The thymic microenvironment supports the differentiation and proliferation of bone marrow-derived progenitors as they enter and migrate through the thymus. Immature thymocytes that lack the expression of the surface molecules CD4 and CD8 (referred to as double negative, DN) can be further subdivided into four distinct developmental stages (DN1, DN2, DN3 and DN4) based on the differential expression of surface markers CD44 and CD251, 2
. The developmental progression of DN thymocytes from DN1 to DN4 is influenced by their localization within the thymic architecture as well as signals from surface receptors1, 3
. DN1 and DN2 thymocytes begin an outward migration from the cortico-medullary junction toward the outer cortex, with the DN3 subset localizing mainly at or near the sub-capsular zone (SCZ)4
. The β-selection developmental checkpoint, which screens for productive Tcrb
rearrangement and assembly of a surface pre-T cell receptor (pre-TCR) complex, begins at the DN3 stage and is competed during the DN4 stage. This β-selection process coincides with a movement of the DN4 cells from the cortex back toward the medulla. Thymocytes that successfully progress through this checkpoint begin to express both CD4 and CD8 (referred to as double positive, DP), undergo further maturation and differentiate into cells expressing only CD4 or CD8 (single positive, SP).
Chemokines and their receptors regulate the carefully orchestrated migration patterns of thymocytes, and may provide additional signals that influence thymocyte differentiation5, 6
. However, the specific chemokines that function just prior to or during the β-selection checkpoint, and how they might influence pre-TCR signals are not well understood. CXCR4 (http://www.signaling-gateway.org/molecule/query?afcsid=A000636
) and its natural ligand SDF-1α (also called CXCL12) are widely expressed in tissues and play a major role in embryonic development7
, organogenesis and vascularization9, 10
. In addition to regulating trafficking11
and homing of hematopoietic progenitors, CXCR4 acts as a co-receptor for human immunodeficiency virus 1 (HIV-1)12
. SDF-1α and CXCR4 have been linked to egress of mature single positive thymocytes from the thymus13, 14
. CXCR4 also potentiates responses of peripheral T cells to TCR signals15, 16
. However, the role of CXCR4 in regulating the β-selection checkpoint is unknown. An earlier study using a bone marrow chimera approach and Lck-Cre-mediated deletion of CXCR4 reported a developmental block at the DN1 stage17
. However, this observation is puzzling as the developmental arrest seen by these authors occurred before the stage at which Lck-Cre-mediated deletion of CXCR4 expression would be predicted to occur. This observation may be due to technical aspects of the bone marrow chimera approach, possibly related to colonizing niches in the thymus or competition between transferred and host progenitors. Thus, the role of CXCR4 in DN thymocyte development, specifically at the β-selection step, and how CXCR4 signals may integrate with pre-TCR signals remain to be defined.
As DN thymocytes undergo β-selection, signals emanating from the pre-TCR and potentially other receptors promote immature thymocyte survival, proliferation and differentiation18
. Pre-TCR and Notch receptors expressed on DN thymocytes critically influence proliferation and differentiation during β-selection19
. Early DN3 survival can be regulated by the pre-TCR, Notch and interleukin 7 (IL-7) receptor20
. Although a function for p53 in rescuing cellularity in CD3γ–deficient thymocytes was initially suggested21
, the induction of anti-apoptotic Bcl-2 family proteins and the suppression of pro-apoptotic signals is now thought to influence DN cell survival22
Among the Bcl-2 family members, Bcl2a1
mRNA expression is upregulated by the pre-TCR during β-selection in a manner dependent on p65 NF-κB, which also has been linked to cell survival signals in early thymic development23, 24
. While chemokines may regulate events beyond the localization of thymocytes within the thymic architecture, the role of chemokine receptors in regulating the survival, proliferation and differentiation signals during β-selection remains unclear.
In this report, by disrupting CXCR4 expression during DN2 and DN3 stages, we identified a key role for CXCR4 during β-selection. We also defined a functional interplay between CXCR4 and pre-TCR, designating CXCR4 as a non-redundant costimulator regulating pre-TCR dependent signals in DN thymocytes.