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The Journal of Immunology, 1999, 163: 5338-5345.
Copyright © 1999 by The American Association of Immunologists

Heterogeneity of NK1.1+ T Cells in the Bone Marrow: Divergence from the Thymus1

Defu Zeng*, Gadi Gazit*, Sussan Dejbakhsh-Jones*, Steven P. Balk{dagger}, Scott Snapper{ddagger}, Masaru Taniguchi§ and Samuel Strober2,*

* Division of Immunology and Rheumatology, Department of Medicine, Stanford Medical School, Stanford, CA 94305; {dagger} Department of Hematology and Oncology, Beth Israel Deaconess Medical Center, and {ddagger} Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02215; and § CREST and Department of Molecular Immunology, Chiba University Graduate School of Medicine, Chiba, Japan


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
NK1.1+ T cells in the mouse thymus and bone marrow were compared because some marrow NK1.1+ T cells have been reported to be extrathymically derived. Almost all NK1.1+ T cells in the thymus were depleted in the CD1-/-, ß2m-/-, and J{alpha}281-/- mice as compared with wild-type mice. CD8+NK1.1+ T cells were not clearly detected, even in the wild-type mice. In bone marrow from the wild-type mice, CD8+NK1.1+ T cells were easily detected, about twice as numerous as CD4+NK1.1+ T cells, and were similar in number to CD4-CD8-NK1.1+ T cells. All three marrow NK1.1+ T cell subsets were reduced about 4-fold in CD1-/- mice. No reduction was observed in CD8+NK1.1+ T cells in the bone marrow of J{alpha}281-/- mice, but marrow CD8+NK1.1+ T cells were markedly depleted in ß2m-/- mice. All NK1.1+ T cell subsets in the marrow of wild-type mice produced high levels of IFN-{gamma}, IL-4, and IL-10. Although the numbers of marrow CD4-CD8-NK1.1+ T cells in ß2m-/- and J{alpha}281-/- mice were similar to those in wild-type mice, these cells had a Th1-like pattern (high IFN-{gamma}, and low IL-4 and IL-10). In conclusion, the large majority of NK1.1+ T cells in the bone marrow are CD1 dependent. Marrow NK1.1+ T cells include CD8+, V{alpha}14-J{alpha}281-, and ß2m-independent subsets that are not clearly detected in the thymus.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Natural killer T cells are defined as T cells bearing the common NK cell marker NK1.1, a member of the NKR-P1 gene family (1, 2). NK1.1+ T cells share several cell surface molecules, such as NK1.1, CD122 (IL-2 receptor ß-chain), CD16 (FcR{gamma}II), and asialo-GM1, with NK cells (1, 2). Their identity as T cells is indicated by expression of CD3-TCR complex, the very early T cell activation Ag CD69, and low levels of L-selectin (CD62L) (1). Unlike conventional T cells, NK1.1+ T cells have displayed a highly restricted TCR repertoire, which consists of an invariant V{alpha}14-J{alpha}281 {alpha}-chain paired preferentially with polyclonal Vß8.2, Vß7, or Vß2 chains (3, 4). This highly skewed TCR{alpha}ß repertoire is positively selected by the nonpolymorphic MHC class I-like CD1d molecule in association with ß2-microglobulin (ß2m)3 (1, 5, 6, 7, 8, 9). It has been reported that NK1.1+ T cells recognize CD1d-bound glycolipid ligands, such as glycosyl phosphatidylinositol and ceramides (10, 11).

NK1.1+ T cells rapidly produce large amounts of IL-4 upon in vivo administration of anti-CD3{epsilon} mAb, and they produce large amounts of IL-4, IL-10, and IFN-{gamma} upon primary in vitro stimulation with anti-CD3{epsilon} mAb, PMA, and calcium ionophore, or anti-NKR-P1 mAb (12, 13, 14). NK1.1+CD4+ T cells have been reported to augment the IgG response to GPI-anchored Ags (15), and to down-regulate autoimmune and alloimmune diseases such as diabetes (16, 17, 18) and acute graft-versus-host disease (GVHD) (19) via IL-4 and/or IL-10. The selective reduction of V{alpha}14-J{alpha}281 NK1.1+ T cells has been reported to be associated with the onset of clinical lupus in a variety of lupus-prone strains of mice (20). The selective reduction of V{alpha}24-J{alpha}Q T cells, a human homologue of V{alpha}14-J{alpha}281 NK1.1+ T cells, and a skewed Th1-like cytokine pattern of the T cells have been associated with human systemic sclerosis and diabetes (18, 21).

In normal mice, NK1.1+ T cells appear to be preferentially distributed in different organs, accounting for 20–30% of T cells in liver and bone marrow and for 0.5–1% of T cells in thymus and spleen. NK1.1+ T cells are rare in lymph nodes and virtually absent among gut intraepithelial lymphocytes (1). A previous report showed that <30% of NK1.1+ T cells in bone marrow but >80% of them in thymus expressed the invariant V{alpha}14-J{alpha}281 {alpha}-chain as determined by quantitative PCR and inverse PCR (22). The developmental relationships among NK1.1+ T cells in thymus, liver, and bone marrow is still controversial. The bone marrow T cells expressing the V{alpha}14-J{alpha}281 rearrangement have been reported to be extrathymically derived from precursors in the bone marrow itself (23, 24). The bone marrow recently has been shown to play the crucial role in restoring the homeostasis of NK1.1+ T cells in liver and spleen after peripheral depletion (25).

In the current study, we isolated NK1.1+ T cell subsets from the bone marrow and thymus of wild-type, CD1-/-, ß2m-/-, J{alpha}281-/- and nu/nu C57BL/6 mice and examined their cytokine secretion profiles. Our results indicate that, consistent with the previous reports (1, 2, 7, 8, 9), almost all NK1.1+ T cells in the thymus express the invariant V{alpha}14-J{alpha}281 TCR and recognize the ß2m-dependent form of CD1, but they do not express CD8. However, NK1.1+ T cells in bone marrow include CD8+ (both CD8{alpha}+/ß+ and CD8{alpha}+/ß-), CD4+, and CD4-CD8- subsets, and the majority do not express the invariant V{alpha}14-J{alpha}281 TCR. Nevertheless, most marrow NK1.1+ T cells are CD1 dependent, but the CD4-CD8- subset is ß2m independent. Bone marrow NK1.1+ T cells expressing the invariant V{alpha}14-J{alpha}281 TCR in the CD4+ and CD4-CD8- subsets are the major source of IL-4 production by these subsets. The CD4+ and CD4-CD8-NK1.1+ T cells that do not express the invariant V{alpha}14-J{alpha}281 TCR have a Th1-like cytokine profile.


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

C57BL/6 wild-type mice were obtained from the breeding facility of the Department of Comparative Medicine at Stanford University. C57BL/6 ß2m-/- mice were purchased from The Jackson Laboratory (Bar Harbor, ME). C57BL/6 nu/nu mice were purchased from Taconic Farms (Germantown, NY). The establishment of J{alpha}281-/- founder mice has been reported previously (26), and those used in the current study were backcrossed nine generations with C57BL/6 mice. CD1-/- founder mice with both CD1d1 and CD1d2 genes deleted were established by S. Balk and S. Snapper (Harvard Medical School, Boston, MA), and those used in the current study were backcrossed five generations with C57BL/6 mice. Littermate CD1+/+ mice were used as controls. All the mice were used at 8–10 wk of age.

mAbs, immunofluorescent staining, and flow cytometric analysis

Single-cell suspensions of thymocytes, obtained from thymus only, or bone marrow cells obtained from the femur and tibia were prepared and stained with mAbs as described previously (27, 28, 29). In most experiments, bone marrow T cells were enriched using immunomagnetic beads (see below) before staining. Stainings were performed in the presence of anti-CD16/32 (2.4G2, PharMingen, San Diego, CA) at saturation to block FcR{gamma}II/III receptors; propidium iodide (Sigma, St. Louis, MO) was added to staining reagents to exclude dead cells. Erythrocytes were excluded by light scatter gating. Three-color FACS analysis was performed using a modified dual-laser FACS Vantage (Becton Dickinson, Mountain View, CA), and data were analyzed using FACS/Desk software (28, 29). The following conjugated Abs were used for staining: FITC-anti-CD8{alpha} (CT-CD8{alpha}) and PE-anti-CD8ß (CT-CD8ß) purchased from Caltag (South San Francisco, CA); Alexia-anti-CD8 (CT-CD8{alpha}), a gift from D. L. Herzenberg (Stanford University School of Medicine, Stanford, CA); and allophycocyanin-anti-TCR{alpha}ß (H57-597), PE-anti-NK1.1 (PK136), and FITC-anti-CD4 (RM4-5) purchased from PharMingen.

Immunomagnetic bead enrichment and sorting of bone marrow NK1.1+ T cells

Sorted CD4+ and CD8+ (collected as a pool and referred to as CD4+/CD8+), CD4+, CD8+, and CD4-CD8-NK1.1+ T cells were obtained from the marrow by flow cytometry after enrichment of bone marrow T cells on immunomagnetic bead columns (Miltenyi Biotec, Auburn, CA). Marrow cells were first incubated with biotinylated anti-Thy-1 (5a-8) mAb (Caltag), then incubated with streptavidin magnetic beads. Thy-1+ cells were positively selected by retention on the magnetic columns and subsequently released. Sorting was performed using a modified dual-laser FACS Vantage (Becton Dickinson) flow cytometer (details of the sorting procedures have been described previously) (27).

In vitro secretion and measurement of cytokines

Sorted NK1.1+ T cells (1 x 105) from bone marrow of wild-type and gene-deficient mice were stimulated in vitro with 20 ng/ml PMA (Sigma) and 1 µM ionomycin (Calbiochem, San Diego, CA) in 10% fetal bovine serum and RPMI 1640 complete medium in 96-well round bottom plates; they were harvested at the peak time point (48 h) as described previously (27, 28). Supernatants were assayed in duplicate for the concentration of IFN-{gamma}, IL-4, and IL-10 using commercial ELISA kits (BioSource International, Camarillo, CA). Assays were developed with avidin-peroxidase and substrate, and plates were read at 450 nm using a microplate reader (27, 28).

Analysis of V{alpha}14-J{alpha}281 RNA expression by RT-PCR

Total RNA was extracted from unfractionated thymus cells and from sorted bone marrow CD4-CD8-NK1.1+ T cells from wild-type and ß2m-/- mice with the TRIzol reagent (Life Technologies, Grand Island, NY). For RNA isolation, 2–3 x 104 cells were used. RNA was then reverse-transcribed using random hexamer primers followed by PCR amplification. Optimal conditions for PCR amplification of ß-actin message, to be used as an internal standard, were established using titration of the number of amplification cycles and serial dilution of the cDNA template using primers specific for ß-actin, followed by densitometry analysis to measure ethidium bromide luminescence of the PCR products. The number of amplification cycles to be used for semiquantitative analysis was determined by plotting the log of the amount of PCR product (measured as absorbance units by densitometry analysis) as a function of cycle number, to establish a standard curve. The number of amplification cycles was derived from the linear portion of the standard curve. Thus, for ß-actin, the cDNAs underwent 24 cycles of amplification at 94°C for 40 s, 55°C for 40 s, and 72°C for 40 s, with primers specific for ß-actin. The sequences of the primers used for ß-actin were 5'-TGGGTCAGAAGGACTCCTATG-3' for the forward primer and 5'-ACCAGACAGCACTGTGTTGGC-3' for the reverse primer. Conditions for similar semiquantitative PCR analysis for detection of V{alpha}14-J{alpha}281 transcripts were established as described previously (7). In brief, the cDNAs underwent 40 cycles of amplification at 94°C for 45 s, 64°C for 15 s, and 72°C for 45 s. The sequences of the primers used were 5'-TAAGCACAGCACGTGCACAT-3' for V{alpha}14, and 5'-CAATCAGCTGAGTCCCAGCT-3' for J{alpha}281. The PCR products were resolved on a 3% agarose gel, and the amount of amplified product was quantified by densitometry. The levels of V{alpha}14-J{alpha}281 message were normalized relative to the amount of the ß-actin signal.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
NK1.1+ T cell subsets in the thymus of wild-type and gene-deficient mice

NK1.1+ T cells in the thymus of wild-type, CD1-/-, ß2m-/-, and J{alpha}281-/- C57BL/6 mice were compared by immunofluorescent staining and two-color flow cytometric analysis for the NK1.1 vs TCR{alpha}ß, CD4, and CD8 markers as shown in Fig. 1Go. About 0.6% of the thymus cells from wild-type mice were NK1.1+ {alpha}ß+ T cells (Fig. 1GoA), and they were about equally distributed between CD4+ and CD4-CD8- cells (Fig. 1Go, B and C). CD8+NK1.1+ T cells were not detected above background staining (Fig. 1GoD). The percentages of total NK1.1+ T cells, CD4+NK1.1+ cells, and CD4-CD8-NK1.1+ cells in the thymus of CD1-/- (Fig. 1Go, E–G), ß2m-/- (Fig. 1Go, I–K), and J{alpha}281-/- (Fig. 1Go, M–O) mice were reduced about 7- to 10-fold compared with those in wild-type mice (Fig. 1Go, A—C). The reduced percentages of NK1.1+ T cells in the thymus were reflected in the reduced mean absolute numbers which were 83–88% below the levels in the wild-type mice (Table IGo). The staining patterns of thymus NK1.1+ T cells in wild-type and littermate control CD1+/+ C57BL/6 mice were similar (data not shown).



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  		FIGURE 1. Two-color flow cytometric analysis of NK1.1+ TCR{alpha}ß+ T cell subsets in the thymus of wild-type and gene-deficient mice. Thymocytes of wild-type (A–D), CD1-/- (E–H), ß2m-/- (I–L) and J{alpha}281-/- (M–P) were stained for NK1.1 vs TCR{alpha}ß, CD4 and CD8 together, or CD4 and CD8 markers separately. All NK1.1+ TCR{alpha}ß+ cells are enclosed in the boxes in A, E, I and M. The CD4-CD8-NK1.1+ and CD4+/CD8+ NK1.1+ cells are enclosed in the left and right boxes, respectively, in B, F, J, and N. The CD4+NK1.1+ T cells are enclosed in boxes in C, G, K, and O. The CD8+ NK1.1+ cells are enclosed in the boxes in D, H, L, and P. The numbers above the boxes are the percentages of different NK1.1+ T cell subsets among all thymocytes. Each panel represents at least four replicate experiments.

 

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  		Table I. The absolute numbers1 of NK1.1+ T cell subsets in thymus and bone marrow of wild-type and gene-deficient C57BL/6 mice

 
NK1.1+ T cell subsets in the bone marrow of wild-type and gene-deficient mice

Analyses of NK1.1+ T cell subsets in the bone marrow of wild-type and gene-deficient mice were performed as described above. As shown in Fig. 2GoA, about 2% of whole bone marrow cells in the wild type stained brightly for TCR{alpha}ß, and about 30% of TCR{alpha}ß+ cells were NK1.1+. T cells were enriched 10- to 20-fold using anti-Thy1.2 mAb and immunomagnetic beads to facilitate further T cell subset analyses. The enrichment procedure did not significantly change the percentage of NK1.1+ T cells among TCR{alpha}ß+ T cells (~30%; Fig. 2GoB), and the yield of NK1.1+ T cells per mouse was 75–85% after enrichment (data not shown). Compared with the wild-type marrow, the percentage of NK1.1+ T cells in whole or enriched CD1-/- bone marrow (Fig. 2Go, E and F) was reduced about 4-fold; this was reflected in a 76% decrease in absolute numbers (Table IGo). Thus, the NK1.1+ T cells in the marrow include a predominant CD1-dependent subset, and a minority CD1-independent subset. Although the CD1-/- mice were backcrossed on the C57BL/6 background for five generations, it is possible that genes other than that encoding CD1 contributed to the reduction of the NK1.1+ T cells in the marrow. Accordingly, we analyzed the marrow from CD1+/+ littermates (Fig. 2Go, C and D) of the CD1-/- mice and found that the percentage of NK1.1+ T cells was similar to that of wild-type C57BL/6 mice. The percentage of NK1.1+ T cells in the enriched ß2m-/- marrow (Fig. 2GoG) was reduced by about 50%, but this reduction was not detected in the whole bone marrow. This discrepancy may be due to the improved resolution and quantitation of subsets in the enriched marrow. The absolute number of NK1.1+ T cells in the ß2m-/- marrow was reduced by 53% (p < 0.01, two-tail Student’s t test; Table IGo). The percentages of NK1.1+ T cells among all marrow T cells in the ß2m-/- and wild-type mice (Fig. 2Go, B and H) remained about the same due to a reduction in the absolute numbers of CD8+ NK1.1- T cells (data not shown). The percentage of NK1.1+ T cells in J{alpha}281-/- marrow (Fig. 2Go, I and J) was similar to that in the wild-type marrow (Fig. 2Go, A and B), and the absolute numbers were not significantly different (Table IGo). The percentage of NK1.1+ T cells in age-matched (8–10 wk) nu/nu marrow (Fig. 2Go, K and L) was reduced 50- to 100-fold to the level of background staining.



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  		FIGURE 2. Two-color flow cytometric analysis of NK1.1+ TCR{alpha}ß+ T cells in the bone marrow of wild-type and gene-deficient mice. The panels in the left column show the pre-enriched bone marrow cells, and those in the right column show the postenriched bone marrow cells of C57BL/6 wild-type (A and B), CD1+/+ littermate control (C and D), CD1-/- (E and F), ß2m-/- (G and H), J{alpha}281-/- (I and J) and nu/nu (K and L) mice stained for NK1.1 vs TCR{alpha}ß markers. The TCR{alpha}ß+ NK1.1+ T cells are enclosed in the upper boxes and the TCR{alpha}ß+ NK1.1- T cells in the lower boxes. The numbers beside the boxes show the percentages of NK1.1+ or NK1.1- TCR{alpha}ß+ T cells among all live nucleated cells.<./>

 
Gated TCR{alpha}ß+ T cells in the enriched marrow cells of wild-type mice (Fig. 3GoA, enclosed in box) were analyzed for CD4 and CD8 markers together, and CD4 and CD8 markers separately (Fig. 3Go, B–D). Three discrete populations of NK1.1+ T cell subsets including CD4-CD8-, CD4+, and CD8+ were observed. The percentage of the CD8+ subset among T cells was about twice that of the CD4+ subset and was similar to that of the CD4-CD8- subset. This was reflected in the absolute numbers of the NK1.1+ T cell subsets harvested from marrow (Table IGo). The ratio of all CD8+ vs CD4+ cells among all TCR{alpha}ß+ T cells in the wild-type marrow was about 2:1 using the absolute numbers (Table IGo). In additional experiments, gated TCR{alpha}ß+ NK1.1+ cells (Fig. 4GoA) from the wild-type enriched marrow were analyzed for CD8{alpha} vs CD8ß markers (Fig. 4GoB). About 70% of the CD8+NK1.1+ T cells stained brightly for both markers (Fig. 4GoB, enclosed in upper box), and about 30% stained dully for only the CD8{alpha} marker (Fig. 4GoB, enclosed in lower box). Staining for CD8 markers was also performed on gated NK1.1+ TCR{alpha}ß+ cells from ß2m-/- bone marrow and on gated NK1.1- TCR{alpha}ß+ cells from wild-type spleen (Fig. 4Go, C and E, respectively). However, the cells did not express levels of either marker above background controls in the case of the ß2m-/- marrow cells (Fig. 4GoD). In the case of the wild-type spleen, almost all CD8+ cells expressed both the CD8{alpha} and CD8ß markers, and few, if any, expressed only the CD8{alpha} marker (Fig. 4GoF).



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  		FIGURE 3. Two-color flow cytometric analysis of NK1.1+ TCR{alpha}ß+ T cell subsets in the bone marrow of wild-type and gene-deficient mice. Enriched marrow cells of wild-type (A), CD1-/- (E), ß2 m-/- (I), and J{alpha}281-/- (M) were stained for CD4/CD8 vs TCR{alpha}ß. Gated TCR{alpha}ß+ cells (enclosed in the boxes) were analyzed for NK1.1 vs CD4/CD8 in B, F, J, and N, for NK1.1 vs CD4 in C, G, K, and O, and for NK1.1 vs CD8 in D, H, L, and P. CD4-CD8-NK1.1+ T cells are enclosed in the upper left boxes in B, F, J, and N. The CD4+NK1.1+T cells are enclosed in the upper right boxes in C, G, K, and O. The CD8+ NK1.1+ T cells are enclosed in the upper right boxes in D, H, L, and P.

 


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  		FIGURE 4. Two-color flow cytometric analysis of NK1.1+ TCR{alpha}ß+ CD8+ T cell subsets in the bone marrow (BM) of wild-type mice and ß2m-/- mice. Enriched bone marrow cells of wild-type (A) and ß2m-/- (C) mice were stained for NK1.1 vs TCR{alpha}ß. Gated NK1.1+ TCR{alpha}ß+ cells (enclosed in the boxes) were stained for CD8ß vs CD8{alpha} receptors in B and D. Upper boxes in B and D enclose CD8{alpha}+ß+ cells, and lower boxes enclose CD8{alpha}+ß- cells. Similar analysis of gated NK1.1- TCR{alpha}ß+ cells from unfractionated wild-type spleen (SPL) cells is shown for comparison in E and F.

 
In additional experiments, the changes in the percentages and absolute numbers of CD4+, CD8+, and CD4-CD8-NK1.1+ T cells were compared in wild-type and gene-deficient marrow. The percentages of all three NK1.1+ T cell subsets in CD1-/- marrow were reduced about 4-fold (Fig. 3Go, E–H) compared with wild type (Fig. 3Go, A–D), and their absolute numbers were reduced by 72–80% (p < 0.001, two-tail Student’s t test; Table IGo). Surprisingly, the percentage of CD4-CD8-NK1.1+ T cells in ß2m-/- marrow (Fig. 3GoJ) was increased compared with wild type, and the absolute numbers were similar (Table IGo). In contrast, there was a marked depletion of CD8+NK1.1+ T cells in ß2m-/- mice (Fig. 3GoL), and the reduction of the absolute number of CD8+ NK1.1+ T cells was 98% (p < 0.001, two-tail Student’s t test, Table IGo). The percentage of CD4+ NK1.1+ T cells in ß2m-/- marrow was reduced (Fig. 3GoK) compared with the wild type, and the absolute numbers were reduced by about 3-fold (p < 0.01, two-tail Student’s t test, Table IGo). The percentage (Fig. 3GoN) and the absolute number of CD4-CD8-NK1.1+ T cells in J{alpha}281-/- mice were not significantly reduced (p > 0.05, two-tail Student’s t test; Table IGo) compared with those in wild-type mice. The percentage (Fig. 3GoP) and absolute number of CD8+NK1.1+ T cells were significantly increased by 38% (p < 0.05, two-tail Student’s t test; Table IGo). On the other hand, the percentage of CD4+NK1.1+ T cells in J{alpha}281-/- mice was reduced about 2-fold (Fig. 3GoO), and the absolute number was reduced by 43% (p < 0.05, two-tail Student’s t test; Table IGo).

Cytokine secretion patterns of NK1.1+ T cell subsets in marrow

It has been reported that CD4+ and CD4-CD8-NK1.1+ T cells from thymus and peripheral tissues such as spleen and liver produced large amounts of IFN-{gamma}, IL-4, and IL-10 in response to in vitro stimulation with anti-CD3{epsilon} mAb and PMA plus ionomycin (1, 12, 14, 30). Because some subsets of NK1.1+ T cells in the marrow were not identified in the thymus, the cytokine secretion profiles of the NK1.1+ T cell subsets in the bone marrow of wild-type and gene-deficient C57BL/6 mice were studied. Sorted CD4+/CD8+ (CD4+ and CD8+ T cells as a pool), CD4+, CD8+, and CD4-CD8-NK1.1+ T cells were stimulated with PMA and ionomycin for 48 h, and the culture supernatants were assayed for concentrations of IL-4, IL-10, and IFN-{gamma}. As shown in Table IIGo, control sorted CD4+/CD8+NK1.1- T cells from the peripheral blood of wild-type mice produced large amounts of IFN-{gamma}, but little IL-4 or IL-10, and the ratio of IFN-{gamma} to IL-4 was 56:1. In contrast, the sorted CD4+/CD8+ and CD4-CD8-NK1.1+ T cells from the marrow produced large amounts of IFN-{gamma}, IL-4, and IL-10. The ratio of IFN-{gamma} to IL-4 was 1.1:1 for CD4+/CD8+ and 1.9:1 for CD4-CD8-NK1.1+ T cells. It is of interest that the sorted CD8+NK1.1+ T cells were similar to the sorted CD4+NK1.1+ T cells and also produced large amounts of IFN-{gamma}, IL-4, and IL-10 (Table IIGo).


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  		Table II. Cytokine secretion profiles of NK1.1+ T cell subsets in bone marrow of wild-type and gene-deficient C57BL/6 mice

 
The cytokine pattern of the NK1.1+ T cells from the marrow of ß2m-/- and J{alpha}281-/- mice was different from that of wild-type mice. Although the CD4-CD8-NK1.1+ T cells from the marrow of both ß2m-/- and J{alpha}281-/- mice produced comparable amounts of IFN-{gamma} compared with the CD4-CD8-NK1.1+ T from wild-type marrow, they produced 10- to 20-fold less IL-4 and IL-10 (p < 0.001, two-tail Student’s t test; Table IIGo). The IFN-{gamma} secretion of the CD4+/CD8+NK1.1+ T cells from the marrow of ß2m-/- and J{alpha}281-/- mice was also similar to that of CD4+/CD8+ NK1.1+ T cells from wild-type marrow, but the IL-4 production of the CD4+/CD8+NK1.1+ T cells was reduced 8-fold from ß2m-/- marrow, and 3-fold from J{alpha}281-/- marrow. In the case of ß2m-/- marrow, the CD4+ cells were the source of cytokines of the sorted CD4+/CD8+NK1.1+ T cells, because CD8+ cells were not easily detected. Thus, changes in the cytokine profile are due to changes on the CD4+NK1.1+ T cells. In the case of the J{alpha}281-/- marrow, the CD8+cells were about 5-fold more numerous than CD4+ cells, and the changed cytokine profile reflects the contribution of both CD4+ and CD8+NK1.1+ T cells.

Changes in V{alpha}14-J{alpha}281 TCR gene expression in ß2m-/- mice

Because the cytokine profiles were markedly different in the CD4-CD8-NK1.1+ T cells in marrow from wild-type and ß2m-/- mice, we examined changes in the expression of the V{alpha}14-J{alpha}281 TCR {alpha}-chain gene in these mice using a semiquantitative RT-PCR assay. Primers were designed to amplify the invariant TCR {alpha}-chain cDNA as well as the ß-actin cDNA. PCR conditions were set so that the intensity of the amplified cDNA products was on the linear portion of titration curves comparing band intensity with the PCR cycle number. RNA was isolated from 2 to 3 x 104 cells from wild-type and ß2m-/- mice, and intensity of the V{alpha}14-J{alpha}281 TCR signal was normalized relative to the ß-actin signal. Fig. 5Go shows the V{alpha}14-J{alpha}281 and ß-actin amplified products and signal intensities obtained from wild-type and ß2m-/- whole thymus cells, and those of sorted CD4-CD8-NK1.1+ T cells from the bone marrow. Lanes 1 and 2 show a marked reduction in the levels of amplified V{alpha}14-J{alpha}281 cDNA from wild-type and ß2m-/- whole thymus cells, respectively, as judged by the intensity of the bands and densitometry values. This was expected due to the reduced number of NK1.1+ T cells in the ß2m-/- thymus. Lane 3 shows that an amplified V{alpha}14-J{alpha}281 signal was easily detectable using sorted CD4-CD8-NK1.1+ T cells from wild-type marrow with an intensity similar to that of whole thymus. This suggests that only a small percentage of CD4-CD8-NK1.1+ T cells in the bone marrow express the V{alpha}14-J{alpha}281 gene. Lane 4 shows that the intensity of this band and the associated densitometry values were markedly reduced in ß2m-/- marrow compared with wild-type marrow, despite equal numbers of sorted CD4-CD8-NK1.1+ T cells. The intensities of the ß-actin bands in lanes 1–4 were comparable.



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  		FIGURE 5. Semiquantitative RT-PCR analysis of V{alpha}14-J{alpha}281 and ß-actin transcripts in wild-type and ß2m-/- mice. The PCR products were amplified using amounts of cDNA template generated from RNA obtained from 2 to 3 x 104 cells of each type. Products were resolved on a 3% agarose gel. Lane 1, wild-type thymus; lane 2, ß2m-/- thymus; lane 3, sorted wild-type CD4-CD8-NK1.1+ T cells from bone marrow; lane 4, sorted ß2m-/- CD4-CD8-NK1.1+ T cells from bone marrow; lane 5, no template; lane 6, 1-kb ladder. The intensity of the V{alpha}14-J{alpha}281 TCR signals was normalized relative to the ß-actin signal for each sample, and relative densitometry units were 9.3 in wild-type thymus, 1.0 in ß2m-/- thymus, 6.7 in sorted wild-type CD4-CD8-NK1.1+ T cells from bone marrow, and <0.1 for sorted ß2m-/- CD4-CD8-NK1.1+ T cells from bone marrow.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
NK1.1+ T cells are a specialized population of {alpha}/ß T cells that co-express receptors of the NK lineage and have the unique potential to very rapidly secrete large amounts of cytokines such as IL-4 (1). Previous studies showed that >85% of NK1.1+ T cells in thymus express the invariant V{alpha}14-J{alpha}281 TCR and that this unique TCR recognizes CD1 in association with ß2m (1, 7, 8, 9). The latter molecule has been shown to positively select the thymic NK1.1+ T cells (5). The CD8+NK1.1+ T cells in thymus are theorized to be depleted by negative selection (1). The current study also showed that the thymus of wild-type mice contained only CD4+ and CD4-CD8-NK1.1+ subsets. The absolute numbers of CD4+ and CD4-CD8-NK1.1+ T cells in the thymus of CD1-/-, ß2m-/-, and J{alpha}281-/- mice were reduced by about 10-fold, as in previous studies (7, 8, 9, 26).

The pattern of NK1.1+ T cell subsets in bone marrow is different from that in thymus. NK1.1+ T cells make up about 30% of T cells in bone marrow compared with 0.5–1% in thymus and include CD4+, CD8+, and CD4-CD8- subsets. The CD8+ subset was twice as numerous as the CD4+ subset and similar in number to the CD4-CD8- subset. Judging from flow cytometric analyses, the CD8+NK1.1+ T cells were made up of about 70% expressing the CD8{alpha}ß ({alpha}+ß+) heterodimer, and 30% expressing the CD8{alpha}{alpha} ({alpha}+ß-) homodimer. CD8{alpha}{alpha} homodimeric T cells are also found among intestine epithelial lymphocytes and are extrathymically derived (31, 32). The presence of CD8+NK1.1+ T cells in the marrow but not in the thymus indicates that those NK1.1+ T cells are extrathymically derived. It is also possible that CD8{alpha} is an activation marker on CD4-CD8-NK1.1+ T cells and that it does not reflect the tissue origin of the CD8{alpha}+ß-NK1.1+ T cell subset. All NK1.1+ T cell subsets including the CD8+ subset were markedly depleted in athymic nu/nu mice. Thus, the bone marrow CD8+NK1.1+ T cells were clearly dependent on the presence of the thymus in the 2-mo-old mice used in the current study. Possible explanations of this apparent paradox are that the wing helix nude (nu) mutation expressed in epithelial cells of nu/nu mice (33, 34) interferes with extrathymic development of the CD8+NK1.1+ T cells, or that the thymus facilitates extrathymic development of these cells by exporting humoral substances or facilitating cells. Alternatively, the CD8+NK1.1+ T cells in the marrow may be derived from thymic emigrants that alter their phenotypic and functional characteristics when they localize to the bone marrow. Some V{alpha}24-J{alpha}Q+ T cells, the human homologue of murine NK1.1+ T cells (35), have been reported to be CD8{alpha}dim/ß- T cells (36), and may be homologues of the CD8{alpha}+/ß-NK1.1+ T cells (which also had dim staining for CD8{alpha}) observed in the mouse marrow. The human CD8{alpha}dim/ß- T cells represented the majority of V{alpha}24-J{alpha}Q+ Vß11+ T cells in some donors, but in others the majority of the cells were CD4-CD8-, the phenotype reported initially for most V{alpha}24-J{alpha}Q+ T cells (35).

Evidence that the bone marrow is a direct source of NK1.1+ T cells expressing the V{alpha}14-J{alpha}281 TCR includes the presence of DNA deletion circles specific for this TCR {alpha}-chain gene rearrangement, and the expression of RAG-1 and RAG-2 genes in marrow cells which are not members of the B cell lineage (23, 24). In the current study, the bone marrow of nu/nu mice older than 3 mo was not investigated for the content of NK1.1+ T cells. Because T cells slowly develop in the lymphoid tissues of nu/nu or thymectomized irradiated mice and are easily detected by 3–6 mo of age (37, 38), it is possible that NK1.1+ T cells gradually develop in the marrow of the older mice. Late development may account for previous reports of the presence of NK1.1+ CD3+ T cells in the lymphoid tissues of such athymic mice (23, 24, 39) and for the lack of detection in the current study.

CD8+NK1.1+ T cells were considerably reduced (by ~72%) in the CD1-/- bone marrow compared with those in the wild-type marrow, indicating that the predominant subset is CD1 dependent, as previously reported for NK1.1+ T cells in other tissues. However, a minority subset of CD1-independent CD8+ NK1.1+ T cells persisted and accounted for about 30% of the wild-type number. In contrast, the absolute number of CD8+NK1.1+ T cells was significantly increased in J{alpha}281-/- marrow, and almost completely depleted in ß2m-/- marrow. These results indicate that few CD8+NK1.1+ T cells express the invariant V{alpha}14-J{alpha}281 TCR, and the CD8+ NK1.1+ cells still recognize the ß2m-dependent form of CD1. The latter molecule presumably is required for positive selection of the marrow CD8+NK1.1+ T cells, but it does not negatively select the marrow cells, as proposed for this subset in the thymus (1). This dichotomy may be due to differences in tissue specific ligands that bind to CD1 and interact with predominantly V{alpha}14-J{alpha}281 receptors in the thymus, and with other {alpha}-chain receptors in the marrow (40, 41, 42).

The number of CD4-CD8-NK1.1+ T cells was not significantly reduced in either J{alpha}281-/- or ß2m-/- bone marrow, indicating that a substantial fraction of these CD4-CD8-NK1.1+ cells do not express the invariant V{alpha}14-J{alpha}281 TCR and may recognize a ß2m-independent form of CD1. The latter form is likely to have an identical amino acid sequence to that of the ß2m-dependent form, but the CD4-CD8-NK1.1+ TCR may interact with different segments of CD1 or with different CD1 ligands, based on differences in conformation engendered by the ß2m association. An alternative explanation is that some subsets of marrow CD4-CD8-NK1.1+ T cells recognize a ß2m-independent molecule other than CD1, and that these NK1.1+ T cells expand in ß2m-/- mice. The presence of a small residual population of NK1.1+ T cells in the marrow of CD1-/- mice (Fig. 3Go) is consistent with this notion. Ongoing experiments, beyond the scope of the current study, will test this explanation, including those that will test the reactivity of sorted marrow CD4-CD8-NK1.1+ T cells to ß2m-deficient CD1-transfected cells, and those that will search for marrow CD4-CD8-NK1.1+ T cells in ß2m-/CD1-/- (double gene deficient) mice.

The presence of V{alpha}14-J{alpha}281- T cells among the CD8+ and CD4-CD8-NK1.1+ T cells in marrow is consistent with the previous reports that more than 70% of NK1.1+ T cells in bone marrow do not express the invariant V{alpha}14-J{alpha}281 TCR (22). Of the <30% of NK1.1+ T cells in the marrow that express the invariant TCR, the large majority are likely to be contained within the CD4+ NK1.1+ T cell subset, and only a small percentage are within the CD4-CD8-NK1.1+ T cell subset. The similar PCR signals in the whole thymus (of which ~0.6% are NK1.1+ T cells) and in the sorted marrow CD4-CD8-NK1.1+ T cells is consistent with the latter distribution. Previous studies of the V{alpha}14-J{alpha}281 expression in marrow NK1.1+ T cells did not examine the expression within isolated NK1.1+ T cell subsets (22, 26). In one of these studies (26), the percentage of NK1.1+ T cells among all T cells in the marrow of both J{alpha}281+/+ and J{alpha}281-/- mice was considerably lower than in the current study. This discrepancy is most likely due to the use of J{alpha}281-/- and control mice in the present study after nine backcross generations, whereas the previous study used mice after only three backcross generations (26). The mixture of NK1.1+ T cells in the marrow is consistent with two different types of NK1.1+ T cell hybridomas; some anti-CD1 V{alpha}14-J{alpha}281- T cell hybridomas recognize a ß2m-independent form of CD1, whereas the V{alpha}14-J{alpha}281+ hybridomas only recognize a ß2m-dependent form of CD1 (43). A CD4-CD8- TCR{alpha}ß+ V{alpha}4.4 anti-CD1 T cell clone also has been reported to recognize a ß2m-independent form of CD1 (27, 44). In addition, a ß2m-independent form of CD1 previously has been reported to be expressed by human intestinal epithelium cells (45).

Although the CD4-CD8-NK1.1+ T cells were abundant in the J{alpha}281-/- and ß2m-/- marrow, they were not detected in the thymi of these mice. Studies of the cytokine secretion profile of the CD4-CD8-NK1.1+ T cells in the marrow of the wild-type and gene-deficient mice showed that a marked change occurred in the latter mice. Whereas the CD4-CD8-NK1.1+ T cells in the wild-type mice secreted high levels of IFN-{gamma}, IL-10, and IL-4, those in the J{alpha}281-/- and ß2m-/- mice secreted high levels of IFN-{gamma} but about 10-fold reduced levels of IL-10 and IL-4 (Th1-like pattern). This suggests that marrow CD4-CD8-NK1.1+ T cells expressing the V{alpha}14-J{alpha}281 TCR and recognizing the ß2m-dependent form of CD1 secrete high levels of all three cytokines, but that the V{alpha}14-J{alpha}281- CD4-CD8-NK1.1+ T cells that recognize a ß2m-independent ligand secrete a Th1-like pattern. Both types of NK1.1+ T cells are present among CD4-CD8-NK1.1+ T cells in the marrow of wild-type mice, as judged by the expression of this TCR {alpha}-chain gene by PCR analysis of the sorted CD4-CD8- subset in the latter mice. The reduced PCR signal in the sorted cells from ß2m-/- mice showed that few, if any, of the residual cells express this TCR. The cytokine pattern of the V{alpha}14-J{alpha}281- subset is only revealed in the ß2m-/- and J{alpha}281-/- mice, and the cytokine pattern of the V{alpha}14-J{alpha}281+ subset is dominant in the wild-type mice.

The CD4+NK1.1+ T cells in the marrow are also made up of a combination of V{alpha}14-J{alpha}281+ and V{alpha}14-J{alpha}281- subsets, based on the results using ß2m-/- and J{alpha}281-/- mice. The reduction of the absolute number of CD4+NK1.1+ T cells by 73% and 43%, respectively, in these gene-deficient mice supports this notion. In addition, the cytokine pattern of the residual CD4+ NK1.1+ T cells in ß2m-/- mice showed a Th1-like pattern similar to that of the CD4-CD8-NK1.1+ T cells in the same mice. Thus, the Th1-like pattern appears to be secreted by both V{alpha}14-J{alpha}281- CD4+ and V{alpha}14-J{alpha}281- CD4-CD8-NK1.1+ T cells, that recognize a ß2m-independent ligand. Because there was a residue of NK1.1+ T cells in the CD1-/- mice, some fraction of the NK1.1+ T cells may use molecules other than CD1 for positive selection.

After the submission of our manuscript, a report by Eberl et al. (46) was published which also showed that CD8+NK1.1+ T cells were more numerous than CD4+NK1.1+ T cells in bone marrow, despite the limited detection of CD8+NK1.1+ T cells in the thymus. However, the marrow CD8+NK1.1+ T cells were reported to be CD1 independent, whereas the current report found both CD1-independent and CD1-dependent CD8+NK1.1+ T cell subsets. Explanations of the different findings include the use of nylon wool columns for marrow cell enrichment (46) that may have depleted the CD1-dependent subset, and the use of CD1-/- mice backcrossed to the C57BL/6 background for three generations compared with those backcrossed for five generations in the current report. Flow cytometric patterns showing analyses of the CD8+NK1.1+ T cells in the marrow of CD1-/- mice or in their littermate controls were not shown by Eberl et al. (46), so that a direct comparison with patterns in the current report cannot be made.

In conclusion, almost all the NK1.1+ T cells in the thymus express the V{alpha}14-J{alpha}281 TCR, whereas the bone marrow NK1.1+ T cells are more heterogeneous and have a different balance of both V{alpha}14-J{alpha}281+ and V{alpha}14-J{alpha}281- NK1.1+ T cells, each of which expresses different cytokine profiles and different accessory molecules, such as CD8. The marrow V{alpha}14-J{alpha}281- NK1.1+ T cells appear to arise extrathymically, in view of their paucity in the thymus and their unusual phenotypes (i.e., CD8{alpha}+ß-), but the V{alpha}14-J{alpha}281+ NK1.1+ T cells are likely to arise from both thymic and extrathymic sources. Recent studies show that the NK1.1+ T cells in the marrow suppress GVHD induced by NK1.1- T cells, and that this NK1.1+ T cell function, especially in the CD4-CD8- subset, is mediated by IL-4 (19). The current study indicates that the suppressive activity in these CD4-CD8-NK1.1+ T cells is confined to the V{alpha}14-J{alpha}281+ cells, because the latter are the key source of IL-4. The selective loss of V{alpha}14-J{alpha}281+ NK1.1+ T cells in lupus-prone mouse strains (20) may result in a shifted pattern of cytokine secretion (Th1-like) in the residual CD1-recognizing T cells. CD1-recognizing T cells with a Th1-like pattern have been reported to induce lupus (27). Thus, the balance of V{alpha}14-J{alpha}281+ and V{alpha}14-J{alpha}281- NK1.1+ T cells described herein may influence autoimmune and alloimmune diseases.


   Acknowledgments
 
We thank Jun-Chuan Xu for excellent technical assistance, Dr. James Tung for helpful discussions for RT-PCR analysis, and Vibeke Cleaver for preparation of the manuscript.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants AI40093, HL58250, and HL57443. Back

2 Address correspondence and reprint requests to Dr. Samuel Strober, Division of Immunology and Rheumatology, Stanford University School of Medicine,300 Pasteur Drive, Room S105B, Stanford, CA 94305. E-mail address: Back

3 Abbreviations used in this paper: ß2-m, ß2-microglobulin; GVHD, graft-versus-host disease. Back

Received for publication April 13, 1999. Accepted for publication September 7, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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