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Integrin Functions Play a Key Role in the Differentiation of Thymocytes In Vivo¹

Peter J. Schmeissner^*, Haichun Xie^*, Lubomir B. Smilenov^*, Fengyu Shu and Eugene E. Marcantonio^2,*

^* Departments of Pathology and Anatomy and Cell Biology, Columbia University College of Physicians and Surgeons, New York, NY 10032; and Department of Microbiology, University of Alabama, Birmingham, AL 35294

	Abstract

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T cells express a variety of surface proteins as they develop to maturity in the thymus. In addition to the TCR-CD3 complex and the two major coreceptors, CD4 and CD8, other surface proteins expressed include receptors for cytokines, growth factors, counterreceptors, and extracellular matrix molecules. To determine the role of integrin adhesion receptors in T cell development, we have expressed a trans-dominant inhibitor of integrin function in the thymus. This inhibitor leads to a block of adhesion to fibronectin due to reduced activation of integrin receptors. This reduced adhesion leads to a partial block in differentiation from CD4^-CD8^- cells to CD4⁺CD8⁺ cells, after the CD25⁺ stage, suggesting that integrins are important during Lck-mediated differentiation. Furthermore, the overall production of CD4⁺ cells is reduced compared with that of CD8⁺ cells without changes in negative selection, suggesting that integrins may be involved in the determination of the fate of the cell as well. These results demonstrate that integrin receptor function is required for proper thymocyte development in vivo.

	Introduction

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T cells proceed through a series of developmental stages in the thymus involving both cell proliferation and differentiation. The expression patterns of various cell surface receptors help to identify the stages of development. The earliest stage is defined by a lack of surface expression of a mature TCR-CD3 complex and a lack of CD4 and CD8 coreceptor surface expression (reviewed in Ref. 1). Within this stage, T cell development is subdivided according to the expression of CD25 (the IL-2R subunit), and CD44 (a proteoglycan involved in cell-cell adhesion). First, T cells only express CD44, then they up-regulate CD25 to express both receptors. Next, T cells down-regulate CD44. Finally, CD25 also is down-regulated, so the T cells appear as CD3^-CD4^-CD8^-CD44^-CD25^- (2). As the cells continue to mature, they first weakly up-regulate the CD8 coreceptor. Following a burst of proliferation at this stage, both the CD4 and CD8 coreceptors are up-regulated concomitant with a gradual up-regulation of the mature TCR-CD3 receptor complex (3). Finally, when TCR/CD3 expression is maximal, a down-regulation of either coreceptor occurs, and the T cells then are fully mature and ready to exit the thymus.

Numerous studies have linked the expression of the major T cell surface receptor, the TCR-CD3 complex, to the cellular changes that occur during T cell development. In the absence of expression of the subunit of the TCR, thymocyte development is halted at the CD44^-CD25⁺ stage. In the absence of expression of the subunit of the TCR, thymocyte development is halted at a later stage, before the up-regulation of both CD4 and CD8 (4, 5). However, the absence of other receptors, such as the CD25 molecule, has no apparent effect on thymocyte development (6, 7).

In addition to the above-mentioned major surface receptors, a number of other proteins are differentially expressed on T cells as the cells develop in the thymus. These include receptors for cytokines and growth factors or receptors that mediate cell-cell or cell-matrix adhesion. Some of the surface proteins that mediate thymocyte adhesion belong to the integrin family of proteins. A large variety of integrin heterodimers exist that show redundancies in ligand binding (reviewed in Ref. 8). In addition to ₂ integrins that mediate cell-cell contacts, ₁ integrins such as ₄₁ and ₅₁, which bind fibronectin (FN),³ and ₃₁ and ₆₁, which bind laminin and merosin, are expressed on immature thymocytes (9, 10). In more mature thymocytes, during the late CD4⁺CD8⁺ and CD4⁺ or CD8⁺ stages, the surface expression levels and the abilities of some integrins to bind ligand are down-regulated (11). As with other surface receptors, integrins, upon binding ligand, generate a number of intracellular signals that lead to cytoskeletal reorganization, the formation of focal adhesions, and changes in gene expression. The intracellular signaling pathways stimulated by integrin-ligand binding are shared by a number of other surface receptors expressed on thymocytes, including growth factors, the TCR-CD3 complex, CD4, and CD8. However, the contribution of integrin functions, including integrin-mediated signaling, to the progression of T cell development is not known.

A number of in vitro studies have demonstrated that the engagement of integrins is required for the differentiation of CD4^-CD8^- cells to become CD4⁺CD8⁺ cells (12, 13, 14). Furthermore, the Ab-mediated engagement of integrin receptors in addition to the engagement of the CD3 complex on CD4⁺CD8⁺ thymocytes is required for cell proliferation in vitro (15, 16). To undertake a more thorough examination of the role of integrin receptors in thymocyte development, we chose to study thymocyte development in vivo using a mouse system that transgenically expresses a chimeric molecule shown to have dominant negative effects on integrin function. The chimera’s effects can act in trans to affect many different integrin heterodimers. This system has an advantage over gene disruption systems for several reasons. First, gene disruptions of integrins hinder the development of the whole animal or the thymocyte precursor cells before the seeding of the thymus. Knockouts of the ₁, ₃, ₄, ₅, and ₆ integrin subunits, for example, result in embryonic or perinatal lethality (reviewed in Ref. 17). Second, gene disruptions of specific integrins can be masked by redundant functions of other integrin subunits.

Using a transgenic trans-dominant inhibitor construct, we show here that integrins are required for the development of thymocytes. Specifically, integrin-mediated activities are required for the differentiation of CD4^-CD8^- cells to CD4⁺CD8⁺ thymocytes. Our results suggest that integrins function in concert with signals generated from pre-TCR-CD3 complexes, which also are required for the same points of transition in thymocyte development. In addition, mice transgenic for the dominant negative integrin chimera and for a single TCR reveal that integrin functions may be required for generation of CD4⁺ cells, but are not essential for the production of CD8⁺ cells. These results support a model in which integrin engagement of ligand modulates or contributes to signals generated by the engagement of other surface receptors to regulate the differentiation of thymocytes.

	Materials and Methods

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Transgene

The chimeric Tac1 molecule was constructed by overlapping PCR as previously described (18). Tac1 was subcloned into the p1017 expression vector using a unique BamHI restriction site. p1017 uses the proximal region of the promoter for the lck gene, allowing for thymus-specific expression of the transgene (19). Additionally, p1017 contains an intron/exon sequence from the human growth hormone gene that maintains splice acceptor sites for a more efficient expression of the transgene (20). The p1017 expression vector has been used successfully in many studies involving the thymus-specific expression of dominant negative molecules. Furthermore, studies have shown that the expression of the human Tac extracellular domain alone in murine thymocytes has no effect on the development of the thymocytes in vivo (5).

Transgenic mice

The expression construct was injected into fertilized oocytes isolated from F₁ hybrid (C57BL/6J x CBA/J) females before pronuclear fusion. Eight mice of 30 offspring were found to be positive for the transgene, with varying copy numbers of the transgene present for each founder, as analyzed by Southern blot (data not shown). Of these eight founder mice, three were bred successfully to C57BL/6J or CBA/J wild-type (WT) mice to obtain separate transgenic mouse lines: C-line mice exhibit the highest amount of Tac1 surface expression, and B- and D-line mice exhibit a decrease in Tac1 surface expression. The results presented here are primarily from C-line mice (primarily heterozygous; homozygous where indicated); however, other lines exhibited similar phenotypes in a dose-response fashion. Tac1 mice were genotyped using tail DNA for PCR with Lck- and Tac1-specific primers. AND mice were obtained from A. Abbas (University of California, San Francisco, CA) and bred with Tac1 heterozygotes, which had been screened for the absence of the Mtv-6 superantigen gene. The resulting progeny were genotyped for Tac1 and AND expression via PCR and flow cytometry for the surface expression of Tac1, V3 (of the AND TCR), and CD4. All animals were cared for under a protocol approved by the Columbia University institutional review board.

Flow cytometry

Thymi were isolated from 8-wk-old mice and were ground between the frosted ends of glass slides to release the cells. RBC lysis was performed using hypotonic solution (Sigma, St. Louis, MO). Cells then were resuspended in PBS, 5% calf serum, and 0.1% sodium azide at a concentration of 2.5 x 10⁷ cells/ml and stained (6.5 x 10⁵ cells/sample). Abs used included CD4-FITC, -allophycocyanin, -PE, (RM4-5), CD8-biotin-FITC, -PE (53-6.7), CD25-FITC, -biotin (7D4), CD44-PE (IM7), CD3-PE, biotin, -FITC (145-2C11), CD24-PE (M1/69), V3-biotin (KJ25; recognizes the AND-transgenic TCR as well as endogenous V3 receptors), CD69-biotin, -FITC (H1.2F3; all from BD PharMingen (San Diego, CA)), CD44-biotin, IM7.8.1, Caltag Laboratories (Burlingame, CA), and Ki-67 (Mib-5, Coulter, Miami, FL). For the haplotyping of mouse strains, H-2K^k-FITC (CTKk; Caltag Laboratories) and H-2D^b-PE (CTDb; Caltag Laboratories) were used. For analyses of ₄ integrins in thymocytes, rat mAbs against the ₄ subunit (SG31, PS/2, R1-2) were a gift from J. Kearney (University of Alabama, Birmingham, AL). Data were collected before the gated analyses of subpopulations using a FACStar^Plus cytometer (BD Biosciences, San Jose, CA). For all analyses, between 10,000 and 200,000 cells were collected, allowing for a minimum of 1,000 cells/subpopulation analyzed. Gating was constructed based upon negative and positive controls, cross-sample comparisons, and Ab titrations on splenocytes. Compensation controls were included in all analyses performed. Dead cells were gated out on the basis of propidium iodide incorporation, with live cells excluding this dye. Population percentages and numbers were generated for gated populations from each experiment using CellQuest software (BD Biosciences). Means and SEs of data from age-, litter-, and sex-matched groupings of mice of identical genotypes were then calculated using StatView software (SAS Institute, Cary, NC).

Adhesion experiments

All adhesion assays were performed in triplicate. FN was purified from bovine plasma by sequential gelatin and heparin affinity chromatography (21), diluted in PBS, and coated onto non-tissue culture-treated 96-well plates (Sarstedt, Newton, NC) overnight at 4°C. The remaining protein binding sites in the wells then were blocked with 1% BSA (heat treated at 70°C for 20 min) in PBS for 2 h at room temperature. Thymocytes were isolated and resuspended in cold DMEM and 0.2% BSA. Thymocytes then were added to each well (2 x 10⁶ cells/100 µl medium/well) and incubated at 37°C for 45 min. Following the addition of 100 µl PBS to each well, plates were inverted, and nonadherent cells were removed by centrifugation at 700 rpm (7 x g) for 7 min. Remaining liquid was aspirated, and adherent cells were fixed in 70% ethanol for 20 min and stained with crystal violet (0.1% in water). Excess stain was removed with water, bound color was dissolved in Triton X-100 (0.2% in distilled water), and the OD was read at 595 nm.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Thymocyte-specific expression of the Tac1 transgene in mice

To study the requirements of integrin function for the progression of T cell development, we generated mice transgenic for a chimeric integrin protein, Tac1, that exhibits dominant negative activity on the function of multiple integrins (at least _1–3) when expressed in cell lines (18, 22, 23, 24). Tac1 is comprised of the extracellular and transmembrane domains of the human CD25 molecule (known as Tac) fused to the avian cytoplasmic domain of the integrin ₁ subunit. Transcriptionally controlled by the proximal promoter for the murine lck gene, this monomeric chimera was expressed uniformly only in thymocytes (data not shown). Tac1 expression began early in T cell development, in the CD44⁺CD25^- subpopulation, and was expressed maximally in the CD44⁺CD25⁺ subpopulation (Fig. 1A). This maximal expression level was maintained throughout T cell development, decreasing only when the thymocytes were fully mature and able to exit the thymus (Fig. 1B). This thymocyte-specific expression pattern is consistent with patterns from previous studies that have used the p1017 expression vector (20, 25).

View larger version (49K): [in this window] [in a new window]   FIGURE 1. Expression patterns of the Tac1-transgenic protein and the endogenous integrin 1 subunit in thymocyte populations throughout thymocyte development. A, Surface expression of Tac1 and the endogenous integrin 1 subunit in CD44/CD25 thymocyte subpopulations within the CD4-CD8- population. Thymocytes from Tac1-transgenic mice were stained with mAbs against the human Tac Ag or the extracellular domain of the murine integrin 1 subunit, followed by fluorescein-conjugated rat anti-mouse IgG1 Abs. Thymocytes also were stained for CD3, CD4, CD8 (all the same fluorophore), CD44, and CD25 using mAbs directly conjugated to fluorophores. Following four-color flow cytometric data acquisition using a FACStarPlus cytometer, gating was performed to analyze the expression of Tac1 and endogenous 1 in CD3-CD4-CD8- subpopulations based upon the expression of CD44 and CD25. B, Surface expression patterns of Tac1 and the endogenous integrin 1 subunit in CD4/CD8 populations. Thymocytes from Tac1-transgenic mice were stained with mAbs against the human Tac Ag and the extracellular domain of the murine integrin 1 subunit and simultaneously stained for CD4 and CD8. Following four-color data acquisition, gating was performed to analyze the Tac1 and endogenous integrin 1 expression levels within CD4/CD8 populations through thymocyte development. Negative control peaks represent thymocytes stained with the secondary fluorescein-conjugated Abs only, without primary Abs.

Comparison of Tac1 expression to endogenous ₁ integrin expression

Since Tac1 is expected to act as a dominant negative inhibitor, we decided to compare its expression pattern in heterozygous mice with that of the target molecule, endogenous ₁ integrins. In the CD44⁺CD25⁺ and CD44^-CD25⁺ subpopulations of the CD4^-CD8^- population, the surface expression of endogenous ₁ integrins is maximal (Fig. 1B). In the CD44^-CD25^-, CD4⁺CD8⁺, CD4⁺, and CD8⁺ populations, the surface expression of endogenous ₁ integrins steadily decreases, while the expression of Tac1 remains constant (Fig. 1). Thus, in these later populations the ratio of Tac1 surface expression to endogenous ₁ integrin expression is greater than the ratio in the more immature populations. The expression levels of endogenous ₁ integrins do not change as a result of Tac1 expression when comparing thymocytes from WT and Tac1 mice (data not shown).

Dominant negative effects of Tac1 on endogenous ₁ integrin function

In vitro and in vivo studies have shown that the expression of chimeric integrin proteins, consisting of only the cytoplasmic domain of the integrin subunit, can have a dominant negative inhibition on the function of endogenous integrins (18, 22, 23, 25, 26, 27). This trans-dominant inhibition by a single family member can affect multiple endogenous integrin family members (most likely due to the high degree of homology among many different integrin subunits). To test for dominant negative activity of Tac1 in vivo, we analyzed integrin function from thymocytes isolated from Tac1-transgenic and WT mice. Thymocytes from the early developmental populations (CD4^-CD8^- and CD4⁺CD8⁺) are able to bind extracellular matrix molecules upon isolation (28, 29). These results indicate that the endogenous integrins that mediate the binding (mainly the ₁ family of integrins) are in an active conformation, capable of binding ligand without further treatment in these early populations. This finding is in contrast with later populations (single positives; CD4⁺ or CD8⁺) that have a decreased ability to bind ligand and maintain latent integrin extracellular conformations (29) (data not shown). In the presence of Tac1, however, there is a dramatic decrease in the ability of all thymocytes to bind to FN, one of the major extracellular matrix molecules found in the thymus (Fig. 2A).

View larger version (38K): [in this window] [in a new window]   FIGURE 2. The dominant negative effect of Tac1 on integrin receptor function. A, Adhesion of thymocytes from WT and Tac1 mice to increasing concentrations of FN. Thymocytes were isolated and adhered in triplicate to increasing concentrations of FN immobilized in 96-well plates. Following the removal of unbound cells, bound cells were fixed and stained with crystal violet and analyzed for the total cell retention of the dye at A595. To test for integrin-mediated specificity of the adhesion, EDTA was added to the cell culture medium. In the absence of divalent cations, integrin-FN binding is greatly reduced. B, The mAb SG31 recognizes the integrin 4 subunit extracellular domain only early in thymocyte development, when 4 is in an activated state. PS/2 is a mAb that can recognize the integrin 4 subunit extracellular domain constitutively throughout thymocyte development. Thymocytes from WT mice were stained with either SG31 or PS/2, followed by a secondary Ab conjugated to a fluorophore. Thymocytes then were stained with mAbs against CD4 and CD8, both directly conjugated to different fluorophores. C, In the presence of Tac1 expression, the recognition of the integrin 4 subunit by the activation-sensitive mAb SG31 is substantially reduced, early in thymocyte development. In populations of later stages of thymocyte development where SG31 binding already is minimal, no substantial changes in Ab recognition occur in the presence of Tac1. Thymocytes from WT and Tac1-transgenic mice were stained with SG31, CD4, and CD8 as described in B. Overlay of the analyzed data was performed using CellQuest software (BD Biosciences). D, In the presence of Tac1 expression, the recognition of the integrin 4 subunit by the mAb PS/2 is not affected throughout thymocyte development. Thymocytes from WT and Tac1-transgenic mice were stained with PS/2, CD4, and CD8 as described in C. Overlay of the analyzed data was performed using CellQuest software (BD Biosciences). Negative control peaks were from thymocytes stained with the secondary Ab alone. B–D, similar analyses were performed a total of seven times, with similar results in all cases. E, In the presence of the phorbol ester PMA, the adhesion of thymocytes expressing Tac1 to 10 µg/ml FN is rescued to the levels of WT thymocytes adhering to 10 µg/ml FN. The adhesion experiment was performed as described in A. Each bar represents an average of 10 samples.

Mechanism of the dominant negative effect of Tac1

The loss of adhesive ability in the Tac1 thymocytes could be due to a number of factors. However, since endogenous integrin ₁ surface expression does not change in the presence of Tac1 surface expression (data not shown), we suspected that there was a decreased level of integrin activation, which has been shown to occur with Tac1 in Chinese hamster ovary cells (22). To determine whether this disruption of cell adhesion was the result specifically of trans-dominant inhibition by Tac1 on endogenous integrin activation, we used mAbs to assay the extracellular conformations of endogenous ₄₁ integrins. The mAb PS/2 constitutively recognizes the ₄ integrin subunit. The mAb SG31, however, recognizes the ₄ subunit only when ₄₁ is capable of binding to FN in the earlier developmental populations (Fig. 2B) (30). In CD4^-CD8^- thymocytes expressing Tac1, there is no change in the binding of PS/2 to thymocytes (Fig. 2D), but there is a decrease in the amount of SG31 binding to the cells compared with thymocytes from WT mice (Fig. 2C). Since integrin activation states can be regulated by extracellular conformational changes, these results suggest that one of the mechanisms of Tac1’s dominant negative activity is to disrupt integrin activation. If so, then a strengthening of the signals that drive integrin activation might help to restore some of the loss of function mediated by Tac1 expression.

Signals that drive integrin activation often rely on protein kinase C (31, 32, 33). In the presence of phorbol esters, protein kinase C activity is enhanced, leading to an enhancement of integrin activation as well. When Tac1-expressing thymocytes are exposed to phorbol esters, cell adhesion to FN is restored to the normal WT thymocyte levels (Fig. 2E). Endogenous ₁ and Tac1 expression levels do not change in the presence of PMA (data not shown), consistent with its known effects on integrin inside-out signaling. Thus, we conclude that Tac1 reduces thymocyte adhesion to FN by blocking integrin activation, and this effect can be rescued with protein kinase C activation.

Effect of dominant negative activity of Tac₁ on T cell development

Several changes occur in T cell developmental populations in the heterozygous mice expressing Tac1. First, while the total cell number of the thymus is slightly decreased, an increase is observed in the numbers of CD4^-CD8^- cells, with decreases observed in the CD4⁺CD8⁺, CD4⁺, and CD8⁺ populations (Tables I–III). By concurrent staining with CD24/HSA and Thy-1, we demonstrate that the increase in CD4^-CD8^- cells is specific to thymocytes and not due to fibroblasts and thymic epithelial cells (data not shown). The increase in the CD4^-CD8^- population also does not concur with any increase in CD4^-CD8^-CD3^highCD24^- thymocytes, which may represent thymocytes that have progressed further in development but then failed to proceed to a mature state, instead reverting back to a CD4^-CD8^- phenotype (Fig. 3) (34). Finally, an analysis of thymocytes expressing the TCR indicates that the increase in CD4^-CD8^- cells in Tac1 mice is not due to increases in TCR cells (data not shown). Thus, the increased cells are immature thymocytes destined to become TCR T cells.

View larger version (18K): [in this window] [in a new window]   FIGURE 3. A representative comparison of CD4-CD8- thymocytes from WT and Tac1 mice for the expression of CD24 and CD3. Thymocytes were isolated and stained for CD4, CD8, CD3, and CD24, all directly conjugated to fluorophores. After data acquisition, CD4-CD8- thymocytes were isolated by gating and the percentages of CD3-, CD3+, CD24-, and CD24+ cells from the CD4-CD8- population were determined. CD24+CD3- cells account for the majority of the increase in the CD4-CD8- population in Tac1 mice.

Tac1-mediated disruption of thymocyte differentiation in embryonic development

We decided to test the idea that Tac1 expression leads to a disruption of thymocyte differentiation from the CD4^-CD8^- to the CD4⁺CD8⁺ stage by examining thymocyte development in embryonic mice. On embryonic day 14 the thymocytes are essentially all in the immature CD4^-CD8^- population (35). Between embryonic days 14–17 there is a dramatic differentiation of these immature cells into CD4⁺CD8⁺ cells. On day 15, the CD4^-CD8^- population is fully developed, and by day 17 the CD4⁺CD8⁺ population has fully developed. Tac1 is expressed on day 14, and the expression remains constant throughout the remainder of fetal development (data not shown). Comparing embryonic thymus development between WT and Tac1 mice, no changes are apparent in the development of the CD4^-CD8^- population at early time points. However, as the CD4⁺CD8⁺ population begins to emerge, increases in the CD4^-CD8^- cell numbers and decreases in the CD4⁺CD8⁺ population cell numbers arise in the Tac1 mice compared with their WT littermates (Fig. 4). Since no differences are apparent in the proliferating CD4^-CD8^- population until the cells begin to differentiate, we conclude that the expression of Tac1 leads to a block in the differentiation of CD4^-CD8^- cells to CD4⁺CD8⁺.

View larger version (17K): [in this window] [in a new window]   FIGURE 4. Analysis of thymocyte populations from day 15 to 17 embryonic WT and Tac1 mice. Thymocytes were isolated from embryonic WT or Tac1 littermates and stained for CD4 and CD8. Following data acquisition, dual-parameter histograms were constructed, and percentages of the CD4-CD8- and CD4+CD8+ populations were determined and averaged (n = 4). By embryonic day 17 increases occur in the CD4-CD8- population from Tac1 mice, while decreases occur in the CD4+CD8+ population from Tac1 mice.

Until this point, all data concerning the phenotype of Tac1 mice were collected using heterozygous mice. Although a clear phenotype emerged in these mice, the effect of Tac1 expression was not strong, requiring a large number of analyses to verify the effect. Since we only knew the ratios of the surface levels of Tac1 vs the endogenous ₁ integrins within populations, we decided to analyze mice homozygous for the Tac1 transgene to determine whether the phenotype would increase with a higher ratio of Tac1 to endogenous ₁ integrins. Homozygous mice were analyzed for the expression of Tac1, and compared with heterozygous littermates. These mice had a 60% higher surface expression of Tac1 (data not shown). When the thymocytes were stained for CD4 and CD8 expression, the comparison with WT thymocytes showed results similar to those for the heterozygotes; however, the changes were greater (Fig. 5). There are increases in the CD4^-CD8^- population and a decreased CD4⁺ population, but, surprisingly, slight increases in the CD8⁺ population emerged. Thus, increasing the amount of Tac1 has a profound effect on thymocyte differentiation, suggesting that the expression of this trans-dominant inhibitor was the limiting factor for the appearance of the dominant negative effect.

View larger version (35K): [in this window] [in a new window]   FIGURE 5. CD4/CD8 dual-parameter histograms of thymocyte populations. Thymocytes were isolated from WT, Tac1 heterozygote (+/-), and Tac1 homozygote (+/+) mice and stained with mAbs for CD4 and CD8 directly conjugated to fluorophores. The numbers are based on averages from seven analyses of WT, +/-, and +/+ littermates.

The CD4^-CD8^- population can be further analyzed for subpopulations according to the expression of CD25 and CD44, as described above. Analysis of the subpopulations was performed on WT, heterozygous as well as homozygous Tac1 mice. The results show that there is an increase in the CD44^-CD25⁺ subpopulation, with the final CD44^-CD25^- subpopulation showing a modest decrease in size (Fig. 6). The increases in the CD44^-CD25⁺ subpopulation followed by a reduction in CD44^-CD25^- cells suggests that Tac1 blocks the differentiation between these last two steps.

View larger version (41K): [in this window] [in a new window]   FIGURE 6. CD44/CD25 dual-parameter contour plot histograms of thymocytes from WT, heterozygous Tac1 mice (+/-), and homozygous Tac1 (+/+). Thymocytes were stained for CD44, CD25, CD3, CD4, and CD8. Following data acquisition, cells positive for CD3, CD4, and CD8 were excluded, and gating was performed using positive and negative controls and cross-sample comparisons. For the direct comparison of WT, +/-, and +/+ littermates, there were two analyses completed. For the comparison of WT and +/- littermates, there were 17 analysis performed, all showing the same trend, i.e., an increase in the CD25+ population in the presence of Tac1.

Tac1 effects on later stages of thymocyte development

As shown above, the homozygous Tac1 mice also clearly exhibit decreased numbers of CD4⁺ cells in the thymus, with a slight increase in CD8⁺ (Fig. 5). These effects on CD4⁺ cells in the thymus were mirrored by changes in the periphery as well. We assayed the number of CD4⁺ or CD8⁺ T cells in the spleens of WT vs Tac1 homozygous mice and found that there was a change in the ratio of CD4⁺:CD8⁺ cells (Table IV). In Tac1 mice, there was a marked decrease in the percentage of CD4⁺ cells, with an increase in CD8⁺ cells. We wanted to know whether these changes resulted from the disruptions of differentiation in early stages of development as described above, or whether Tac1 was affecting later stages of thymocyte development as well. In the later stages of development following the period of cell proliferation, thymocytes are positively selected on the basis of the ability of the cell’s TCR to recognize and bind with sufficient affinity self-MHC receptors on APC. For those thymocytes that are positively selected, they then migrate into the medullary regions of the thymus, where they are negatively selected again on the basis of the TCR-MHC interactions. Cells that fail to be positively selected or cells that are negatively selected will die before they are able to exit the thymus (3, 36, 37).

Tac1 effects in AND mice

In the earlier stages of T cell development, the lack of expression of the TCR-CD3 complex allows each cell to undergo very similar patterns of development. However, since thymocytes express a variety of TCR conformations due to rearrangements of the TCR gene, there is heterogeneity in the positive or negative selection of each thymocyte. To follow a single pattern of development, mice transgenic for a TCR against pigeon cytochrome c were crossed with mice transgenic for Tac1. The TCR-transgenic mice express the AND TCR that drives the cell to undergo positive selection and avoid negative selection to become a CD4⁺ cell (38). This effect is dependent on the nature of the MHC molecules as well. MHC molecules of the k haplotype allow for a large degree of positive selection, MHC molecules of the b/k haplotype allow for a lesser degree of selection, and MHC molecules of the b haplotype allow for the least positive selection of CD4⁺CD8⁺ thymocytes to become CD4⁺ cells (39).

In the presence of Tac1, the AND mice exhibit different changes compared with WT mice. There is a greater expansion of the CD4^-CD8^- population, and there are decreases in the CD4⁺CD8⁺ and CD4⁺ populations. The decrease in the CD4⁺ is proportionally greater than the decrease in the CD4⁺CD8⁺ population. Although the increase in the CD4^-CD8^- population is similar over all the different haplotype backgrounds, the decrease in the CD4⁺ population is proportionally greater on the higher selecting backgrounds and decreases on the b background. The CD8⁺ population increases slightly on all MHC haplotype backgrounds (Tables I–III and Fig. 7). The additional expansion of the CD4^-CD8^- population in AND/Tac1 mice compared with Tac1 mice most likely results from the fact that the expression of the AND TCR leads to more CD4^-CD8^- cells that express lower levels of endogenous ₁ integrins (data not shown; see also Discussion).

View larger version (45K): [in this window] [in a new window]   FIGURE 7. Dual-parameter histograms of thymocyte developmental populations. CD4/CD8 dual-parameter contour plot histograms of thymocytes from AND and AND/Tac1 mice. Thymocytes were stained with mAbs directly conjugated to fluorophores. After data acquisition, gating was performed using positive and negative controls and cross-sample comparisons. Numbers for these samples, including n of analyses, are listed in Tables I–III.

The decreases in the CD4⁺ population could result from a disruption of positive selection or an increase in negative selection or could be due to the earlier block in differentiation. The fact that the number of CD4⁺CD8⁺ cells, which are precursors to the mature CD4⁺ cells, does not increase (in fact, it decreases slightly) suggests that a simple block in positive selection of CD4⁺ cells alone is not likely (see Discussion). However, an increase in negative selection of CD4⁺ cells could account for the phenotype. To test whether there is an increase in the negative selection of thymocytes in the presence of Tac1, thymocyte populations were stained for the presence of annexin V binding to the cell membrane. A vital dye such as 7-amino-actinomycin D (7-AAD) or propidium iodide was added in conjunction with the staining. Annexin V binding is a measurement of cells in the early stages of apoptosis, the cell death associated with negative selection. The incorporation of a vital dye then measures cells whose membranes have permeabilized and are in later stages of apoptosis. In the presence of Tac1, there is no increase in the numbers of thymocytes either in the early or later stages of apoptosis (Fig. 8).

View larger version (43K): [in this window] [in a new window]   FIGURE 8. Annexin V binding and the inclusion of 7-AAD in thymocytes from WT and Tac1 mice. Thymocytes were incubated with annexin V directly conjugated to a fluorophore and 7-AAD. Following data acquisition on a FACSCalibur cytometer, gates were applied for mature populations; dual-parameter histograms were constructed, and the percentages of cells that are annexin V+ or annexin V+ 7-AAD+ were determined. Slight decreases in these percentages occur in thymocytes from Tac1 mice compared with WT mice. The numbers shown are representative for this analysis; however, four separate analysis were performed with similar results; that is no increase in apoptosis seen in Tac1 mice.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Previously, in vitro systems have shown a requirement for integrins and integrin-related proteins for the maturation of CD4^-CD8^- to CD4⁺CD8⁺ cells (12, 13, 14). Most of the in vitro data in mice point to the CD4^-CD8^- cells as being adhesive cells with activated integrins. These cells are strongly adherent to FN and VCAM and bind well in vitro without the addition of activators, such as PMA (11, 13). It has been reported (13) that CD4^-CD8^- cells have high levels of constitutively active ₄₁. Since it has been shown in Chinese hamster ovary cells that Tac1 can act as a trans-dominant inhibitor of integrin inside-out signaling (22), we considered the possibility that Tac1 thymocytes may have a reduced adhesion via decreased activation, particularly in the CD4^-CD8^- population. In this case we consider activation as an increased ability to bind ligand regardless of whether it is due to conformation changes, clustering, or both.

Indeed, we found that Tac1 thymocytes in vitro have a marked reduction in FN adhesion compared with their negative littermates. This reduced adhesion is associated with a decrease in expression of the epitope for the anti ₄ Ab SG31, which is associated with an active conformation. This Ab binds when the cells are most adhesive to ligands for ₄₁, or when integrins are stimulated via Mn²⁺ treatment (30). It may be indicative of a ligand-occupied conformation, rather than a probe of all active conformations (however, ligands can "activate" integrins (40), making it difficult to be sure which conformations are measured). We believe this Ab is a useful probe that demonstrates the presence in vivo of ₄₁, which is highly capable of binding ligand. Although we have used SG31 as an assay for suppression of activation, many integrins besides ₄₁ could be affected by Tac1. Most likely ₅₁, another FN receptor and ₆₁, a receptor for laminin and merosin, as well as other integrins are affected by Tac1.

Despite the significant reduction in vitro of the adhesion of Tac1 heterozygous thymocytes to FN, in vivo these thymocytes showed a very modest phenotype, including an increase in CD4^-CD8^- cells and a decrease in CD4⁺ cells. Is this due to a limited role in vivo for integrins, or is this a result of insufficient inhibition in vivo by this trans-dominant inhibitor of integrins? Our results suggest the latter. When we increased the amount of Tac1 expression by using homozygous mice, the phenotype increased. Furthermore, in the AND mice in which there are more CD4^-CD8^- cells that express lower levels of endogenous ₁ than in WT mice (presumably due to inappropriate early expression of the TCR; data not shown), the Tac1 phenotype is increased. Thus, we believe that integrins do play a significant role in the differentiation of immature thymocytes in vivo, and we are just beginning to see that role as the ratio of Tac1 to endogenous integrins is increased. In fact, when we combine the decrease in endogenous integrins in the AND mice with a Tac1 homozygous gene dose, we see a 10-fold increase in CD4^-CD8^- cells (data not shown). Consistent with the increased abilities of the immature thymocyte populations to bind extracellular matrix molecules, the requirements for integrin functions seem to be greatest for those populations.

The decrease in mature CD4⁺ cells seen in Tac1 mice was investigated using TCR-transgenic mice to analyze the role of integrins in positive selection. In vitro, in both peripheral T cells and thymocytes, integrins are costimulatory with CD3. Since selection through the TCR stimulation in vivo is thought to be similar to these in vitro assays, we expected that integrins may be costimulatory in positive selection as well. Thus, by lowering integrin signaling, we could have reduced the costimulatory signal necessary for some TCRs to undergo positive selection. However, our studies with AND mice suggest that a perturbation of positive selection is not the major reason for the decrease in CD4⁺ cells.

In the AND mice, changing the class II background leads to changes in the degree of selection. Thus, when the mice are on a strong positive selection background, such as b/k, they show a large CD4⁺, with a smaller CD4⁺CD8⁺ population. As the selection gets weaker, there is a concomitant decrease in CD4⁺ and an increase in the CD4⁺CD8⁺ population, somewhat akin to a precursor-product relationship. In the presence of Tac1, in the stronger selection background there is a decrease of CD4⁺ cells compared with WT, but also a modest decrease in CD4⁺CD8⁺ cells. As the selection strength gets weaker, the Tac1 effect is also less. Thus, while the AND system demonstrates a relationship between the Tac1 phenotype and the strength of selection, the block between CD4^-CD8^- cells and CD4⁺CD8⁺ cells appears to exert a greater effect on subsequent populations.

Due to the strong allelic exclusion of the AND TCR, there is limited production of CD8⁺ cells. Interestingly, however, there is an increase in the number of CD8⁺ cells in the Tac1 mice compared with their negative littermates. Thus, it appears that Tac1 affects the production of CD4⁺ precursor cells, with little effect or a relative increase in the production of CD8⁺ precursors. These results are consistent with those of Hedrick and coworkers (41), who showed that a strong mitogen-activated protein kinase signal is required for CD4⁺ differentiation, while blocking that signal favored CD8⁺ production. They propose that the CD4⁺ lineage should be referred to as the primary cell fate, and the CD8⁺ lineage as the secondary one. Thus, the results for the homozygous Tac1 and Tac1/AND mice suggest that a disruption of integrin function also leads to a shift from the primary to the secondary fate. This model could explain the shift within the spleens of these animals to an increased CD8⁺:CD4⁺ ratio (Table IV and data not shown). Since the changes in CD8⁺ cells do not exactly mirror the changes in CD4⁺ cells, cell fate determination probably does not fully account for the decrease in the CD4⁺ population.

Integrin-mediated signaling has been shown to act through signaling molecules used by other cell surface receptors. Most notably, integrin signaling can act through Src-related kinases and the Ras pathway (42, 43). It is likely, then, that, in addition to acting as mediators of cell adhesion as thymocytes migrate through the thymus, integrins act to generate signaling pathways that drive or regulate the maturation of the thymocytes. In fact, since no real defects are seen in thymocytes migration in situ (data not shown), and since the Tac1-mediated disruptions can be placed at distinct points of development, it is likely that the major role of integrins for T cell development is to generate intracellular signals. Although we do not believe that Tac1 would have direct effects on other (nonintegrin) receptors, we cannot rule out indirect effects on those receptors, since cell adhesion is required for full growth factor responses (43).

Since we know that CD4^-CD8^- cells are receiving inside-out signals to stimulate adhesion, it is not surprising that the effects on differentiation appear to be focused in this population. However, what is surprising is the fact that there appears to be a specific delay at the CD25⁺ stage within this group. This stage is interesting because it is associated with Lck-mediated pre-TCR signaling. Thus, these results suggest that integrin signals and Lck signals may be corequirements for progression through this stage of development. Interestingly, knockouts of the negative regulator of Src family kinases, Csk, allow for Ag-independent development of CD4⁺ cells, but not CD8⁺ cells (44). However, a constitutively activated lck transgene drives the production of both CD4⁺ and CD8⁺ cells (45). Thus, the effects on CD4, but not CD8, production seen in the Tac1 mice could also be consistent with partial effects on Lck/Fyn signaling. In fact, homozygous Tac1 mice bred with Lck-null mice lead to a synergistic block of CD4^-CD8^- cells, suggesting that integrins are necessary for the residual Fyn activity that allows some CD4^-CD8^- differentiation to occur in the absence of Lck (H. Xie and E. E. Marcantonio, unpublished results). We are currently performing a number of experiments to test the model that a major role for integrins in T cell development involves Src family kinase signaling, as it does in fibroblasts (46).

	Acknowledgments

 
We gratefully acknowledge Dr. John Kearney for the anti-₄ mAbs and Dr. Roger Perlmutter for the p1017 expression vector. Special thanks to Dr. Gerald Siu for all of his basic immunology advice. We also thank Drs. Gerald Siu and Paul Rothman for critical reading of this manuscript. E.E.M. dedicates this paper to the memory of Dr. Ramzi Cotran, a friend and mentor.

	Footnotes

 
¹ This work was supported by National Institutes of Health Grant AI41600 (to E.E.M.).

² Address correspondence and reprint requests to Dr. Eugene E. Marcantonio, Department of Pathology, Columbia University College of Physicians and Surgeons, 630 West 168th Street, Black Building 1422, New York, NY 10032. E-mail address: eem2{at}columbia.edu

³ Abbreviations used in this paper: FN, fibronectin; 7-AAD, 7-amino-actinomycin D; WT, wild type.

Received for publication February 9, 2001. Accepted for publication July 24, 2001.

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