B-cell development in the thymus is limited by inhibitory signals from the thymic microenvironment

doi:10.1182/blood-2002-03-0733Prepublished online July 5, 2002;

Kenneth DorshkindYoshiko Hashimoto, Encarnacion Montecino-Rodriguez, Hyosuk Leathers, Robert P Stephan and thymic microenvironmentB cell development in the thymus is limited by inhibitory signals from the

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1

B Cell Development in the Thymus is Limited By Inhibitory Signals from the Thymic

Microenvironment

Running Head: Inhibition of Thymic B Lymphopoiesis

Scientific Section: Hematopoiesis

Yoshiko Hashimoto*, Encarnacion Montecino-Rodriguez*, Hyosuk Leathers, Robert P.

Stephan+, and Kenneth Dorshkind

*These authors contributed equally to this work

Department of Pathology and Laboratory Medicine and the Jonsson Comprehensive Cancer

Center, UCLA School of Medicine, Los Angeles, CA 90095, +Department of Developmental and

Clinical Immunology, University of Alabama, Birmingham, AL 35294

Supported by National Institutes of Health Grant HL60658

Correspondence: Kenneth Dorshkind

Department of Pathology and Laboratory Medicine and the Jonsson Comprehensive Cancer

Center 173216

10833 Le Conte Avenue

Los Angeles, CA 90095

310 206-9535/206-9391 FAX

[email protected]

Total Text: 4757 words/Abstract: 151 words

Copyright 2002 American Society of Hematology

Blood First Edition Paper, prepublished online July 5, 2002; DOI 10.1182/blood-2002-03-0733 For personal use only. by guest on June 7, 2013. bloodjournal.hematologylibrary.orgFrom



2

SUMMARY

B cell precursors are present in the thymus, and the thymic microenvironment is the

source of lymphopoietic factors that include IL-7. Despite the fact that they are bone marrow

derived cells, the data in this report demonstrate that intrathymic B cell progenitors accumulate

at an early pro-B cell stage of development, cycle less than their bone marrow counterparts, and

fail to differentiate efficiently. Additional studies presented herein indicate that these effects are

mediated, at least in part, by soluble factors produced by the thymic microenvironment and

suggest that they affect the ability of pro-B cells to respond optimally to interleukin 7. Taken

together, these observations demonstrate a specific inhibition of intrathymic B lymphopoiesis,

which in turn may explain why lymphoid cell production in the thymus is largely restricted to

production of T lineage cells despite the fact that B cell precursors and B lymphopoietic stimuli

are present in that organ.

Corresponding Author e-mail: [email protected]




3

INTRODUCTION

Sustained T cell development in the thymus is thought to be dependent on continuous

migration of bone marrow derived precursors to that organ (1-4). While T lineage committed

progenitors could be included among these cells (5), experimental evidence suggests that at least

some intrathymic lymphoid precursors are multi-potent and retain the capacity to generate B

cells (6,7). The recently characterized bone marrow common lymphoid precursor (CLP; 8),

which has B and T cell developmental potential, could be the bone marrow population that

migrates to the thymus and sustains thymopoiesis.

Despite the fact that B cell precursors are present in the thymus, B lymphopoiesis in that

organ is minimal. One reason for this is that entry of lymphoid precursors into the B lineage may

be limited. In this regard, it has been proposed that binding of the Notch1 receptor on

multipotential lymphoid precursors, such as the CLP, to ligands expressed by thymic stromal

cells (9) results in commitment to the T lineage and a block in B cell development (10-12).

Activation of Notch1 signaling pathways may be a critical means by which entry of

multipotential precursors into the B cell lineage is blocked within the thymus. However, some

thymic lymphoid precursors may fail to do so, thereby allowing pro-B cells to develop. Indeed, it

has been reported that 10-13% of cells within the CD3-CD4-CD8- triple negative (TN) pool of

intrathymic progenitors express the CD45R (B220) B lineage associated cell surface determinant

(13).

Nevertheless, B lineage cells in the thymus account for less than 1% of total lymphocytes

(14-20). Such a low frequency is surprising, because B cell precursors present among TN

thymocytes are exposed to many of the same microenvironmental stimuli as their counterparts in

the bone marrow. For example, the interaction between lymphoid cells and stromal cells in both

organs is mediated through similar ligand-receptor complexes, such as VLA-4/VCAM-1 and




4

stromal cell derived cytokines that include IL-7 (21). IL-7 stimulates pro-B cell proliferation and

potentiates immunoglobulin heavy chain gene rearrangements in those precursors (22-24).

Because it has been established that they are bone marrow derived cells (25), intrathymic B cell

progenitors would be expected to respond to IL-7 and other lymphopoietic factors.

These observations suggest that mechanism(s) which limit the maturation of intrathymic

B cell progenitors exist. Such regulatory pathway(s) would ensure that expansion of B lineage

cells able to compete for microenvironmental niches required for T lymphopoiesis does not

occur. There may be additional reasons for limiting intrathymic B cell production. A case has

been made that thymic B cells are involved in selection of the T cell repertoire (26). Thus, an

inordinate increase in their numbers might be detrimental to the thymic education process.

Therefore, it is likely that signals from the thymic microenvironment minimize the development

of B lineage cells that develop in that organ.

The data in this report support this hypothesis and demonstrate that B cell progenitors in

the thymus accumulate at an early pro-B cell stage of development and fail to differentiate

efficiently. These effects are consistent with findings demonstrating that intrathymic pro-B cells

respond inefficiently to IL-7. Taken together, these results document an unappreciated inhibitory

effect on B lymphopoiesis by the thymic microenvironment that helps to explain why lymphoid

cell production in the thymus is largely restricted to production of T lineage cells.

MATERIALS AND METHODS

Mice. BALB/cJ mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and

housed in the vivarium of the UCLA Division of Laboratory Animal Medicine. Timed pregnant

Swiss/Webster mice were purchased from Taconic Farms (Germantown, NY). Ifnar1-/- mice

were bred and maintained in the vivarium of the University of Alabama, Birmingham, AL.




5

Preparation of cell suspensions. Single cell suspensions of thymus were prepared by gently

pushing tissues through a fine mesh screen into α-Minimal Essential Medium (MEM; GIBCO,

Grand Island, NY) supplemented with 5% fetal calf serum (Atlanta Biologicals, Atlanta, GA).

Bone marrow cells were flushed from femurs using a syringe fitted with a 23-gauge needle. Cells

were counted with a hemacytometer and viability was determined by eosin dye exclusion.

Fetal Thymic Organ Cultures. Fetal thymic organ cultures (FTOC) were established according

to the protocol described by Jenkinson et al. (27). Briefly, fetal thymic lobes from 15 day old

Thy-1.1 Swiss/Webster embryos were harvested aseptically, dissected free from extraneous

tissue, and cultured for 5 days in the presence of 1.35mM deoxyguanosine to deplete endogenous

thymocytes. The lobes were then rinsed and incubated with donor cells in hanging drop cultures

in Terasaki plates for 48 hr. Subsequently, lobes were transferred to FTOC on filter/gelfoam

rafts in RPMI-1640 supplemented with 10% FCS, 5 x 10-5M 2-β mercaptoethanol, 100 U/ml

streptomycin, and 100 U/ml penicillin and placed in a humidifed, 5%CO2/air incubator at 37oC.

Cell proliferation and differentiation in FTOC was assessed at regular intervals following culture

initiation as described below.

Long-term bone marrow cultures. Long-term myeloid bone marrow cultures (LTBMC) were

initiated as described by Dexter et al. (28). Briefly, the contents of a femur were flushed into a 25

cm2 tissue culture flask in α-MEM supplemented with 20% horse serum and 10-6 M sodium

hydrocortisone succinate. Cultures were incubated at 33oC in a 5% CO2/air incubator. After two

weeks, cultures were recharged with 106 bone marrow cells per flask. B cell development was

induced by switching cultures to the B lymphoid permissive conditions (RPMI-1460




6

supplemented with 5% FCS and 5x10-5 M β-mercaptoethanol) described by Whitlock and Witte

(29,30).

Preparation of bone marrow and thymus stromal cells. Confluent, heterogeneous bone

marrow stromal cell cultures were established by treating LTBMC with 5µg/ml mycophenolic

acid for 2 to 4 weeks to eliminate hematopoietic cells as described (31). The adherent stromal

cells were then maintained in α-MEM supplemented with 10% FCS in a 37oC, 5% CO2 /air

incubator and were passaged minimally to retain the characteristics of primary stroma.

Hetergenous thymic stromal cell cultures were established as described (32). Thymuses were

digested with trypsin/EDTA and collagenase dispase in Ca++, Mg++ free medium for 35 min at

37oC. The confluent thymic stromal cells were the fed weekly with MEM D-valine supplemented

with 10% FCS and maintained in a 37oC, 5% CO2 /air incubator. These cells were passaged

minimally to retain the characteristics of primary stroma. The generation of the S17 stromal cell

line has been described (33). The S17 bone marrow stromal cell line was fed weekly with α-

MEM supplemented with 10% FCS.

Diffusion chamber cultures. In some experiments, heterogeneous bone marrow or thymic

stromal cells were grown to confluency in transwells fitted with membranes containing 0.4 µm

pores (Becton-Dickinson). Inserts were inserted into wells of six-well plates in which myeloid

LTBMC had been established. Cultures were then switched to B lymphoid permissive

conditions, fed twice weekly, and cells assayed for B cell production by immunophenotyping.

Immunofluorescence and cell sorting. Bone marrow cell suspensions were treated with Tris-

ammonium chloride (pH 7.2) to lyse red blood cells. Cells were then incubated with an anti-




7

CD16/CD32 antibody (FcγRII/III, clone 2.4G2, Pharmingen, San Diego, CA) to reduce non-

specific labeling prior to staining. The following monoclonal anti-mouse antibodies used for

staining were all obtained from Pharmingen: CD45R (B220, clone RA3-6B2), CD127 (IL-7Rα,

clone SB/14), CD43 (clone S7), CD24 (HSA, clone 30-F1), and Ly-51 (BP-1, clone 6C3). These

antibodies were conjugated to FITC, PE, TriColor (TC), or biotin. Biotinylated antibodies were

revealed with TC-conjugated streptavidin (Southern Biotechnology). Optimal working dilutions

were determined for each antibody prior to use. All incubations were performed in Ca++, Mg++

free PBS at 4oC for 30 min. Following the last wash, 104 - 105 live cells per sample were

analyzed by flow cytometry on a FACScan (Becton Dickinson, San Jose, CA) with CELLquest

software (Becton Dickinson).

BM cells were incubated with FITC conjugated anti-CD45R, PE conjugated anti-CD43 or

PE conjugated anti-IgM as described above prior to sorting on a FACStarplus flow cytometer

(Becton Dickinson) located in the Jonsson Comprehensive Cancer Center Flow Cytometry Core

at UCLA. Reanalysis showed that the purity of the sorted CD45R+ IgM- population was

routinely above 99%, while we routinely achieved 95% purity for CD45R+CD43+ cells. In the

experiments described herein, 5 x 104 CD45R+sIgM- or 2.5 x 104 CD45R+CD43+ cells were

seeded into fetal thymic lobes or onto S17 bone marrow stroma.

Cell Cycle Analysis. Cells were fixed in 0.5% paraformaldehyde for 15 min at room

temperature and then permeabilized with 70% ethanol and resuspended in Ca++, Mg++, free PBS

supplemented with 1 µg/ml 7-amino actinomycin D (7-AAD; Calbiochem, San Diego, CA). At

least 105 cells were acquired with a FACScan and their cycling status was estimated using

Modfit LT software.




8

RESULTS

Thymic B Cell Differentiation is Blocked at an Early Stage In Vivo

Multiple stages of B cell differentiation in the bone marrow can be defined based on the

differential expression of various cell surface determinants. Since thymic B lineage cells are

bone marrow derived (25), their expression of the CD45R, CD43, Ly51(BP-1), and CD24 cell

surface determinants was assessed by flow cytometry. This combination of reagents allows the

pro-B cell pool to be subdivided into fractions A, B, C, and C’ as defined by Hardy et al. (34).

Fraction A includes the most immature pro-B cells while those in fraction C’ are completing

immunoglobulin (Ig) heavy chain gene rearrangements before their transition into pre-B cells.

As expected, cells in the bone marrow pro-B cell compartment were present at the

frequency described previously (35) and included populations that had matured to the C’ stage of

development (Figure 1A). In contrast, very few thymic pro-B cells had matured to the fraction C’

stage (Figure 1A). Because the most actively proliferating B lineage cells are those in the pro-B

cell compartment, the cell cycle status of intrathymic CD45R+CD43+ cells was also determined.

As shown in Figure 1B, the frequency of cycling thymic pro-B cells was approximately three-

fold lower than observed with comparable populations from the bone marrow.




9

Figure 1. B cell development in the

thymus is blocked at the pro-B cell stage.

(A) Phenotype of CD45R+CD43+ pro-B

cells harvested directly from the bone

marrow (BM) or thymus (THY) of BALB/c

mice. The frequency of cells in Fractions A,

B, C and C’ stages of development was

assessed by analysis of Ly51 (BP-1) and

CD24 expression on the gated

CD45R+CD43+ cells. (B) Cell cycle status

of pro-B cells in the bone marrow and

thymus. Data in the figures are

representative of three experiments.

B Cell Development is Blocked in Fetal

Thymic Organ Culture

The above results indicate that B cell production in the thymus is blocked at a relatively

early stage of development. In order to assess the fate of isolated B cell progenitors in the thymic

environment in more detail, a modification of the fetal thymic organ culture (FTOC) system was

used. Fetal thymic lobes from day 15 embryos were treated with deoxyguanosine to remove

endogenous thymocytes prior to seeding with FACS purified CD45R+ sIgM- bone marrow cells.

Thymic lobes prepared in this manner are fully functional and support T cell development (36).

Other aliquots of CD45R+sIgM- cells were seeded on the bone marrow stromal cell line S17

which has been shown to support B cell differentiation in vitro (33). Seven days following

initiation of these cultures, cells were recovered and analyzed phenotypically as described above.

B. A-C THY

5.1

CD

45R

A-C25.1 6.4

BM THY0.4

CD43

A B C C’15.5 50 .0 14.3 12.8

Ly-

51 /

BP

-1

A B

C C’

CD24

A B C C’39.6 21.2 27.0 1.7




10

Figure 2 demonstrates that while B lineage cells were recovered from thymic lobes, they

did not efficiently mature past the Fraction C stage. These data are consistent with observations

made on freshly harvested thymic pro-B cells (Figure 1A) and indicate the validity of the FTOC

system for analyzing thymic B lymphopoiesis. The fate of CD45R+sIgM- cells seeded in FTOC

contrasts with that of other aliquots of CD45R+sIgM- cells seeded onto the S17 stromal cells. In

this case, cells that were recovered seven days later had efficiently matured to the C’ stage of

development (Figure 2A). Similar results were obtained when fetal thymic lobes were seeded

with FACS purified CD45R+CD43+ (fractions A-C) pro-B cells. Again, when lobes were

processed seven days after seeding, few pro-B cells had matured past the fraction C stage of

development (Figure 2B).

Figure 2. Phenotype

of CD45R+CD43+

pro-B cells following

culture on S17

stromal cells or in

FTOC. (A) 5 x 104

CD45R+sIgM- cells

were seeded into

FTOC or onto S17

stroma. Cells were

phenotyped as

described in Figure 1

seven days later. (B) 2.5 x 104 FACS purified CD45R+CD43+ pro-B cells were seeded into

FTOC. Phenotypic analysis was performed on cells harvested from lobes seven days after

initiation of cultures. Data in the figures are representative of three experiments.

84.19.2L

y-51

(BP

-1)

CD

45R

A. CD45R+ IgM- B. CD45R+ CD43+

77.513.5

d7 FTOC

A B C C’ 11.9 5.5 67.2 8.2

CD43

CD24

CD

45R

Ly-

51(B

P-1

)

47.5 24.9

S17

A B C C’ 1.4 53.8 8.3 38.3

CD

45R

CD43

Ly-

51(B

P-1

)

CD24

A B

C C’




11

Exposure to the Thymic Environment Renders B Cell Progenitors Unresponsive to

Lymphopoietic Signals

Since cytokines such as IL-7 are produced in the thymus (37,38), it seemed unlikely that

failure of pro-B cells to mature was due to the absence of B lymphopoietic factors. Instead, the

possibility existed that the thymic microenvironment rendered pro-B cells unresponsive to B

lymphopoietic signals. In order to test this hypothesis, cells harvested from FTOC at various

times after seeding lobes with bone marrow derived CD45R+sIgM- cells were re-seeded onto the

S17 bone marrow stromal cell line under B cell permissive conditions.

As shown in Table 1 and Figure 3, cells harvested two days after culture in FTOC

established vigorous long-term B cell cultures when re-seeded on S17 stroma. However by 7

days of culture in FTOC, they were no longer able to do so. These results are consistent with data

from two experiments demonstrating that CD45R+sIgM- cells directly isolated from the adult

thymus did not establish long-term B cell cultures following seeding on S17 stromal cells (data

not shown).

Table I. B lineage cells harvested from FTOC do not respond to bone marrow stromal cell signals.a

Week after seeding frequency of CD45R+ cells on S17 stroma S17 stroma FTOC� S17 stroma

(x 104) (x 104)d2 d7 d11 d16

1 11.8 3.9 1.9 0.3 ND

2 6.6 4.7 <0.2 5.7 ND

3 11.1 28.1 <0.1 0.6 <0.1

a CD45R+ sIgM- cells were isolated from the bone marrow. Aliquots of 5 x 104 cells from this pool were used to establish long-term cultures on S17 stroma (S17 stroma) or to seed FTOC. Following 2, 7, 11 or 16 days of incubation in these FTOC, cells were harvested and reseeded on S17 stroma (FTOC� S17 stroma). At weekly intervals for 3 weeks thereafter,cells were harvested from the cultures and examined for CD45R+ expression by flow cytometry. Data representative of 5 independent experiments. ND: no cells were not harvested as too few cells were present in the cultures.




12

Figure 3. Exposure to the thymic microenvironment renders pro-B cells unresponsive to B

lymphopoietic stimuli. (A) Cells harvested from FTOC two days after seeding lobes with 5 x

104 CD45R+sIgM- bone marrow cells can still establish long-term cultures on S17 stroma. (B)

CD45R+sIgM- cells can no longer establish long-term bone marrow cultures following 7 days of

culture in FTOC.

Pro-B Cells in the Thymus Are Hyporesponsive to IL-7

IL-7 is required for murine pro-B cells to proliferate and complete Ig heavy chain gene

rearrangements and mature into pre-B cells (39). Because thymic pro-B cells accumulate in

Fraction C and cycle at a lower rate than their bone marrow counterparts, their capacity to

respond to IL-7 was examined. Following seven days of culture in FTOC, cells were harvested

from the lobes and their proliferative response to IL-7 was compared to that of CD45R+sIgM-

cells from fresh bone marrow. The frequency of CD45R+CD43+ cells, which includes the most

IL-7 responsive cells, in each population was determined by FACS. Based on this information,

cultures were initiated using the same total number of CD45R+CD43+ cells from each source.

As shown in Figure 4A, cells harvested from thymic lobes responded to IL-7, but the magnitude

of their proliferative response was lower than that of the freshly harvested bone marrow

population.

This observation prompted an analysis of IL-7 receptor α chain (IL-7Rα (CD127))

expression on thymic B lineage cells. Cells were harvested from thymic lobes seven days after

A. 2 days in FTOC 7 days in FTOC




13

seeding with CD45R+sIgM- cells, and the level of CD127 expression on CD45R+CD43+ pro-B

cells was compared to that on freshly isolated CD45R+CD43+ bone marrow cells. The results

indicated that both the frequency of pro-B cells that express the IL-7Rα chain and those which

express it at relatively high levels are reduced following 7 days of culture in FTOC (Figure 4B,

top).

Figure 4. IL-7 responsiveness and IL-7R�receptor levels are decreased on B lineage cells

exposed to the thymic microenvironment.. (A)

CD45R+sIgM- cells were cultured in FTOC and

seven days later their proliferative response to

increasing concentrations of IL-7 was compared

to that of CD45R+sIgM- cells harvested from the

bone marrow. The frequency of CD45R+CD43+

cells in each population was determined by FACS

and based on this information, cultures were

initiated using the same total number of

CD45R+CD43+ cells from each source. (B)

Seven days after seeding thymic lobes with 5 x

104 CD45R+sIgM- bone marrow cells, CD127

expression on CD45R+CD43+ pro-B cells was

examined by FACS. Freshly isolated

CD45R+CD43+ pro-B cells from the bone

marrow were examined in parallel. Studies were

performed using cells from BALB/c (top), Ifnar1-

/- (middle), and 129 strain mice. The frequency of

high and low CD127 expressing cells is indicated

in each plot. (C) Frequency of B lineage cells in

the thymus of BALB/c and Ifnar1-/- mice.

C. Ifnar1-/-

CD

45R

0.43 0.370.02

CD

45R

CD43

A.

0

500

1000

1500

2000

2500

CD45R+IgM- BMCD45R+IgM- BM FTOC

B.30.6 6.1

CD127

BALB/c

Ifnar1-/-

BM17.3 0.4

CD127

24.3 1.1

d7 FTOC

35.4 11.5

129

33.0 4.0 12.8 1.0




14

CD

45R

+ ce

ll nu

mbe

r x

104

0

50

100

150

0 1 2 3

week of Culture

B . CD45R+ cells no transwellheterogenousbone marrow stroma

heterogenousthymic stroma

Dexter cultures

Transwell (with stromal cells)

- harvest cells - FACS analysis

1-3wks

A.

B cell culture conditions

Thymic Stromal Cell Derived Factors Inhibit B Lymphopoiesis

The above observations indicate that the thymic microenvironment is a source of

mediators that render pro-B cells hypo-responsive to IL-7. To determine whether such inhibitors

were soluble molecules, initial experiments used a modification of the myeloid to lymphoid

long-term bone marrow switch culture

system (29) to test whether thymic

stomal cells could inhibit B cell

development in the absence of direct

cell contact.

Figure 5. Inhibitory signals from

thymic stromal cells can counteract

B lymphopoietic stimuli. (A) shows

the experimental protocol used to

introduce transwells containing

heterogeneous bone marrow or thymic

stromal cells into myeloid (Dexter)

long-term bone marrow cultures at the

time of their transfer to B lymphoid

permissive conditions. (B) Non-

adherent cells were harvested weekly

from the cultures and analyzed for

expression of CD45R. Data are

representative of three experiments.




15

Within a week following transfer of long-term bone marrow cultures established under

the myeloid conditions described by Dexter and colleagues (28) to B lymphoid permissive

conditions (30), pro-B cells emerge, and by three weeks B lineage cells predominate in the

cultures. As shown in Figure 5A, heterogeneous populations of bone marrow or thymic stroma

growing in transwells were introduced into these cultures. By three weeks following transfer of

long-term myeloid bone marrow cultures to B lymphoid permissive conditions, vigorous cultures

containing CD45R+ cells established (Figure 5B). This same pattern of growth occurred when

empty transwells (data not shown) or transwells containing heterogeneous bone marrow stroma

(Figure 5B) were placed in the cultures at the time of their transfer to the lymphoid conditions.

However, when transwells containing heterogeneous preparations of thymic stromal cells were

introduced into the cultures, CD45R+ cell production was dramatically reduced (Figure 5B),

indicating that soluble mediators produced by the thymic microenvironment can inhibit

intrathymic B lymphopoiesis.

Downregulation of IL-7Rαααα Occurs Through a Type 1 IFN Receptor Independent

Mechanism

Type 1 interferons (IFN) have been shown to inhibit the response of B lineage cells to IL-

7 (40-43). In view of the above data indicating the involvement of soluble factors in limiting

intrathymic B lymphopoiesis, it was of interest to assess their potential involvement. In order to

do so, CD45R+sIgM- cells from Type 1 IFN receptor deficient (Ifnar1-/-) mice were used to seed

fetal thymic lobes. CD45R+sIgM- cells from 129 mice, the background strain of the Ifnar1-/-

mice, were assayed in parallel. Seven days later CD127 expression on CD45R+CD43+ cells

harvested from these FTOCs was compared to that on bone marrow pro-B cells. As shown in

figure 4B, exposure to the thymic microenvironment resulted in a greater than 90% reduction of




16

CD127 high expressing cells in Ifnar1-/- mice. This level of inhibition was comparable to that in

BALB/c and greater than that in 129 strain mice. A prediction based on this observation is that

the frequency of B lineage cells in the thymus of Ifnar1-/- mice should not be elevated. This is in

fact the case, as the frequency of B lineage cells in the thymus of Ifnar1-/- mice was comparable

to that in control animals (Figure 4C).

Resumption of B Cell Maturation in the Presence of Exogenous IL-7

Taken together, the above data suggest that the growth and development of pro-B cells in

the thymus is limited through IL-7Rα downregulation. This event may in turn limit their capacity

to compete with T cell progenitors for the IL-7 produced by the thymic microenvironment. A

prediction based on this hypothesis is that increasing intrathymic IL-7 levels may in turn allow

their growth. In order to test this premise, FTOC were initiated with CD45R+sIgM- bone

marrow cells in the presence of 50 U/ml of exogenous IL-7. As shown in Figure 6A,

approximately half of the CD45R+ cells no longer expressed CD43 after seven days of culture,

suggesting that they had matured to the pre-B cell stage. This result contrasts with the finding

that the majority of B lineage cells in non-IL-7 supplemented FTOC were CD45R+CD43+. In

addition, although B lineage cells harvested from day 7 FTOC were not able to establish long-

term bone marrow cultures on S17 bone marrow stromal cells (Figure 3), they were able to do so

if the medium was supplemented with IL-7 (Figure 6B). These cultures could be maintained for

at least three weeks and phenotypic analysis confirmed that the cultures contained B lineage cells

(data not shown).

Since IL-7 is required for T cell development (44), it was important to determine how levels

of IL7Rα expression on thymic B and T cell progenitors compared. As shown in Figure 6C, the

CD3-CD4-CD8- triple negative (TN) fraction of cells was isolated from the fresh thymus and




17

analyzed for expression of CD127 and CD45R. This allowed levels of CD127 on CD45R+ B

lineage cells to be compared to that on CD45R- cells, which primarily include T cell progenitors.

The data demonstrate that the frequency of cells that express CD127 is higher in the CD45R- TN

thymocyte population and that they express it

at higher levels than is found among CD45R+

cells.

Figure 6. Responsiveness of thymic B cells

to exogenous IL-7. (A) Phenotype of cells

harvested seven days after seeding FTOC

with 5 x 104 CD45R+sIgM- bone marrow

cells in the presence or absence of 50 U/ml of

IL-7. (B) Cells harvested 7 days after seeding

FTOC with CD45R+sIgM- bone marrow cells

could establish long-term cultures when

seeded on S17 bone marrow stromal cells in

the presence of IL-7. (C) Expression of

CD127 and CD45R on CD3-CD4-CD8- triple

negative (TN) thymocytes harvested from the

fresh thymus.

DISCUSSION

Both bone marrow and thymic stromal cells express ligands required for direct

interactions with developing B lineage cells and secrete cytokines required for B cell

development. Despite these similarities, B cell development in the thymus is limited. Studies

aimed at investigating this phenomenon revealed that B cell development is blocked at a

C.

30.7

6.0

0.8

0.1

CD

127

CD45R

TN thymocytes

B. no IL-7 + IL-7

A.

0

20

40

60

80

% c

ells

CD45R+ CD43+ CD45R+ CD43-

50U/ml IL-7

no IL-7




18

relatively early stage of development in the thymus because signals produced the thymic

microenvironment inhibit that process.

Initial analyses focused on characterizing B lineage cells harvested from the thymus.

These studies demonstrated that the frequency of pro-B cells in cycle was lower in the thymus

than in the bone marrow and that B lineage cells did not efficiently mature past the Fraction C

stage of development. When similar analyses were performed on bone marrow B cell progenitors

seven days after being seeded in fetal thymic lobes in vitro, the same results were obtained. In

addition to corroborating the results obtained with primary cells, this result established the fetal

thymic organ culture system as a model for analyzing thymic B cell production.

It is paradoxical that culture in the thymic microenvironment in the FTOC system

inhibited B cell development while other studies have reported the isolation of thymic stromal

cell lines that can support B cell differentiation. Indeed, our own laboratory characterized a

thymic stromal cell line that efficiently supported the pre-B to B cell transition (45). However, it

is important to emphasize that the thymic stromal cell line in question only potentiated this latter

phase of development but not the short or long-term growth of pro-B cells. In fact, our

characterization of numerous thymic stromal cell lines has so far failed to identify any capable of

supporting long-term B cell development. Furthermore, even if thymic stromal cells with such

potential were isolated, they do not represent the thymic microenvironment as a whole. That is

why, in order to assess the effects of the thymic microenvironment on B cell development,

primary cultures of heterogeneous thymic stromal cells rather than thymic stromal cell lines were

used in the diffusion chamber studies.

That a mechanism to inhibit intrathymic B cell development exists at all might seem

puzzling, since the overwhelming majority of lymphoid cells in the thymus are T lineage cells.

Instead, the inefficient expansion and maturation of thymic pro-B cells could result from their




19

failure to compete effectively for microenvironmental niches. However, it is important to focus

discussion on the most immature thymocytes contained within the TN compartment. While the

majority of cells within this population are likely committed to the T lineage, it has been reported

that 10-13% are CD45R+ (13). If even a fraction of these cells did come into contact with thymic

stromal cells that supported their growth and development, this in turn could compromise overall

levels of T cell production, particularly if enough mature B cells that affected the process of

thymic education subsequently developed. Thus, it is logical to propose the existence of a

mechanism(s) to specifically inhibit(s) thymic pro-B cell development.

That such signals exist is supported by recent studies in which selective inactivation of

Notch1 by gene targeting was performed. Radtke et al. (11) and Wilson et al. (12) demonstrated

that T cell development was effectively blocked in mice in which Notch1 was conditionally

inactivated and that there was an expansion in the number of B lineage cells in the thymus.

However, while their incidence was higher than in control mice, a critical point is that the total

number of B lineage cells in the thymus of these mice increased to only about four million cells.

This number is considerably lower than the 100 million thymocytes that are present in the

thymus of young animals. More recently, Han et al. (46) demonstrated that mice in which the

RBP-J transcription factor, which associates with the intracellular domain of Notch to allow

DNA binding, has been inactivated, also exhibit impaired T cell development and increased

intrathymic B lymphopoiesis. However, even though Notch signaling in these mice was

effectively blocked, the total number of thymic B lineage cells was again only about 4 x 106

cells. Taken together, these results indicate that even when not competing for environmental

niches with developing T cells, B cell precursors do not undergo extensive expansion in the

thymic microenvironment. Importantly, no differences between bone marrow B lineage cells in




20

mice in which Notch1 expression was conditionally inactivated and their control littermates were

reported.

A recent study of mice that expressed a lunatic fringe transgene, which results in the

inhibition of Notch1 activation, showed that their thymus contained up to 50 million B lineage

cells (13). That report would also seem to contradict the conclusion that the thymus actively

inhibits B lymphopoiesis. However, while the results of that study could be interpreted to infer

that inhibitors of thymic B lymphopoiesis do not exist, that conclusion may be too simplistic in

view of the minimal level of B lymphopoiesis in the Notch1-/- and RBP-J-/- mice. Instead, it is

important to consider that lunatic fringe normally functions as an intracellular mediator, but its

transgenic expression resulted in secretion of the protein. This fact, combined with findings that

the lunatic fringe protein can have effects beyond the inhibition of Notch1 activity (47), raises

the distinct possibility that normal thymus physiology was altered in lunatic fringe transgenic

mice. That this in turn impacted upon one or more regulatory pathways, such as those which

inhibit B cell development, must be considered.

In any case, the studies presented herein clearly demonstrate that exposure to the thymic

microenvironment renders B cell precursors unresponsive to B lymphopoietic stimuli. After

seven days in thymic lobes, B lineage cells could no longer proliferate and differentiate on bone

marrow stromal cells. Further analysis showed that this occurs because pro-B cells that have

been exposed to the thymic microenvironment are hyporesponsive to IL-7. This seems to occur

through IL-7Rα chain down-regulation. Proposing that interference with IL-7 signaling

pathways is ultimately responsible for the inhibition of intrathymic B cell development is

entirely consistent with the observation that thymic pro-B cells or pro-B cells exposed to the

thymic environment do not efficiently mature from the fraction C to the C’ stage of development.

As described by Hardy et al. (34), Fraction C cells have undergone Ig heavy chain gene




21

rearrangements at the D-J loci but V-DJ rearrangements have not yet occurred. IL-7 is required

for cells to complete the latter stage of Ig gene rearrangement and to transition from fraction C to

the pre-B (fraction D) stage of development.

The long-term bone marrow culture experiments described in this study suggest that

soluble factors produced by thymic stromal cell(s) are at least partially responsible for the

inhibition of thymic B cell production. In fact, thymic stromal cells present in diffusion chambers

were able to inhibit the emergence of B lineage cells, even when precursors were in contact with

a supporting layer of bone marrow stromal cells. While aspecific effects of thymic stromal cells

on B lymphopoiesis in this system through excessive consumption of nutrients can not be

excluded, it does not seem likely. First, there is no a priori reason to assume that confluent

thymic and bone marrow stroma differ significantly in the nutrients they consume. Second,

cultures were fed twice weekly and neither pH fluctuations nor excessive cell death were

observed in cultures in which thymic stroma was present. Therefore, it is logical to propose that

soluble factors from the thymic stroma are able to inhibit B cell development and that their

effects are potent enough to counteract positively acting B lymphopoietic signals.

It has been reported that type 1 interferons (IFN) can inhibit the response of B cell

progenitors to IL-7 (40-43). Since IFNs are produced in the thymus, it was of interest to

determine if they were responsible for the observed effects by seeding pro-B cells from type 1

IFN receptor deficient mice into fetal thymic lobes. This analysis revealed that the frequency of

CD45R+CD43+ cells that expressed CD127 at high levels had decreased by over 90% after

seven days in FTOC. This finding, combined with the fact that the frequency of B lineage cells

in the thymus of these knockout mice was not elevated, strongly suggests that signaling through

the type 1 IFN receptor is not responsible for the inhibition of intrathymic B lymphopoiesis.




22

These findings would also seem to exclude the involvement of a new type 1 IFN family member,

limitin, which has been proposed as a selective inhibitor of B cell production (48).

Taken together, the data in this and other studies make it possible to formulate a model in

which checkpoints operative at multiple levels act in concert to limit B cell development in the

thymus. Initially, intrathymic lymphoid precursors, such as the CLP, might receive signals that

preferentially potentiate their development along the T rather than the B cell lineage, and Notch1

activated signaling pathways may be critical at this juncture (10,11,49). Nevertheless, some pro-

B cells develop and their growth and differentiation is severely limited by thymic

microenvironmental signals that render them hyporesponsive to IL-7. While IL-7 is not critical

during human B lymphopoiesis (50), similar or alternative thymic stromal cell factors can be

postulated to limit pro-B cell expansion in the human thymus. The inhibition of intrathymic B

lymphopoiesis may not be absolute, however, and some pro-B cells may mature into pre-B cells

from which B lymphocytes may be generated. A few of these may be retained in the thymus as

antigen presenting cells (26) while others are exported to the periphery (19).

It is becoming increasingly appreciated that negative regulators play an important role in

the regulation of hematopoiesis. In this regard, numerous mediators have been described which

specifically inhibit B cell production (51). However, these factors have generally been

considered in terms of their effects on bone marrow B lymphopoiesis. The findings herein

describe an unexpected role of negative regulatory factors in the homeostasis of thymic

lymphocyte production. These data contribute to the understanding of how an organ which has

the potential to support B cell production limits that process and suggest that further comparisons

of bone marrow and thymic lymphopoiesis will provide additional insights into the regulation of

primary lymphocyte production.




23

ACKNOWLEDGEMENTS

The authors appreciate the helpful discussions with Drs. Ellen Rothenberg, Max Cooper, David

Rawlings, and Andrew Farr. This work was supported by grant HL60658 from the National

Institutes of Health.

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B-cell development in the thymus is limited by inhibitory signals from the thymic microenvironment

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