Comparison of thymocyte development and cytokine production in CD7-deficient, CD28-deficient and CD7/CD28 double-deficient mice

Craig S. Heinly1, Gregory D. Sempowski1,2, David M. Lee1, Dhavalkumar D. Patel1,2, Patrice M. McDermott1, Richard M. Scearce1, Craig B. Thompson3 and Barton F. Haynes1,2

1 Division of Rheumatology, Allergy and Clinical Immunology, Department of Medicine, and
2 Department of Immunology and the Duke University Human Vaccine Institute, Duke University Medical Center, Durham, NC 27710, USA

Correspondence to: Correspondence to: B. F. Haynes, Box 3703, Duke Hospital, Durham, NC 27710, USA


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
CD7 and CD28 are Ig superfamily molecules expressed on thymocytes and mature T cells that share common signaling 0mechanisms and are co-mitogens for T cell activation. CD7-deficient mice are resistant to lipopolysaccharide (LPS)-induced shock syndrome, and have diminished in vivo LPS-triggered IFN-{gamma} and tumor necrosis factor (TNF)-{alpha} production. CD28-deficient mice have decreased serum Ig levels, defective IgG isotype switching, decreased T cell IL-2 production and are resistant to Staphylococcus aureus enterotoxin-induced shock. To determine synergistic roles CD7 and CD28 might play in thymocyte development and function, we have generated and characterized CD7/CD28 double-deficient mice. CD7/CD28-deficient mice were healthy, reproduced normally, had normal numbers of thymocyte subsets and had normal thymus histology. Anti-CD3 mAb induced similar levels of apoptosis in CD7-deficient, CD28-deficient and CD7/CD28 double-deficient thymocytes as in control C57BL/6 mice (P = NS). Similarly, thymocyte viability, apoptosis and necrosis following ionomycin or dexamethasone treatment were the same in control, CD7-deficient, CD28-deficient and CD7/CD28-deficient mice. CD28-deficient and CD7/CD28-deficient thymocytes had decreased [3H]thymidine incorporation responses to concanavalin A (Con A) stimulation compared to control mice (P <= 0.01 and P <= 0.05 respectively). CD7/CD28 double-deficient mice had significantly reduced numbers of B7-1/B7-2 double-positive cells compared to freshly isolated wild-type, CD7-deficient and CD28-deficient thymocytes. Con A-stimulated CD4/CD8 double-negative (DN) thymocytes from CD7/CD28 double-deficient mice expressed significantly lower levels of CD25 when compared to CD4/CD8 DN thymocytes from wild-type, CD7-deficient and CD28-deficient mice (P < 0.05). Anti-CD3-triggered CD7/CD28-deficient thymocytes also had decreased IFN-{gamma} and TNF-{alpha} production compared to C57BL/6 control, CD7-deficient and CD28-deficient mice (P <= 0.05). Thus, CD7 and CD28 deficiencies combined to produce abnormalities in the absolute number of B7-1/B7-2-expressing cells in the thymus, thymocyte IL-2 receptor expression and CD3-triggered cytokine production.

Keywords: apoptosis, co-stimulatory molecules, thymus


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Human CD7 is a 40 kDa member of the Ig gene superfamily that is expressed on most mature T and NK cells as well as on bone marrow-derived T, B, NK and myeloid lineage precursors (18). CD7 is one of the earliest surface molecules for cells of the T and NK cell lineages (4,7,8).

Human CD28 is a 44 kDa homodimeric glycoprotein which, like CD7, is also a member of the Ig gene superfamily (3,912). CD28 is expressed at low density on most immature thymocytes, at higher density on most mature thymocytes (10,13) and has been implicated in regulating thymocyte negative selection (1416). CD28 is present on ~95% of peripheral CD4+ T cells and on 50% of peripheral CD8+ T cells (10,13,17). Activation through CD28 is a critical co-stimulatory pathway for T cells. Cross-linking of CD28 synergizes with TCR-mediated signals in enhancing T cell proliferation, cytokine production and effector function (10,1823).

Construction of mouse strains deficient in either CD7 or CD28 has shown that thymocyte development is normal, although overproduction of double-positive thymocytes has been suggested in both mouse strains (15,24,25). Impaired apoptosis following TCR–CD3 ligation on thymocytes has been reported in CD28-deficient mice (15), although positive and negative T cell selection in CD7-deficient mice was normal (D. Lee et al., unpublished). Co-stimulation of T cells via CD28 confers an enhanced ability to expand the peripheral T cell pool (26), and is important in augmenting peripheral T cell production of IL-2, IFN-{gamma} and tumor necrosis factor (TNF)-{alpha} (2729). Similarly, CD7-deficient peripheral T cells have defects in peripheral T cell antigen-induced IFN-{gamma} and TNF-{alpha} production (24). Moreover, CD7-deficient mice are resistant to LPS-induced shock, and have decreased in vivo IFN-{gamma} and TNF-{alpha} production in this setting (30).

Even though both CD7 and CD28 are expressed early in T cell ontogeny, the roles that CD7 and CD28 might play in T cell development remain unclear. Studies in CD7-deficient and CD28-deficient mice have suggested key roles for these molecules in mediating peripheral T cell co-stimulation (CD28) and cytokine production (CD28 and CD7) (20,31), and in mediating shock induced by lipopolysaccharide (LPS) (CD7) and Staphylococcus aureus toxin, TSST-1 (CD28) (30,32).

Thus, to study the synergistic roles that CD7 and CD28 may play in thymocyte development and function, we have bred CD7-deficient and CD28-deficient mice to produce CD7/CD28 double-deficient mice, and evaluated their thymocyte development and function. We found that while thymocyte numbers and induced thymocyte apoptosis was normal in CD7-deficient, CD28-deficient and CD7/CD28 double-deficient mice, deficiency in both CD7 and CD28 induced abnormalities in thymocyte cytokine production.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
C57BL/6 mice were obtained from Jackson Laboratories (Bar Harbor, ME). Homozygous CD7-deficient (CD7–/–) mice were generated as previously described (24). Homozygous CD28-deficient (CD28–/–) mice were generated as previously described (33). Both CD7-deficient and CD28-deficient mouse lines were backcrossed five generations onto C57BL/6. Homozygous CD7/CD28 double-deficient (CD7–/–/CD28–/–) mice were generated by crossing the CD7–/– and CD28–/– mice to achieve F1 heterozygotes at both loci, and then by mating the F1 to obtain homozygosity. Detection of the disrupted genes was performed using specific primers to amplify genomic tail DNA (24,33).

Antibodies
Hybridoma 145-2C11 (anti-murine CD3{varepsilon}) was generously provided by Dr Jeff Bluestone (UCSF, San Francisco, CA) (34). mAb 145-2C11 was purified from culture supernatant using an Immunopure Protein A/G column according to the manufacturer's protocol (Pierce, Rockford, IL). mAb 37.51 (anti-murine CD28) was the generous gift of Dr James P. Allison (UC Berkeley, Berkeley, CA) (35). Hybridomas were cultured in serum-free medium (Life Technologies, Grand Island, NY).

Flow cytometry
Thymus tissues were teased with a 1 cm3 tuberculin syringe plunger (Becton Dickinson, Franklin Lakes, NJ) to achieve a single-cell suspension. Thymocytes were washed in RPMI 1640 medium with L-glutamine (Life Technologies) supplemented with 10% FCS (Hyclone, Logan, UT), 5.5x10–5 M 2-mercaptoethanol and 10 µg/ml gentamicin (BioWhittaker, Walkersville, MD), and counted on a Coulter counter (Coulter Electronics, Hialeah, FL). Then 1x106 thymocytes were incubated (30 min, 4°C) with saturating amounts of antibody. mAb used for immunofluorescent staining were rat anti-mouse CD3 (Sigma, St Louis, MO), rat anti-mouse CD4 (Caltag, Burlingame, CA), rat anti-mouse CD80 (PharMingen), rat anti-mouse CD86 (PharMingen), rat anti-mouse CD25 (PharMingen) and rat anti-mouse CD8{alpha} (Caltag). Cells were washed 2 times with 3 ml PBS wash (1xPBS, 1% BSA and 0.1% NaN3) and resuspended (1x106 cells/ml in PBS wash with 0.4% paraformaldehyde). Samples were analyzed on a FACStar Plus flow cytometer (Becton Dickinson, Mountain View, CA) in the Duke University Center for AIDS Research Flow Cytometry Facility (Durham, NC).

Thymocyte viability assay
Thymocytes (1x106 cells/ml) were cultured in 48-well tissue culture plates (Costar, Cambridge, MA) in RPMI 1640 medium with L-glutamine (Life Technologies) supplemented with 10% FCS (Hyclone), 5.5x10–5 M 2-mercaptoethanol and 10 µg/ml gentamicin (BioWhittaker), with and without 20U/ml murine rIL-2 (R & D Systems, Minneapolis, MN) for 0–96 h. Following incubation at 37°C in a 5% CO2 humidified incubator, 5x105 thymocytes were harvested and the percentage of viable cells determined using multiparameter flow cytometry with Annexin V–FITC and propidium iodide (PI) (Immunotech/Coulter, Marseille, France), according to the manufacturer's protocol (36,37). Samples were analyzed on an Epics XL/MCL flow cytometer (Coulter, Miami, FL) in the Duke University Center for AIDS Research Flow Cytometry Facility (Durham, NC).

Thymocyte apoptosis/necrosis assays
Thymocytes (1x106 cells/ml) were cultured in 48-well tissue culture plates (Costar) in RPMI 1640 medium with L-glutamine (Life Technologies) supplemented with 10% FCS (Hyclone), 5.5x10–5 M 2-mercaptoethanol and 10 µg/ml gentamicin (BioWhittaker). Cells were incubated alone or in the presence of immobilized anti-CD3 (145-2C11; 10 µg/ml in 1xPBS), dexamethasone (Sigma; 10–7 to 10–9 M in DMSO) or ionomycin (Calbiochem, La Jolla, CA; 5 to 0.2 µg/ml in DMSO). Following incubation at 37°C in a 5% CO2 humidified incubator, 5x105 thymocytes were harvested and the percentage of viable, apoptotic and necrotic cells were determined using multiparameter flow cytometry with Annexin V–FITC and PI (Immunotech/Coulter) according to the manufacturer's protocol.

Thymocyte [3H] thymidine incorporation assays
Single-cell suspensions of thymocytes (1x106 cells/ml) in RPMI 1640 with L-glutamine (Life Technologies) supplemented with 10% FCS, 5.5x10–5 M 2-mercaptoethanol and 10 µg/ml gentamicin (BioWhittaker) were cultured in 96-well plates (Costar, Cambridge, MA). Then 1x105 thymocytes were incubated in the presence of either medium alone, immobilized anti-CD3 (145-2C11; 0–10 µg/ml) or concanavalin A (Con A; Sigma; 0–10 µg/ml) with and without 20U/ml murine rIL-2 (R & D Systems) at 37°C in 5% CO2 in air for 3 days. Six h prior to harvesting, 0.4 µCi [3H]thymidine (NEN, Boston, MA) was added to each well. Plates were harvested with a PHD cell harvester (Cambridge Technology, Watertown, MA) onto glass fiber filters. The c.p.m. values were determined using a Minaxiß Tri-CARB 4000 series liquid scintillation counter (Packard Instrument, Downers Grove, IL).

Assays of cytokine production
Quantification of murine IFN-{gamma} and TNF-{alpha} present in thymocyte culture supernatants was determined using Duoset cytokine-specific ELISA kits (Genzyme, Cambridge, MA).

Statistics
Experimental and control groups were compared using Student's t-test.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Thymocyte subset analysis and in vitro thymocyte proliferation to Con A and anti-CD3 mAb
CD7/CD28 double-deficient mice were healthy and reproduced normally. In contrast to the initial characterizations of CD7-deficient (24) and CD28-deficient (15) mice, no significant differences were seen in absolute thymocyte numbers among the four groups of animals (n>=20) (Table 1Go) in this present study of expanded numbers of mice. In addition, no abnormalities were observed in thymic histology in the three groups of animals (data not shown).


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Table 1. Comparison of thymocyte total cell number in CD7-deficient, CD28-deficient and CD7/CD28 double-deficient and control mouse thymusa
 
Similarly, no differences were found in the CD4/CD8 double-positive (DP) and CD8 single-positive (SP) thymocyte subsets among the four mouse lines (Table 2Go). In addition, the percentages and absolute numbers of CD3+ cells were not significantly different among the four mouse strains (Table 2Go). However, CD7-deficient mice had fewer CD4 SP thymocytes than both the CD28-deficient and CD7/CD28 double-deficient mice, although none of these groups showed a significant difference in CD4 SP thymocyte numbers from control C57BL/6 mice (Table 2Go). These modest differences in CD4 SP thymocytes did not translate into fewer CD4 SP T cells in the periphery (data not shown). CD7-deficient mice also had fewer CD4/CD8 DN thymocytes than control C57BL/6 mice (Table 2Go).


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Table 2. Comparison of thymocyte subsets in CD7-deficient, CD28-deficient and CD7/CD28 double-deficient and control mouse thymusa
 
The role of CD28 in T cell co-stimulation has been well described (1821,28,38). Proliferative defects to Con A, which are not fully restored with rIL-2, have been reported in CD28-deficient splenocytes (33). However, thymocyte proliferation to mitogens has not been studied in CD28-deficient mice. Therefore, the proliferative response to Con A and CD3 mAb of cultured thymocytes from CD7-deficient, CD28-deficient and CD7/CD28-deficient mice was evaluated.

When thymocytes from the four mouse lines were stimulated with Con A in the absence of rIL-2, CD7-deficient thymocytes proliferated normally compared to control C57BL/6 thymocytes. In contrast, thymocytes from CD28-deficient mice had diminished Con A-triggered [3H]thymidine incorporation compared to C57BL/6 thymocytes (P<=0.01; Fig. 1AGo). CD7/CD28 double-deficient thymocytes proliferated to Con A greater than CD28-deficient thymocytes (P<=0.002) and less than CD7-deficient thymocytes (P<=0.027). After addition of rIL-2 to the Con A proliferation assay, both CD28-deficient and CD7/CD28-deficient thymocytes proliferated normally (Fig. 1BGo). Taken together, these data suggested that in the setting of Con A stimulation, CD7 may play a subtle negative regulatory role in thymocyte proliferation. We found no detectable endogenous IL-2 production following Con A stimulation by thymocytes from the four strains.



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Fig. 1. [3H]Thymidine incorporation following Con A stimulation of thymocytes isolated from C57BL/6 control, CD7-deficient, CD28-deficient and CD7/CD28 double-deficient mice. Thymocytes were cultured at 106/ml in tissue culture medium without (A) or with (B) 20 U/ml rIL-2, as described in Methods. [3H]Thymidine incorporation, as an indicator of proliferation, was determined following 72 h of in vitro culture. Data are expressed as c.p.m./106 thymocytes. Data are the mean ± SEM of six age-matched male mice.

 
To investigate the ability of thymocytes from C57BL/6 control, CD7-deficient, CD28-deficient and CD7/CD28 double-deficient mice to proliferate in response to direct CD3–TCR complex triggering, immobilized anti-CD3 antibody with or without rIL-2 were used to activate thymocytes. We found no significant differences in [3H]thymidine incorporation among the four mouse lines following stimulation of thymocytes with immobilized anti-CD3 in the presence or absence of rIL-2 (Fig. 2Go).



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Fig. 2. [3H]Thymidine incorporation following immobilized anti-CD3 stimulation of thymocytes isolated from C57BL/6 control, CD7-deficient, CD28-deficient and CD7/CD28 double-deficient mice. Thymocytes were cultured at 106/ml in tissue culture medium without (A) or with (B) 20 U/ml rIL-2, as described in Methods. [3H]Thymidine incorporation, as an indicator of cell proliferation, was determined following 72 h of in vitro culture. Data are expressed as c.p.m./106 thymocytes. Data are the mean ± SEM of six age-matched male mice.

 
B7-1 and B7-2 levels in thymocyte preparations
Critical to our understanding of the mechanisms at play in our in vitro cultures is the level of expression of B7-1 (CD80) and B7-2 (CD86), the ligands for CD28. We found that CD7-deficient, CD28-deficient and CD7/CD28 double-deficient freshly isolated thymocyte preparations contained significantly less B7-1 SP cells, when compared to C57BL/6 wild-type cells (Table 3Go). In addition, we observed significantly decreased numbers of B7-1/B7-2 DP cells in thymocyte preparations from CD28-deficient and CD7/CD28-deficient mice. The CD7/CD28 double-deficient mice had significantly reduced numbers of B7-1/B7-2 DP cells compared to wild-type, CD7-deficient and CD28-deficient mice.


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Table 3. Expression of B7-1 and B7-2 on fresh wild-type CD7-deficient, CD28-deficient and CD7/CD28 double-deficient thymocytesa
 
IL-2 receptor expression (CD25) on thymocyte subsets
We observed that thymocytes from CD7-deficient, CD28-deficient and CD7/CD28-deficient mice are defective in their proliferative response to Con A, but not to anti-CD3 triggering. Our data indicate that there are no significant differences in the endogenous IL-2 production by thymocytes from these four strains of animals; therefore, we examined the level of expression of the IL-2 receptor (CD25) on thymocyte subsets. IL-2 receptor is at its highest on CD4/CD8 DN thymocytes, is down-regulated on CD4/CD8 DP thymocytes, and then its expression is increased on CD4 and CD8 SP thymocytes prior to emigration to the periphery. Freshly isolated CD4/CD8 DN thymocytes from CD7/CD28 double-deficient mice (554 ± 158) had significantly reduced expression of CD25 compared to wild-type (1050 ± 73) and CD7-deficient mice (1125 ± 110) (P < 0.05). Interestingly, CD25 expression on fresh CD28-deficient thymocytes (713 ± 126) was not significantly different than wild-type nor CD7/CD28 double-deficient thymocytes. Thus, suggesting a combined effect of CD7 and CD28 deficiency on basal CD25 expression.

CD25 expression was also markedly defective on Con A-stimulated cultures of all thymocyte subpopulations from CD7/CD28 double-deficient (Table 4Go). Most notably, CD4/CD8 DN thymocytes from CD7/CD28 double-deficient mice expressed significantly lower levels of CD25 when compared to CD4/CD8 DN thymocytes from wild-type, CD7-deficient and CD28-deficient mice (P < 0.05). It is interesting to note that reduced levels of CD25 were also observed on CD4/CD8 DP thymocytes from CD7/CD28-deficient mice when triggered with anti-CD3 (data not shown).


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Table 4. Expression of CD25 on Con A-stimulated wild-type, CD7-deficient, CD28-deficient and CD7/CD28 double-deficient thymocyte subsetsa
 
Thymocyte viability and induction of apoptosis in CD7/CD28-deficient thymocytes
Thymocytes from C57BL/6 control, CD7-deficient, CD28-deficient and CD7/CD28-deficient mice were placed in culture for 0–96 h and assayed at various time points with Annexin V–FITC plus –PI to determine viability of thymocytes in culture. No difference in viability of cultured thymocytes was found among C57BL/6 control, CD7-deficient, CD28-deficient and CD7/CD28-deficient mice over the 96 h time course (Fig. 3AGo). Similarly, the percent of apoptotic and necrotic thymocytes over the same time period was the same among the four groups (Fig. 3B and CGo respectively).



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Fig. 3. Viability, apoptosis and necrosis of isolated thymocytes from C57BL/6 control, CD7-deficient, CD28-deficient and CD7/CD28 double-deficient mice over 96 h in vitro culture. Thymocytes were cultured at 106/ml in tissue culture medium as described in Methods. Percent viable (A), apoptotic (B) and necrotic (C) thymocytes from each sample was determined using Annexin V–PI staining and flow cytometry. Data are the mean of three age-matched male mice and are representative of two separate experiments.

 
Normal mouse thymocytes undergo apoptosis when cross-linked with immobilized anti-CD3 mAb (39). Noel et al. have shown decreased apoptosis in CD28-deficient mice expressing an ovalbumin-specific TCR transgene in response to ovalbumin antigen triggering and CD28-deficient DP thymocytes were resistant to in vivo anti-CD3 mAb-induced cell death (15). Thus, we analyzed the response of fresh unfractionated thymocytes to immobilized anti-CD3 mAb ligation with regard to apoptosis and necrosis. Thymocytes from C57BL/6 control, CD7-deficient, CD28-deficient and CD7/CD28-deficient mice were cultured for 0–24 h, and thymocyte viability, apoptosis and necrosis determined (Table 5Go). After 12 h in culture, there was no difference between the control and experimental groups in terms of percent viable, apoptotic or necrotic cells. By 18 h all groups had a decrease in overall viability in the presence of immobilized anti-CD3, when compared to PBS controls (P <=0.04) (Table 5Go). Thymocyte apoptosis and necrosis in response to CD3 triggering was the same in all four groups (Table 5Go).


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Table 5. Comparison of thymocyte viability (%), apoptosis (%) and necrosis (%) in CD7-deficient, CD28-deficient and CD7/CD28 double-deficient and control mouse thymusa
 
Just as anti-CD3 ligation induces apoptosis, calcium ionophores and steroids have also been shown to induce thymocyte death via apoptosis (3942). We have previously shown that CD7 surface expression on human peripheral blood lymphocytes is increased following non-mitogenic doses of ionomycin (43). Thus, we chose to analyze thymocyte viability, apoptosis and necrosis over time in response to stimulation with ionomycin and dexamethasone. There was no difference in the viability among C57BL/6 control (Fig. 4A and EGo), CD7-deficient (Fig. 4B and FGo), CD28-deficient (Fig. 4C and GGo) and CD7/CD28-deficient (Fig. 4D and HGo) thymocytes in response to dexamethasone (Fig. 4A–DGo) or ionomycin (Fig. 4E–HGo). The percentage of apoptotic and necrotic thymocytes at each time point did not differ among the mouse lines. It should be pointed out that the decrease in viability following exposure to dexamethasone was largely due to thymocyte apoptosis, while the decrease in viability of thymocytes following exposure to ionomycin was mainly due to necrosis (data not shown).



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Fig. 4. Viability of isolated thymocytes from C57BL/6 control, CD7-deficient, CD28-deficient and CD7/CD28 double-deficient mice stimulated with either dexamethasone or ionomycin. (A–D) Thymocytes were cultured at 1x106/ml in tissue culture medium with DMSO 1:200, dexamethasone (DEX) 10–7, 10–8 or 10–9 M. (E–H) Thymocytes were cultured at 1x106/ml in tissue culture medium with DMSO 1:200, ionomycin 5, 2 or 0.2 µg/ml. Data are the mean of three age-matched male mice and are representative of two separate experiments.

 
Cytokine production by CD7/CD28-deficient thymocytes
Next, IFN-{gamma} and TNF-{alpha} cytokine production by immobilized CD3 mAb-triggered thymocytes from C57BL/6 control, CD7-deficient, CD28-deficient and CD7/CD28 double-deficient mice was examined. Specific ELISAs were used to quantify thymocyte supernatant IFN-{gamma} and TNF-{alpha} levels following three days in culture with immobilized anti-CD3 mAb in the presence or absence of rIL-2. In cultures with rIL-2, CD7-deficient (P <= 0.02) and CD7/CD28-deficient (P <= 0.01) thymocyte supernatants had a significantly reduced level of IFN-{gamma} in comparison to the control mice (Fig. 5AGo). In the absence of rIL-2, CD3-triggered thymocytes from CD7/CD28 double-deficient mice produced significantly less IFN-{gamma} than thymocytes from C57BL/6 control (P <= 0.02) and CD7-deficient (P <= 0.03) mice.



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Fig. 5. Cytokine production by anti-CD3 stimulated thymocytes from C57BL/6 control, CD7-deficient, CD28-deficient and CD7/CD28 double-deficient mice. Thymocytes were cultured at 106/ml in tissue culture medium with or without 20 U/ml rIL-2 in 48-well tissue culture-treated plates pre-coated with either PBS or anti-CD3 at 10 µg/ml as labeled. IFN-{gamma} (A) and TNF-{alpha} (B) production (pg/ml) after 3 days was determined by cytokine-specific ELISA. Data are the mean ± SEM of three age-matched male mice. {dagger}P <= 0.05 compared to CD7–/– mice. *P <= 0.05 compared to C57BL/6 control mice.

 
TNF-{alpha} production by thymocytes in response to immobilized anti-CD3 showed similar differences among the four mouse strains (Fig. 5BGo). In response to immobilized anti-CD3 (in the absence of rIL-2) CD7/CD28 double-deficient thymocytes produced significantly less TNF-{alpha} compared to C57BL/6 (P <= 0.01) and CD7-deficient mice (P <= 0.05). Stimulation of thymocytes with the combination of anti-CD3 mAb plus rIL-2 revealed a significant decrease in TNF-{alpha} secreted by CD7/CD28-deficient thymocytes, compared to C57BL/6 wild-type control mice (P <= 0.03). It is interesting to note that equivalent levels of endogenous IL-2 were produced following anti-CD3 stimulation of thymocytes from the four strains (data not shown). Taken together, these data suggested that CD7 is involved in regulating thymocyte production of IFN-{gamma} and TNF-{alpha}, and that the double-deficiency of CD7 plus CD28 yields a greater defect in IFN-{gamma} and TNF-{alpha} production than CD7 deficiency alone.


    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In this paper we have shown that CD7 and CD28 deficiencies lead to normal thymocyte development. However, thymocyte IFN-{gamma} and TNF-{alpha} production was abnormal. Thymocyte proliferation to Con A stimulation was abnormal in CD28-deficient and CD7/CD28-deficient thymocytes, while TCR complex mediated apoptosis and necrosis was normal in CD7/CD28 double-deficient mice. Thymocyte preparations from CD7/CD28 double-deficient mice had significantly reduced numbers of B7-1/B7-2 co-stimulatory molecule double positive cells compared to freshly isolated wild-type, CD7-deficient and CD28-deficient thymocytes. In addition, Con A-stimulated CD4/CD8 DN thymocytes from CD7/CD28 double-deficient mice expressed significantly lower levels of CD25 when compared to CD4/CD8 DN thymocytes from wild-type, CD7-deficient and CD28-deficient thymocytes (P < 0.05).

It was originally thought that both CD7 and CD28 were likely involved in regulating thymocyte maturation (10,17,44,45). A patient with CD7 deficiency and SCID syndrome has been described (46), and CD28 and its ligand B7 have been demonstrated to be present in thymus (47). Both CD7-deficient and CD28-deficient mice have been previously reported to have elevated numbers of CD4/CD8 DP thymocytes (15,24), initially suggesting a defect in regulation of cell death or a defect in regulation of thymocyte proliferation. CD28-deficient mice carrying a TCR transgene specific for ovalbumin (D011.10) have been reported to have abnormal negative selection of thymocytes (15). However, Bonilla et al. did not find overproduction of thymocytes in CD7-deficient mice (48) and other studies in CD28-deficient mice have shown normal negative selection (25).

In this study we observed a slight increase in the number of CD4 SP thymocytes in CD28-deficient and CD7/CD28 double-deficient mouse strains; however, these results were not statistically significant when compared to control C57BL/6 animals. Previous reports characterizing CD7-deficient and CD28-deficient mice have reported elevated numbers of DP thymocytes versus control (15,24). In this study we did not observe increases in DP thymocytes in either knockout strain. We cannot rule out the possibility that if additional animals were analyzed we would see a significant increase in DP thymocytes in these animals. It has been previously demonstrated that CD28-deficient thymocytes have normal apoptosis in response to dexamethasone treatment (15), and we have found similar results with CD7-deficient and CD7/CD28 double-deficient mice. Thus, despite reported increases in DP thymocytes in these mice, we found that animals deficient in both CD7 and CD28 have normal thymocyte development.

The effects of CD7, CD28 and CD7/CD28 deficiency in the thymus appear to be most pronounced on thymocyte production of cytokines. The role of CD28 as a key T cell co-stimulatory molecule is well established. The ligands for CD28 are the B7 molecules (CD80/CD86) (49). Ligation of T cell CD28 by CD80 on antigen-presenting cells up-regulates CD40 ligand on T cells that bind to CD40 on antigen-presenting cells, thus achieving optimal antigen presentation and T cell activation (50). Ligation of CD28 by B7 induces T cell cytokine production (2729). B7-1+, B7-2+ and B7-1+/B7-2+ DP accessory cells were present in our preparations of thymocytes from wild-type, CD7-deficient, CD28-deficient and CD7/CD28-deficient mice (Table 3Go). We found that CD7/CD28 double-deficient mice had significantly reduced numbers of B7-1/B7-2 DP cells compared to wild-type, CD7-deficient and CD28-deficient mice. This important observation suggests that the combined deficiencies of CD7 and CD28 lead to either decreased development or migration to the thymus of co-stimulatory molecule expressing cells. Reduced amounts of co-stimulation is one possible mechanism for the reduced in vitro proliferation and cytokine production by CD7/CD28 double-deficient thymocytes.

The reduced proliferative and cytokine responses seen in CD7/CD28 double-deficient thymocytes could also be explained by decreased endogenous IL-2 production or decreased IL-2 receptor (CD25) expression. We observed no significant differences in endogenous IL-2 production by thymocytes isolated from wild-type, CD7-deficient, CD28-deficient and CD7/CD28-deficient mice. However, we saw a combined effect of CD7 and CD28 deficiency on basal IL-2 receptor expression, and a marked decrease in IL-2 receptor expression on Con A-stimulated cultures of all thymocyte subpopulations from CD7/CD28 double-deficient mice (Table 4Go). Thus, these data suggest that CD7 and CD28 collaborate to prepare T lymphocytes for response to IL-2 by regulating IL-2 receptor expression.

Previous work has suggested multiple functions for CD7. In human peripheral TCR{alpha}ß T cells, cross-linking of CD7 leads to increased intracellular calcium mobilization (3,9). Moreover, ionomycin treatment has been shown to induce a transmembrane calcium flux which results in increased CD7 surface expression (10). Immobilized anti-CD7, in the presence of submitogenic anti-CD3, induces a co-mitogenic signal leading to T cell proliferation, IL-2 production and IL-2 receptor expression. Cross-linking of CD7 on peripheral TCR{alpha}ß T cells also induces association of the cytoplasmic domain of CD7 with phosphatidylinositol 3-kinase and modulates T cell adhesion (5154). Triggering of CD7 on TCR{gamma}{delta} T cells leads to cell activation and induction of TNF-{alpha}, TNF-ß and granulocyte macrophage colony stimulating factor mRNA (19). Activation of CD7 on NK cells leads to proliferation, IFN-{gamma} production, increased ability to kill NK targets and increased cell adhesion to fibronectin via ß1 integrins (7,20,55). In addition, granulocyte macrophage colony stimulating factor production is induced following cross-linking of CD7 in bone marrow T and myeloid progenitor cells (23,56).

Ligation of CD28 and CD7 initiates binding of phosphatidylinositol 3-kinase within the CD28 and CD7 cytoplasmic tails, and this binding is critical for CD28-deficient and CD7-mediated T cell activation (5759). Signaling through CD28 with anti-CD28 mAb is able to provide a co-stimulatory signal which prevents the induction of anergy in T cell clones (60). Data suggest that the co-stimulatory signal generated by CD28 ligation induces intracellular calcium mobilization in activated splenic T cells and appears to be mostly limited to the CD4+ lineage (26). A recent study has shown that stimulation of resting human T cells with a CD28-specific antibody can induce proliferation and cytokine synthesis without co-signaling through the TCR (61).

Our hypothesis in this study was that CD7 and CD28 might be complimentary co-stimulatory molecules and therefore it would require simultaneous deletion of both genes to see disruption of T cell development. As mentioned, however, we found that T cell development was normal in CD7/CD28 double-deficient mice. Rather, the effects of CD7, CD28 and CD7/CD28 double-deficiency were primarily manifest in the thymus as defects in thymocyte cytokine production and Con A/[3H]thymidine incorporation responses. Since no differences in endogenous IL-2 production were observed between the four mouse strains, the difference in proliferative response to Con A and cytokine response to anti-CD3 cannot be explained by differences in endogenous IL-2 production. Thus, suggesting possible involvement of other cytokines (e.g. IL-7 and IL-15) which will be the subject of future investigations. Interestingly, these are functions primarily seen in mature thymocytes after T cell selection processes have occurred. Thus, for both CD7 and CD28 molecule deficiencies, the effects of CD7/CD28 deficiencies are more on thymocyte function than on T cell development in the thymus.

In summary, our studies strongly suggest that neither CD7 nor CD28 thymocyte signaling is centrally involved in the early stages of thymocyte development. Moreover, our study confirms an important role for CD7 and CD28 molecules in regulation of thymocyte cytokine production, by demonstrating an additive effect of CD7 and CD28 deficiencies on thymocyte IFN-{gamma} and TNF-{alpha} production.


    Acknowledgments
 
We acknowledge the expert technical assistance of Jonathan Baron and secretarial assistance of Kim R. McClammy. This work was supported by NIH grants CA28936 and T32-CA09058.


    Abbreviations
 
Con A concanavalin A
DN double negative
DP double positive
LPS lipopolysaccharide
PI propidium iodide
SP single positive
TNF tumor necrosis factor

    Notes
 
3 Abramson Family Cancer Research Institute, University of Pennsylvania, Philadelphia, PA 19104, USA Back

Transmitting editor: J. F. Kearney

Received 17 January 2000, accepted 24 October 2000.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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