SEARCH

SEARCH BY CITATION

Keywords:

  • Human;
  • Thymus;
  • NKT cell;
  • CD161;
  • CD1d

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Materials and methods
  6. Acknowledgements

NKT cells are a CD1d-restricted T cell subset with strong immunoregulatory properties. Human NKT deficiencies are associated with autoimmune diseases such as type 1 diabetes and several types of cancer, yet there is little understanding of how the human NKT cell pool develops or is maintained. In this study, we present the first detailed analysis of human NKT cells from donor-matched postnatal thymus and blood samples. In mice, NKT cells are a thymus-dependent population that migrates to the periphery at an immature stage. Our data show that human NKT cells also undergo early stages of development in the thymus, forming a CD4+CD161–/low population that predominates neonatal thymic and blood NKT cell pools. CD4 and CD161+ NKT cells accumulate with age in the blood, but not thymus, to the point that they dominate the NKT cell compartment in adult blood. This is consistent with the post-thymic maturation of NKT cells exported from the thymus at the putatively immature CD4+CD161–/low stage. Interestingly, while thymus and peripheral NKT cell frequencies vary widely between patients and are relatively stable between age groups, there is no clear relationship between the NKT cell frequency in thymus and blood.

Abbreviations:
αGC:

α-Galactosylceramide

TREC:

TCR excision circle

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Materials and methods
  6. Acknowledgements

NKT cells are an immunoregulatory subset of CD1d-restricted T cells that express a semi-invariant T cell receptor (TCR) consisting of an invariant TCR-α chain (Vα14Jα18 in mice, Vα24Jα18 in humans) linked with a limited number of TCR-β chains (Vβ8.2, 7, or 2 in mice, Vβ11 in humans). They include CD4+ and CD4 subsets, respond to glycolipids presented by CD1d, and are capable of rapid and prolonged cytokine production following activation 13. In mouse models, NKT cells can suppress a number of different autoimmune diseases 4, including type 1 diabetes and experimental autoimmune encephalitis (EAE), yet also enhance pro-inflammatory immune responses that promote tumor rejection and clearance of some microbial infections 57. Several studies have linked NKT cell deficiencies with cancer and autoimmune disease in humans 811, with a few exceptions 12. In NKT cell-deficient NOD mice, the systemic NKT cell deficiency appears to originate in the thymus 13, but whether the highly variable frequency of NKT cells in human blood is also linked to NKT cell levels in the thymus is unknown, because no human studies have directly compared thymus and blood NKT cells from the same donors.

NKT cells are often defined by the expression of CD161 (NK1.1 or CD161c in mice), which is usually associated with NK cells. However, studies in mice have shown that NKT cells pass through an immature NK1.1 (CD161) stage of development and that most NKT cells are exported from the thymus at this immature stage, presumably to continue their maturation in the periphery 1416.

While the differentiation of NKT cells in the thymus of mice is becoming increasingly well defined 1719, only three studies have examined NKT cells in the human thymus. The earliest study utilized fresh thymus tissue from infants, and the SCID-hu model, in which T cells develop within mice engrafted with human thymus tissue and progenitor lymphocytes 20. NKT cells were not detected within fresh or grafted thymus, leading to the conclusion that peripheral NKT cells may develop independently of the thymus. Another group studied human thymic NKT cells derived from human embryos aged up to 20 weeks gestation 21. This report showed that Vα24+Vβ11+ NKT cells are present in human thymus from a gestational age of 12 weeks, and that an inverse correlation existed between the frequency of thymic NKT cells and gestational age. Based on the falling NKT cell frequency in embryonic thymus, and the lack of a clear NKT cell population in a postnatal thymus sample, the authors proposed that the human thymus would have little or no role in generating peripheral NKT cells after birth. This was very recently challenged by a second study of NKT cells in postnatal human thymus 22. Using tissue removed during cardiac surgery of otherwise healthy infants, NKT cells were identified within each thymus (n=10) at levels similar to those at week 20 of gestation. They determined that the average frequency of NKT cells in postnatal thymus (∼0.005%) was approximately 10–20-fold lower than that of adult blood and cord blood (albeit of different donors). Despite the different ages of subjects, both studies reported that a high proportion (>80%) of thymic NKT cells were CD4+, which raised important questions about the origin of the CD4 NKT cells, which are a major population of NKT cells in adult blood 21, 22. Furthermore, these studies also concurred that many thymic NKT cells were CD161–/low, in contrast to those in cord blood or adult peripheral blood, suggesting that CD161 is also a maturation marker for NKT cells in humans, but one that is expressed prior to NKT cell emigration from the thymus. An important shortcoming of these studies is that thymus, cord blood and peripheral blood each came from different donors of different ages, thus it was not possible to directly correlate thymus and blood NKT cell levels or phenotype for any given individual.

Here, we present a comparison of NKT cells in donor-matched thymus and blood samples at ages ranging from 1 day to 9 years of age from over 30 donors. We clearly demonstrate that NKT cells are present in postnatal thymus up to at least 9 years of age, and offer the first comprehensive analysis of the frequency and phenotype of NKT cells from thymus and blood of the same donor. By further comparing blood NKT cells from infants and adults, the developmental changes that take place in the NKT cell compartment with age are also defined.

Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Materials and methods
  6. Acknowledgements

Detecting low frequency NKT cells in the human thymus

In human blood, NKT cells can be clearly identified using a staining combination of anti-Vα24 (or 6B11 mAb, which recognizes the CDR3 α-chain loop of Vα24 23) and anti-Vβ11, or by staining with α-galactosylceramide (αGC)-loaded CD1d tetramer in combination with anti-CD3 or anti-αβ-TCR. As with most instances of flow cytometry, the putative NKT cell region can include some autofluorescent, or non-specifically stained cells, but the frequency of NKT cells is usually sufficiently high to make this level of ‘contamination’ inconsequential to analysis (Fig. 1, top panels). In the human thymus, however, we found the frequency of NKT cells is so low (in the order of 1/100,000 events) that similar gating can result in the proportion of contaminant cells in the NKT cell region being significantly higher, and NKT cell levels being overestimated as a result (Fig. 1; bottom panels). Although one strategy could be to ‘gate’ more conservatively, this risks excluding some legitimate NKT cells (particularly in the thymus where TCR levels are more variable) and can cause NKT cell levels to be underestimated.

thumbnail image

Figure 1. Removal of nonspecific events from NKT cell gate by use of ‘dump’ channel. A comparison of methods to exclude nonspecific events from flow cytometry analysis is shown. The top row shows the analysis of a representative sample from blood, while the bottom row shows analysis from a single sample of thymus. All profiles were subject to standard forward and side scatter discrimination of live lymphoid events. The standard method used forward and side scatter discrimination to exclude most nonspecific events (left), while exclusion gating employed the use of a separate ‘dump’ channel in which cells binding unloaded CD1d-tetramer were excluded (‘exclusion gating’, right). Each pair of thymus and blood density profiles are from one acquisition. Acquisition gating was applied to each plot shown (as depicted with the dashed line in the top left dot plot) because of the large number of events analyzed (blood: >2×106 events; thymus: >3×107 events).

Download figure to PowerPoint

The most effective way to maximize the stringency of NKT cell identification is to intentionally exclude autofluorescent or non-specifically stained cells during flow cytometry using a ‘dump’ channel 12. The detector for the dump channel is specific for a fluorochrome conjugated to unloaded CD1d-tetramer, so that cells binding the unloaded tetramer, and/or autofluorescent cells, can be detected and excluded from further analysis, a technique we refer to as exclusion gating (Fig. 1). In previous studies of NKT cells from human thymus, NKT cell staining was performed without the use of a separate dump channel 2022. Although this strategy effectively identifies NKT cells in the blood, the published thymus FACS profiles appear consistent with our own experiences in which the absence of exclusion gating allows low levels of background staining to encroach upon the NKT cell region. As a consequence, the absence of exclusion gating can create uncertainty about the precise level and phenotype of thymus NKT cells – a problem highlighted by the authors of the most recent study 22, who stated that the frequency of NKT thymocytes (defined by αGC-loaded CD1d tetramer) in humans does not exceed tetramer background staining. Consistent with the work of others 12, it should be made clear that staining with unloaded CD1d tetramer alone did not produce a profile with a distinct ‘NKT cell-like’ population, indicating that legitimate NKT cells were not removed from analysis by use of the dump channel (data not shown).

Stringency of NKT cell detection in the human thymus

Using a range of NKT cell-specific reagents including αGC-loaded CD1d tetramer, anti-Vα24, anti-Vβ11, anti-αβ-TCR and anti-CD3, combined with exclusion gating of cells non-specifically stained by unloaded CD1d tetramer, we specifically identified NKT cells in all human thymus and blood samples. A six-color LSRII flow cytometer was used to enable three or more channels to be dedicated to identifying NKT cells. Typically, these channels were: (1) αGC-loaded CD1d tetramer, (2) unloaded CD1d tetramer (dump channel), and (3) anti-CD3 or anti-αβ-TCR. In many instances, we confirmed the stringency of this process by also staining for the Vα24 and Vβ11 TCR chains. Although co-staining for multiple TCR-related markers sometimes led to a slight reduction in staining intensity (probably caused by minor steric hindrance), all three variations of this analysis (αGC-loaded CD1d tetramer versus anti-CD3; αGC-loaded CD1d tetramer versus Vα24; and Vα24 versus Vβ11) consistently isolated the same proportion of cells from one donor sample (Fig. 2A). These cells typically displayed an NKT-like phenotype, confirming that this was a reliable means of collecting NKT cells for analysis. Two files were collected for each thymus sample, the first containing ∼4×106 events of unfractionated thymocytes to determine the percentage of NKT cells, and the second where an acquisition gate was applied to exclude the majority of non-NKT cells to collect a file derived from >30×106 total thymocytes (Fig. 2B). This was necessary to collect sufficient thymus NKT cells to clearly examine their phenotype.

thumbnail image

Figure 2. Stringency of NKT cell gating using αGC-loaded CD1d tetramer and anti-CD3 staining. (A) Human NKT cells were identified by virtue of their dual reactivity to αGC-loaded CD1d tetramers and anti-αβ-TCR following exclusion of nonspecific event. The putative NKT cell gate identifies the same proportion of blood NKT cells defined by three alternate NKT cell phenotypes (αGC-loaded CD1d tetramer+CD3+, αGC-loaded CD1d tetramer+Vα24+ and, Vα24+Vβ11+). (B) Analysis of human thymus involved two separate data acquisitions per antibody cocktail. An initial acquisition of approximately 4×106 cells was made as a representative sample of all cells (bottom middle), then an acquisition gate (dashed line) that excluded most non-NKT cells was applied to a separate analysis of (typically) 3×107 additional thymocytes to collect sufficient NKT cells for analysis (bottom right). A stained blood sample from the same donor is shown for comparison (bottom left).

Download figure to PowerPoint

Frequency of human NKT cells in thymus and blood

Our analysis revealed that the high variability reported for NKT cell levels in adult blood (e.g.12, 22) also applies to the blood and more importantly, the thymus, of infants. In fact, this variability appears to be a hallmark of NKT cell levels in humans, highlighting the need to determine how levels are established and maintained. For this reason, it is important to emphasize that the average NKT cell frequency of pooled data was not necessarily indicative of the actual frequency for any one donor. Therefore, data are presented showing the individual results from each donor alongside the average NKT cell frequency for each group.

The mean frequency of NKT cells in the thymus was 0.0016% (n=30), with no significant difference between groups aged younger (0.0018%; n=17), or older (0.0012%; n=13) than 6 months (Fig. 3). This is more than twofold lower than frequencies reported by Baev and colleagues 22 and may reflect the higher stringency of NKT cell identification afforded by exclusion gating.

thumbnail image

Figure 3. The incidence of human NKT cells in thymus and blood. The frequency of NKT cells in human thymus and blood is shown. The consistency of NKT cell levels with age as a proportion of total lymphocytes within the compartment is shown. Each circle represents one donor. The dotted lines indicate the mean frequency for each group. Donor numbers are shown for each group.

Download figure to PowerPoint

In blood, NKT cell levels were significantly higher than in thymus, ranging from 0.003 to 0.78% of lymphocytes (Fig. 3). The mean frequency of NKT cells in adult blood (>18 years) (0.202%, n=10) was higher than in blood from children (0–9 years) (0.064%, n=29), but the variability in each group meant the differences were not statistically significant. There was also no correlation between age and NKT cell frequency in children's blood. For example, the average frequency among children aged 0–6 months was 0.066% (n=16), and for children aged 6 months to 9 years, it was 0.062% (n=13) (Fig. 3). Among adults, blood NKT cell frequency ranged from 0.006% to 0.78%, which was slightly more variable than among children (0.021% to 0.182%). The variability between individuals of all ages emphasizes the importance of determining how NKT cell levels are established and maintained, given the potential immune disadvantage conferred by low NKT cell numbers.

Correlating NKT cell levels in human thymus and blood

NKT cells were present in every thymus tested and postnatal levels did not fall with age. This is consistent with the study from Baev and colleagues 22, but contrasts with earlier suggestions that NKT cells may be absent from the postnatal thymus 20, 21. The difference in NKT cell frequency between thymus and blood from the same donor was surprisingly high. It averaged 71-fold (n=28) and ranged from 8-fold to as high as 285-fold. There was no correlation with age, but blood levels were always higher than thymus, regardless of whether frequency was expressed in terms of total lymphocytes or CD3hi T cells (Fig. 4). We also compared the frequency of NKT cells relative to CD3hi cells within the thymus and blood to exclude the possibility that the different composition of each compartment had skewed the results. Although the mean difference fell from 71-fold to 31-fold, the disparity in frequencies was maintained and there remained no correlation with age (Fig. 4B).

thumbnail image

Figure 4. Correlating human NKT cell levels in thymus and blood. (Top) Correlation between thymus and blood NKT cell levels of donors where both compartments were tested. NKT cell frequencies are expressed as a function of total lymphocytes (left), or CD3hi cells (right). (Bottom) Correlation (lack thereof) between NKT cell frequencies in thymus and blood, expressed as a function of total lymphocytes (left), or CD3hi cells (right). Samples were prepared fresh. Each circle represents one donor and group sizes are listed for each element.

Download figure to PowerPoint

Given the large difference between NKT cell levels in the thymus and blood, but the relative stability of NKT cell frequencies with age, NKT cells may accumulate in the periphery prior to birth, with levels thereafter maintained through thymic output and/or peripheral turnover. We saw no evidence that blood:thymus NKT cell ratios increased significantly with age. The youngest donors (aged 0–6 months, n=16) averaged peripheral blood NKT levels 74-fold higher than levels in the thymus (33-fold higher for CD3hi cells), whereas donors aged over 6 months (n=12) averaged blood NKT cells levels 67-fold greater (28-fold higher for CD3hi cells). Although adult thymus was not available, levels in adult blood were comparable to those of children. This suggests that the disparity between thymus and blood NKT cell levels is established prior to birth and maintained thereafter in an age-independent manner, consistent with an earlier hypothesis drawn from embryonic studies 21.

If the peripheral NKT cell frequency is established prior to birth, there are important implications for the potential treatment of diseases associated with NKT cell deficiency. It is possible, for instance, that therapeutic manipulation of thymic NKT cell development would have little effect on peripheral numbers – regardless of whether NKT cell production was boosted. Instead, it may be more effective to expand existing NKT cells in the periphery, or harness their immunoregulatory function with specific agonists.

The authors of a previous study hypothesized that peripheral NKT cell numbers were probably maintained through peripheral cell division, with thymic export playing a progressively less important role 22. Although this is consistent with the activated/memory phenotype of human NKT cells at all ages in blood 24, 25 and, to a lesser extent, thymus (Fig. 5), our data indicated that virtually no NKT cells in the blood of children, or adults, expressed the Ki67 antigen expressed by proliferating cells (Fig. 5C) 2628. In contrast, a significant proportion of NKT cells in the thymus were Ki67+ (∼5%), suggesting that a proportion of thymus NKT cells are cycling. As a positive control for Ki67 staining, 30–40% of non-NKT thymocytes were Ki67+, as expected, given the high cycling frequency of developing T cells. We do not exclude the possibility that very low levels of proliferation could support peripheral NKT cell numbers, but given our data, and evidence of low proliferation by NKT cells in adult blood 22, the ongoing role of thymic export should not be discounted.

thumbnail image

Figure 5. CD25 and Ki67 expression by NKT cells in thymus and blood. NKT cells from thymus and blood were analyzed for CD25 and Ki67 expression. (A) Representative profiles for CD25 expression on CD4+ mainstream T cells (as a control) and gated NKT cell populations. (B) Distribution of CD25 expression by thymus and blood NKT cells is shown for all donors. The proportion of the ‘immunoregulatory’ CD25+CD4+ subset is shown for comparison. Each circle represents one donor. Dotted lines indicate the mean frequency for each group. Donor numbers are provided at the top right corner. (C) Expression of Ki67 as a marker of cell proliferation. The left panel illustrates the expected high level of Ki67+ cell among normal thymocytes. Other panels show low levels of Ki67 expression by NKT cells in the thymus and blood. Data is representative of three separate experiments involving three donors aged less than 2 years (thymus and blood) and four adult donors (blood only).

Download figure to PowerPoint

It is interesting to note that despite the lack of proliferation, markers such as CD25 (Fig. 5) and CD45RO (data not shown) were more highly expressed by NKT cells than mainstream T cells. In the case of CD25, this was especially striking in the blood. Given the high proportion of CD4+ NKT cells, our data support an earlier observation that CD25+CD4+ NKT cells may be a previously unrecognized element of the more broadly defined immunoregulatory CD25+CD4+ T cell pool 24.

CD4 and CD8 expression by human NKT cells in thymus and blood

Analysis of 28 paired samples of thymus and blood showed that the thymus NKT cell compartment was phenotypically distinct from that of blood (Fig. 6). Over 80% of thymus NKT cells were CD4+CD8 (CD4+), with less than 10% being CD4CD8 (DN) or CD4+CD8+ (DP) cells. CD4CD8+ (CD8+) cells were either absent, or present at very low numbers (0–5%). CD4+ cells were also the dominant NKT cell subset in blood from children aged less than 6 months where the NKT cell compartment strongly resembled that of the thymus. The blood NKT cell compartment of this age group was noticeably distinct from children (and adults) older than 6 months where there were clear populations of CD8+ and DN NKT cells.

thumbnail image

Figure 6. Expression of CD4 and CD8 by human NKT cells. Thymus and blood NKT cells were analyzed for CD4 and CD8 expression. (A) Representative profiles for gated NKT cell populations. (B) Distribution of NKT cell subsets defined by CD4 and CD8 expression for all donors. Circles represent one donor sample. Groups of different ages are placed side by side for ease of analysis. Dotted lines indicate the mean frequency for each group.

Download figure to PowerPoint

While NKT cells in the blood of younger donors were almost exclusively CD4+ (>95%), the proportion of CD4+ NKT cells in older donors (6 months to 9 years) was far more variable, generally comprising ∼60% of the total NKT cell pool, but sometimes as low as 25%. In adult blood, less than 40% of NKT cells expressed CD4, with levels of CD4 NKT cells correspondingly higher, consistent with earlier analyses 24, 29, 30. The emerging CD4 NKT cell subset in children was dominated by DN cells, with CD8+ cells typically accounting for less than 20% of NKT cells (Fig. 6B). Interestingly, CD8+ NKT cells were heterogeneous in the intensity of CD8 expression compared to mainstream T cells. Mainstream CD8+ T cells expressed uniformly high levels (data not shown), but as previously reported, CD8 expression by NKT cells ranged in intensity from intermediate to high 30, 31.

Most studies of human NKT cells have been restricted to cells isolated from adult blood, where three distinct NKT cell subsets have been identified: CD4+, CD4CD8, and CD8+29. Our finding that NKT cells from the blood of very young donors (<6 months) more closely resembles the thymus compartment of the same donors, and is clearly distinct from the blood of adults, raises questions about the origin of the DN and CD8+ subsets. No previous study has directly examined NKT cells from thymus and blood of the same donors, but previous reports showing very few DN and CD8+ NKT cells in cord blood and thymus (of different donors) led to speculation that these subsets arise in the periphery 21, 22. The possibilities raised in those studies were that the establishment of peripheral NKT cell subsets occurred by preferential expansion of CD4 NKT cells 22, down-regulation of CD4 by CD4+ NKT cells (and presumably up-regulation of CD8 in some cases) 21, or thymus-independent production of CD4 NKT cells 20.

Our cohort of samples indicates that it is only after 6 months of age that prominent DN and CD8+ subsets begin to appear. Previous embryonic studies showed that most NKT cells that emerge from the thymus prior to birth are CD4+21, 22, and the authors suggested that increasing numbers of DN and CD8+ subsets may emerge through the preferential expansion of these subsets following export from the thymus. This was supported by data showing that TCR excision circle (TREC) levels, which decrease as cells proliferate, were lower in CD4 NKT cells compared to the CD4+ fraction 22, although these data showed that the TREC levels of CD4+ and CD4 adult NKT cell subsets were both very low (less than 2 per 1,000 cells), suggesting that both populations had divided extensively. However, our results show that it is only after 6 months of age that DN and CD8+ subsets begin to appear, and staining with Ki67 showed little evidence of blood NKT cell proliferation at any age. This was in contrast to thymus NKT cells, which clearly included proliferating cells, nearly all of which were CD4+ (Fig. 5 and data not shown). While we cannot easily explain this discrepancy, it remains possible that very low (undetectable) levels of selective proliferation or survival of peripheral CD4 NKT cells contribute to their accumulation with age, or that NKT cell subsets are only dividing in tissues that were not tested. Given the very consistent profile of NKT cells in the human thymus from donors of different ages, and the clear presence of DN and CD8+ subsets in the blood of some children whose thymus was dominated by CD4+ NKT cells, the possibility that CD4 subsets are produced by older thymuses seems less likely. An alternative explanation is that some CD4+ NKT cells are induced to down-regulate CD4, perhaps as a consequence of activation as observed in mouse NKT cells 32. This might also explain the difference in TREC levels between these two subsets 22. We also cannot formally exclude the possibility that DN and CD8+ NKT cells are a thymus-independent lineage, but given their functional and phenotypic similarities to CD4+ NKT cells, and the thymus dependence of all NKT cell subsets in the mouse, this possibility seems remote.

Differential CD161 expression by thymus and blood NKT cells

The clear differences between thymus and blood NKT cell compartments led us to examine the expression of CD161, the human homologue of mouse NK1.1. Consistent with its importance as a marker of NKT maturity in mice 1416, CD161 was expressed by a significantly higher proportion of NKT cells in blood compared to thymus (n=24; p<0.001) (Fig. 7).

thumbnail image

Figure 7. CD161 expression on NKT cells from thymus and blood. (A) Representative profiles of CD161 expression by gated NKT cells. CD161 expression changed significantly with age on blood (two profiles), but not thymus (one profile) NKT cells. (B) Correlation of CD161 expression with age for all donors. (C) Correlation between CD161 expression by thymus and blood NKT cells of the one donor. Each circle represents one donor and dotted lines indicate the mean frequency.

Download figure to PowerPoint

Previous studies suggested that CD161 may be up-regulated on NKT cells prior to exiting the thymus 21, 22. However, as <50% of blood NKT cells in donors aged less than 6 months are CD161+ (Fig. 7), CD161 expression is clearly not a prerequisite for emigration, nor does it occur immediately on entry to the peripheral pool. These data are consistent with previous studies showing that many cord blood NKT cells are CD161–/lo21, 22. CD161 expression did not necessarily coincide with the age-associated appearance of CD4 NKT cells in blood, as CD161CD4 NKT cells were present in blood at all ages. Nevertheless, CD161 expression was significantly lower on blood NKT cells from children aged less than 6 months compared to older children (p<0.05), and lower on the thymus NKT cells compared to those in blood of matched donors (p<0.005) (Fig. 7 and data not shown). This provides the first direct evidence that CD161 expression by NKT cells correlates with the maturity of NKT cells in the same individual.

Conclusions

The distinct phenotypes of NKT cells from matched thymus and blood samples of the same donor strongly support a thymus-dependent developmental pathway that is completed in the periphery. Interestingly, the thymus NKT cell frequency in humans is far lower than that of blood, with peripheral NKT cell levels apparently established from birth and simply maintained thereafter. When compared to NKT cell development in the mouse, the human thymus NKT cell compartment is more heavily skewed toward CD4+ cells. However, if CD161–/low (NK1.1–/low) mouse NKT cells are examined, they too are predominantly CD4+13. Therefore, one explanation may be the extent to which NKT cells differentiate in the human thymus, as more NKT cells reach CD161+ stages in the mouse thymus, often giving rise to CD4CD161+ NKT cells at a late stage in thymic NKT cell development 14.

Data from very young children suggests that most thymus emigrant NKT cells are also CD4+, with the DN and CD8+ subsets seen in adult blood emerging with age through mechanisms that are yet to be defined, but not appearing to involve altered thymic export. Given the functional distinction between CD4+ and CD4 NKT cells 24, 29, 30, and their potential therapeutic importance, it is critical to identify the trigger(s) for these developmental/maturational steps to fully understand the causes of NKT cell deficiency and dysfunction – particularly when considering recent reports that DN and CD8+ NKT cells are the most numerically variable NKT cell subsets in human patients 33. Although NKT cell development is clearly thymus dependent, a large part of the answer may lie with understanding how NKT cells complete their maturation in the periphery. The poorly defined nature and role(s) of natural ligands for NKT cells makes it difficult to speculate on what may cause these late maturational events but recent advances in these areas, including the identification of a possible natural glycolipid ligand for NKT cells, are likely to provide important clues 34, 35.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Materials and methods
  6. Acknowledgements

Human tissue samples

Thymus and blood samples (5 ml) were obtained from 30 otherwise healthy children undergoing corrective cardiac surgery. Adult blood samples were obtained from the Red Cross Blood Bank (Melbourne, Australia). The limited amount of blood from infant donors and the low frequency of NKT cells in general meant that not all tests were conducted on all samples. Hence, the experimental group size (see Results) varies between assays. The project was approved by The Ethics in Human Research Committee at the Royal Children's Hospital (Melbourne, Australia) and the Health Sciences Human Ethics sub-committee (University of Melbourne). Informed consent was obtained from the legal guardian of each child. Blood samples were collected in heparinized tubes and lymphocytes isolated by Ficoll-mediated density centrifugation. Thymocyte suspensions were prepared by pushing fragments of freshly excised thymus through a metal sieve.

Flow cytometry

All antibodies and secondary reagents were purchased from BD Biosciences (San Diego, CA). The generation and use of fluorochrome-labeled αGC-loaded, or unloaded, CD1d tetramer has been described in detail elsewhere 36. Surface staining was performed using standard techniques; however, larger numbers of cells were collected than usual to collect sufficient NKT cells. For thymus, typically 30×106 cells were used per staining cocktail. As a consequence, cells were stained in v-bottom tubes and proportionally increased volumes of staining cocktail and washing buffer were used. Blood from cardiac patients was evenly split between cocktails to maximize cell numbers, but was typically 1×106–3×106 cells per sample. Cells were fixed prior to data acquisition on a LSRII flow cytometer (BD Biosciences) and data analyzed using CellQuestPro software (BD Biosciences). Intracellular Ki67 staining was performed using a BD cytofix/cytoperm Plus Kit (BD Biosciences).

Statistical analysis

Quantitative differences between groups were assessed by Mann-Whitney U test or Kruskal-Wallis test using InStat statistical analysis software.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Materials and methods
  6. Acknowledgements

S.P.B. was supported by a fellowship from the Human Frontiers Science Program. 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. Special thanks to M. Kronenberg for expert advice and provision of constructs to generate CD1d tetramer reagents.

  • 1

    WILEY-VCH

  • 2

    WILEY-VCH

  • 3

    WILEY-VCH

  • 4

    WILEY-VCH

  • 5

    WILEY-VCH

  • 6

    WILEY-VCH

  • 7

    WILEY-VCH

  • 1
    Kronenberg, M. and Gapin, L., The unconventional lifestyle of NKT cells. Nat. Rev. Immunol. 2002. 2: 557568.
  • 2
    Taniguchi, M., Harada, M., Kojo, S., Nakayama, T. and Wakao, H., The regulatory role of Valpha14 NKT cells in innate and acquired. Annu. Rev. Immunol. 2003. 21: 483513.
  • 3
    Godfrey, D. I. and Kronenberg, M., Going both ways: immune regulation via CD1d-dependent NKT cells. J. Clin. Invest. 2004. 114: 13791388.
  • 4
    Hammond, K. J. and Kronenberg, M., Natural killer T cells: natural or unnatural regulators of autoimmunity? Curr. Opin. Immunol. 2003. 15: 683689.
  • 5
    Smyth, M. J., Crowe, N. Y., Hayakawa, Y., Takeda, K., Yagita, H. and Godfrey, D. I., NKT cells – conductors of tumor immunity? Curr. Opin. Immunol. 2002. 14: 165171.
  • 6
    Gumperz, J. E. and Brenner, M. B., CD1-specific T cells in microbial immunity. Curr. Opin. Immunol. 2001. 13: 471478.
  • 7
    Park, S. H. and Bendelac, A., CD1-restricted T cell responses and microbial infection. Nature 2000. 406: 788792.
  • 8
    Sumida, T., Sakamoto, A., Murata, H., Makino, Y., Takahashi, H., Yoshida, S., Nishioka, K., Iwamoto, I. and Taniguchi, M., Selective reduction of T cells bearing invariant V alpha 24J alpha Q antigen receptor in patients with systemic sclerosis. J. Exp. Med. 1995. 182: 11631168.
  • 9
    van der Vliet, H. J. J., von Blomberg, B. M. E., Nishi, N., Reijm, M., Voskuyl, A. E., van Bodegraven, A. A., Polman, C. H., Rustemeyer, T., Lips, P., van den Eertwegh et al., Circulating V alpha 24(+) V beta 11(+) NKT cell numbers are decreased in a wide variety of diseases that are characterized by autoreactive tissue damage. Clin. Immunol. 2001. 100: 144148.
  • 10
    Wilson, S. B., Kent, S. C., Patton, K. T., Orban, T., Jackson, R. A., Exley, M., Porcelli, S., Schatz, D. A., Atkinson, M. A., Balk et al., Extreme Th1 bias of invariant Valpha24JalphaQ T cells in type 1 diabetes. Nature 1998. 391: 177181.
  • 11
    Dhodapkar, M. V., Geller, M. D., Chang, D. H., Shimizu, K., Fujii, S., Dhodapkar, K. M. and Krasovsky, J., A reversible defect in natural killer T cell function characterizes the progression of premalignant to malignant multiple myeloma. J. Exp. Med. 2003. 197: 16671676.
  • 12
    Lee, P. T., Putnam, A., Benlagha, K., Teyton, L., Gottlieb, P. A. and Bendelac, A., Testing the NKT cell hypothesis of human IDDM pathogenesis. J. Clin. Invest. 2002. 110: 793800.
  • 13
    Hammond, K. J., Pellicci, D. G., Poulton, L. D., Naidenko, O. V., Scalzo, A. A., Baxter, A. G. and Godfrey, D. I., CD1d-restricted NKT cells: an interstrain comparison. J. Immunol. 2001. 167: 11641173.
  • 14
    Pellicci, D. G., Hammond, K. J., Uldrich, A. P., Baxter, A. G., Smyth, M. J. and Godfrey, D. I., A natural killer T (NKT) cell developmental pathway iInvolving a thymus-dependent NK1.1(-)CD4(+) CD1d-dependent precursor stage. J. Exp. Med. 2002. 195: 835844.
  • 15
    Benlagha, K., Kyin, T., Beavis, A., Teyton, L. and Bendelac, A., A thymic precursor to the NK T cell lineage. Science 2002. 296: 553555.
  • 16
    Gadue, P. and Stein, P. L., NK T cell precursors exhibit differential cytokine regulation and require Itk for efficient maturation. J. Immunol. 2002. 169: 23972406.
  • 17
    MacDonald, H. R., Development and selection of NKT cells. Curr. Opin. Immunol. 2002. 14: 250254.
  • 18
    Berzins, S. P., Uldrich, A. P., Pellicci, D. G., McNab, F., Hayakawa, Y., Smyth, M. J. and Godfrey, D. I., Parallels and distinctions between T and NKT cell development in the thymus. Immunol. Cell Biol. 2004. 82: 269275.
    Direct Link:
  • 19
    Pear, W. S., Tu, L. and Stein, P. L., Lineage choices in the developing thymus: choosing the T and NKT pathways. Curr. Opin. Immunol. 2004. 16: 167173.
  • 20
    Gurney, K. B., Yang, O. O., Wilson, S. B. and Uittenbogaart, C. H., TCR gamma delta(+) and CD161(+) thymocytes express HIV-1 in the SCID-hu mouse, potentially contributing to immune dysfunction in HIV infection. J. Immunol. 2002. 169: 53385346.
  • 21
    Sandberg, J. K., Stoddart, C. A., Brilot, F., Jordan, K. A. and Nixon, D. F., Development of innate CD4+ alpha-chain variable gene segment 24 (Valpha24) natural killer T cells in the early human fetal thymus is regulated by IL-7. Proc. Natl. Acad. Sci. USA 2004. 101: 70587063.
  • 22
    Baev, D. V., Peng, X. H., Song, L., Barnhart, J. R., Crooks, G. M., Weinberg, K. I. and Metelitsa, L. S., Distinct homeostatic requirements of CD4+ and CD4 subsets of V{alpha}24-invariant natural killer T cells in humans. Blood 2004. 104: 41504156.
  • 23
    Thomas, S. Y., Hou, R., Boyson, J. E., Means, T. K., Hess, C., Olson, D. P., Strominger, J. L., Brenner, M. B., Gumperz, J. E., Wilson, S. B. and Luster, A. D., CD1d-restricted NKT cells express a chemokine receptor profile indicative of Th1-type inflammatory homing cells. J. Immunol. 2003. 171: 25712580.
  • 24
    Lee, P. T., Benlagha, K., Teyton, L. and Bendelac, A., Distinct functional lineages of human V(alpha)24 natural killer T cells. J. Exp. Med. 2002. 195: 637641.
  • 25
    D'Andrea, A., Goux, D., De Lalla, C., Koezuka, Y., Montagna, D., Moretta, A., Dellabona, P., Casorati, G. and Abrignani, S., Neonatal invariant Valpha24+ NKT lymphocytes are activated memory cells. Eur. J. Immunol. 2000. 30: 15441550.
  • 26
    Starborg, M., Gell, K., Brundell, E. and Hoog, C., The murine Ki-67 cell proliferation antigen accumulates in the nucleolar and heterochromatic regions of interphase cells and at the periphery of the mitotic chromosomes in a process essential for cell cycle progression. J. Cell Sci. 1996. 109: 143153.
  • 27
    Schluter, C., Duchrow, M., Wohlenberg, C., Becker, M. H., Key, G., Flad, H. D. and Gerdes, J., The cell proliferation-associated antigen of antibody Ki-67: a very large, ubiquitous nuclear protein with numerous repeated elements, representing a new kind of cell cycle-maintaining proteins. J. Cell Biol. 1993. 123: 513522.
  • 28
    Sachsenberg, N., Perelson, A. S., Yerly, S., Schockmel, G. A., Leduc, D., Hirschel, B. and Perrin, L., Turnover of CD4+ and CD8+ T lymphocytes in HIV-1 infection as measured by Ki-67 antigen. J. Exp. Med. 1998. 187: 12951303.
  • 29
    Gumperz, J. E., Miyake, S., Yamamura, T. and Brenner, M. B., Functionally distinct subsets of CD1d-restricted natural killer T cells revealed by CD1d tetramer staining. J. Exp. Med. 2002. 195: 625636.
  • 30
    Rogers, P. R., Matsumoto, A., Naidenko, O., Kronenberg, M., Mikayama, T. and Kato, S., Expansion of human Valpha24+ NKT cells by repeated stimulation with KRN7000. J. Immunol. Methods 2004. 285: 197214.
  • 31
    Takahashi, T., Chiba, S., Nieda, M., Azuma, T., Ishihara, S., Shibata, Y., Juji, T. and Hirai, H., Cutting edge: Analysis of human V alpha 24(+)CD8(+) NK T cells activated by alpha-galactosylceramide-pulsed monocyte-derived dendritic cells. J. Immunol. 2002. 168: 31403144.
  • 32
    Chen, H., Huang, H. and Paul, W. E., NK1.1+ CD4+ T cells lose NK1.1 expression upon in vitro activation. J. Immunol. 1997. 158: 51125119.
  • 33
    Bollyky, P. L. and Wilson, S. B., CD1d-restricted T cell subsets and dendritic cell function in autoimmunity. Immunol. Cell Biol. 2004. 82: 307314.
    Direct Link:
  • 34
    Godfrey, D. I., Pellicci, D. G. and Smyth, M. J., Immunology. The elusive NKT cell antigen–is the search over? Science 2004. 306: 16871689.
  • 35
    Zhou, D., Mattner, J., Cantu Iii, C., Schrantz, N., Yin, N., Gao, Y., Sagiv, Y., Hudspeth, K., Wu, Y., Yamashita, T. et al. Lysosomal glycosphingolipid recognition by NKT cells. Science 2004
  • 36
    Matsuda, J. L., Naidenko, O. V., Gapin, L., Nakayama, T., Taniguchi, M., Wang, C. R., Koezuka, Y. and Kronenberg, M., Tracking the response of natural killer T cells to a glycolipid antigen using CD1d tetramers. J. Exp. Med. 2000. 192: 741754.