Mature Human Thymocytes Research Articles

Sphingosine-1-phosphate/sphingosine-1-phosphate receptor 1 signaling is required for migration of naive human T cells from the thymus to the periphery

The mechanisms that govern the egress of mature thymocytes from the human thymus to the periphery remain understudied yet are of utmost importance to the field of basic immunology, as well as T-cell reconstitution in various immunodeficiencies. We examined the expression and function of sphingosine-1-phosphate (S1P) receptors in human thymocyte egress. We aimed to determine whether S1P receptors (S1P-Rs) play a role in mature human thymocyte egress and to identify the thymocyte population or populations that express S1P-Rs and respond to S1P by migrating across a concentration gradient. Human thymocytes were exposed to S1P in Transwell plate migration assays coupled to flow cytometry to evaluate the response to S1P of thymocytes at different stages of maturation. Constitutive S1P-R expression was quantified by means of real-time PCR in sorted thymocyte subsets and flow cytometry. S1P-R1 and Kruppel-like factor 2 expression were monitored after S1P exposure by using flow cytometry and quantitative PCR. S1P-R1 was the prevalent S1P receptor on mature human thymocytes (CD3(hi)CD27(+)CD69(-)), the population that also demonstrated the greatest response to S1P in migration assays. Pretreatment with FTY720, an S1P-R1 nonselective modulator significantly reduced migration and suggested a role for S1P-R2 in retaining thymocytes in the tissue. Lastly, surface S1P-R1 expression, as well S1PR1 and Kruppel-like factor 2 (KLF2) transcripts, were significantly decreased in mature thymocytes on exposure to S1P. Mature human thymocytes rely on S1P-R1 to migrate toward S1P. Taken in the context of murine work demonstrating that S1P is required for thymocyte egress to the periphery, our data highlight a new key chemokine for human thymocyte egress.

HIV-1 Infection increases sphingosine-1-phosphate receptor 1 expression in the human thymus (CAM4P.162)

Abstract Sphingosine-1-phosphate receptor 1 (S1P-R1) is essential in mice for homing of lymphoid cells as well as egress of mature thymocytes from the thymus to the periphery. We have recently demonstrated that in the human thymus, response of S1P-R1 to its ligand, Sphingosine-1-phosphate (S1P), is required for egress of mature human thymocytes to the periphery. Herein we demonstrate that immune dysregulation accompanying Human Immunodeficiency Virus (HIV) infection alters the expression of S1P-R1 on human thymocytes, increasing expression of the receptor at both the mRNA and protein levels. Moreover, the function of the receptor is maintained, as assayed by flow cytometry based detection of phosphorylated Akt (pAkt). Finally, we found that several cytokines including Tumor Necrosis Factor Alpha and Interferon Alpha are perturbed during infection of the thymus, and exogenous treatment of human thymocytes with these cytokines increases or attenuates S1P-R1 expression. Therefore we have identified a contributing mechanism to alteration of S1P-R1 expression during HIV infection of the human thymus, which may have important implications for therapeutics for HIV infected individuals.

Opposing chemokine gradients control human thymocyte migration in situ

The ordered migration of thymocytes from the cortex to the medulla is critical for the appropriate selection of the mature T cell repertoire. Most studies of thymocyte migration rely on mouse models, but we know relatively little about how human thymocytes find their appropriate anatomical niches within the thymus. Moreover, the signals that retain CD4+CD8+ double-positive (DP) thymocytes in the cortex and prevent them from entering the medulla prior to positive selection have not been identified in mice or humans. Here, we examined the intrathymic migration of human thymocytes in both mouse and human thymic stroma and found that human thymocyte subsets localized appropriately to the cortex on mouse thymic stroma and that MHC-dependent interactions between human thymocytes and mouse stroma could maintain the activation and motility of DP cells. We also showed that CXCR4 was required to retain human DP thymocytes in the cortex, whereas CCR7 promoted migration of mature human thymocytes to the medulla. Thus, 2 opposing chemokine gradients control the migration of thymocytes from the cortex to the medulla. These findings point to significant interspecies conservation in thymocyte-stroma interactions and provide the first evidence that chemokines not only attract mature thymocytes to the medulla, but also play an active role in retaining DP thymocytes in the cortex prior to positive selection.

An In vitro Model for the Study of HIV Infection of Thymocytes

The study of human thymocytes requires an appropriate matrix to enable the proper function of thymocytes. Although OP9-DL1 cells have been well established as an ideal co-culture system for the generation of T-cells from their progenitors; their ability to support a mixed population of mature human thymocytes for in vitro HIV infection studies has yet to be established. We assessed the effects of co-culturing a heterogeneous population of mature human thymocytes with a mouse derived cell line (OP9) transduced with the notch ligand delta like 1 (OP9-DL-1) and compared this to standard co-culture with human thymic epithelial cells (TEC). Co-culturing thymocytes with OP9- DL1 cells resulted in higher viability and lower apoptosis when compared to TEC co-cultures. The subset distribution and CD127 expression of thymocytes varied slightly between conditions. Thymocytes co-cultured with OP9-DL1 cells had a lower proportion of CD3+DP cells and higher proportion of SP4 cells compared to TEC co-cultures. The mature CD3+CD4+CD8- (SP4) cells also had lower levels of CD127 expression in OP9-DL1 cultures when compared to TEC. Interleukin-7 stimulation of thymocytes resulted in a decrease in CD127 expression in OP9-DL1 co-cultures, as previously observed with TEC co-cultures. Thymocytes co-cultured with OP9-DL1 tended to have higher levels of IL-7 induced STAT-5 phosphorylation and had higher levels of Interleukin-7 induced Bcl-2 expression. OP9-DL1 cells provide a microenvironment which is permissive to HIV infection in thymocytes in vitro. Co-culturing thymocytes with OP9-DL1 will facilitate the study of human thymocytes and aid in the study of exogenous stimuli or infection on individual thymocyte subsets.

Mature human thymocytes migrate on laminin-5 with activation of metalloproteinase-14 and cleavage of CD44.

We have previously shown that laminin-5 is expressed in the human thymic medulla, in which mature thymocytes are located. We now report that laminin-5 promotes migration of mature medullary thymocytes, whereas it has no effect on cortical immature thymocytes. Migration was inhibited by blocking mAbs directed against laminin-5 integrin receptors and by inhibitors of metalloproteinases. Interactions of thymocytes with laminin-5 induced a strong up-regulation of active metalloproteinase-14. However, we found that thymocytes did not cleave the laminin-5 gamma(2) chain, suggesting that they do not use the same pathway as epithelial cells to migrate on laminin-5. Interactions of thymocytes with laminin-5 also induced the release of a soluble fragment of CD44 cell surface molecule. Moreover, CD44-rich supernatants induced thymocyte migration in contrast with supernatants depleted in CD44 by immunoadsorption. CD44 cleavage was recently reported to be due to metalloproteinase-14 activation and led to increased migration in cancer cells. Thus, in this study, we show that laminin-5 promotes human mature thymocyte migration in vitro via a multimolecular mechanism involving laminin-5 integrin receptors, metalloproteinase-14 and CD44. These data suggest that, in vivo, laminin-5 may function in the migration of mature thymocytes within the medulla and be part of the thymic emigration process.

CD6: expression during development, apoptosis and selection of human and mouse thymocytes.

CD6, a 130-kDa surface glycoprotein, is expressed primarily on cells of T lineage. A co-stimulatory role for CD6 in mature T cells has been shown, but the function of CD6 during thymocyte development is unknown. Since CD6 ligands are expressed on thymic epithelium, their interactions with CD6 could be important in thymic selection. In this report we show that CD6 is developmentally regulated in human and mouse thymocytes, and further demonstrate that increase in the level of CD6 expression correlates with expression of the selection marker CD69. We also show that activation via CD2 induces CD6 expression on mature human thymocytes and on a subset of immature human thymocytes that are resistant to apoptosis. In human and mouse thymocytes that express heterogeneous TCR, CD6 increases occur as double-positive thymocytes are selected to a single-positive stage. In contrast, in thymocytes from TCR transgenic mice, CD6 is barely increased following selection, suggesting that as functional avidity increases, requirements for CD6-dependent co-stimulation decrease. Taken together, these results indicate that during thymic development CD6-dependent signals may contribute both to thymocyte survival, and to the overall functional avidity of selection in both man and mouse.

CCR8 on human thymocytes functions as a human immunodeficiency virus type 1 coreceptor.

To determine whether human immunodeficiency virus type 1 (HIV-1) coreceptors besides CXCR4 and CCR5 are involved in HIV-1 infection of the thymus, we focused on CCR8, a receptor for the chemokine I-309, because of its high expression in the thymus. Similar levels of CCR8 mRNA were detected in immature and mature primary human thymocytes. Consistent with this, [(125)I]I-309 was shown to bind specifically and with similar affinity to the surface of immature and mature human thymocytes. Fusion of human thymocytes with cells expressing HIV-1 X4 or X4R5 envelope glycoprotein was inhibited by I-309 in a dose-dependent manner. In addition, I-309 partially inhibited productive infection of human thymocytes by X4, R5, and X4R5 HIV-1 strains. Our data provide the first evidence that CCR8 functions as an HIV-1 coreceptor on primary human cells and suggest that CCR8 may contribute to HIV-1-induced thymic pathogenesis.

T-cell receptor J beta gene segment usage in immature and mature human thymocytes.

Immature double positive (DP, CD4+CD8+) and mature single positive (SP, CD4+CD8- and CD4-CD8+) human thymocytes from nine thymi were analysed for their complete patterns of relative TCR J beta multigene member usage in relation to six rearranged V beta family exons (V beta 5.1, 6.1-3, 8, 9, 12 and 18). Each sample tested contained mRNA transcripts corresponding to all potential V beta(D beta)J beta combinations. Individual J beta gene segments were expressed in a similar, highly non-random manner both in SP and DP thymocytes, irrespective of original genomic position of the individual associated V beta exon. In addition, ranges of family usage and frequency of individual over-representations of J beta gene segments, as determined in DP and SP thymocyte populations, displayed no significant differences. Upon comparison of DP and SP thymocytes, however, a discrepancy in one aspect of J beta gene utilization was established: decreasing J beta family 1/J beta family 2 ratios were determined to be positively correlated with increasing maturity of thymocytes, a condition further supported by data previously obtained from studies of PBL T cells. At the individual J beta gene level, the observed gradual modification of the relative family usage can largely be explained by a significant shift from a higher J beta 1.1/J beta 2.7 ratio in DP to a higher J beta 2.7/J beta 1.1 ratio in SP thymocytes. Altogether, the present results imply that selectional processes in the thymus appear to have only minor consequences on the distribution pattern of expressed J beta exons. Hence, the disproportionate pattern of TCR J beta gene usage seems to be established mainly at the recombinatorial level followed by minor adjustments during thymic and post-thymic events.

Human thymocyte development in mouse organ cultures

A novel system to study human thymocyte development is described in which embryonic mouse thymic rudiments are seeded with human precursor cells in vitro. In these cultures human thymocytes proliferate extensively (greater than 20-fold increase in cell number) and mature, as evidenced by the accumulation of double and single positive (CD4+ and/or CD8+) cells. Data presented here suggest that the survival and ordered development of the mature human thymocytes in chimeric thymuses is dependent on human stromal elements. Immature CD4-CD8- human thymocytes failed to colonize or minimally recolonized mouse thymic lobes unless provided with high density (greater than 1.077 g/ml) human thymic cell fractions. These fractions contain multicellular complexes of epithelial/nurse cells, thymocytes, and dendritic cells/macrophages which dramatically enhanced the recolonizing capacity of purified CD4-CD8- thymocytes. The chimeric organ culture system described here provides not only a new approach for studying human T cell ontogeny but also a direct means for the future dissection of stromal interactions necessary for successful transition of precursor cells (CD4-CD8-) to immature double positive (CD4+CD8+) and mature single positive cells (CD4+ or CD8+) in the thymus.

Human thymocytes do not respond to interleukin-2 after removal of mature “bright” CD5 positive cells

Interleukin-2 receptors (IL-2R) are expressed on minor populations of immature and mature human thymocytes. These studies were designed to determine if immature T cells could respond to the mitogen phytohemagglutinin (PHA-P) plus IL-2 in vitro by increasing the expression of IL-2R and by proliferation. Using monoclonal antibodies to CD5 and magnetic immunobeads we were able to remove all mature, “bright” CD5 + cells from nylon wool-purified thymocytes and to obtain less mature cells which consisted almost completely of cells with the CD4 +CD8 + phenotype. These immature cells were mostly “dim” CD5 + and less than 5% CD5 − and a small percentage expressed the IL-2R. After culture in serum-free medium with PHA-P, these cells showed only a slight increase in the percentage of IL-2R + cells and the addition of IL-2 did not increase the percentage of IL-2R + cells and no proliferation was observed. Unseparated, nylon wool-purified thymocytes contained 14% bright CD5 + cells. These bright CD5 + cells had a mature phenotype of CD4 +CD8 − (52%) and CD4 −CD8 + (27%) cells. A small percentage of these cells were IL-2R +. These bright CD5 +IL-2R + cells were predominantly mature CD4 +CD8 − cells as measured by three-color flow cytometry. After culture with PHA-P and IL-2, the percentage of IL-2R + cells increased and they were now found not only on CD4 +CD8 − but also on CD4 − CD8 + and on CD4 +CD8 + cells. IL-2 plus PHA-P increased proliferation of these cells as compared to those cultured in medium with PHA-P without IL-2. Thus, we show that human immature thymocytes in contrast to mature thymocytes are not responsive to IL-2 as measured by a lack of IL-2R expression and proliferation. These data indicate that mature thymocytes can express a functional high affinity receptor for IL-2 and suggest that immature thymocytes may not possess a (functional) p75 chain of the IL-2R.

A Monoclonal Antibody with Selective Reactivity with Functionally Mature Human Thymocytes and All Peripheral Human T Cells

Abstract A monoclonal antibody directed at a determinant on human peripheral blood T cells was produced and characterized. This hybridoma antibody, termed OKT1, was reactive by indirect immunofluorescence with the entire human peripheral blood T cell population and a subset of human thymocytes. In contrast, OKT1 was unreactive with normal B cells, Null cells, macrophages, and ≥90% of human thymocytes. These findings suggested that OKT1 defines a mature T cell differentiation antigen. In support of this notion was the observation that T cell acute lymphoblastic leukemia cells were nonreactive with OKT1, whereas T cell chronic lymphocytic leukemia cells were reactive. Concomitant functional studies on FACS-separated lymphocytes showed that the T cell proliferative responses to mitogens and soluble and cell surface antigens were contained in the OKT1+ population. Fractionation of peripheral blood T cells into strongly and weakly reactive OKT1+ subgroups uncovered no functional T cell heterogeneity. In addition, when the thymocyte population was separated into OKT1+ and OKT1- subsets, only the OKT1+ thymocytes were MLC responsive. However, unlike peripheral T cells, neither the OKT1+ or OKT1- thymocytes proliferated to the mitogens PHA and Con A. Thus, OKT1+ thymocytes are functionally distinct from OKT1+ peripheral blood T cells. These studies show that a hybridoma antibody can be produced that detects a human differentiation antigen that appears during late intrathymic T cell ontogeny and persists on peripheral T cells.