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Originally published online as doi:10.1189/jlb.0404262 on December 23, 2004

Published online before print December 23, 2004
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(Journal of Leukocyte Biology. 2005;77:513-521.)
© 2005 by Society for Leukocyte Biology

CD38 is expressed selectively during the activation of a subset of mature T cells with reduced proliferation but improved potential to produce cytokines

Claudia Sandoval-Montes and Leopoldo Santos-Argumedo1

Departamento de Biomedicina Molecular, Centro de Investigación y Estudios Avanzados, I.P.N., México

1 Correspondence: Departamento de Biomedicina Molecular, CINVESTAV-IPN, Av. IPN #2508, Col. Zacatenco, Apartado Postal 14-740, CP 07360, México, D.F., México. E-mail: lesantos{at}cinvestav.mx


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CD38 is an ~45-kDa type II transmembrane glycoprotein expressed by hematopoietic and nonhematopoietic cells. Its surface expression is under complex control and varies during lymphocyte development, activation, and differentiation, suggesting an important role in these processes. Murine CD38 has been mainly characterized on B lymphocytes, and in humans, the molecule has been studied in T cells. This paper provides evidences that murine CD38 is regulated tightly during T cell activation and differentiation. On the periphery, a subset of mature T lymphocytes was identified by the expression of CD38. These cells showed an activated phenotype; they were larger and more granular than their negative counterparts. In accord with this observation, in vitro-activated T cells up-regulated CD38. Memory T lymphocytes also were CD38-positive. It is interesting that T cells expressing high levels of CD38 had a reduced, proliferative capacity but displayed an improved potential to produce interleukin-2 and interferon-{gamma}, suggesting a role of this molecule during T cell activation and differentiation.

Key Words: subpopulations • naïve • memory • T lymphocytes


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CD38 is a widely distributed molecule in hematopoietic and nonhematopoietic cells. It was originally described as a human cell-surface molecule using monoclonal antibody (mAb) T10; however, since then, it has been used as a marker in the study of T and B cell activation and differentiation [1 2 3 ]. CD38 belongs to the nicotinamide adenine dinucleotide glycohydrolase/adenosine 5'-diphosphate-ribosyl cyclase gene family, and its product is an ~45-kDa single-chain type II glycoprotein. Several mAb against human and mouse CD38 have shown agonistic properties, suggesting a role as a cell-surface receptor whose putative ligand in humans has been claimed to be CD31 [4 5 6 7 ].

Human CD38 is highly expressed on early T cell precursors and on CD4+veCD8+ve double-positive thymocytes [8 ]. In contrast, mature T cells have low levels of CD38, but upon mitogenic activation, they up-regulate their expression [2 , 9 , 10 ]. Similarly, human B cell progenitors have surface CD38, whereas mature, naïve B lymphocytes are CD38low [9 , 11 ]. Activation of mature, naïve B lymphocytes induces the re-expression of CD38, which has been used to describe an activation stage of germinal center reaction B lymphocytes [12 ]. Several lymphocyte activators have been characterized for their ability to enhance the expression of CD38. Among these, phorbol 12-myristate 13-acetate (PMA) and anti-CD3 or anti-immunoglobulin M (IgM) plus interleukin (IL)-4 up-regulate CD38 on human T and B lymphocytes, respectively. Other agents such as vitamins A and D3, interferons (IFNs), and cyclic adenosine monophosphate-elevating drugs also promote CD38 expression in activated human T and B cells or in human cell lines [9 , 13 ].

Cross-linking CD38 with a selected set of mAb induces activation and proliferation of human T lymphocytes [4 , 14 , 15 ]. CD38 signaling in T cells is initiated within a subset of membrane rafts [16 ]. Molecules involved in the signal transduction pathway include {zeta}-associated protein-70, CD3{varepsilon}, phospholipase C-{gamma}, Raf-1/mitogen-activated protein kinase, and calcium mobilization [17 18 19 ]. CD38 signaling requires a functional T cell receptor (TCR)/CD3 receptor complex, suggesting a functional association and dependency of CD38 to the TCR signal transduction machinery [14 , 17 18 19 ]. CD38 is also associated with the CD21/CD19 complex in B cells and CD16 in natural killer (NK) cells. CD38 ligation promotes growth arrest in human B cell progenitors, and it induces killing in human NK cells. The signaling involved in these phenomena is unclear, but it has been suggested that CD38 may use the B cell receptor (BCR) or the NK signaling machinery [9 , 20 ].

The analysis of murine CD38 began with the description of the agonistic antibody NIM-R5 and allowing the cloning of the gene from WEHI-231 murine B cell lymphoma [21 , 22 ]. Since then, mouse CD38 has been analyzed mainly as a B cell surface marker/receptor. The NIM-R5 antibody induces activation and proliferation of murine B cells through a not-yet defined pathway, involving Btk, Lyn, Fyn, and calcium mobilization [22 23 24 ]. CD38 is expressed early in ontogeny (B220+ve, BCR–ve) and maintained by all mature B lymphocytes [25 ]. In contrast, resting T lymphocytes do not express CD38, its expression having been reported only by {alpha}ß-TCR+veCD4–veCD8–ve thymocytes and by a population of T cells with immunoregulatory functions [26 , 27 ]. Therefore, the main aim of this work was to analyze the expression of CD38 in peripheral T lymphocytes. Our results clearly demonstrate that CD38 is expressed on subsets of activated T lymphocytes of the naïve and memory phenotypes. It is interesting that T cells, expressing high levels of CD38, displayed a reduced, proliferative capacity but exhibited an improved potential to produce IL-2 and IFN-{gamma}, suggesting a role of this molecule during T cell activation and differentiation. The integrated analysis of CD38 in T lymphocytes from mice may provide new tools to study the biological role of this molecule.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice
BALB/c and C57BL/6 mice (6–8 weeks of age) were used in all experiments. They were produced at the Centro de Investigación y Estudios Avanzados (CINVESTAV, México) animal facility, and the Animal Care and Use Committee of CINVESTAV approved all the experiments.

Medium
RPMI 1640 (Gibco, Grand Island, NY) was supplemented with 1% nonessential amino acids (Gibco), 5 x 10–5 M 2-mercaptoethanol (Sigma Chemical Co., St. Louis, MO), 1 mM sodium pyruvate (Sigma Chemical Co.), 2 mM glutamine (Sigma Chemical Co.), and 10% (v/v) fetal calf serum (Gibco).

Surface staining
Cells freshly obtained from spleen, lymph nodes, peritoneal cavity, Peyer’s patches, peripheral blood, and tissue cultures were labeled directly with combinations of the following mAb: anti-CD3-fluorescein isothiocyanate (FITC) or -allophycocyanin (APC), anti-B220-phycoerythrin (PE) or -peridinin chlorophyll protein (PerCP), anti-CD4-PE, anti-CD8-PE or -PerCP, anti-CD25-PerCP, anti-CD44-FITC or -PE, anti-CD62L-APC, anti-CD69-biotin, anti-IgG2a-biotin, streptavidin-APC, or PerCP (all from BD PharMingen, San Diego, CA) and anti-CD38-PE (from Southern Biotechnology Associates, Inc., Birmingham, AL). mAb NIM-R5 (rat anti-mouse CD38) was conjugated with biotin or fluorescein in our laboratory using standard procedures [22 ]. The cells were incubated for 15 min with the antibodies at room temperature and then washed three times with phosphate-buffered saline (PBS) containing 0.5% bovine serum albumin (BSA; Sigma Chemical Co.) and 0.02% sodium azide [Sigma Chemical Co.; PBS+0.5% BSA+0.02% sodium azide (PBA)]. If required, the cells were incubated further with APC- or PerCP-labeled streptavidin, washed, and then analyzed in a FACSCalibur cell sorter (BD Biosciences, San José, CA).

CD38 expression after in vitro T cell stimulation
One million splenocytes were obtained using Ficoll-Hypaque (Sigma Chemical Co.) gradient separation and incubated in complete medium with one of the following stimuli: anti-CD3 (1 µg/ml), concanavalin A (Con A; 2.5 µg/ml), phytohemagglutinin (PHA; 10 µg/ml), or PMA (25 ng/ml). All cells were incubated at 37°C. At different times, the cells were harvested, labeled as described above, and then analyzed using flow cytometry.

Analysis of the proliferation of CD38+ve and CD38–ve T lymphocytes
For proliferation, splenocytes were resuspended at 5 x 107/ml in PBS containing 5 µM 5 (and 6-)- carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes, Eugene, OR), incubated for 10 min at 37°C, and then washed three times in cold complete medium. As a control, 2 x 106 CFSE-labeled cells were activated for 24 h, 48 h, and 72 h with Con A (2.5 µg/ml) at 37°C or cultured with complete medium only. At the end of each incubation period, cells were harvested and labeled with anti-CD3-APC and anti-CD38-PE. The cells were analyzed using flow cytometry.

CD38 expression on T lymphocytes from mice immunized with hen egg lysozyme (HEL)
BALB/c mice were immunized intraperitoneally with 200 µg HEL (Sigma Chemical Co.) in complete Freund’s adjuvant. Two weeks later, mice received the same amount of HEL but were emulsified in incomplete Freund’s adjuvant and finally received a boost with 200 µg HEL in saline. Mice were killed 1 week after the last immunization, and spleens were removed to purify lymphocytes using Ficoll-Hypaque gradient. The cells were labeled with anti-CD3-FITC, anti-CD44-PE, anti-CD62L-APC, anti-CD38-biotin, and streptavidin-PerCP and were analyzed using flow cytometry.

Panning and magnetic cell sorting (MACS)
Splenocytes were incubated 2 h at 37°C and 5% CO2 in a plate precoated with 20 µg/ml mAb B220. After two rounds of panning, the unbound cells were removed and labeled with CFSE as described above. The cells were then labeled with biotinylated anti-CD38 and incubated for 20 min on ice. After incubation, the cells were washed with PBS containing 5 mM EDTA (PBS-EDTA) and resuspended in 90 µl PBS-EDTA with 10 µl MACS streptavidin microbeads. The cells were incubated for 20 min on ice, washed with PBS-EDTA, and resuspended in 500 µl PBS-EDTA. The cell suspension was passed through a MACS column (Miltenyi Biotec, Auburn, CA) mounted into the MACS separator, and the CD38–ve T cells were obtained in the unbound fraction. The column was then washed and removed from the magnet, and the CD38+ve T cells were obtained from the bound fraction. Finally, 5 x 104 CD38–ve or CD38+ve T cells were incubated for 24 h, 48 h, or 72 h at 37°C and 5% CO2 with 2.5 µg/ml Con A or medium only (control). At the end of each incubation period, the cells were harvested and labeled with anti-CD3-APC, anti-CD38-PE, and B220-PerCP and then analyzed using flow cytometry.

Fluorescein-activated cell sorting (FACS)
Splenocytes (1x108) were labeled with anti-CD3-PE, anti-CD38-FITC, and B220-PerCP and then incubated for 15 min on ice in the dark. After incubation, the cells were washed with PBS containing 10% BSA, resuspended in 4 ml PBS containing 10% BSA, and sorted through a FACSVantage (Becton Dickinson, San Jose, CA).

Sorted CD38–ve or CD38+ve T cells (2x105) were incubated for 24 h or 48 h at 37°C and 5% CO2 with 2.5 µg/ml Con A or only medium (control). The cells were harvested at the times indicated and labeled with anti-CD3-PE, anti-CD38-FITC, anti-CD69-biotin, and anti-CD25-PerCP. After incubation and three washes with PBA, the cells were further incubated with streptavidin-APC. Finally, cells were analyzed using flow cytometry.

Sorted CD38–ve or CD38+ve T cells (5x104) were incubated for 48 or 72 h at 37°C and 5% CO2 with 2.5 µg/ml Con A or only medium (control), and 8 h before harvesting, the cells were pulsed with 1 µCi [3H]-thymidine. The cells were harvested, and the [3H]-thymidine incorporated into DNA was measured with a multipurpose scintillation counter (Beckman, Fullerton, CA).

Detection of intracellular cytokines
The production of cytokines was measured using intracellular staining [28 , 29 ]. Briefly, the splenocytes were obtained using Ficoll-Hypaque (Sigma Chemical Co.) gradient separation; 2 x 106 cells were incubated for 5 h at 37°C and 5% CO2 with 25 ng/ml PMA (Sigma Chemical Co.), 1 µg/ml ionomycin (Sigma Chemical Co.), and 10 µg/ml brefeldin A (BFA; Sigma Chemical Co.) or BFA only with medium (control). After incubation, the cells were harvested and labeled with antibodies to cell-surface markers anti-CD3-APC and anti-CD38-FITC by incubating for 15 min at room temperature in the dark. After incubation, the cells were washed with PBS containing 0.5% BSA (Sigma Chemical Co.) and 0.02% sodium azide (Sigma Chemical Co.; PBA) and centrifuged for 5 min at 500 g. The supernatants were removed, and then FACS permeabilizing solution (Becton Dickinson) was added to the pellets and incubated for 10 min at room temperature in the dark. For intracellular staining, the cells were centrifuged for 5 min at 500 g, and the pellets were incubated with anti-IL-2-PE or anti-IFN-{gamma}-PE at room temperature for 15 min in the dark. Finally, the samples were washed with PBA, fixed with PBS containing 1% paraformaldehyde, and analyzed in a FACSCalibur (Becton Dickinson).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
CD38 was expressed by a subset of T lymphocytes from different lymphoid compartments
Earlier reports stated that CD38 was absent or poorly expressed on peripheral T lymphocytes from mice [22 , 25 , 26 ]. These results were in contradiction to the findings in human T lymphocytes in which activated T cells express the molecule. To examine this discrepancy, the distribution of CD38+ve T lymphocytes was analyzed in different lymphoid compartments in the mice. These results are shown in Figure 1a , where it can be seen that CD38 is expressed on the surface of T lymphocytes from all the compartments analyzed. BALB/c and C57BL/6 mice showed similar results in the percentages of CD38+ve T cells: BALB/c (n=7; X±SD; spleen 9±3; lymph node 5±2; peripheral blood 2±2; Peyer’s patches 20±5; and peritoneal cells 14±2); C57BL/6 (n=2; X±SD; spleen 11±2; lymph node 3±1; peripheral blood 3±1; Peyer’s patches 30±7; and peritoneal cells 9±3). It is interesting that the cells expressing CD38 on their surface included larger and more granular cells than the CD38–ve T cell population (data not shown).



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  		Figure 1. CD38 expression on peripheral T lymphocytes. (a) Lymphocytes, isolated from spleen, lymph node, peripheral blood, Peyer’s patches, and the peritoneal cavity from BALB/c mice, were stained with anti-CD3-FITC, anti-CD38-biotin, and streptavidin-APC. Dot plots show CD3+ve and CD38+ve lymphocytes. These results are representative of seven independent experiments conducted in the same manner and with similar results. (b) A similar staining was performed with the addition of anti-CD4-PE or anti-CD8-PerCP, and results from five independent experiments are presented (mean±SD). The percentages shown correspond to CD3+veCD4+veCD38+ve cells taking the CD3+veCD4+ve subpopulation as 100%. The same calculations were completed for the CD3+veCD8+ve subpopulation. One mouse was used for each experiment.

 
As only a subset of T lymphocytes expressed CD38 on their surface, the presence of this molecule was evaluated in CD4+ve or CD8+ve T cells. The expression of CD38 in these T cells is shown in Figure 1b , where the percentages shown correspond to CD3+veCD4+veCD38+ve cells taking the CD3+veCD4+ve subpopulation as 100%. The same calculations were completed for the CD3+veCD8+ve subpopulation. The percentage of CD8+ve T lymphocytes with CD38 on their surface was higher when compared with the CD4+ve T cell subpopulation in all the compartments analyzed; however, the mean fluorescence intensity (MFI) between these two subpopulations was equivalent (data not shown).

In vitro-activated T lymphocytes up-regulated the expression of CD38
As described above, many CD38-expressing T lymphocytes had an activated phenotype (larger and more granular cells). Therefore, to better analyze whether activation and expression of CD38 were correlated, whole splenic lymphocytes were activated in vitro. The cells were activated with different stimuli, and depending on the stimulus used, CD38 was up-regulated upon activation, reaching its highest expression between 4 h and 24 h (Fig. 2a ). All stimuli induced a steady-state expression of CD38 once the cells reached their highest expression.



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  		Figure 2. CD38 is up-regulated in in vitro-activated T lymphocytes. (a) Lymphocytes isolated from the spleen of BALB/c mice were incubated with one of the following stimuli: anti-CD3 (1 µg/ml), Con A (2.5 µg/ml), PHA (10 µg/ml), or PMA (25 ng/ml). The cells were harvested at the times indicated and labeled with anti-CD3-FITC, anti-CD38-PE, anti-CD69-biotin, anti-CD25-PerCP, and streptavidin-APC. The comparison among the expression of CD38, CD25, and CD69 for each stimulus is shown. These results represent eight independent experiments (mean±SD). (b) Lymphocytes stimulated 24 h with Con A were stained with anti-CD3-FITC, anti-CD38-PE, and independently, with one of the following biotinylated antibodies: anti-CD25, anti-CD69, or anti-CD44 and were then counterstained with streptavidin-APC. Density plots comparing the expression of CD38 to CD25, CD69, or CD44 are shown. These results are representative of five independent experiments with similar results. Three mice were used for each experiment.

 
Upon activation, T cells increased the expression of several other molecules on their surface. Among these molecules were CD69, an early marker, and CD25, a later marker, which are frequently used as markers of activation. To compare the pattern of expression of CD38 to these two markers, cells were stimulated, and the expression of CD69 and CD25 was analyzed simultaneously with CD38. As it can be seen in Figure 2a , CD38 increased before CD69 and CD25. It is noticeable that all cells expressing CD38 also expressed the activation markers CD25, CD69, or CD44 (Fig. 2b) . Similar results were obtained with BALB/c and C57BL/6 mice (data not shown).

A general observation is that none of the stimuli used for in vitro activation induced more than 50% CD38+ve T lymphocytes. In other words, activated T cell subpopulations could be differentiated by the presence or absence of CD38 on their surface. This difference apparently did not reside in the ability of the cells to become activated, as CD38–ve T lymphocytes up-regulated CD69, CD25, and CD44 and became larger and granular after activation.

The expression of CD38 was analyzed for in vitro-activated, CD38+ve- and CD38–ve-sorted T cells. These subpopulations were activated 24 h and 48 h with Con A. During activation, the CD38+ve subpopulation did not decrease the expression of CD38, and the CD38–ve subpopulation expressed very little CD38 (Fig. 3 ). These results are in agreement with previous observations, where highly purified T lymphocytes did not up-regulate the expression of CD38 [30 ] (C. Sandoval-Montes, unpublished observations). We believe that some soluble or membrane-bound factors from other cells in the spleen may be necessary to up-regulate the CD38 surface expression in the CD38–ve T cell subpopulation. It is too early to make any clear statement regarding this hypothesis, as we do not have any additional supporting data. However, these highly purified cells had similar activating capabilities, as the expression of CD69 and CD25 was comparable with CD38+ve and CD38–ve T cell subpopulations.



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  		Figure 3. CD38 is not down-regulated after T lymphocyte activation. Splenocytes from three mice were labeled with anti-CD3-PE, anti-CD38-FITC, and B220-PerCP and sorted with a FACSVantage sorter. CD38–ve or CD38+ve T cells were incubated for 24 h or 48 h with Con A or only medium (control). The cells were harvested at the times indicated and labeled with anti-CD3-PE, anti-CD38-FITC, anti-CD69-biotin, anti-CD25-PerCP, and streptavidin-APC. The dot plots show the expression of CD3 and CD38, and the histograms, the expression of CD25 and CD69. These results are representative of three independent experiments conducted in the same manner and with similar results.

 
Lymphocytes from immunized mice had more T cells expressing CD38
As described above, in vitro activation caused T lymphocytes to up-regulate CD38 expression. For this reason, CD38 expression was evaluated after in vivo immunization with HEL. Cells were gated into CD38+ve, and then three regions were drawn based on the expression of CD44 and CD62L to distinguish naïve, activated, and memory T lymphocytes. Analysis of CD62LhighCD44low naïve and CD62LhighCD44high activated T cells showed two subpopulations, one expressing high levels of CD38 and another not expressing this marker (Fig. 4 ). Size and granularity were analyzed in these two populations, demonstrating that CD38+ve contained larger and more granular T cells than the CD38–ve population, as it was described earlier for in vitro-activated cells.



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  		Figure 4. CD38 is highly expressed on in vivo-activated T lymphocytes, which when from HEL-immunized and -nonimmunized BALB/c mice, were labeled with anti-CD3-FITC, anti-CD44-PE, anti-CD62L-APC, anti-CD38-biotin, and streptavidin-PerCP. Cells were gated into the CD3+ve population and selected as naïve (CD62LhighCD44low), activated (CD62Llow CD44high), and memory (CD62L–veCD44high) T lymphocytes (upper panels). These populations were further analyzed for CD38 expression for each T cell subset as indicated in the histograms. These results are representative of five independent experiments. One mouse was used for each experiment.

 
The analysis of CD62L–veCD44high memory T cells also demonstrated CD38 expression. The expression of CD38 was intermediate between resting (CD38–ve) and activated (CD38+ve) T cells, demonstrating that CD38 was expressed in recently activated and memory T lymphocytes (Fig. 4) . Nonimmunized mice had more naïve T cells with minor percentages of activated and memory T cells; however, the expression of CD38 was similar among naïve, activated, and memory T cells from nonimmunized and immunized mice.

Activated T lymphocytes expressing high levels of CD38 did not proliferate
There are few papers describing the characterization of CD38+ve T lymphocytes in mice. In humans, CD38 is considered to be functionally associated to CD3, as it uses its signaling molecules to induce T cell activation [18 , 20 ]. In mice, CD38 identifies a subpopulation of CD45RBlowCD4+ve T cells. The CD45RBlowCD4+ve population contains regulatory T cells that have an impaired ability to proliferate [27 ]; therefore, the proliferative capacity of CD38-positive and -negative T cells was evaluated upon stimulation with Con A. Whole splenocytes were labeled with CFSE and were activated with Con A. Cells were gated into the CD3+ve population and examined for expression of CD38 and CFSE. T cells expressing high levels of CD38 did not proliferate (Fig. 5a ). A separation between CD38+ve and CD38–ve T cells was clearly seen at 48 h when CD38–ve started to dilute the amount of CFSE, whereas CD38+ve remained unchanged. Later, the CD38high T cells decreased, and only proliferating CD38low or CD38–ve T cells were detected in the culture.



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  		Figure 5. CD38+ve T lymphocytes are less able to proliferate. (a) Lymphocytes isolated from the spleen were labeled with CFSE and then activated with Con A (2.5 µg/ml). The cells were harvested at the times indicated, labeled with anti-CD3-APC and anti-CD38-PE. T lymphocytes were selected by CD3 expression, and contour plots were drawn to describe the expression of CD38 and CFSE. These results are representative of three independent experiments. Three mice were used for each experiment. (b) T lymphocytes isolated from panning were stained with CFSE labeled with biotinylated anti-CD38 and streptavidin microbeads. The cells were separated through a MACS column (Miltenyi Biotec), and the CD38–ve or CD38+ve T cell fractions were obtained. CD38–ve or CD38+ve T cells were incubated for 24 h, 48 h, or 72 h with Con A or only medium (control). At the end of each incubation period, the cells were harvested and labeled with anti-CD3-APC, anti-CD38-PE, and B220-PerCP. CFSE expression for each population is presented as histograms. These results are representative of three independent experiments. Two mice were used for each experiment. (c) Splenocytes were labeled with anti-CD3-APC, anti-CD38-PE, and B220-PerCP and sorted with a FACSVantage sorter (left). CD38–ve or CD38+ve T cells were incubated for 48 h or 72 h with Con A or only medium (control) and pulsed with [3H]-thymidine, which when incorporated into the DNA, was counted (right). These results are presented as mean and SD of three independent experiments. Five mice were used for each experiment.

 
From the experiments described in Figure 5a , it was difficult to determine whether CD38+ve T cells did not proliferate and die. In an attempt to answer this question, splenic CD38+ve and CD38–ve T cells were purified using panning and MACS and were then labeled with CFSE. The purified subpopulations were then activated with Con A. Aliquots of these suspensions were harvested at 24 h, 48 h, and 72 h and analyzed as described above. T cells expressing CD38 proliferated poorly (Fig. 5b) . Again, at 48 h, CD38–ve T cells started to proliferate, whereas CD38+ve cells remained unchanged. Later, the CD38+ve T cells showed poor proliferation, and most of the cells were dying, making the analysis impossible.

The proliferative capacity of CD38+ve and CD38–ve T cells was also evaluated using the ability of the sorted populations to incorporate [3H]-thymidine as a gold standard test for proliferation. Whole splenocytes were sorted to purify CD38+ve and CD38–ve T lymphocytes, resulting in a greater than 95% purity for each population (Fig. 5c , left panel). The purified populations were stimulated with Con A, and to evaluate proliferation, the cells were pulsed with [3H]-thymidine 8 h before harvesting. As shown in Figure 5c and in close agreement with the results shown in Figure 5 a and b , CD38+ve T lymphocytes proliferated poorly when compared with CD38–ve T cells. Similar results were obtained with BALB/c and C57BL/6 mice (data not shown).

The results from Figure 5 clearly show that T cells expressing high levels of CD38 had a reduced proliferative capacity.

A higher percentage of CD38+ve T lymphocytes produce cytokines
As described earlier, CD38+ve was up-regulated after activation within a subpopulation of T lymphocytes. These cells did not proliferate, or if they did, the proliferation was considerably reduced when compared with the CD38–ve population. To look for differences in the ability of CD38+ve and CD38–ve T lymphocytes to produce cytokines, whole splenocytes were activated 5 h with PMA plus ionomycin in the presence of brefeldin A. Figure 6a shows that CD38+ve and CD38–ve T cells were equally able to produce IL-2 and IFN-{gamma}. However, a higher percentage of the cells expressing CD38 on their surface was able to produce cytokines when compared with cells lacking CD38 (Fig. 6b) . More CD38+ve T lymphocytes produced IL-2 and IFN-{gamma}, and their MFI was also higher (Fig. 6b) ; however, secreted cytokines measured using an enzyme-linked immunosorbent assay did not explain these differences, and they were not statistically significant (data not shown). When these data were normalized, taking the percentage of the cytokine-producing CD38–ve cells as one, it was found that a ratio of 2.3 and 1.98 CD38+ve T cells was producing IL-2 and IFN-{gamma}, respectively.



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  		Figure 6. A higher percentage of CD38+ve T lymphocytes is committed to produce IL-2 and IFN-{gamma}. The production of cytokines was measured using intracellular staining. The splenocytes from C57BL/6 mice were incubated for 5 h with 25 ng/ml PMA, 1 µg/ml ionomycin (ION), and 10 µg/ml BFA or BFA only with medium (control). After incubation, the cells were harvested and labeled with anti-CD3-APC and anti-CD38-FITC and then permeabilized, fixed, and intracellular-stained with anti-IL-2-PE or anti-IFN-{gamma}-PE. (a) The dot plots are representative of six independent experiments conducted in the same manner and with similar results. One mouse was used for each experiment. SSC, Side-scatter. (b) The results from six independent experiments were graphed, showing mean and SD of the percentages of cytokine-producing T cells and the MFI for each cytokine produced by the T cell subpopulations. One mouse was used for each experiment.

 
Comparisons between BALB/c and C57BL/6 mice revealed similar percentages of cytokine-producing CD38+ve and CD38–ve T cells: BALB/c (n=3; X±SD; IL-2+CD38–ve 26±8; CD38+ve 45±3; IFN-{gamma}+CD38–ve 19.5±5; CD38+ve 39±6); C57BL/6 (n=6; X±SD; IL-2+CD38–ve 14±5; CD38+ve 29±12; IFN-{gamma}+CD38–ve 17±7; CD38+ve 31±5).

These results show that CD38+ve represents activated but nonproliferative lymphocytes with an enhanced ability to produce IL-2 and IFN-{gamma}.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
CD38 expression on mouse T lymphocytes has been studied insufficiently. Previous results from our group indicated that a small subset (~7%) of T lymphocytes in the spleen was CD38+ve [25 ]. These results were confirmed and extended in this work. T lymphocytes expressing CD38+ve on their surface were inconsistently observed in previous studies, probably as mice bred under specific, pathogen-free conditions have low numbers of CD38+ve T lymphocytes. Mice bred under clean but normal conditions exhibit an increased number of these cells at the levels reported in this work. This observation was verified easily using in vitro activation, where all the polyclonal stimuli used increased CD38 expression. Remarkably, none of the stimuli tested here induced more than 50% of the cells to express CD38 on their surface. CD38 was expressed very early on stimulation and remained on the cell surface with all the stimuli used. Cells expressing CD38 were also positive for CD25 and CD69, therefore confirming their activated status. However, more than half of the cells expressing CD25 and CD69 were CD38–ve. The biological meaning of this finding is not yet clear; however, the characterization of these two subsets of activated T lymphocytes will certainly improve the understanding of the events occurring during T cell activation and differentiation.

CD38 expression in the naïve population from nonimmunized and immunized mice confirmed that CD38 labels a subset of activated T lymphocytes, probably representing recently activated T cells. Therefore, in vivo-activated T cells can also be identified by the expression of this molecule, which can provide tools for the analysis of this molecule during the activation process. The analysis of immunized mice also showed that memory T lymphocytes are CD38+ve, suggesting a role of this glycoprotein in the maintenance and survival of these T cell subsets.

The biological role of CD38 has been elusive, and neither its enzymatic activity nor its receptorial function has been clearly elucidated [10 ]. The results from CD38-deficient mice have been disappointing so far, as no clear defects have been found [31 ]. CD38-deficient mice presented mild deficiencies in the B cell compartment, and it was only recently that Partida-Sanchez et al. [32 , 33 ] showed defects in neutrophil and dendritic cell chemotaxis. Until now, no defects in the T cell compartment have been shown in CD38-deficient mice. Moreover, work in progress has not found any major deficiencies in T cell development, activation, or differentiation (C. Sandoval-Montes, unpublished results). Therefore, if CD38 is required in any of these processes, its presence might be important but not indispensable, which may indicate a redundant activity of CD38 with other molecules [34 ].

Recently, it has been published that CD38-expressing thymocytes are more prone to suffer apoptosis in the human thymus [8 ]. The only indication of a function of CD38 in mice is that activated T lymphocytes expressing high levels of CD38 on their membranes did not proliferate. These cells disappeared from the cultures and so far, no evidence has been found to confirm if the cells died from apoptosis. Research is being conducted to clarify this possibility. It is important to note that almost all the experiments were conducted with BALB/c and C57BL/6 mice with identical results; therefore, the differences found in this work can be equally applied, at least, to the two strains of mice studied.

The analysis of cytokine production revealed that there were two subpopulations of T cells, the first being CD38–ve with fewer cells producing IL-2 and IFN-{gamma} and the other, CD38+ve with more cells producing IL-2 and IFN-{gamma}. CD38+ve T cells may represent already-activated lymphocytes more prone to the effector functions than to proliferate. Preliminary results from our group indicate that mice experimentally infected with Mycobacterium tuberculosis, showed an increase of CD8+ve and CD38+ve T cells in the lung infiltrates during the chronic stage of the disease (Claudia Sandoval-Montes and Leopoldo Santos-Argumedo, unpublished observations). Also in humans, there is a substantial proportion of human immunodeficiency virus (HIV)-specific CD8+ve T cells expressing CD38 on their surface at the chronic stages of the disease, correlating with a poor prognosis outcome [35 , 36 ].

A recent paper has shown that a substantial proportion of CD8+ve T cells expressing CD38 in HIV-infected individuals with active viral replication are susceptible to spontaneous and Fas-mediated cell death [35 ]. This observation may explain why CD38+ve T cells die in culture. The CD38+ve T lymphocytes may be ready for action, unable to proliferate, but dispensable once their function has been performed. Of course, further work will be needed to determine if these assumptions regarding CD38+ve T lymphocytes can be illustrated.

In summary, we have identified a T cell subpopulation with an activated phenotype, which failed to proliferate but was efficient in producing cytokines in response to polyclonal stimulation. These cells can be phenotypically distinguished based on the expression of CD38, indicating that the expression of this molecule is tightly regulated during lymphocyte activation. These data provide new routes for the analysis of the CD38 function in mice.


   ACKNOWLEDGEMENTS
 
This work was supported by grants from the Consejo Nacional de Ciencia y Tecnología (CONACYT), México (33497N and 40218Q). The authors thank MSc. Héctor Romero-Ramírez (from our laboratory), QFB Víctor Rosales-García (from the FACS unit), and MVZ Ricardo Gaxiola-Centeno (from the animal facility) for their technical assistance.

Received April 30, 2004; revised November 24, 2004; accepted November 26, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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