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Department of Anatomy, University of Birmingham Medical School, Edgbaston, Birmingham, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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ßTCR
ligation by peptide/MHC complexes expressed on thymic stromal cells, it
is clear that positive selection is a multistage process involving
transition through an intermediate
CD4+8+69+ phase as well as
subsequent postselection phases. By analyzing the development of
preselection CD4+8+69- and
intermediate CD4+8+69+ thymocytes
in the presence of MHC class I-deficient, MHC class II-deficient, and
MHC double-deficient thymic stromal cells, we investigated the role of
MHC molecules at three distinct points during positive selection.
Although the initiation of positive selection is critically dependent
upon MHC interactions, we find the that later stages of maturation,
involving the differentiation of CD4+8- and
CD4-8+ cells from
CD4+8+69+ thymocytes, occur in the
absence of MHC molecules. Moreover, an analysis of the postselection
proliferation of newly generated CD4+8- and
CD4-8+ thymocytes shows that this also
occurs independently of MHC molecules. Thus, our data provide direct
evidence that, although positive selection is a multistage process
initiated by TCR-MHC interactions, continuation of this process and
subsequent postselection events are independent of ongoing engagement
of the TCR. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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ß T cell development,
essential for the generation of functionally mature single-positive
(SP)3
CD4+8- and CD4-8+
cells from immature CD4+8+ thymocytes (1, 2).
Interactions between MHC molecules and the ßTCR complex upon the
preselection CD4+8+ thymocytes are known to be
essential for the initiation of positive selection, with interactions
involving MHC class I or MHC class II molecules determining whether
maturation proceeds to the SP CD8 or CD4 lineage, respectively (3, 4). Recently, it has become clear that positive selection of CD4+8+ thymocytes requires sustained interactions with thymic epithelium, and that these epithelial cells are unique in their capacity to mediate and support efficient positive selection. Thus, we (5, 6) and others (7, 8) have shown that CD4+8+ thymocytes which have initiated positive selection still require interactions with thymic stromal cells to complete their maturation, whereas Dyall and Nikolic-Zugic (9) showed that CD4+8low cells, representing a late stage in positive selection, still require the thymus to acquire functional competence. However, although a number of studies have investigated the mechanisms involved in the initiation of positive selection, the nature of the signals and support provided by the thymic epithelium in the later stages of positive selection are less clearly defined.
Interestingly, by analyzing the development of CD4+8+TCRhighCD69+ thymocytes from H-Y-specific TCR transgenic (TCRtg) mice, Kisielow and Miazek (7) provide evidence that the continued maturation of these cells to the CD4-8+ stage, and so, the terminal stages of positive selection, involves sustained interactions with positively selecting MHC molecules. However, we recently showed that CD4+8+69+ cells which have initiated positive selection on thymic stromal cells of the H-2d haplotype are capable of completing their maturation in the presence of stroma of an H-2b haplotype (6). Thus, although these studies clearly define a need for sustained epithelial interactions during positive selection, the role of MHC molecules throughout this process is controversial.
In this study, by isolating stromal cells from the thymuses of MHC class I-deficient, MHC class II-deficient, and MHC double-deficient mice, we have investigated the requirements for interactions with MHC molecules at three distinct points in the maturation of CD4+8+ thymocytes, using an in vitro reaggregate thymic organ culture (RTOC) system that closely mimics the thymocyte-stromal cell interactions seen in vivo (10, 11). First, by isolating wild-type (wt) CD4+8+69-TCR- thymocytes, representing a population of cells that have not yet initiated positive selection (12), we show that under RTOC conditions, the initiation of positive selection (and in particular expression of CD69 on CD4+8+ thymocytes) is dependent upon interactions with MHC molecules expressed on thymic epithelial cells. In marked contrast however, using thymocytes from wt, MHC class I- (13), and MHC class II- (14) restricted TCRtg mice, we find that CD4+8+69+ cells are less dependent upon interactions involving MHC molecules and generate CD4+8-TCRhigh and CD4-8+TCRhigh cells in the absence of MHC class I and MHC class II. Finally, we also show that the SP cells generated from CD4+8+69+ thymocytes in the absence of MHC molecules undergo a wave of cell division that is independent of TCR-MHC interactions. Thus, although positive selection involves sustained interactions with thymic stromal cells, our findings provide the first direct evidence that the requirement for MHC molecules is restricted to the initial pre-CD69 phase of positive selection, with terminal stages of positive selection and early postselection events occurring in an MHC-independent manner.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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AND TCRtg (H-2b) mice, P14 TCRtg (H-2b) mice, ß2-microglobulin (ß2m)-/- (H-2b) mice (The Jackson Laboratory, Bar Harbor, ME), MHC class II-/- mice, and MHC-deficient (ß2m-/- x MHC class II-/-) mice (Taconic, Germantown, NY) were bred and maintained at the Biomedical Sciences Unit of the University of Birmingham. Embryos from these mice and from wt C57BL/6 (H-2b) mice were obtained at day 15 of gestation and used as a source of embryonic thymuses for the preparation of thymic stromal cells. The day of detection of the vaginal plug was designated as day 0 of gestation.
Antibodies
The following Abs were used for flow cytometric analysis and immunomagnetic isolation of cell types as described previously (all from PharMingen, San Diego, CA, unless stated otherwise): anti-CD3 (C363.29b; Southern Biotechnology Associates, Birmingham, AL), anti-CD4 phycoerythrin (GK1.5), anti-CD4 (GK1.5), anti-CD8 FITC (53-6.7), anti-CD8 APC (53-6.7), anti-CD8 (YTS 169.4; Sera-Lab, Sussex, U.K.), anti-CD45 (M1/9; American Type Culture Collection, Manassas, VA), anti-CD69 (H1.2f3), anti-Vß3 TCR (KJ25), and anti-Vß8 TCR (F23.1). Thymocytes were analyzed using a dual laser Coulter Epic Elite machine (Coulter, Hialeah, FL), with forward and side scatter gates set so as to exclude nonviable cells (12).
Preparation of cell types
CD4+8+TCR- thymocytes. Thymocyte suspensions from neonatal BALB/c mice were prepared as described previously (12). Briefly, suspensions were depleted of CD3+ cells using multiple rounds of anti-CD3-coated magnetic beads. CD4+8+ thymocytes were further purified from these CD3- preparations by positive selection using anti-CD8-coated rat IgG Dynabeads (Dynal, Wirral, U.K.); beads were removed by a brief exposure to trypsin. Such a procedure results in a population of CD4+8+TCR- thymocytes at >98% purity (Ref. 12 and data not shown).
CD4+8+69+ thymocytes. Thymocyte suspensions were prepared from either BALB/c, P14 TCRtg, or AND TCRtg neonatal mice, as indicated. Cells expressing CD69 were selected from such preparations using streptavidin-coated Dynabeads (Dynal) coated with biotinylated anti-CD69 Abs. Beads were removed from CD69+ cells with Detachabead (Dynal) according to the manufacturer’s instructions. As with all methods involving a positive selection of cell types, we cannot formally exclude the possibility that direct isolation of cells on a particular cell surface molecule invokes signaling within the target cell population. The isolation of CD4+8+69+ cells from this CD69+ population was then achieved by further selection using anti-rat IgG Dynabeads coated with anti-CD4 for enrichment of CD4+8+69+ P14 thymocytes or coated with anti-CD8 for BALB/c or AND CD4+8+69+ thymocytes, as described previously (5, 6). Such a procedure results in the efficient isolation of CD4+8+69+ thymocytes that contain no contaminating SP CD4+ or CD8+ thymocytes (Ref. 5 and data not shown).
Thymic stromal cells. Mouse embryo thymuses from indicated strains (15 days gestation) were cultured for 5–7 days in 1.35 mM 2-deoxyguanosine and trypsinized (0.25% trypsin in 0.02% EDTA; Sigma, Poole, Dorset) to form a single-cell suspension. Residual hemopoietic elements were depleted with anti-CD45- (M1/9) coated Dynabeads as described previously (10).
Reaggregate organ cultures
Freshly prepared thymocytes and appropriate stromal cells were mixed together in 1.5-ml Eppendorf tubes at a ratio of 1:1 and pelleted by centrifugation. The resultant cell slurry was transferred to the surface of a 0.8-µm nucleopore filter in organ culture, using a finely drawn glass pipette. Thymocytes were harvested from RTOCs after the indicated time period by gently teasing apart with fine knives.
Analysis of cell division using 5- (and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE)
CFSE, an FITC-based lipophilic membrane-binding dye, has been used to identify a wave of multiple cell divisions immediately following the completion of positive selection (6). Thus, thymocytes were pulsed with 1 µM of CFSE in PBS for 10 min at 37°C before their incorporation into reaggregate cultures. Thymocytes harvested from such cultures were analyzed for CD4 or CD8 expression, together with CFSE content, to allow separate study of the proliferation of CD4+8- and CD4-8+ cells.
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Maturation of CD4+8+ precursors to SP
cells involves interactions between the ßTCR and MHC molecules,
with interactions with MHC class I and MHC class II leading to the
generation of SP CD4-8+ and
CD4+8- cells, respectively (4). However,
because positive selection is a multistage process that involves
sustained contact with thymic epithelial cells (5, 6, 15), the
potential importance of the continued engagement of MHC molecules
throughout positive selection is unclear. Therefore, in an initial
series of experiments, we investigated the MHC requirements during the
initiation of positive selection in reaggregate cultures to provide a
comparative basis for MHC requirements later in the selection process.
Thus, purified wt CD4+8+69-
(preselection) thymocytes were reaggregated with thymic stromal cells
lacking either MHC class II, MHC class I
(ß2m-/-), or both MHC class I and II, and
maturation was analyzed after 5 days.
As expected, wt thymic stromal cells induced the maturation of
CD4+8+69- thymocytes to both the
CD4+8- and CD4-8+
stage (Fig. 1
a), together with the induction
of CD69 expression (Fig. 1a, inset). However, in the absence
of MHC class II, CD4+8+69-
thymocytes failed to generate significant numbers of SP
CD4+8- cells, although a normal generation of
CD4-8+ cells was observed (Fig. 1b). Interestingly, and in agreement with the findings of
Ernst et al. (16), CD4+8+69-
thymocytes generate both SP CD4+8- and
CD4-8+ cells in the presence of
ß2m-/- thymic epithelial cells (Fig. 1c), indicating that CD4-8+ cells
may be selected by both MHC class I and MHC class II molecules. Of
note, although differences in the level of coreceptor expression are
evident on CD8+ cells generated with the various stromal
cell types (Fig. 1, a–c), it is likely that this
difference represents heterogeneity in the developmental kinetics of
individual RTOCs, because following the initiation of positive
selection at the CD4+8+ stage, thymocytes
transit through intermediate stages where coreceptor molecules may be
partially down-regulated before the appearance of true SP thymocytes
(4). In marked contrast however, thymocytes cultured with MHC
double-deficient (i.e., ß2m-/-, MHC class
II-/-) stromal cells did not give rise to either of the
mature SP CD4+8- or
CD4-8+ subsets, although a large population of
viable CD4+8+ thymocytes were recovered (Fig. 1d). Notably, these CD4+8+
thymocytes showed no evidence of induction of CD69 expression (Fig. 1d, inset), indicating that they have remained at the
preselection CD69- stage of development and thereby
confirming the importance of MHC molecules in initiating positive
selection.
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Having shown that the initiation of positive selection is
dependent upon interactions with the MHC molecules expressed on thymic
epithelium, and that these interactions are necessary for the induction
of CD69 expression, we used
CD4+8+69+ thymocytes as an
intermediate population in the positive selection process to determine
the requirements for MHC molecules in the later stages of maturation.
Thus, CD4+8+69+ thymocytes were
isolated from either wt, MHC class II-restricted (AND) TCRtg
(13), or MHC class I-restricted (P14) TCRtg (14) neonates and analyzed
for their continued maturation and subsequent completion of positive
selection in the presence of a variety of thymic stromal cell
preparations. In the case of wt
CD4+8+69+ thymocytes, as expected,
analysis of CD4 and CD8 expression after a 5-day culture period in the
presence of wt (MHC class 1+, MHC class II+)
thymic stromal cells revealed that
CD4+8+69+ cells had undergone
further development, resulting in the generation of both SP
CD4+8- and CD4-8+
cells (Fig. 2a). Interestingly, in the
presence of MHC-deficient thymic stromal cells, a similar pattern of
development occurs, with the maturation of both mature thymocyte
subsets being observed (Fig. 2b), although to a somewhat
reduced extent in terms of actual cell numbers (Fig. 3,
a and b). The reasons for this reduction are
unclear, although it may indicate a degree of heterogeneity within the
CD69+ population with regard to MHC dependence. Recently
however, we have shown that
CD4+8+69+ thymocytes can be
subdivided into two subsets on the basis of the expression of CD132,
the common 
-chain of cytokine receptors, with CD132 expression
occurring after the expression of CD69 (17). Additional experiments are
underway to determine whether
CD4+8+69+132- and
CD4+8+69+132+
thymocytes differ in their requirements for MHC molecules in maturation
to the SP stages.
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c) and CD4-8+Vß8high
cells (Fig. 2e) from AND and P14
CD4+8+69+ thymocytes, respectively.
Gating on cells bearing high levels of TCR transgenes (shown in Fig. 2), as used by others (7), eliminates the confusion of
including in the analysis any cells that may bear endogenously
rearranged TCRs, and allows a direct analysis of the development of
mature SP thymocytes expressing high TCR levels. Moreover, a similar
pattern of development of AND
CD4+8+69+ thymocytes was observed
in the presence of MHC-deficient thymic stromal cells (Fig. 2d), with mature
CD4+8-Vß3high cells being
generated, albeit at reduced numbers (Fig. 3a). Accordingly,
analysis of development of
CD4+8+69+ thymocytes from P14
neonates generated
CD4-8+Vß8high cells in the
presence of MHC-deficient thymic stromal cells (Fig. 2f),
again at reduced numbers compared with control cultures (Fig. 3b).
As the expression of high levels of ßTCR can be used as an
indication of complete maturation during positive selection, we
analyzed the expression of transgenic V11 on AND TCRtg
CD4+8-Vß3high cells (Fig. 4, a and b) and of V2 on P14
TCRtg CD4-8+Vß8high cells (Fig. 4, c and d) generated from TCRtg
CD4+8+69+ thymocytes in the
presence (Fig. 4, a and c) or absence (Fig. 4, b and d) of MHC molecules. In all cases,
CD4+8- and CD4-8+
thymocytes were found to uniformly express high levels of the
appropriate transgenic TCR-chain, characteristic of mature SP
thymocytes, irrespective of whether they had completed their maturation
in the presence of wt or MHC-deficient thymic stromal cells. As
mentioned above, this experiment thus provides an important control,
proving that transgenic thymocytes completing their maturation in the
presence of MHC-deficient stromal cells express both and ß
components of the transgenic TCR, thereby ruling out the possibility
that the maturation observed here is a consequence of pairing of
transgenic TCRß-chains with endogenously rearranged TCR-chains.
Importantly, we also found that SP cells generated from
CD4+8+69+ thymocytes in the absence
of MHC molecules undergo TCR-mediated activation and proliferation in a
manner that is indistinguishable from that seen with cells generated in
the presence of MHC-bearing stromal cells (data not shown), indicating
that functional as well as phenotypic maturation has occurred.
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Postpositive selection cell division is independent of MHC molecules
It has been shown, both by bromodeoxyuridine incorporation and by CFSE labeling, that SP cells newly generated from double-positive CD4+8+ precursors are out of cycle but subsequently show evidence of cell division (6, 18, 19). This postpositive selection cellular expansion has recently been quantified to suggest that in fact multiple cell divisions are involved, and that these divisions are dependent upon association with thymic epithelial cells (6). Because the data summarized above indicate that the later stages of positive selection show an independence of interactions with MHC molecules for the phenotypic and functional differentiation of SP thymocytes, we investigated the requirements for MHC molecules in the proliferation following the emergence of SP cells, which is epithelial cell-dependent (6).
Thus, CD4+8+69+ thymocytes were
isolated from neonatal wt, AND, and P14 mice, labeled with CFSE, and
placed in RTOC either with wt stroma or with stromal cells deficient in
both MHC class I and II molecules. After a 3-day culture period,
thymocytes were recovered and analyzed for cell division based upon
their dilution of CFSE (6). Subsequently, an individual analysis of
CD4+8- and CD4-8+
populations revealed that SP thymocytes generated from either wt (Fig. 5, a and b), AND TCRtg (Fig. 5c), or P14 TCRtg (Fig. 5d) precursors in the
absence of MHC molecules, although reduced in absolute cell numbers (as
shown in Fig. 3), still undergo a wave of multiple cell divisions to a
similar extent as that observed of cells developing in the presence of
MHC molecules (wt stroma) (Fig. 5, a–d).
Interestingly, Fig. 5c shows that the postselection
expansion of CD4+8- cells generated from AND
TCRtg CD4+8+69+ thymocytes appears
to be somewhat reduced compared with the proliferation observed in wt
CD4+8- cells (Fig. 5a). The reason
for this difference is unclear, but it is important to note that,
although it is somewhat reduced, proliferation occurs in the AND system
to a similar degree in the presence of wt and MHC-deficient stromal
cells (Fig. 5c). Thus, these findings suggest that the
proliferation of newly generated wt and transgenic
CD4+8- and CD4-8+
thymocytes does not require continued signaling through the TCR beyond
that initially triggering positive selection.
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Positive selection is a crucial stage in thymocyte development, resulting in the generation of functionally competent CD4+8- and CD4-8+ T cells. In this study, we have analyzed the requirement for interactions with MHC molecules during initial, intermediate, and postpositive selection events. Our data show that, although the initiation of positive selection is critically dependent upon interactions with MHC molecules expressed by the thymic epithelium, subsequent events in positive selection (although requiring interactions with epithelium) occur in an MHC-independent manner. Thus, MHC-deficient stromal cells are sufficient to induce the maturation of CD4+8+69+ thymocytes to the CD4+8- and CD4-8+ stage. Collectively, these findings indicate that although positive selection involves sustained interactions with thymic epithelial cells, the requirement for MHC molecules is limited to the initiation of positive selection and to the subsequent expression of CD69.
Finally, we have also shown that the wave of proliferation following positive selection occurs independently of the MHC molecules expressed by thymic epithelium. However, the underlying mechanisms regulating the proliferation of newly selected CD4+8- and CD4-8+ thymocytes remain unclear. Interestingly, we have found that newly selected thymocytes express a variety of cytokine receptors, such as IL-7R, IL-2R, and IL-6R (data not shown). In addition, by analyzing cytokine mRNA expression in RTOCs, we have found IL-7 and IL-15 mRNA in thymic epithelial cells (Ref. 20 and data not shown) and IL-2, IL-4, and IL-6 mRNA in CD4+8- and CD4-8+ cells (Ref. 12 and data not shown). As many of these cytokines have been shown to be involved in the proliferation associated with mature T cell activation, a more detailed analysis may reveal a role for these molecules in the proliferation following positive selection.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Katherine Hare, Department of Anatomy, University of Birmingham Medical School, Edgbaston, Birmingham, B15 2TT, United Kingdom. E-mail address:
3 Abbreviations used in this paper: SP, single positive; RTOC, reaggregate thymic organ culture; TCRtg, TCR transgenic; ß2m, ß2-microglobulin; CFSE, 5- (and 6-) carboxyfluorescein diacetate succinimidyl ester; wt, wild type.
Received for publication October 16, 1998. Accepted for publication January 13, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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ß T cell receptors. Curr. Opin. Immunol. 9:250.[Medline]
ß and 
T cell receptor-expressing mature T cells. Int. Immunol. 2:859.This article has been cited by other articles:
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