By
From the * Division of Immunology and Rheumatology, In the principal pathway of Since the landmark discoveries that lymphocytes initiate
cellular immune responses and that the thymus is the
principal source of these immune cells, the maturation of
lymphocytes in the thymus has been studied extensively,
and the principal intermediary stages of development have
been identified (1, 2). Extrathymic maturation of T cells also occurs, and contributes to the pool of T cells in the lymph nodes, spleen,
marrow, liver, and intestines of athymic nude mice and
thymectomized lethally irradiated mice injected with a
source of hematopoietic progenitor cells (15). The expression of the genes encoding the recombinase enzymes
recombination-activating gene (RAG)-1 and RAG-2, as well as the presence of circular DNA products of TCR- The nature of the extrathymic microenvironments that
support T cell maturation and the intermediate stages of
maturation in these sites are presently unknown. Previous
studies have shown that T cell-depleted cultures of single
cell suspensions of bone marrow from euthymic and athymic mice and from thymectomized, lethally irradiated mice
reconstituted with stem cells are capable of generating T
cells in vitro (19, 22). In this study, we show that these
bone marrow cultures can give rise to Animals.
Department of Pathology, Stanford University School of Medicine, Stanford, California 94305
Abstract
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
/
T cell maturation, T cell precursors from the bone marrow
migrate to the thymus and proceed through several well-characterized developmental stages
into mature CD4+ and CD8+ T cells. This study demonstrates an alternative pathway in which the bone marrow microenvironment also supports the differentiation of T cell precursors into
CD4+ and CD8+ T cells. The marrow pathway recapitulates developmental stages of thymic
maturation including a CD4+CD8+ intermediary cell and positive and negative selection, and
is strongly inhibited by the presence of mature T cells. The contribution of the marrow pathway in vivo requires further study in mice with normal and deficient thymic or immune function.
Introduction
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
/
T cell precursors emigrate from
the bone marrow to the thymus and the earliest precursors in the thymus do not express the CD4, CD8, or CD3 surface receptors, and have not rearranged the TCR-
or -
chain genes (3). Shortly after the latter genes are rearranged during thymic maturation, the predominant intermediary CD4+CD8+ (double positive) T cells develop and
give rise to CD4+ and CD8+ (single positive)
/
T cells
via processes of positive and negative selection (6). The
latter cells emigrate to the periphery and, after stimulation
by antigen, generate effector cells of cell-mediated immunity or helper cells that regulate humoral immunity (10, 11). The predominance of the thymus in T cell maturation
is thought to be related to its unique stromal microenvironment, which has been shown to support differentiation in
vitro in thymic organ cultures or in heterogeneous thymic
stromal cell cultures (12).
chain
gene rearrangements, indicate that T cell development occurs in several extrathymic sites, including the bone marrow, adult and fetal liver, and yolk sac (20, 21).
/
T cells with the
mature CD4+CD8
and CD4
CD8+ surface phenotype
via a CD4+CD8+ intermediary stage that recapitulates the
principal features of thymic maturation, including negative
selection of autoreactive cells. Although the marrow microenvironment is able to support the maturation of T
cells, the presence of mature T cells strongly inhibits the
process, and the marrow must be stringently depleted of
the latter cells in order to observe the process. These results indicate that T cell production might be under complex
homeostatic controls that regulate the differentiation of T
cell precursors in the marrow and thymic pathways.
Materials and Methods
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
Antibodies.
The following monoclonal antibodies used in immunofluorescent staining were purified and conjugated to FITC as previously described (14): KT31.1 (anti-CD3); 6B2 (anti-B220, CD45R); GK1.5 (anti-CD4); 53-6.7 (anti-CD8Cell Preparation and Immunofluorescent Staining.
Single cell suspensions of spleen and bone marrow cells were obtained as previously described (22). Fresh single cell suspensions were incubated with various combinations of fluorochrome- or biotin-conjugated antibodies at saturation for 30 min on ice. In the case of biotin-conjugated reagents, counterstaining for 15 min with fluorochrome-conjugated streptavidin was performed. After staining, cells were washed and resuspended in fresh medium containing PBS without calcium or magnesium (Biowhittaker, Walkersville, MA) with 1% fetal bovine serum (FBS; Hyclone, Logan, UT) before sorting or analysis. Cultured bone marrow samples were stained as described above, except that cells were pretreated with unconjugated anti-CD16/32 mAb at saturation to block FcCell Sorting and FACS® Analysis.
All fresh and cultured marrow samples were gated by light scatter and propidium iodide uptake to exclude dead cells and erythrocytes from the analysis of fluorochrome staining or from cell sorting. Sorting of stained bone marrow cells to stringently deplete T cells was performed using a FACStar® (Becton Dickinson, Mountain View, CA). Thresholds for TCR-Bone Marrow Cell Cultures.
T cell-depleted bone marrow cells were cultured in RPMI-1640 (Biowhittaker) supplemented with 10% FBS (Hyclone), penicillin/streptomycin (Biowhittaker), glutamine (Biowhittaker), and 2-mercaptoethanol (GIBCO BRL, Bethesda, MD). 4-8 × 106 sorted cells were cultured at 37°C, for up to 48 h in a humidified incubator with 5% CO2 in a vented, upright T12.5 flask (Falcon, Franklin Lakes, NJ) at 2 × 106 cells/ml. For time course analysis, the culture was stopped at the indicated time. At the end of the culture period, cells were harvested and washed twice in staining medium before immunofluorescent staining. The nucleated cell yields of all cultures included in the Tables and Figures were at least 80%, but did not exceed the input cell number. ![]() |
Results |
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Previous studies showed that T cell-depleted
cultures of bone marrow cells from a variety of euthymic
and athymic mice generated CD4CD8
/
T cells after
a 48-h incubation in tissue culture medium, supplemented
with FBS but without growth factors (19, 23). In this
study, use of the same culture system with an appropriate source of FBS generated CD4+ and CD8+
/
T cells
without CD4
CD8
/
T cells as shown in Fig. 1. Fresh
C57BL/Ka marrow cells were stringently depleted of cells
expressing CD4, CD8, and TCR-
/
markers by immunofluorescent staining and flow cytometry. Fig. 1 A shows
the staining pattern of the marrow cells using PE-conjugated anti-CD4 and anti-CD8 monoclonal antibodies, and
FITC-conjugated anti-TCR-
/
antibodies. The box
shows the gating thresholds used for cell sorting, and reanalysis of the sorted cells is shown in B. T cell-depleted
(sorted) cells were cultured for 48 h and stained again for
CD4 and CD8 versus TCR-
/
surface markers or for
CD4 versus CD8 markers. C shows that a population of
bright CD4+ or CD8+ TCR-
/
+ T cells, accounting for
12.3% of cells (enclosed in box), appeared in the cultures
after 48 h. Less than 0.01% of cells were enclosed within
the same box in B. Analysis of CD4 versus CD8 receptors in the same cultured cells as in C showed that almost all
/
T cells were either CD4+ (8.3%, enclosed in left box) or
CD8+ (5.3%; Fig. 1 D). Less than 1% of cultured cells in C
were TCR-
/
+ and CD4
and CD8
. In control experiments, bone marrow cells were stained as in A, and cultured for 48 h without depletion of T cells by flow cytometry. The latter cells were restained after culture for CD4 and CD8 versus TCR-
/
receptors, and the staining pattern is shown in E. Fewer than 1% of bright CD4+ or
CD8+
/
T cells were generated in these cultures.
|
In similar cultures, marrow cells from BALB/c mice
were stained for CD4, CD8, and TCR-/
markers, and
T cells were depleted by flow cytometry. Fig. 1 F shows
the staining pattern of the unfractionated BALB/c marrow
cells, and G shows the reanalysis of the T cell-depleted
cells after cell sorting. Culture of the sorted cells for 48 h
generated a population of bright CD4+ or CD8+
/
+ T
cells (21.0%) enclosed in the box in Fig. 1 H. Analysis of CD4 and CD8 receptors of the cultured cells showed that
12.2% were CD4+ and 8.3% were CD8+ (Fig. 1 I). Staining of the control culture of unsorted marrow cells is
shown in J. Three different batches of fetal bovine serum from the same commercial source were tested and all supported the outgrowth of CD4+ and CD8+ T cells. However, the vigor of outgrowth varied from ~5-30% of nucleated cells harvested.
To determine the
kinetics of T cell outgrowth from marrow cultures, sorted
CD4CD8
/
marrow cells from C57BL/Ka mice
were cultured for 36, 40, 44, and 48 h, and cells were harvested and restained for CD4, CD8, and TCR-
/
markers at each time point as shown in Fig. 2. A and B show
that little or no bright CD4+ and/or CD8+
/
+ T cells
developed after 36 h of culture, and 0.1% of the cultured cells were enclosed in the box in B. Between 40 and 48 h,
the percentage of CD4+ and/or CD8+
/
+ T cells increased rapidly, and the percentages of cells enclosed in the
boxes in Fig. 2, C, D, and E were 2.5, 3.5, and 7.8%, respectively. Analysis of CD4 versus CD8 markers at the later
time points showed that at 40 and 44 h (F and G), the majority of T cells were CD4+CD8+ (enclosed in boxes).
However, at 48 h the latter cells accounted for only 0.2%
of cultured cells (panel H). CD4+ T cells were observed
also at 40 and 44 h, but a discrete population of CD8+ cells
was not observed until 48 h (panel H). Fig. 2, I, J, and K show
CD4 versus CD8 receptor expression at 40, 44, and 48 h in
a repeat experiment with a more robust outgrowth of CD4+
CD8+ T cells. The transient appearance of CD4+CD8+
T cells was observed again at 40 and 44 h (27.5 and 29.6%
of cells, respectively), and the marked loss of these cells at
48 h (0.4%) was associated with the appearance of a discrete
population of CD8+ T cells. At 40 h (Fig. 2 I), a discrete
population of CD4+CD8med cells was observed, and at 48 h
the intensity of CD8 expression on CD4+ cells was reduced
(Fig. 2 K) as reported in heterogeneous thymic stromal cell
cultures (14). The rapid development of CD4+CD8+ T
cells, which are the immediate precursors of CD4+ and
CD8+ T cells, has been described also in the thymic stromal cell cultures, but the double positive T cells persisted as
the single positive T cells were generated.
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During thymic maturation, CD4+ and CD8+ T
cells expressing V receptors, which are reactive to endogenous superantigens, undergo clonal deletion via a process of
negative selection (9, 26). The pattern of deletion of V
receptors has been characterized for many mouse strains and
is dependent on their expression of MHC gene haplotypes
(9, 27, 28). Comparison of the C57BL (H-2b) and BALB/c
(H-2d) strains have shown that the V
4, V
6, V
8.1/8.2, and V
9 receptors are not deleted in both strains, and the V
3, V
5.1, V
11, and V
12 receptors are deleted in the
BALB/c but not C57BL strain (29). V
2 receptors are deleted in some strains of BALB/c mice, but not others (30). To determine whether the CD4+ and CD8+ T cells generated in marrow cultures undergo negative selection, the
percentage of T cells expressing nondeleted and deleted V
receptors was compared in both strains after harvesting cells at 48 h (see Fig. 1). In each case, the percentage of T cells expressing each V
receptor was measured by two-color
analysis after staining for TCR-
/
or CD3 versus V
receptors with the appropriate fluorochrome-conjugated
monoclonal antibodies. Since the V
8.1/8.2 T cells represent the largest subset of nondeleted T cells in both strains,
the ratio of T cells expressing other V
receptors versus
V
8.1/8.2+ T cells was determined to normalize the variation of the levels of all T cells generated in each culture.
Table 1 compares the mean Vn:V
8 ratios of T cells in
the normal spleens of C57BL/Ka and BALB/c mice in
three to seven experiments. The mean ratios of the nondeleted V
receptors (V
2, V
4, V
6, and V
9) were similar in the two strains, and the ranges were 0.13-0.45 (C57BL/
Ka) and 0.11-0.47 (BALB/c). The mean ratios of V
receptors deleted in BALB/c mice (V
3, V
5.1, V
11, and
V
12) were uniformly reduced in BALB/c (range: 0.03-
0.08) versus C57BL/Ka (range: 0.24-0.42) spleen cells.
The ranges for the nondeleted V
receptors in marrow culture-derived T cells from the two strains were similar, and
varied from 0.10 to 0.39 in C57BL/Ka and from 0.08 to
0.38 in BALB/c. On the other hand, the mean ratios for
deleted V
receptors were uniformly lower again in BALB/c mice (range: 0.05-0.12) versus that in C57BL/Ka mice
(range: 0.19-0.39).
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During thymic maturation, CD4+ and CD8+ T cells expressing certain V receptors are positively selected by endogenous superantigens from their CD4+CD8+ T cell precursors. CD4+ T cells expressing V
17a receptors are
strongly selected in I-E+ strains of mice that express the H-2q
haplotype and are weakly selected in those that express the
H-2b haplotype (14, 31, 32). Accordingly, the mean percentages of V
17a+ T cells were compared in CD4+CD8+
T cells harvested at 40 and 44 h and in CD4+ T cells harvested at 44 and 48 h in marrow cultures from SWR (H-2q)
and C57BL/Ka (H-2b) mice in three experiments. Table 2
shows that the mean percentage of V
17a+ cells within the
gated CD4+CD8+, CD4+CD8med, or CD4+CD8
subsets
at 40, 44, and 48 h was <1% in cultures of C57BL/Ka marrow cells. In contrast, the mean percentage of V
17a+ cells
rose from 2.9% in the CD4+CD8+ subset at 40 and 44 h to
14% in the CD4+CD8med subset at 44 h and 19% in the
CD4+CD8
subset at 48 h in the SWR cultures.
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Another indication of positive selection is the transient
appearance of the CD69 T cell activation marker on
CD4+CD8med intermediary cells during maturation from
CD4+CD8+ precursors to their CD4+CD8 progeny (33).
Accordingly, the percentage of CD69+ cells was determined in the gated C57BL/Ka T cell subsets at 40, 44, and
48 h. Fig. 3 A shows that the percentage of CD69+ cells
was 30.7% in the CD4+CD8med subset (box 1) and 1.5% in
the CD4+CD8+ subset (box 2) at 40 h. At 44 h (Fig. 3 B),
the percentage of CD69+ cells increased to 51.4% in the
CD4+CD8med subset (box 3) and 2.8% in the CD4+CD8+
subset (box 4). At 48 h (Fig. 3 C), the percentage of
CD69+ cells within the CD4+CD8
subset (box 5) had
fallen to 5.2% and was 3.7% in the CD4
CD8+ subset (box
6). Stages of thymic development between the TCRlo double positive and the TCRhi single positive cells are CD69+
in mice, and are similar to the bone marrow T cell development described here (14).
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Sorted CD4CD8
/
marrow cells from C57BL/Ka mice were cultured for 48 h as
before, and the
/
+ T cells were counterstained for a variety of other markers as shown in Fig. 4. In panels A-L,
cultures were gated for
/
+ cells and one- or two-color
analysis of other markers are shown. Almost all of the
/
+
T cells expressed the other usual T cell markers CD3,
CD2, CD5, and Thy1.2 at bright staining levels (A-D; solid
lines). The intensity of staining of these markers, as well as
CD4 and CD8, was similar to that of splenic T cells as
shown in Table 3. Heterogeneous staining for both L-selectin (CD62L) and CD44 was observed in both cultured and
splenic T cells (E and F). Two-color analysis resolved several discrete populations of T cells as shown in G and H. Two subsets of CD44hi cells that stained with either bright
or intermediate intensity for CD62L are enclosed in the
box in H, and accounted for 5.8% of T cells. Less than 1%
of the normal splenic T cells were TCR-
/
+CD44hi
CD62L+, and no discrete contours for this CD44hi subset
were observed (G). The remaining CD62L+ cells showed a
staining intensity that was similar to that of normal splenic
T cells (Table 3) with a minor population of CD44int cells,
and a major population of CD44dull/
cells, reported to be
virgin T cells (34). Further experiments showed that the
CD44hi subset in the cultured marrow cells expressed
CD16+ also (I). The remaining cultured cells were CD16
.
Almost all of the CD8+
/
T cells derived from the marrow cultures expressed both the CD8
and CD8
markers,
and few if any showed the CD8
/
phenotype (J). Less
than 2% of the cultured T cells exceeded the background staining for the CD25 or NK1.1 markers (K and L) found
on activated or NK1.1+ T cells (35, 36). Markers of immature thymocytes (HSA and cKit), which are not present on
single positive CD4+ and CD8+ thymic T cells, were also
not present on the cultured marrow T cells (M and N).
The staining of conventional markers of B cells, granulocytes, macrophages, and erythroid cells (B220, GR-1,
Mac-1, and Ter119) were not above background on the
marrow-derived T cells (O-R).
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Since the
outgrowth of the bright CD4+ and CD8+ T cells from cultures shown in Fig. 1 was dependent upon depletion of
marrow T cells before culture, several experiments were
performed to determine whether the addition of T cells to
T cell-depleted cultures prevented the outgrowth of the
bright T cells. Fig. 5, A-D shows an experiment in which
C57BL/Ka marrow cells were stained for CD4, CD8, and
TCR-/
markers, and sorted into CD4
CD8
/
and
/
+ cells as shown by the gating thresholds of boxes
1 and 2 (left and right), respectively, in Fig. 5 A. Reanalysis
of the cells in box 1 are shown in B, and culture of 4 × 106
of the depleted marrow cells and restaining are shown in C. Bright CD4+ or CD8+
/
+ T cells enclosed in the box of
C accounted for 13.4% of cultured cells at 48 h. Addition
of 3 × 105 sorted TCR-
/
+ T cells from box 2 of A to 4 × 106 depleted marrow cells from box 1 of A at the initiation of the culture inhibited the outgrowth of bright CD4+
or CD8+
/
T cells after the 48-h culture, and <1% of
the cultured cells are enclosed in the box shown in D.
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To determine whether the inhibition was mediated by
CD4+ and CD8+ /
T cells and/or CD4
CD8
/
T
cells, subsequent experiments were performed in which
C57BL/Ka marrow cells were sorted using threshold gates
shown in Fig. 5 E. Box 1 (left) enclosed CD4
CD8
/
marrow cells, box 2 (upper right) enclosed CD4+ and CD8+
/
+ T cells, and box 3 (lower right) enclosed CD4
CD8
/
+ T cells. Fig. 5 F shows the reanalysis of cells from
box 1 before culture, and G shows the restaining after culture. Bright CD4+ and CD8+
/
T cells accounted for
14% of cultured cells (enclosed in the box in G). In further
experiments, 0.3 × 105 T cells from box 2 or 2 × 105 from
box 3, (Fig. 5 E) were added to 4 × 106 depleted marrow
cells in box 1, and the combined cells were cultured for 48 h.
Fig. 5, H and I, show the staining of the cultured cells with
CD4+ and CD8+ or CD4
CD8
T cells added back, respectively. The boxes in H and I enclosed <1% of bright cells.
Thus, the CD4+ and/or CD8+
/
+ T cells, as well as
CD4
CD8
/
+ T cells, from the marrow markedly inhibited the outgrowth of bright T cells from the marrow
cultures.
NK1.1+ T cells have been reported to have an unusual
cytokine secretion pattern and V and V
repertoire (20,
37, 38), and their percentage among T cells is increased in
the marrow as compared to the spleen and thymus (39).
We tested the ability of NK1.1+ and NK1.1
marrow T
cells to inhibit the outgrowth of bright CD4+ and CD8+ T
cells from the marrow cultures. Fig. 5 J shows C57BL/Ka
marrow cells stained for NK1.1 and TCR-
/
surface
markers. Box 1 (left) encloses TCR-
/
NK1.1
marrow
cells. Boxes 2 and 3 (upper and lower right) enclose the TCR-
/
+ NK1.1+ and TCR-
/
+ NK1.1
T cells, respectively. Reanalysis of the sorted cells within box 1 is
shown in Fig. 5 K. Culture of the latter cells for 48 h, and
staining for CD4, CD8, and TCR-
/
markers after culture is shown in panel L. Bright CD4+ or CD8+
/
+ T
cells (enclosed in box) represented 22.3% of the harvested cells. Addition of 0.5 × 105 of the sorted T cells from box
2 or 2.5 × 105 from box 3 from J were added to 4 × 106
cells from box 1, and the staining patterns of the cultured
cells are shown in M and N, respectively. The combined
cell cultures contained <1% of bright CD4+ and CD8+
/
+ T cells (enclosed in boxes) after 48 h.
Since CD4+ and CD8+, CD4CD8
, NK1.1+, and
NK1.1
marrow T cells strongly inhibited the outgrowth
of bright T cells, we determined whether the inhibitory
function was a unique property of marrow T cells or a
property of peripheral T cells as well. Accordingly, sorted
CD4+ and CD8+ T cells from the lymph nodes of C57BL/6
mice expressing the Ly5.2 marker were added to T cell-
depleted marrow cultures from C57BL/6 congenic mice
expressing the Ly5.1 marker. Fig. 6 A shows the staining
pattern of the marrow cells from Ly5.1 mice for CD4 and CD8 versus TCR-
/
markers, and reanalysis of sorted
CD4
CD8
/
marrow cells is shown in B. Cells (4 × 106) in Fig. 6 B were cultured for 48 h and stained for the
Ly5.2 versus CD4 and CD8 markers. Bright CD4+ and
CD8+
/
+ T cells (16.1%) were generated from these
cultures, and, as expected, these cells did not express the
Ly5.2 marker (see box in C).
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In subsequent experiments, 1.2 × 105 sorted Ly5.2+ /
+
T cells from the lymph nodes of congenic Ly5.2 mice were
added to 4 × 106 T cell-depleted marrow cells from Ly5.1
mice and cultured for 48 h. Fig. 6 D shows the staining
pattern of the lymph node cells for Ly5.1 versus Ly5.2
markers after gating on TCR-
/
+ cells. As expected,
most all of these cells expressed the Ly5.2 marker (enclosed
in box). Sorted
/
+ T cells from D were added to those
from B, and the cultured cells were stained for Ly5.2 versus
CD4 and CD8 markers as shown in E. The latter cells contained <1% of CD4+ and CD8+ cells (enclosed in left box)
derived from the Ly5.1+ marrow cells, but included a discrete population of CD4+ and CD8+ Ly5.2+ T cells
(19.2% enclosed in right box) derived from the added lymph node cells. Comparison of Fig. 6, C with E, indicated that the addition of the lymph node T cells to marrow cultures markedly inhibited the outgrowth of Ly5.2
T cells derived from the marrow.
The experimental results showed that all of the sorted
mature T cells from the marrow and lymph nodes that expressed the TCR-/
were able to inhibit the outgrowth
of bright CD4+ and CD8+ T cells from the marrow cultures. Bone marrow cells which express the Thy1.2+
TCR-
/
phenotype of the earliest T cell precursors in
the thymus (40, 41) also were tested for the ability to inhibit the marrow cultures. C57BL/Ka marrow cells were
stained for Thy1.2 versus TCR-
/
markers. Fig. 6 F
showed that bright Thy1.2+ cells that are bright
/
+ T
cells (enclosed in box 2, upper right) account for 1.3% of cells, and bright Thy1.2+ cells that are dull or negative for
the TCR-
/
marker (enclosed in box 3, lower right) account for 1.5% of cells. The Thy1.2+
/
cells were
added to C57BL/Ka marrow cells, which were stringently depleted of Thy1.1+ and TCR-
/
+ cells as shown in Fig.
6 G. The latter cells (4 × 106) were cultured for 48 h as in
previous experiments. Staining of the cultured cells from G
for CD4, CD8, and TCR-
/
receptors showed that a
population of bright CD4+ and/or CD8+
/
+ T cells
were present and accounted for 18% of cells (enclosed in
box) in H. Fig. 6 I shows that the addition of 3 × 105
Thy1.2+
/
cells (Fig. 6 F, box 3) to the cultures did
not inhibit the outgrowth of the bright CD4+ and/or
CD8+
/
+ T cells at 48 h, and 19.4% of the cultured cells
are enclosed in the box. Thus, inhibition was associated
with the expression of the TCR-
/
, but not the Thy1.2
marker.
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Discussion |
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Several lines of evidence have supported the notion that
/
T cells can develop in the absence of the thymus microenvironment, including studies of in vivo T cell development in nontransgenic and TCR-
/
transgenic athymic nude mice, and in irradiated thymectomized fetal liver,
bone marrow, or hematopoietic stem cell-reconstituted mice (15, 18, 19, 23, 24). In vitro studies of T cell development have demonstrated that single cell agar gel cultures of
marrow precursors, or dispersed cultures of hematopoietic
progenitor cells, can give rise to T cells (42, 43). Our previous reports of the generation of T cells from dispersed
bone marrow cultures showed that mature T cells must be
stringently depleted from nontransgenic or TCR-
/
transgenic marrow cells before culture to allow for in vitro T cell
maturation (19, 24). In the latter studies, almost all the T cells
generated within 48 h expressed the CD4
CD8
/
+ surface phenotype, and few if any CD4+ or CD8+ T cells were
observed (19, 24). In this study, almost all the T cells generated in the marrow cultures were CD4+ or CD8+ at 48 h,
using a different source of FBS to supplement the culture medium. The nature of the substances in the different sources of serum that dramatically alter the maturation of T cells is unknown. Candidate molecules include thymic hormones
that have been shown to facilitate T cell maturation (44).
Our experiments show that few if any /
+ T cells were
generated in the T cell-depleted marrow cultures during
the first 36 h, and a rapid maturation of CD4+ and CD8+
(single positive) T cells occurred during the ensuing 12 h.
The first expression of bright TCR expression occurred at
40 h, and was predominantly on CD4+CD8+ (double positive) T cells and on a minority of CD4+CD8med T cells.
The transition from the predominant double positive T
cells at 40 h to the almost complete predominance of single positive T cells at 48 h recapitulates the transition from
double positive to single positive T cells in the thymus (1,
2). In the heterogeneous thymic stromal cell culture system, the transition from CD4
CD8
CD3
thymus precursor cells to single positive cells also occurs in 48 h, and,
as in the bone marrow cultures, the transition from double
positive to single positive cells occurs within 12 h. In both
the thymic stromal and bone marrow cultures, the appearance of CD4+CD8med T cells preceded that of the CD8+ T
cells (14). It is likely that rearrangements of the TCR genes occurred in the marrow cultures before TCR expression at
40 h, since previous studies showed that no functional rearrangements of the TCR-
chain genes were detected in T
cell-depleted marrow cultures at time zero due to the presence of stop codons in the junctions or out of frame reading of the J
segments. However, functional rearrangements were plentiful at 48 h (19). Thus, the outgrowth of
T cells in these cultures does not represent the upregulation
of expression of previously rearranged TCR genes. A similar robust outgrowth of extrathymically derived CD4+
CD8+ T cells in the peripheral lymphoid tissues has been
observed in athymic mice reconstituted with bone marrow
obtained from mice expressing the bovine oncostatin-M
transgene under the control of the p56lck proximal promoter (45). The alternate pathway described here is a likely
source for the development of the transgenic T cells.
As in the thymic stromal cultures (14), evidence for positive selection of the CD4+CD8 T cells was obtained by
comparing their expression of V
17a to that of the
CD4+CD8+ T cells in the cultures from SWR (H-2q) and
C57BL/Ka (H-2b) strains. As expected, a marked increase
in the percentage of V
17a+ cells within the CD4+CD8
as compared to the CD4+CD8+ subset was observed in the
SWR but not in the C57BL cultures. Additional evidence
for positive selection was the transient expression of the
CD69 activation antigen on CD4+CD8med T cells at 40 and 44 h, but not on CD4+CD8
at 48 h. Comparison of
superantigen-deleted and nondeleted V
receptors on the
single positive T cells in 48-h cultures of C57BL/Ka and
BALB/c marrow cells showed that the latter cells had undergone negative selection, and the pattern of V
receptor
expression was similar to that of T cells in the spleen of
both strains.
The surface phenotype of single positive T cells generated in the marrow cultures was similar to that of mature
(HSA) single positive T cells in the thymus and peripheral
lymphoid tissues, since the typical T cell markers Thy1.2,
CD5, CD2, and CD3 were expressed in the absence of
markers of B cells, NK cells, or other hematopoietic cells.
The marrow-derived T cells contained subsets usually associated with virgin (CD44
CD62L+) and memory
(CD44+CD62L
) T cells in normal spleen (34, 46). An
unusual minor subset of CD44hiCD16+CD62L+ marrow-derived T cells was not found in the spleen, and more closely resembles the immature CD44hi subsets of T cells in
thymus (47).
The marrow-derived CD8+ T cells expressed both the
CD8 and CD8
chains, and few if any expressed only
CD8
/
homodimers. The expression of the latter homodimers has been found on extrathymically derived
CD8+TCR-
/
+ intraepithelial lymphocytes in the intestines (16, 50). Since there were no surface markers on most
marrow-derived single positive T cells that clearly distinguished them from thymus-derived single positive T cells
in the periphery, the contribution of marrow-derived T
cells to the peripheral T cell pool in vivo is unclear. The almost complete absence of single positive T cells in the
spleen and lymph nodes of nude mice in the first 3 mo of
life and the slow accumulation thereafter (15) suggest that
the marrow contribution to the peripheral pool is minimal.
However, the thymus may augment the marrow production of T cells by the secretion of humoral factors that promote T cell maturation (44). Thus, the rate of production of single positive T cells by the marrow and their contribution to the peripheral pool of euthymic mice may not be
accurately determined by studies of athymic mice.
Several subsets of mature T cells inhibited the outgrowth
of CD4+CD8+, CD4+, and CD8+ T cells from the marrow cultures. Adding back sorted subsets of marrow T cells
showed that marked inhibition was achieved with CD4+
and CD8+, CD4CD8
, NK1.1+, and NK1.1
/
+ T
cells. These
/
+ T cells prevented T cell maturation at a
ratio as low as 1:100 of sorted T cells to T cell-depleted
marrow cells. A similar low ratio of mature
/
+ T cells
obtained from the lymph nodes prevented the outgrowth of T cells from marrow cultures. Although marrow-derived
and peripheral T cells expressing the TCR-
/
were inhibitory, sorted Thy1.2+
/
marrow cells failed to inhibit T cell maturation. Bright Thy1.2+ cells that do not
express the TCR are found in the thymus also, and are able
to differentiate into the more mature Thy1.2+
/
+ thymic
T cells (49, 51, 52). The differences in the inhibitory activity of the Thy1.2+
/
and Thy1.2+
/
+ marrow cells
indicates that only mature T cells prevent marrow T cell
maturation by a feedback inhibition process. Maturation is
blocked before the double positive stage, since neither
double positive nor single positive T cells were detected at
36, 40, 44, or 48 h in the nondepleted marrow cultures
(data not shown). Feedback inhibition may contribute to
the disappearance of double positive cells at 48 h in marrow cultures, whereas these cells persist in thymic stromal
cell cultures (14). Alternatively, the in vitro lifespan of double positive cells, most of which are postselected cells and
are destined to die, may be longer in heterogeneous thymic
stromal cell cultures than those in bone marrow cultures for
reasons other than feedback inhibition, e.g., the presence of
survival factors. Bone marrow and thymic stromal cell culture mixing experiments should help resolve these alternatives.
Our results suggest that a microenvironment that supports CD4+ and CD8+ /
+ T cell maturation from precursors is not unique to the thymus, and that the marrow
microenvironment is capable of carrying out this function,
presumably by specialized stromal cells. The predominance of the thymus as the source of T cells in the lymphoid tissues may be explained better by the differences in the regulation of T cell maturation in different generative sites than
by differences in the capacities of microenvironments in
these generative sites to support maturation. In particular,
the presence of mature T cells may strongly inhibit maturation in the marrow but not in the thymus. T cell maturation in the marrow may be regulated in part by thymus-derived emigrants such that the rate of marrow T cell production and export is increased in disease states in which
mature T cells are depleted, such as acquired immunodeficiency syndrome in humans, or after myeloablation and
stem cell transplantation in hosts with poor thymic function.
Is T cell feedback inhibition of T cell development a
process limited to bone marrow cultures? The heterogeneous thymic stromal cell cultures (13, 14) were always initiated in the absence of mature T cells, and feedback inhibition was not tested. This process might be operative also
in the thymus in situ; thymic CD4CD8
CD3
cells are
located in the subcapsular cortex (1, 10, 51), whereas single
positive CD4+ and CD8+ T cells are in the juxtamedullary
cortex and the medulla. Thus, the separation of immature
and mature cells in the thymus could prevent short-range
inhibitory effects. However, in some circumstances activated T cells recirculate to the thymic cortex (53) and may participate in negative feedback. These potential feedback loops in the thymus and the bone marrow in situ have
yet to be tested.
In conclusion, the microenvironment of the bone marrow provides necessary and sufficient signals for the maturation of single positive T cells from T cell precursors in the marrow. The process is tightly regulated by the presence of mature T cells in the microenvironment, and appears to recapitulate the main elements of thymic maturation through a double positive intermediary T cell. Differences in the functional capacities of marrow-derived and thymus-derived T cells remain to be elucidated.
![]() |
Footnotes |
---|
Address correspondence to Samuel Strober, Division of Immunology and Rheumatology, Department of Medicine, Stanford University School of Medicine, 300 Pasteur Drive, S105B, Stanford, California 94305-5111. Phone: 650-723-6500; Fax: 650-725-6104; E-mail: sstrober{at}leland.stanford.edu
Received for publication 9 September 1997 and in revised form 26 March 1998.
M.E. García-Ojeda and S. Dejbakhsh-Jones contributed equally to the research described in this paper.We thank V. Braunstein for antibody preparation, A. Mukhopadhyay for technical assistance, M. Amano for FACS® assistance, T. Knack for FACS® support, and V. Cleaver for clerical assistance.
This work was supported by grants HL-57443 and HL-58250 from the National Institutes of Health and grant CA-42551 from the United States Public Health Service. M.E. García-Ojeda is supported by minority grant supplement AI-37683 from the National Institutes of Health.
1Abbreviations used in this paper FBS, fetal bovine serum; HSA, heat-stable antigen.
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