevents the continued growth of the peripheral T cell pool in young lambs
Newborn (1–2 days old) lambs were thymectomized, and the weights and the total number of cells in spleen, peripheral LN (PLN), caudal mediastinal LN and MLN were determined 3 months later. Neonatal thymectomy had profound effects on both the size and cellularity of the secondary lymphoid tissues (Fig. 1Go). The caudal mediastinal LN, PLN and MLN of neonatally thymectomized (NTx) lambs weighed significantly less than non-thymectomized lambs when examined at 3 months of age although neonatal thymectomy had little effect on the size of the spleen, as the spleen weights were the same in both NTx and thymus intact (Ti) lambs 3 months after birth (Fig. 1Go). Although the weight of the spleen was not affected by neonatal thymectomy at birth (possibly due to an increase in size of the red pulp although this was not examined), significantly fewer lymphocytes were recovered from the spleen in NTx compared with Ti lambs (Fig. 1Go). The smaller weight of PLN of NTx lambs was associated with reduced numbers of cells being recovered from the PLN of NTx lambs compared with Ti lambs. Furthermore, whereas 120x109 cells were recovered from the MLN of 3-month-old Ti lambs, only 53x109 cells were recovered from MLN of NTx lambs 3 months later (Fig. 1Go).



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Fig. 1. Reduced growth of the secondary lymphoid tissues following neonatal thymectomy in postnatal lambs. Lambs were thymectomized 1–2 days after birth, and the weights and cellularity of LN and spleen measured 3 months later. Values represent the mean ± SEM of seven animals in the Ti group (open bars) and eight animals in the NTx group (solid bars). *Statistically significant (P < 0.05) difference using a paired Student's t-test.

 
When cell populations within individual tissues were examined, it was found that NTx resulted in changes in the composition and total number of T cells, T cell subsets and B cells in all peripheral lymphoid tissues. Figure 2Go shows the total number of all lymphocyte subsets recovered in the blood, spleen, PLN and MLN of Ti and NTx lambs at 3 months of age. The number of T cells found in the blood, spleen and LN of NTx lambs was significantly less (P < 0.05) than the number found in these tissues in Ti lambs (Fig. 2Go). Overall, the number of T cells found in NTx lambs was reduced by ~70% compared to age-matched Ti controls. The total number of B cells in the periphery of NTx lambs was also reduced compared with Ti lambs. In Ti lambs 50x109 B cells were found in the blood, spleen and LN, in contrast to NTx lambs in which 34x109 B cells were recovered in these tissues (Fig. 2Go). It has been shown previously that following fetal thymectomy the ileal Peyer's patch was reduced in size in NTx lambs compared with Ti lambs (7). The reduced B cell numbers could have been due to reduced numbers of memory T cells because of a lack of appropriate T help or reduced export of naive B cells from the Peyer's patch. Analysis of the phenotype of B cells might help to determine which was most likely.



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Fig. 2. NTx largely prevents the continued growth of all T cell subsets in secondary lymphoid tissues. The total number of T cells, T cell subsets and B cells in blood, spleen, PLN and MLN is shown in 3-month-old Ti (open bars) and NTx animals (solid bars). Values represent the mean ± SEM of seven animals in the Ti group and eight animals in the NTx group. *Statistically significant (P < 0.05) difference using a paired Student's t-test.

 
The growth of peripheral CD4+, CD8+ and {gamma}{delta} TCR+ T cell populations is impaired following neonatal thymectomy
The effects of neonatal thymectomy were more dramatic on CD8+ and {gamma}{delta} TCR+ peripheral T cells than on CD4+ T cells. CD8+ and {gamma}{delta} TCR+ T cells in 3-month-old NTx lambs were reduced by 75 and 80% respectively when compared with 3-month-old Ti lambs, whereas CD4+ T cells in 3-month-old NTx lambs were reduced by 64% compared to 3-month-old Ti lambs (Fig. 2Go). CD8+ T cells were uniformly reduced in all tissue compartments as a consequence of NTx. CD4+ T cells in the blood and spleen of 3-month-old NTx lambs were ~50% of the number of CD4+ T cells recovered in the blood and spleen of 3-month-old Ti lambs (Fig. 2Go). NTx had a slightly greater affect on CD4+ T cell populations in PLN and MLN, where their numbers were reduced to 60% of the number of CD4+ T cells recovered from PLN and MLN in age-matched control lambs.

NTx had a much greater impact on {gamma}{delta} T cells populations in the blood, spleen and MLN compared with PLN (Fig. 2Go). {gamma}{delta} TCR+ T cells were reduced in the blood (88%), spleen (76%) and MLN (79%), whereas the number of {gamma}{delta} TCR+ T cells in the PLN of 3-month-old thymectomized lambs were only reduced by ~40% (Fig. 2Go). {gamma}{delta} T cells in the PLN may therefore have a longer lifespan or might be preferentially expanded post-thymically, compared with {gamma}{delta} T cells in spleen and MLN.

Neonatal thymectomy has different effects on the size of the sessile and recirculating T cell pools, suggesting they might be under separate homeostatic control
Lymphocytes are distributed widely throughout the body, although they are concentrated in secondary lymphoid tissues, such as the spleen and LN (6,28). The majority of lymphocytes in the spleen and LN are sessile, i.e. they do not normally leave these organs (6,28,29). In contrast, there exists a second mobile population, which continuously recirculates between the blood, peripheral tissues, LN and lymph. The size of this recirculating lymphocyte pool (RLP) is determined by chronic lymphatic drainage (usually thoracic duct) and is based on the total number of lymphocytes collected in lymph following drainage for 3–5 days (15,29,30). The RLP (which consists of 75–80% T cells) has been estimated to comprise ~10% of the total number of lymphocytes present in young adult sheep (31), and is a key integrating feature of the adaptive immune system allowing for immune surveillance and the dissemination of immunological memory (32).

The sizes of the sessile and recirculating lymphocyte pools were examined in 3-month-old lambs which had been thymectomized at birth and in aged-matched nonthymectomized lambs (Table 1Go). It was found that neonatal thymectomy had a much greater effect on the postnatal growth of recirculating than of sessile lymphocyte populations. The recirculating T cell pool was reduced by ~75% in NTx lambs compared with Ti lambs. The number of sessile T cells in spleen and LN was reduced by 56% in NTx lambs compared with Ti lambs (Table 1Go). In contrast to T cells, the number of B cells was reduced to the same degree in both the sessile and RLP (38–42% reduction).


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Table 1. The effect of neonatal thymectomy on the growth of sessile and recirculating lymphocyte populations in young lambs
 
The reduced size of the recirculating T cell pool in NTx lambs resulted in a reduced cell output from LN of NTx lambs (Fig. 3Go). The efferent lymph output from the prescapular LN and MLN was significantly (P < 0.05) reduced in NTx lambs (Fig. 3Go). The cell output from the prescapular LN in 3-month-old NTx lambs was 0.3x108 cells/h; in contrast, the cell output in 3-month-old Ti lambs was 2.6x108 cells/h. The cell output from the MLN of 3-month-old Ti lambs was 9.4x108 cells/h, while in 3-month-old NTx lambs, the cell output was 3.5x108 cells/h (Fig. 3Go). Neonatal thymectomy had a much greater impact on the output from PLN compared to MLN. The output from the MLN in NTx lambs was reduced by ~3.5-fold compared with Ti lambs. In contrast, the cell output from the prescapular LN of NTx lambs was reduced by ~10-fold compared with Ti lambs.



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Fig. 3. The reduced size of the recirculating lymphocyte pool following NTx resulted in a reduced cell output from PLN and MLN efferent lymph. Values represent the mean ± SEM of three animals in the Ti (open bars) and five animals in the NTx group (solid bars). *Statistically significant (P < 0.05) difference using a paired Student's t-test.

 
NTx affected the growth of all T cell populations in the sessile and recirculating T cell pools, with the growth of the recirculating T cell populations being more dramatically influenced than the growth of the corresponding populations within the sessile lymphoid tissues. For example, the number of CD4+ T cells in the sessile lymphoid tissues in NTx lambs was about half the number found in the spleen and LN of Ti lambs. In contrast, the number of CD4+ T cells in the RLP of NTx lambs was reduced by 70% compared with the number of recirculating CD4+ T cells in Ti lambs (Table 1Go). Similar findings were observed for both CD8+ and {gamma}{delta} TCR+ T cells in the sessile and recirculating lymphocyte pools (Table 1Go). Not only were total T cell numbers reduced following NTx, the normal CD4/CD8 ratios found in Ti lambs were not conserved. CD4+ T cells were the dominant population in PLN and RLP, and CD8+ T cells were the major subset in spleen and MLN in Ti lambs (Fig. 4Go). After NTx there was a marked increase in the CD4/CD8 ratios in both sessile and recirculating populations. These findings suggest that some homeostatic expansion of CD4+ T cells was occurring and that CD8+ T cells were more thymic dependent than CD4+ T cells (Fig. 4Go).



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Fig. 4. CD4:CD8 ratios in 3-month-old lambs. Values represent the mean ± SEM of three animals in the Ti (open bars) group and five animals in the NTx group (solid bars) group.

 
Differential post-thymic expansion of sessile and recirculating T cell subsets in the postnatal lamb suggests the sizes of these two pools might be independently regulated
The size of the peripheral lymphocyte pool in 3-month-old NTx lambs was compared with the size of the peripheral lymphocyte pool of 2-week-old non-thymectomized lambs (Fig. 5Go). The sizes of the T cell pools were similar in both 3-month-old NTx lambs and 2-week-old lambs, suggesting that there had been very little growth of the peripheral T cell pool in NTx lambs. This was not the case for the peripheral B cell pool, which was ~6 times larger in 3-month-old NTx (34x109 B cells) compared with 2-week-old lambs (6x109 B cells) (Fig. 5Go).



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Fig. 5. Differences in the post-thymic expansion of sessile and recirculating T cell subsets in the postnatal lamb. The sizes of the total, sessile and recirculating lymphocyte pools were determined in 2-week-old Ti (open bars) and 3-month-old NTx (solid bars) lambs. The values for the total lymphocyte pool represent the mean ± SEM of seven animals in the Ti group and eight animals in the NTx group. The size of the non-recirculating T cell pool was determined from the number of T cells remaining in the blood, spleen and LN following chronic lymphatic drainage (5–7 days) of both prescapular LN efferent and MLN efferent lymphatics. The size of the recirculating lymphocyte pool was determined from the total number of lymphocytes collected in lymph during chronic lymphatic drainage. The sizes of the sessile and recirculating T cell pools were determined using a smaller cohort of animals. The values for the sessile and recirculating pools represent mean ± SEM of three animals in the Ti group and five animals in the NTx group. *Statistically significant difference using a paired Student's t-test.

 
When the sizes of the sessile and recirculating lymphocyte T cell populations were examined it was found that there was little difference in the sizes of these pools in 3-month-old NTx and 2-week-old lambs except for the sessile CD4+ T cell population in spleen and LN (Fig. 5Go). While the size of the recirculating CD4+ T cell pool was similar in both 3-month-old NTx and 2-week-old lambs (11x109 and 14x109 cells respectively), the size of the sessile CD4+ T cell pool was significantly greater (P < 0.05) in 3-month-old NTx lambs compared with 2-week-old lambs (Fig. 5Go). Approximately 3x109 CD4+ sessile T cells were present in the spleen and LN of 2-week-old lambs, whereas 8x109 CD4+ T cells were recovered from the spleen and LN in 3-month-old NTx lambs following lymphatic depletion (Fig. 5Go). There was no growth of CD8+ and {gamma}{delta} TCR+ T cell populations in the sessile or recirculating T cell pools of NTx lambs as shown by comparison of the size of these pools in 3-month-old NTx and 2-week-old lambs (Fig. 5Go).


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The role of the thymus in the development of sessile lymphocyte populations resident in the secondary lymphoid tissues (spleen and LN) and the recirculating lymphocyte population trafficking between blood and lymph during neonatal life in sheep was examined. The peripheral lymphoid system of mammalian species with long gestation periods, such as sheep and humans, reaches an advanced stage of development by the end of fetal life. In both sheep and humans, a period of exponential growth of the peripheral T cell pool during fetal life is followed by a further period of rapid growth after birth (6,33). The growth and development of these tissues during fetal life in sheep is dependent on thymic export (8,9). The contribution of the thymus to T cell homeostasis after birth in sheep has not previously been explored. The results in this report show that the thymus is just as important in early postnatal life, as it is in fetal life, for the continued growth and development of the peripheral T cell pool.

Neonatal thymectomy has been performed in a range of animal species, with underdeveloped peripheral T cell pools at the time of thymectomy such as mice, rats, rabbits and hamsters (34). Some of the main effects observed following neonatal thymectomy in these species have included reduced body weight and the development of a wasting syndrome, fewer recirculating cells in the blood and secondary lymphoid tissues, and impaired cell-mediated immunity (34). Neonatal thymectomy has also been performed in a range of animal species which have well-developed peripheral lymphoid tissues at birth, such as calves (35), pigs (36) and dogs as well as in humans (37–40). In most cases, neonatal thymectomy was reported to have very little impact on the development of the peripheral T cell pool and resulted in variable effects on immune responses.

The results in this report represent the first comprehensive study of neonatal thymectomy in an animal species in which the peripheral immune system reaches a fairly advanced stage of development by the end of fetal life. Although there have been a number of reports on the effects of neonatal thymectomy in species with well-developed T cell pools at birth, these studies did not examine either the effect of thymectomy on T cell population sizes and subset composition or the effect of thymectomy on the growth and development of sessile versus recirculating T cell pools (35,36,39,40). With the exception of one of these studies (36), the majority of these above-mentioned studies on neonatal thymectomy were not performed on neonates, but on animals at various ages after birth. In contrast to neonatal thymectomy in mice, adult thymectomy of mice has been reported to have very little impact on the secondary lymphoid tissues (15,34,41).

Neonatal thymectomy has most frequently been performed in mice, where it has been found that if thymectomy is delayed for a few days after birth, the development of the peripheral T cell pool occurs normally (4,34,42). These observations have led to the suggestion that once formed, the peripheral T cell pool might be maintained independently of the thymus (1,43–45), and that the thymus is not important for the continued growth and development of the peripheral T cell pool once thymic emigrants initially seed it. Although extrathymic sources of de novo T cell production do exist as shown by the presence of intraepithelial T cells in the gut, these cells normally reside at their sites of production and express aberrant cell-surface antigens (46). Such T cells are not the same as T cells found in spleen and LN and were not examined in this study.

In the experiments reported here, the thymus was found to be essential for the continued growth of the peripheral T cell pool after birth. Neonatal thymectomy in mice, however, cannot readily be compared with the experiments reported here. Significant differences exist between mice and sheep (and between mice and humans) in the development of the peripheral T cell pool. Thymic export in mice does not begin until around the time of birth and the peripheral T cell pool does not appear until after birth (2,3). In sheep and humans thymic export begins early during development and both species have a well-developed peripheral T cell pool at birth. Caution must therefore be exercised in attempting to compare the role of the thymus in the development and homeostasis of naive T cells in the periphery during neonatal life in diverse species such as sheep and mice. In our experiments in neonatal lambs, homeostatic expansion of T cell subsets following thymectomy, with the exception of CD4+ T cells in the sessile T cell pool, did not occur. This was in marked contrast to studies in lymphocyte-depleted adult mice where considerable homeostatic expansion occurs (47–49). The simplest explanation for these different results is that the rules that govern T cell homeostasis are different in the neonate and adult.

Experimental distinctions between sessile and recirculating populations have not usually been made when describing peripheral T cell populations. The development of the sessile and recirculating T lymphocyte pools during fetal life in sheep and neonatal life in mice are dependent on thymic export (2,4,8). Sessile and recirculating T lymphocytes present in spleen and LN share a common origin, in that they are both derived from the thymus. To date the relationships between the two pools have not been explored (5,28). Recently, sessile and recirculating T cell populations have been studied in adult rats using thoracic duct cannulation to obtain recirculating T cells. These experiments showed that secondary lymphoid organs such as spleen, LN and Peyer's patches differed considerably in their CD4+ T cell composition due to different ratios of recirculating:sessile CD4+ T cells. It was also found that sessile and recirculating populations displayed a different CD45RC (naive/memory) phenotype (29). The sizes of both sessile and recirculating T cell pools increase dramatically after birth in young lambs; however, the majority of this growth appears to be thymic dependent as very little post-thymic expansion of peripheral T cells occurred in NTx lambs. The only exception was in the sessile CD4+ T cell population which, in marked contrast to recirculating CD4+ T cells, showed a substantial increase in number after birth (Fig. 5Go). Although neonatal thymectomy was associated with a substantially reduced growth rate of both sessile and recirculating peripheral T cell populations, the reduced growth was more marked amongst recirculating T cell populations compared with sessile T cells. The different effects of thymectomy on sessile and recirculating T cell populations strongly suggest that sessile and recirculating T cell populations represent distinct pools, which might be independently regulated and under separate forms of homeostatic control. Further support for this was found in the case of CD4+ T cells, where a significant degree of post-thymic expansion occurred amongst sessile CD4+ T cells, but not recirculating CD4+ T cells.

The source of the increased number of sessile CD4+ T cells in NTx lambs is unknown, but could have resulted from a extra-thymic source of de novo T cell production, antigen-driven expansion or antigen-independent homeostatic expansion. It has now become generally accepted that T cells require constant signaling from self-peptide–MHC complexes in order to maintain their quiescent state and survival in the periphery (47,50–52). Furthermore it has also been reported that under conditions of lymphocyte depletion, naive T cells undergo homeostatic expansion, which helps to maintain their numbers in the periphery (47–49). It remains to be determined why some T cell populations expanded postnatally in thymectomized lambs (e.g. sessile CD4+ T cells) and others did not (e.g. CD8+ T cells, {gamma}{delta} TCR+ sessile T cells and recirculating CD4+ T cells). The results in this report raise the possibility that the thymus exports mixed populations of T cells with different homing properties, lifespans and post-thymic expansion potential. Alternatively, the thymus might export T cells with similar potential, but which is then subsequently modified by events occurring in the periphery, such as through interactions with self-peptide–MHC.


    Acknowledgments
 
Supported by grants from the National Health and Medical Research Council of Australia. C. P. C. was the recipient of a CSL Ltd Special Postgraduate Scholarship.


    Abbreviations
 
LN lymph node
MLN mesenteric lymph node
NTx neonatally thymectomized
PLN peripheral lymph node
RLP recirculating lymphocyte pool
Ti thymus intact

    Notes
 
Transmitting editor: M. Miyasaka

Received 1 January 2001, accepted 24 July 2001.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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