The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Related articles in The JI
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Berzins, S. P.
Right arrow Articles by Godfrey, D. I.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Berzins, S. P.
Right arrow Articles by Godfrey, D. I.
The Journal of Immunology, 2006, 176: 4059-4065.
Copyright © 2006 by The American Association of Immunologists

Long-Term Retention of Mature NK1.1+ NKT Cells in the Thymus1

Stuart P. Berzins2,*, Finlay W. McNab*, Claerwen M. Jones*, Mark J. Smyth{dagger} and Dale I. Godfrey*

* Department of Microbiology and Immunology, University of Melbourne, Parkville, Australia; and {dagger} Cancer Immunology Program, Peter MacCallum Cancer Center, East Melbourne, Australia


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The NKT cell pool in the thymus contains immature (NK1.1–) and mature (NK1.1+) subsets that represent distinct linear stages of a linear developmental pathway. An unexplained paradox is why immature NK1.1– NKT cells are mainly exported to the periphery instead of the more mature and more abundant NK1.1+ NKT cells. In this study we have determined that mature NK1.1+ NKT cells are retained by the thymus to form an extremely long-lived resident population capable of rapid and prolonged production of IFN-{gamma} and IL-4. The retention of mature NKT cells provides an explanation for why the periphery is mainly seeded by immature NK1.1– cells despite mature NK1.1+ NKT cells being more abundant in the thymus. This is the first study to identify a mature T cell subset retained within the thymus and is additional evidence of the distinct developmental pathways of mainstream T cells and NKT cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The NKT cells are a regulatory T cell subset capable of enhancing or suppressing immune responses through potent cytokine release (1, 2). They are regarded as a potential therapeutic target because numerous studies in humans and mice report a strong association between NKT cell defects and an increased susceptibility to many autoimmune diseases and cancers (1, 3, 4). NKT cells use a semi-invariant, CD1d-restricted TCR that enables recognition of the glycolipid Ags responsible for selection in the thymus and activation in the periphery (5, 6). Although NKT cells were initially identified though their dual expression of a TCR and NK receptors, they are now more definitively identified using CD1d tetramers loaded with the pan-NKT cell agonist {alpha}-galactosylceramide ({alpha}GC)3 that targets the distinctive TCR (7, 8).

The earliest tetramer-reactive NKT cells in the thymus are devoid of most NK-associated markers, including the NK1.1 molecule (CD161 in humans) most commonly associated with this lineage (9, 10, 11). The CD1d-dependent up-regulation of NK1.1 is a crucial checkpoint in NKT cell maturation and is often the point at which a developmental block occurs in models of NKT cell deficiency (12, 13, 14, 15). NK1.1 up-regulation also coincides with the expression of functionally significant cell surface molecules, including inhibitory Ly49 receptors and stimulatory receptors such as NKG2D (10, 16, 17, 18). Most importantly, NK1.1 expression signals a maturing of the cytokine response toward high IFN-{gamma} production (9, 10, 11).

In sharp contrast to mainstream T cells, most thymic NKT cells carry a mature NK1.1+ phenotype. Paradoxically though, it is mainly immature NK1.1– NKT cells that are exported to the periphery, where they again complete their maturation in a CD1d-dependent manner (9, 10, 12, 19). This developmental pathway is in stark contrast to mainstream T cells where thymic export is tightly restricted to ensure that only mature cells are released to the periphery (20, 21, 22). It is not clear why NKT cells are the exception to this rule, but the same phenomenon takes place in human NKT cell development (23, 24).

One obvious question is what happens to the mature NK1.1+ cells that make up >75% of NKT cells in the young adult thymus. In this study we use various assays to track these cells long term and resolve fundamental inconsistencies in the current understanding of NKT cell development. Most strikingly, we identify a population of NK1.1+ NKT cells that are resident in the thymus and capable of potent cytokine production. They appear to be the only T cell subset in the thymus not rapidly turned over, strongly suggesting a functional role for these cells within the confines of the thymus microenvironment.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Inbred CD45.2+ and CD45.1+ C57BL/6 mice were bred at the Department of Microbiology and Immunology House, Melbourne University. Chimeras were generated by irradiating CD45.2+ mice with two doses of 550 cGy and immediately reconstituting them via i.v. injection of 5 x 106 CD45.1+ bone marrow cells. All experiments were conducted under the approval of the University of Melbourne animal experimental committee.

Thymus transplantation

Thymic lobes were removed from newborn pups and engrafted beneath the kidney capsule of 5- to 6-wk-old mice in accordance with a previously described technique (22, 25).

Intrathymic injection of FITC dye or cells

The details of this technique have been described previously (20, 22). Briefly, animals were anesthetized, and the chest was opened (or kidney was exposed in the case of grafted mice) to reveal the thymic lobes. Each lobe was injected with ~10 µl of 500 µg/ml FITC (in PBS), which typically resulted in random labeling of 40–60% of the thymocyte population (70–80% for grafted thymi). The wound was closed with a surgical staple, and the mouse was warmed until it recovered from anesthesia. Mice were killed at different time points after injection, with graft thymus and lymphoid organs taken for analysis. Instruments were washed between the removal of each organ, and in the case of FITC injections, the injected thymus was always removed last to avoid cross-contamination of samples.

Flow cytometry

Unfractionated or NKT cell-enriched cell suspensions were labeled with combinations of Abs including: FITC-conjugated anti-{alpha}betaTCR (clone H57-597); FITC-conjugated anti-CD4 (clone RM4-5); FITC- or PerCP-conjugated anti-CD45.1; FITC- or PerCP-conjugated anti-CD45.2; PE-, Pe-Cy7-, or allophycocyanin-conjugated anti-NK1.1 (clone PK-136); PE-conjugated anti-heat-stable Ag; biotinylated anti-CD4 (clone RM4-5); and allophycocyanin-, allophycocyanin-Cy7-, or Pe-Cy7-conjugated anti-{alpha}betaTCR (clone H57-597). Fluorochrome-labeled, {alpha}GC-loaded or unloaded, CD1d tetramer was produced in-house using a construct provided by M. Kronenberg (La Jolla Institute for Allergy and Immunology, San Diego, CA). The generation and use of this reagent have been described in detail previously (7). To avoid nonspecific binding of Abs to FcR-{gamma}, cells were routinely incubated with anti-mouse CD16/32 (clone 2.4G2; grown in-house). All flow cytometry reagents were purchased from BD Pharmingen unless otherwise indicated. Data were collected on an LSR2 flow cytometer (BD Biosciences).

Isolation of liver lymphocytes

Lymphocytes were isolated from perfused liver tissue by being gently pressed through 200-µm pore size mesh sieves into PBS containing 2% FCS (Commonwealth Serum Laboratories). Lymphocytes were separated from hepatocytes and cellular debris via a 33% isotonic Percoll density gradient (Amersham Biosciences) conducted at room temperature, and red cells were lysed with ammonium chloride.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Mature NKT cells are retained in thymus

We were intrigued by the consistent findings of multiple groups (including our own) that immature NK1.1– NKT cells were preferentially exported from the thymus of the adult mouse despite the greater abundance of mature NK1.1+ thymic NKT cells. To directly test whether mature NKT cells were retained in the thymus, we transplanted congenic CD45.2+ thymic lobes beneath the kidney capsule of CD45.1+ recipients. Previous studies had shown a highly predictable rate of reconstitution of transplanted thymus lobes in which donor thymocytes are lost from the thymus as they fail selection or are exported, whereas recipient progenitor cells enter the lobes and eventually repopulate all thymocyte compartments (22). The result is that by 4–6 wk after engraftment, <1% of thymocytes are of donor origin. These kinetics are in harmony with estimates from studies of the endogenous thymus, and similar kinetics were observed throughout this study (data not shown) (26, 27, 28). We also confirmed that transplanted thymic lobes (as well as the endogenous thymus) in recipient mice contained normal numbers of major thymocyte subsets (including NKT cells), and that NKT cells and mature T cells were exported with normal frequency, again consistent with earlier studies (22) (data not shown).

We harvested thymic lobes at various times after engraftment and, as expected, found cells of donor origin to be rare in all thymuses transplanted >6 wk earlier. Nevertheless, there were always low, but consistent, numbers of mature (i.e., TCR+), residual, donor-derived thymocytes. Strikingly, NKT cells made up the majority of this residual population despite representing <1% of thymocytes in the overall thymus (Fig. 1a). In donor thymuses transplanted 6 wk previously, NKT cells accounted for more than half of all residual cells, and similar results were observed 8 and 12 wk after grafting (Fig. 1b). In each instance, NKT cells represented at least half of the residual donor-derived cells in the donor thymus. The non-NKT cell residents were primarily mature ({alpha}beta-TCRhigh) T cells, with CD4+CD8– T cells most abundant, but small numbers of CD4–CD8+ and CD4–CD8– T cells also present (data not shown).


Figure 1
View larger version (48K):
[in this window]
[in a new window]
  		FIGURE 1. Mature NK1.1+ NKT cells are selectively retained in the thymus. Recipient CD45.1+ C57BL/6 mice engrafted with newborn CD45.2+ thymic lobes were harvested at various times and checked for donor thymus-derived (CD45.2+) cells. a, This figure shows a comparison of donor- and recipient-derived cells in the donor thymus 3 and 6 wk after transplantation. Note that NKT cells in the 3 wk thymus are relatively rare and are enriched for the immature NK1.1– phenotype. A and C, NKT cell frequency within the donor- and host-derived compartments of the thymus, whereas B and D illustrate the expression of CD4 and NK1.1 by these NKT cells. Note the over-representation of NK1.1+ NKT cells among residual donor-derived cells at 6 wk (bottom panels, C and D). b, This figure shows the frequency of NKT cells among residual donor-derived cells in the thymic lobes (•) 6, 8, and 12 wk after transplantation. Also shown are frequencies in the endogenous thymus ({blacktriangleup}), spleen ({blacksquare}), and liver ({diamondsuit}). Each symbol represents an individual mouse. c, Recipient CD45.1+ C57BL/6 mice were lethally irradiated and subsequently engrafted with CD45.2+ bone marrow. This figure shows donor- and recipient-derived cells in the endogenous thymus 3 mo and 1 year after bone marrow reconstitution. A and C, The frequency of NKT cells within the donor- and host-derived compartments of the thymus; B and D illustrate the expression of CD4 and NK1.1 by these NKT cells. Note the over-representation of NK1.1+ NKT cells among donor-derived cells at each time point (bottom panels). Profiles from chimeric mice are representative of five mice per group.

 
To exclude any possibility that the persistence of NKT cells was an artifact of the engraftment process, we next examined CD45.1+ mice rendered chimeric by irradiation and subsequent reconstitution with bone marrow from CD45.2+ mice. In such instances, host-derived T cell precursors and progenitors are largely ablated, but some cells persist to produce a limited chimerism between host- and donor-derived, hemopoietically derived cells (26, 27). In the endogenous thymus, this creates a circumstance similar to that in the transplanted thymus, where surviving endogenous thymocytes are exported or otherwise lost as donor-derived progenitors enter and repopulate the thymus microenvironment. We reasoned that this would allow us to confirm that NKT cells were being preferentially retained, this time in the endogenous thymus.

Mice were examined at 3 mo or 1 year after reconstitution; once again, NKT cell levels were greatly over-represented among residual thymocytes of both groups (Fig. 1c). Despite reports that the mainstream population of T cells is essentially turned over every 4–6 wk, some host-derived cells had persisted in the chimeric thymus, even after 1 year. NKT cells made up >20% of these host-derived cells, again consistent with their preferential retention. It should be noted that the irradiation/reconstitution model provides a more variable starting point than the consistent reconstitution kinetics of the transplanted thymus, because the relative susceptibilities of the various NKT cell and mainstream T cell subsets to irradiation are not entirely predictable. Nevertheless, the large proportion of NKT cells among residual cells, even 1 year after reconstitution, confirmed that mature NKT cells were retained in the endogenous thymus.

Most thymic NKT cells are long-term residents

We used the mice analyzed in Fig. 1b to also examine whether long-term resident NKT cells represented a significant proportion of total NKT cells in the thymus. As expected, donor-origin mainstream T cells made up <0.5% of the total T cells in the transplanted thymus 6, 8, and 12 wk after engraftment. In sharp contrast, nearly half of all thymic NKT cells at these time points were donor derived (Fig. 2). This is extremely significant, because it suggests that most mature NKT cells in the thymus are long-lived residents, particularly when residents that are not congenically distinct are taken into account. Again, this is in sharp contrast to mainstream T thymocytes, which are essentially all replaced within 6 wk.


Figure 2
View larger version (17K):
[in this window]
[in a new window]
  		FIGURE 2. Most thymic NKT cells are long-term residents. Recipient CD45.1+ C57BL/6 mice engrafted with newborn CD45.2+ thymic lobes were harvested at various times, and lymphoid organs were examined for donor-derived (CD45.2+) cells. This figure shows the proportion of all NKT cells in the thymic lobes (•) 6, 8, and 12 wk after transplantation that are residual donor-derived cells. Also shown is the contribution of donor-derived cells to the NKT cell compartments in the endogenous thymus ({blacktriangleup}), spleen ({blacksquare}), and liver ({diamondsuit}). Each symbol represents an individual mouse.

 
It is important to note that we can exclude the possibility that donor-derived NKT cells had recirculated back to thymic tissue from the periphery after export. If this were the case, a similar phenomenon would have been observed in the endogenous thymus of grafted recipients. Instead, the number of donor NKT cells in the donor thymus was >10-fold higher than that in the endogenous thymus (Fig. 3). Moreover, although donor NKT cells represented >50% of all NKT cells in the donor thymus, they represented <1% of NKT cells in the endogenous thymus (Fig. 2). We have also previously reported that peripheral NKT cells injected into the thymus are very poorly retained compared with thymic NKT cells (12), consistent with earlier evidence that nonactivated T cells do not re-enter the thymus after export (29, 30).


Figure 3
View larger version (19K):
[in this window]
[in a new window]
  		FIGURE 3. NKT cells of donor origin are maintained in donor thymus. Recipient CD45.1+ C57BL/6 mice engrafted with newborn CD45.2+ thymic lobes were harvested at various times, and lymphoid organs were examined for donor-derived (CD45.2+) cells. This figure shows the number of residual donor-derived NKT cells in the thymic lobes (•) 6, 8, and 12 wk after transplantation. Also shown is the number of donor thymus-derived NKT cells in the endogenous thymus ({blacktriangleup}), spleen ({blacksquare}), and liver ({diamondsuit}). Each symbol represents an individual mouse.

 
Retained NKT cells are all NK1.1+

Long-lived thymic NKT cells generally displayed a similar distribution of NK receptors as surrounding NKT cells (Fig. 4). This is not overly surprising, because our data suggest that long-lived NKT cells represent the bulk of the overall population, making it unlikely that the two groups would be greatly dissimilar. One profound difference, however, was in the expression of NK1.1 (Fig. 5). As mentioned previously, NK1.1 expression is a hallmark of NKT cell maturation, yet it is NK1.1– cells that are preferentially exported to the periphery. As the site of NKT cell development, it is not surprising that the thymus contains a significant population of NK1.1– cells, and this was again the case in our study. Strikingly, however, the retained NKT cells were overwhelmingly NK1.1+. A difference was also observed for NKG2D, with resident NKT cells expressing higher levels of this NK receptor than surrounding NKT cells. The magnitude of the change was very reminiscent of the increased NKG2D expression that follows chronic Ag stimulation of NKT cells, but other changes observed in that report, such as the up-regulation of Ly49 molecules, were not observed in long-term resident NKT cells (31).


Figure 4
View larger version (18K):
[in this window]
[in a new window]
  		FIGURE 4. Expression of NK receptors by resident thymic NKT cells. Recipient CD45.1+ C57BL/6 mice engrafted with newborn CD45.2+ thymic lobes were harvested 8 wk later, and the donor and endogenous thymus were separately examined for donor-derived (CD45.2+) cells. This figure shows the expression of various NK cell-associated markers on NKT cells of donor thymus (•) or host ({circ}) origin. Each symbol represents an individual mouse.

 

Figure 5
View larger version (38K):
[in this window]
[in a new window]
  		FIGURE 5. Resident thymic NKT cells are NK1.1+. Recipient CD45.1+ C57BL/6 mice engrafted with newborn CD45.2+ thymic lobes were harvested 6 or 12 wk later, and the donor and endogenous thymuses were separately examined for donor-derived (CD45.2+) cells. Note that the time point (6 or 12 wk) refers to the length of time the thymus graft was in place, not to the age of the mouse itself. This figure shows the expression of NK1.1 on NKT cells of donor (•) or host ({circ}) origin. The upper figure is a representative sample of the grafted thymus (top panels) and endogenous thymus (bottom panels) that illustrates NKT cell frequency (left panels), the relative proportion of residual Ly5.2+ (donor-derived) cells (middle panels), and the expression of NK1.1 on thymocytes (right panels) 6 wk after thymus grafting. The lower figure is a summary of the data demonstrating the bias toward an NK1.1+ phenotype among resident NKT cells. Each symbol represents an individual mouse.

 
Retained NKT cells are potent cytokine producers

We next assessed the capacity for retained NKT cells to produce cytokines, because the potent production of cytokines is crucial to the immunoregulatory function of NKT cells. In general terms, mature NK1.1+ NKT cells produced higher levels of IFN-{gamma} than their less mature NK1.1– counterparts, but there was an even greater bias when comparing long- and short-term NKT cell residents. IFN-{gamma} production by the long-term resident cells was considerably higher than that of the younger NKT cells of host origin despite the vast majority of those cells also being NK1.1+ (Fig. 6). IL-4 levels were similar in the two groups. The conditions that might lead to intrathymic NKT cell activation are unknown, but resident NKT cells in the thymus clearly possess the capacity for potent cytokine release.


Figure 6
View larger version (29K):
[in this window]
[in a new window]
  		FIGURE 6. High cytokine production by resident thymus NKT cells. Recipient CD45.1+ C57BL/6 mice engrafted with newborn CD45.2+ thymic lobes were harvested 6 wk later, and thymocytes were stimulated with PMA/ionomycin. This figure shows a representative (upper panels) and summarized (lower panels) analysis of gated thymus NKT cells for IFN-{gamma} (left panels), IL-4 (middle panels), and isotype control mAbs (right panels). Note the high IFN-{gamma} production of donor (CD45.2+) NKT cells compared with younger NKT cells of host origin (CD45.2–). Paired samples in the lower panels represent analysis of host and donor NKT cells from the same thymus that were stimulated and analyzed simultaneously.

 
Age-related changes within NKT cell compartments

Two implications of our study are, firstly, that overall NKT cell levels might rise in the thymus with age as NK1.1+ cells accumulate and, secondly, that the population might become increasingly skewed toward a NK1.1+ phenotype as a result. We therefore compared the NKT cell compartments in mice of different ages. The analysis of NKT numbers revealed a reduction of overall NKT cell numbers with age in the thymus that was not observed in peripheral organs, although the significance of this finding is clouded by the well-documented atrophy of the thymus with age (Fig. 7) (32, 33). The frequency of NKT cells as a proportion of all lymphocytes remained relatively stable in all organs tested, including the thymus, arguing against a selective accumulation or loss (Fig. 7b). With regard to the phenotype of NKT cells in the thymus, there was a very clear shift with age toward an NK1.1+ phenotype, consistent with the accumulation of mature cells (Fig. 8). Approximately 50% of thymic NKT cells in 4-wk-old mice were NK1.1–, but the proportion dropped to <5% by 1 year of age. Interestingly, this age-related phenotypic shift was clearly evident by 3 mo. There was no shift observed among mainstream T cells in the thymus to cells of a mature phenotype.


Figure 7
View larger version (24K):
[in this window]
[in a new window]
  		FIGURE 7. Age-related changes in thymic NKT cell number and frequency. C57BL/6 mice of different ages were analyzed for NKT cell levels in the thymus, spleen, and liver. a, Comparison of the total number of NKT cells within each organ. b, The frequency of NKT cells among total lymphocytes. Each symbol represents a single mouse. Note that the liver NKT cell percentage is presented separately because the percentage (but not number) of NKT cells in the liver can vary unpredictably due to blood contamination.

 

Figure 8
View larger version (39K):
[in this window]
[in a new window]
  		FIGURE 8. Changes in NK1.1 expression by NKT cells with age. NKT cells from the thymus, spleen, and liver of C57BL/6 mice of different ages were analyzed for the expression of NK1.1 Ag. a, Representative data from each organ at 1, 3, and 12 mo. b, Summary of the data, with each symbol representing a single mouse.

 
Age-related changes among NKT cell thymic emigrants

Given that the thymic NKT cell compartment became increasingly mature with age, we measured the effect on NKT cells exported from the thymus. Previous studies of NKT cell thymic export have used young mice to show that immature NK1.1– NKT cells are preferentially exported and reach full maturity (i.e., becoming NK1.1+) in the periphery.

We examined NKT cell export from the thymus of mice of various ages, reasoning that the selective export of NK1.1-NKT cells should be maintained if it was a developmental requirement for NKT cells to be exported at an immature stage. Significantly, there was a clear trend toward the export of NK1.1+ NKT cells with age. A reduced NK1.1– bias was still present among emigrant NKT cells from older thymuses, but this phenotype was clearly not a rigid selection criterion for export (Fig. 9).


Figure 9
View larger version (39K):
[in this window]
[in a new window]
  		FIGURE 9. Changed NK1.1 expression by thymic emigrant NKT cells with age. Recent thymic emigrant (RTE) NKT cells from 5-wk-old and 5-mo-old mice were assessed for the expression of NK1.1 Ag. Upper panels, The method used to identify RTEs. The upper middle and upper left panels are typical staining patterns of the thymus and liver, respectively, after intrathymic injection of FITC 24 h previously. The lower left panels are representative FACS plots showing CD4 (y-axis) and NK1.1 (x-axis) expression of resident NKT cells (left) and RTE NKT cells (right). The far right graphs summarize the NK1.1 expression data from one representative experiment (of four total experiments) involving six to eight mice per group.

 
No spontaneous thymic defect in NKT cell-deficient mice

The very specific retention of NK1.1+ NKT cells by both the endogenous and the transplanted thymus suggested that these cells might play a role in the normal functioning of the thymus, particularly since NKT cells are regarded as an immunoregulatory cell. To test this, we compared the frequencies of all major T cell subsets in the thymus of wild-type and the NKT cell-deficient CD1d–/– or Ja18–/– strains; we found no significant differences, other than the lack of NKT cells (data not shown). In addition to the normal frequencies of thymocyte subsets defined by CD4 and CD8, similar proportions of subsets defined by markers, such as heat-stable Ag, CD25, and CD69, were seen in NKT cell-deficient mice. Deletion of immature thymocytes reactive to endogenous mouse mammary tumor virus superantigens was also normal in NKT cell-deficient BALB/c.CD1d–/– and C57BL/6.CD1d–/– mice, showing that there was no gross defect in negative selection (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
This study revealed that many NK1.1+ NKT cells are preferentially retained in the thymus. This was a most unexpected finding, because there is compelling evidence that the vast majority of mainstream thymocytes are usually resident for <4 wk (22, 28, 34), the exception being one early study that described a rare resident population that could not be definitely identified due to the technical limitations of the day (35). Our identification of a resident thymic NK1.1+ NKT cell extends that previous report, but also resolves the paradox of why the thymus exports a high proportion of immature NK1.1– NKT cells to the periphery when most thymic NKT cells are mature NK1.1+ cells. Our findings complement earlier studies (including our own) that show that NKT cell development can involve the periphery being seeded with immature NKT cells (9, 10), but it now appears that many NKT cells are not exported and, instead, develop into extremely long-term thymic residents. The retention of mature NKT cells is consistent with the increased proportion of NK1.1+ NKT cells in the aged thymus and among recent thymic emigrants of older mice. Significantly, neither of these events was evident in previous studies of NKT cell export, because they were conducted on young adult mice <8 wk of age (9, 10).

The mechanism of NKT cell retention probably involves the differential expression of trafficking molecules, because these are known to influence the export of mainstream T cells (36, 37). There are many reported differences between the chemokine receptors expressed by NKT cells in different organs, but of particular significance might be the observation that immature NK1.1– NKT cells, and not mature NK1.1+ NKT cells, traffic in response to the CCR7 ligand SLC/CCL21, because this receptor/ligand pairing is known to be partly responsible for thymic export (36, 38). The resident numbers are also likely to be supported by the low basal proliferation described for peripheral NKT cells (10, 14, 39), although NK1.1+ thymocytes in adult mice proliferate far less than their immature NK1.1– counterparts (10). It is interesting to note, however, that most thymic NK1.1+ NKT cells are proliferating in mice younger than 4 wk (40), suggesting that the proliferation is a function of cellular age and is not directly controlled by the expression of NK1.1.

There are indications that the mature thymic NKT pool may be a functionally distinct subset from its peripheral counterparts. Thymic and peripheral NKT cells differ in their expression of Ly49 receptors (41, 42, 43) and respond differently to trafficking molecules, such as the aforementioned SLC/CCL21 (38). More importantly, however, are the very clear functional differences between thymic and peripheral NKT cells when they are used in models of NKT cell-dependent immunity. The functional competence of thymic NKT cells has been demonstrated by their effective suppression of diabetes in NOD mice after adoptive transfer and in vitro assays (44), yet thymic NKT cells cannot provide the same antitumor protection afforded by liver NKT cells (45).

What then is the possible functional significance of long-term thymic resident NKT cells? Given the potent protection against autoimmunity provided by thymic NKT cells in transfer models, it is tempting to speculate that they could suppress potentially damaging immune responses within the thymus. This is consistent with the suppressive role for thymus NKT-like cells in anterior chamber-associated immune deviation (46) and with a similar role proposed for peripheral NKT cells (4, 47). Alternatively, there is some evidence that thymic NKT cells can participate in mainstream selection events through Fas-mediated deletion of immature CD4+CD8+ thymocytes (48), although our analysis of mouse mammary tumor virus superantigen-mediated deletion in NKT cell-deficient mice would appear to exclude a fundamental role in negative selection.

Resident NKT cells are unlikely to play a day-to-day role in T cell development because they do not constitutively release cytokines, and T cell development is normal in NKT cell-deficient mice and in mice before the appearance of NK1.1+ NKT cells on day 8 of ontogeny. However, given that the importance of peripheral NKT cells is only revealed in NKT cell-deficient mice when they are challenged with NKT-dependent pathogens, a more likely scenario is that resident NKT cell activity is only triggered under very particular circumstances. Theoretically, the most obvious means to assess the function of resident thymic NKT cells is to selectively activate them within the thymus. Unfortunately, although cell suspensions of thymic NKT cells are easily stimulated in vitro, it is not yet possible to selectively activate them in situ, even after direct intrathymic injection of {alpha}GC, or in fetal thymic organ cultures using medium supplemented with {alpha}GC (49).

In summary, we have identified a mature NK1.1+ NKT cell subset that can remain resident in the thymus for >1 year. Their identification sheds considerable light on why immature NK1.1– NKT cells are more readily released to the periphery and why there is an age-related accumulation of NK1.1+ NKT cells in the thymus. Thymic resident NKT cells are not constitutively activated, but their capacity to rapidly release high levels of cytokines is strongly indicative of a potential immunoregulatory role.


   Acknowledgments
 
We thank Ken Field and the Picchi Brothers Foundation Flow Cytometry Facility for assistance with flow cytometry, and David Taylor and the animal house staff for animal husbandry.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 F.M. is a recipient of a Dora Lush postgraduate scholarship. D.I.G. and M.J.S. were supported by Research Fellowships and a program grant from the National Health and Medical Research Council of Australia. Back

2 Address correspondence and reprint requests to Dr. Stuart P. Berzins, Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria 3010, Australia. E-mail address: berzins{at}unimelb.edu.au Back

3 Abbreviations used in this paper: {alpha}GC, {alpha}-galactosylceramide; CD1d/{alpha}-galactosylceramide tetramer, CD1d tetramer. Back

Received for publication November 18, 2005. Accepted for publication January 16, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Godfrey, D. I., M. Kronenberg. 2004. Going both ways: immune regulation via CD1d-dependent NKT cells. J. Clin. Invest. 114: 1379-1388. [Medline]
  2. Kronenberg, M.. 2005. Toward an understanding of NKT cell biology: progress and paradoxes. Annu. Rev. Immunol. 23: 877-900. [Medline]
  3. Brigl, M., M. B. Brenner. 2004. CD1: antigen presentation and T cell function. Annu. Rev. Immunol. 22: 817-890. [Medline]
  4. Godfrey, D. I., K. J. Hammond, L. D. Poulton, M. J. Smyth, A. G. Baxter. 2000. NKT cells: facts, functions and fallacies. Immunol. Today 21: 573-583. [Medline]
  5. Bendelac, A., O. Lantz, M. E. Quimby, J. W. Yewdell, J. R. Bennink, R. R. Brutkiewicz. 1995. CD1 recognition by mouse NK1+ T lymphocytes. Science 268: 863-865. [Abstract/Free Full Text]
  6. Zhou, D., J. Mattner, C. Cantu, III, N. Schrantz, N. Yin, Y. Gao, Y. Sagiv, K. Hudspeth, Y. Wu, T. Yamashita, et al 2004. Lysosomal glycosphingolipid recognition by NKT cells. Science 306: 1786-179. [Abstract/Free Full Text]
  7. Matsuda, J. L., O. V. Naidenko, L. Gapin, T. Nakayama, M. Taniguchi, C. R. Wang, Y. Koezuka, M. Kronenberg. 2000. Tracking the response of natural killer T cells to a glycolipid antigen using CD1d tetramers. J. Exp. Med. 192: 741-754. [Abstract/Free Full Text]
  8. Benlagha, K., A. Weiss, A. Beavis, L. Teyton, A. Bendelac. 2000. In vivo identification of glycolipid antigen-specific T cells using fluorescent CD1d tetramers. J. Exp. Med. 191: 1895-1903. [Medline]
  9. Pellicci, D. G., K. J. Hammond, A. P. Uldrich, A. G. Baxter, M. J. Smyth, D. I. Godfrey. 2002. A natural killer T (NKT) cell developmental pathway involving a thymus-dependent NK1.1–CD4+ CD1d-dependent precursor stage. J. Exp. Med. 195: 835-844. [Abstract/Free Full Text]
  10. Benlagha, K., T. Kyin, A. Beavis, L. Teyton, A. Bendelac. 2002. A thymic precursor to the NK T cell lineage. Science 296: 553-555. [Abstract/Free Full Text]
  11. Gadue, P., P. L. Stein. 2002. NK T cell precursors exhibit differential cytokine regulation and require Itk for efficient maturation. J. Immunol. 169: 2397-2406. [Abstract/Free Full Text]
  12. McNab, F. W., S. P. Berzins, D. G. Pellicci, K. Kyparissoudis, K. Field, M. J. Smyth, D. I. Godfrey. 2005. The influence of CD1d in postselection NKT cell maturation and homeostasis. J. Immunol. 175: 3762-378. [Abstract/Free Full Text]
  13. Hammond, K. J., D. G. Pellicci, L. D. Poulton, O. V. Naidenko, A. A. Scalzo, A. G. Baxter, D. I. Godfrey. 2001. CD1d-restricted NKT cells: an interstrain comparison. J. Immunol. 167: 1164-1173. [Abstract/Free Full Text]
  14. Matsuda, J. L., L. Gapin, S. Sidobre, W. C. Kieper, J. Y. T. Tan, R. Ceredig, C. D. Surh, M. Kronenberg. 2002. Homeostasis of V{alpha}14i NKT cells. Nat. Immunol. 3: 966-974. [Medline]
  15. Stanic, A. K., J. S. Bezbradica, J. J. Park, N. Matsuki, A. L. Mora, L. Van Kaer, M. R. Boothby, S. Joyce. 2004. NF-{kappa}B controls cell fate specification, survival, and molecular differentiation of immunoregulatory natural T lymphocytes. J. Immunol. 172: 2265-2273. [Abstract/Free Full Text]
  16. Gapin, L., J. L. Matsuda, C. D. Surh, M. Kronenberg. 2001. NKT cells derive from double-positive thymocytes that are positively selected by CD1d. Nat. Immunol. 2: 971-978. [Medline]
  17. Berzins, S. P., A. P. Uldrich, D. G. Pellicci, F. McNab, Y. Hayakawa, M. J. Smyth, D. I. Godfrey. 2004. Parallels and distinctions between T and NKT cell development in the thymus. Immunol. Cell Biol. 82: 269-275. [Medline]
  18. Matsuda, J. L., L. Gapin. 2005. Developmental program of mouse V{alpha}14i NKT cells. Curr. Opin. Immunol. 17: 122-130. [Medline]
  19. Wei, D. G., H. Lee, S. H. Park, L. Beaudoin, L. Teyton, A. Lehuen, A. Bendelac. 2005. Expansion and long-range differentiation of the NKT cell lineage in mice expressing CD1d exclusively on cortical thymocytes. J. Exp. Med. 202: 239-248. [Abstract/Free Full Text]
  20. Scollay, R. G., E. C. Butcher, I. L. Weissman. 1980. Thymus cell migration: quantitative aspects of cellular traffic from the thymus to the periphery in mice. Eur. J. Immunol. 10: 210-218. [Medline]
  21. Gabor, M. J., D. I. Godfrey, R. Scollay. 1997. Recent thymic emigrants are distinct from most medullary thymocytes. Eur. J. Immunol. 27: 2010-205. [Medline]
  22. Berzins, S. P., R. L. Boyd, J. F. Miller. 1998. The role of the thymus and recent thymic migrants in the maintenance of the adult peripheral lymphocyte pool. J. Exp. Med. 187: 1839-1848. [Abstract/Free Full Text]
  23. Sandberg, J. K., C. A. Stoddart, F. Brilot, K. A. Jordan, D. F. Nixon. 2004. Development of innate CD4+ {alpha}-chain variable gene segment 24 (V{alpha}24) natural killer T cells in the early human fetal thymus is regulated by IL-7. Proc. Natl. Acad. Sci. USA 101: 7058-7063. [Abstract/Free Full Text]
  24. Berzins, S. P., A. D. Cochrane, D. G. Pellicci, M. J. Smyth, D. I. Godfrey. 2005. Limited correlation between human thymus and blood NKT cell content revealed by an ontogeny study of paired tissue samples. Eur. J. Immunol. 35: 1399-1407. [Medline]
  25. Berzins, S. P., D. I. Godfrey, J. F. Miller, R. L. Boyd. 1999. A central role for thymic emigrants in peripheral T cell homeostasis. Proc. Natl. Acad. Sci. USA 96: 9787-9791. [Abstract/Free Full Text]
  26. Hirokawa, K., T. Sado, S. Kubo, H. Kamisaku, K. Hitomi, M. Utsuyama. 1985. Intrathymic T cell differentiation in radiation bone marrow chimeras and its role in T cell emigration to the spleen: an immunohistochemical study. J. Immunol. 134: 3615-3624. [Abstract]
  27. Penit, C., S. Ezine. 1989. Cell proliferation and thymocyte subset reconstitution in sublethally irradiated mice: compared kinetics of endogenous and intrathymically transferred progenitors. Proc. Natl. Acad. Sci. USA 86: 5547-5551. [Abstract/Free Full Text]
  28. Scollay, R., D. I. Godfrey. 1995. Thymic emigration: conveyor belts or lucky dips?. Immunol. Today 16: 268-273. [Medline]
  29. Agus, D. B., C. D. Surh, J. Sprent. 1991. Reentry of T cells to the adult thymus is restricted to activated T cells. J. Exp. Med. 173: 1039-1046. [Abstract/Free Full Text]
  30. Penit, C., F. Vasseur. 1997. Expansion of mature thymocytes subsets before emigration to the periphery. J. Immunol. 159: 4848-4856. [Abstract]
  31. Hayakawa, Y., S. P. Berzins, N. Y. Crowe, D. I. Godfrey, M. J. Smyth. 2004. Antigen-induced tolerance by intrathymic modulation of self-recognizing inhibitory receptors. Nat. Immunol. 5: 590-596. [Medline]
  32. Hirokawa, K., M. Utsuyama, M. Kasai, C. Kurashima. 1992. Aging and immunity. Acta Pathol. Jpn. 42: 537-548. [Medline]
  33. Aspinall, R., D. Andrew. 2000. Thymic involution in aging. J. Clin. Immunol. 20: 250-256. [Medline]
  34. Tanchot, C., B. Rocha. 1997. Peripheral selection of T cell repertoires: the role of continuous thymus output. J. Exp. Med. 186: 1099-1106. [Abstract/Free Full Text]
  35. Scollay, R.. 1984. The long-lived medullary thymocyte re-visited: precise quantitation of a very small subset. J. Immunol. 132: 1085-108. [Abstract]
  36. Ueno, T., K. Hara, M. Swope Willis, M. Malin, U. E. Hopken, D. Gray, K. Matsushima, M. Lipp, T. Springer, R. Boyd, et al 2002. Role for CCR7 ligands in the emigration of newly generated T lymphocytes from the neonatal thymus. Immunity 16: 205-218. [Medline]
  37. Norment, A. M., M. J. Bevan. 2000. Role of chemokines in thymocyte development. Semin. Immunol. 12: 445-455. [Medline]
  38. Johnston, B., C. H. Kim, D. Soler, M. Emoto, E. C. Butcher. 2003. Differential chemokine responses and homing patterns of murine TCR {alpha}beta NKT cell subsets. J. Immunol. 171: 2960-299. [Abstract/Free Full Text]
  39. Eberl, G., H. R. MacDonald. 1998. Rapid death and regeneration of NKT cells in anti-CD3{epsilon}- or IL-12-treated mice: a major role for bone marrow in NKT cell homeostasis. Immunity 9: 345-353. [Medline]
  40. Coles, M. C., D. H. Raulet. 2000. NK1.1+ T cells in the liver arise in the thymus and are selected by interactions with class I molecules on CD4+CD8+ cells. J. Immunol. 164: 2412-2418. [Abstract/Free Full Text]
  41. MacDonald, H. R., R. K. Lees, W. Held. 1998. Developmentally regulated extinction of Ly-49 receptor expression permits maturation and selection of NK1.1+ T cells. J. Exp. Med. 187: 2109-2114. [Abstract/Free Full Text]
  42. Skold, M., S. Cardell. 2000. Differential regulation of Ly49 expression on CD4+ and CD4–CD8– (double negative) NK1.1+ T cells. Eur. J. Immunol. 30: 2488-2496. [Medline]
  43. Gays, F., K. Martin, R. Kenefeck, J. G. Aust, C. G. Brooks. 2005. Multiple cytokines regulate the NK gene complex-encoded receptor repertoire of mature NK cells and T cells. J. Immunol. 175: 2938-2947. [Abstract/Free Full Text]
  44. Hammond, K. J. L., L. D. Poulton, L. J. Palmisano, P. A. Silveira, D. I. Godfrey, A. G. Baxter. 1998. {alpha}/beta-T cell receptor (TCR)+CD4–CD8– (NKT) thymocytes prevent insulin-dependent diabetes mellitus in nonobese diabetic (NOD)/Lt mice by the influence of interleukin (IL)-4 and/or IL-10. J. Exp. Med. 187: 1047-1056. [Abstract/Free Full Text]
  45. Crowe, N. Y., J. C. Coquet, S. P. Berzins, K. Kyparissoudis, R. Keating, D. G. Pellicci, D. I. Godfrey, M. Smyth. 2005. Differential anti-tumor immunity mediated by NKT cell subsets in vivo. J. Exp. Med. 202: 1279-1288. [Abstract/Free Full Text]
  46. Wang, Y., I. Goldschneider, D. Foss, D. Y. Wu, J. O’Rourke, R. E. Cone. 1997. Direct thymic involvement in anterior chamber-associated immune deviation: evidence for a nondeletional mechanism of centrally induced tolerance to extrathymic antigens in adult mice. J. Immunol. 158: 2150-215. [Abstract]
  47. Bendelac, A.. 1995. Mouse NK1+ T cells. Curr. Opin. Immunol. 7: 367-374. [Medline]
  48. Arase, H., N. Arase, Y. Kobayashi, Y. Nishimura, S. Yonehara, K. Onoe. 1994. Cytotoxicity of fresh NK1.1+ T cell receptor {alpha}/beta+ thymocytes against a CD4+8+ thymocyte population associated with intact Fas antigen expression on the target. J. Exp. Med. 180: 423-432. [Abstract/Free Full Text]
  49. Pellicci, D. G., A. P. Uldrich, K. Kyparissoudis, N. Y. Crowe, A. G. Brooks, K. J. Hammond, S. Sidobre, M. Kronenberg, M. J. Smyth, D. I. Godfrey. 2003. Intrathymic NKT cell development is blocked by the presence of {alpha}-galactosylceramide. Eur. J. Immunol. 33: 1816-1823. [Medline]

Related articles in The JI:

IN THIS ISSUE

The JI 2006 176: 3851-3852. [Full Text]  



This article has been cited by other articles:


Home page
 J. Immunol.Home page
E. Germanov, L. Veinotte, R. Cullen, E. Chamberlain, E. C. Butcher, and B. Johnston
Critical Role for the Chemokine Receptor CXCR6 in Homeostasis and Activation of CD1d-Restricted NKT Cells
J. Immunol., July 1, 2008; 181(1): 81 - 91.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
M. Felices and L. J. Berg
The Tec Kinases Itk and Rlk Regulate NKT Cell Maturation, Cytokine Production, and Survival
J. Immunol., March 1, 2008; 180(5): 3007 - 3018.
[Abstract] [Full Text] [PDF]

Home page
 FASEB J.Home page
M. L. Allende, D. Zhou, D. N. Kalkofen, S. Benhamed, G. Tuymetova, C. Borowski, A. Bendelac, and R. L. Proia
S1P1 receptor expression regulates emergence of NKT cells in peripheral tissues
FASEB J, January 1, 2008; 22(1): 307 - 315.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
F. W. McNab, D. G. Pellicci, K. Field, G. Besra, M. J. Smyth, D. I. Godfrey, and S. P. Berzins
Peripheral NK1.1 NKT Cells Are Mature and Functionally Distinct from Their Thymic Counterparts
J. Immunol., November 15, 2007; 179(10): 6630 - 6637.
[Abstract] [Full Text] [PDF]

Home page
 J. Exp. Med.Home page
T. M. McCaughtry, M. S. Wilken, and K. A. Hogquist
Thymic emigration revisited
J. Exp. Med., October 29, 2007; 204(11): 2513 - 2520.
[Abstract] [Full Text] [PDF]

Home page
 J. Exp. Med.Home page
N. Schrantz, Y. Sagiv, Y. Liu, P. B. Savage, A. Bendelac, and L. Teyton
The Niemann-Pick type C2 protein loads isoglobotrihexosylceramide onto CD1d molecules and contributes to the thymic selection of NKT cells
J. Exp. Med., April 16, 2007; 204(4): 841 - 852.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
L. Cheng, A. Ueno, S. Cho, J. S. Im, S. Golby, S. Hou, S. A. Porcelli, and Y. Yang
Efficient Activation of V{alpha}14 Invariant NKT Cells by Foreign Lipid Antigen Is Associated with Concurrent Dendritic Cell-Specific Self Recognition
J. Immunol., March 1, 2007; 178(5): 2755 - 2762.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Related articles in The JI
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Berzins, S. P.
Right arrow Articles by Godfrey, D. I.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Berzins, S. P.
Right arrow Articles by Godfrey, D. I.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS