Journal of Leukocyte Biology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published online as doi:10.1189/jlb.0303098 on September 12, 2003

Published online before print September 12, 2003
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.0303098v1
74/6/998    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cantó, E.
Right arrow Articles by Vidal, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cantó, E.
Right arrow Articles by Vidal, S.
(Journal of Leukocyte Biology. 2003;74:998-1007.)
© 2003 by Society for Leukocyte Biology

Distinctive response of naïve lymphocytes from cord blood to primary activation via TCR

Elisabet Cantó, Jose Luis Rodriguez-Sanchez and Silvia Vidal1

Department of Immunology, Institut de Recerca Hospital Sant Pau, Barcelona, Spain

1Correspondence: Department of Immunology, Hospital Sant Pau, Pare Claret 167, Barcelona-08025, Spain. E-mail: svidal{at}hsp.santpau.es


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Umbilical cord blood (UCB) is now being considered an alternative to bone marrow for restoring hematopoiesis after myeloablative therapy. The lower risk of acute and chronic graft-versus-host disease in patients who received UCB cells seems related to the nature of UCB–T cells. Phenotypically, UCB–CD3+ cells are mostly naive (CD45RA+) and represent a transitional population between thymocytes and adult T cells. We examined the immune reactivity of highly purified, negatively selected CD4+CD45RA+ cells by mimicking activation via T cell receptor (TCR). All experiments included the extensively characterized adult peripheral blood (APB) cells as reference. On the contrary to APB, naive UCB–CD4+ cells were able to proliferate with anti-CD3 stimulation alone. With addition of interleukin (IL)-2 or costimulatory signal, both populations reached similar proliferation. Forty-eight hours after anti-CD3 stimulation, CD4+CD45RA+ from UCB, but not APB, showed characteristic blastic morphology and significant expression of CD25 on the surface. A low concentration of IL-2 was detected at 24 h by anti-CD3-stimulated UCB CD4+CD45RA+, which rapidly disappeared. By 72 h after activation, CD4+CD45RA+ UCB cells showed extensive apoptosis, whereas CD4+CD45RA+ APB cells showed low levels of apoptosis. Using RNase protection assay, we observed that CD95L levels were significantly higher in naive CD4+ cells from UCB than from APB after activation. However, neutralizing Fas-Fc protein was unable to inhibit anti-CD3-induced apoptosis, suggesting that this was a CD95-independent mechanism. These results indicate that UCB–CD4+CD45RA+ cells are able to start proliferating as a result of early IL-2 production after TCR engagement alone, but probably, as a result of the consumption of this IL-2, they undergo cell death.

Key Words: human • T lymphocytes • cellular activation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Accumulated evidence suggests advantages in the use of hematopoietic stem cells from umbilical cord blood (UCB) as an alternative to unrelated donor bone marrow in the treatment of hematologic disorders. The presence of uncommitted and myeloid-committed precursor cells in UCB provides a durable engraftment with potentially lower risks of graft-versus-host disease (GvHD) and viral transmission than unrelated donor bone marrow transplant [1 , 2 ]. The higher tolerance to human leukocyte antigen mismatch in UCB transplants has been attributed to particular immunophenotypic features of UCB cells and their specific functional properties [3 4 5 6 7 ]. In particular, the GvHD aspect is largely dependent on the nature of CD3+ cells. There are various reports illustrating phenotypical singularities of UCB–T lymphocytes. UCB–T lymphocytes express less CD31 than adult naive CD4+ T cells, and more than 95% of UCB–T cells bear CD38 molecules but do not express CD57 [6 , 8 ]. The fact that the CD38 molecule is found on most thymocytes suggests that UCB–T cells may represent a transitional population between thymocytes and adult T cells [5 , 9 ].

UCB–T lymphocytes respond to primary allostimulation, but they do not proliferate upon rechallenge with alloantigen [10 11 12 ]. This unresponsiveness to secondary stimulation occurs in spite of T cell receptor (TCR) and costimulatory activation, but it can be overcome by treatment with phorbol 12-myristate 13-acetate (PMA) and ionomycin or a very high dose of exogenous interleukin (IL)-2 [13 ]. A defective Ras activation could be the cause of this unresponsiveness. Proliferative responses of UCB– and adult peripheral blood (APB)–T cells to mitogens show no differences in terms of increased cell size, granularity, CD25, and HLA-DR coexpression or percentage of cells in S-phase [14 15 16 ]. Conversely, UCB–T cells are significantly less robust than APB–T cells in their responses to anti-CD3 or -CD2 stimulation [17 , 18 ]. This could be the consequence of an inherent defect in protein phosphorylation after TCR engagement in UCB–T cells, characterized by an impaired phospholipase C activation, reduced levels of lck, and less phosphorylation of {zeta}-associated protein-70 [19 ].

The most common explanation for the differences observed between the APB– and UCB–T cell functions is that UCB has a higher proportion of CD4+CD45RA+ (naive) cells than APB [20 21 22 23 ]. CD4+CD45RA+ cells from UCB have been considered a homogeneous population of unprimed naïve cells, equivalent to CD4+CD45RA+ from APB. However, there are some differences at the expression level of differentiation markers (CD45 and CD31) on CD4+CD45RA+ cells from UCB [24 , 25 ]. In addition, purified UCB–CD4+CD45RA+ cells do not acquire similar activation status as their adult counterparts following anti-CD2 + anti-CD28 stimulation [26 ].

Possible explanations for these differences are that APB–CD4+CD45RA+ cells may be composed of naive cells as well as reverted CD45RO+ cells; bidirectional interconversion of CD45 isoforms has in fact been observed in rats [27 ]; or UCB–T cells represent a particular stage in ontogeny known as recent thymic emigrants, confirmed at a molecular level by the higher level of T cell excision circle markers [9 ].

In general, most reports agree that UCB–T cells are naive and behave as recent thymic emigrants. However, the use of diverse experimental conditions and modes of activation has produced noncomparable and sometimes controversial results. Conversely, heterogeneous cultures with mononuclear or unseparated T cells are complex systems that have complicated the interpretation of the results, as accessory cell activity and the proportion of T cell subsets differ in UCB and APB. Conversely, some separation techniques have resulted in a selective loss of cellular populations or in functional perturbations. In addition, stimulating agents intervening at different signaling levels have provided conflicting reports. To overcome these problems, we planned to examine the in vitro immune reactivity of highly purified negatively selected CD4+CD45RA+ cells. Studies were performed by mimicking activation through TCR with anti-CD3 monoclonal antibodies (mAb), as analysis of antigen-specific responses in freshly isolated populations is limited by the low frequency of T cells specific for any particular antigen. By using mAb specific to costimulatory molecules, we have also avoided variables such as natural substrates interacting with a variety of cell-surface ligands. Primary activation of UCB–CD4+CD45RA+ cells was compared with the well-characterized response of the CD4+CD45RA+ subset from APB.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Peripheral blood and umbilical cord CD3+ and CD4+CD45RA+ cell isolation
Peripheral blood and cord blood were obtained from healthy volunteers after given consent for the collection. All experiments received prior approval from the Hospital de Sant Pau Institutional Ethics Committees (Barcelona, Spain). Mononuclear cells were isolated by Ficoll-Hypaque (Axis-Shield PoC As, Lymphoprep, Oslo, Norway) density-gradient centrifugation. T cells were obtained by negative selection from mononuclear cells using pan–T cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Non-T cells were labeled with a cocktail of hapten-conjugated CD11b, CD16, CD19, CD36, and CD56 mAb. The non-T cells were then magnetically labeled by using magnetic cell sorter (MACS) MicroBeads coupled to an antihapten mAb. After retaining non-T cells in a magnetic column, the purity of T cells was determined by cytometry (>98%). Purified CD4+CD45RA+ T cells were obtained by negative selection from T cells using biotin-conjugated anti-CD8 (Caltag, Burlingame, CA) and anti-CD45RO (Leinco Technologies, Manchester). Afterward, CD8+ and CD45RO+ cells were magnetically labeled by using streptavidin-coupled microbeads. The viability of an untouched population recovered was determined by trypan blue (>99%), and it was >98% CD3+CD4+CD45RA+. The remaining <2% of cells do not have lymphocyte or monocyte markers. Furthermore, purity was confirmed by nonresponsiveness to phytohemagglutinin (PHA) at a final concentration of 2.5 µg/ml, as in the absence of accessory cells, purified naive T cells fail to proliferate to PHA [28 ].

Cell culture
The well plates were coated with 5 µg/ml rabbit anti-mouse immunoglobulins (Igs; Dako, Glostrup, Denmark) in phosphate-buffered saline (PBS) overnight at 4°C, washed three times, and coated with anti-CD3 (0.7 µg/ml; OKT3, American Type Culture Collection, Manassas, VA), and in some cultures, anti-CD28 mAb (Caltag) was added at 0.5 µg/ml in PBS for 1 h at room temperature. After three washes, cells were added to the plates and incubated for indicated time periods in RPMI medium completed with 10% fetal calf serum (previously tested as nonspecific-stimulating batch) at a final concentration of 0.25 x 106 cells/ml. Soluble human recombinant IL-2 (hrIL-2; Roche, Indianapolis, IN) was added at a final concentration of 50 IU/ml in indicated experiments.

DNA synthesis
[3H]-Thymidine incorporation assays were performed to determine DNA synthesis. A total of 0.25 x 106 cells was incubated in 200 µl complete medium with or without immobilized anti-CD3 mAb for indicated times. In some cultures, immobilized anti-CD28 mAb or rIL-2 were added at concentrations of 0.5 µg/ml and 50 IU/ml, respectively. The cultures were pulsed for the last 10 h with 1 µCi [3H]-thymidine. Cells were then harvested, and liquid scintillation counting assessed [3H]-thymidine uptake.

IL-2 measurement by enzyme-linked immunosorbent assay (ELISA)
The concentration of IL-2 in 24 h, 48 h, and 72 h culture supernatants was determined following the manufacturer’s recommendations using a quantitative colorimetric sandwich ELISA kit (Becton Dickinson, San Diego, CA). This technique used combinations of unlabeled capturing (clone 5344.111) and biotin-coupled detecting (clone B33-2) mAb to different epitopes of IL-2. The limit of detection was 40 pg/ml.

Apoptotic cells
The percentage of cells undergoing apoptosis was determined by cellular staining with a rh-Annexin V-fluorescein isothiocyanate (FITC) kit (Bender MedSystem, Vienna, Austria) [29 ]. Resting control and 72 h-activated cells were harvested and washed with PBS. The pellet was resuspended in 200 µl binding buffer and stained for 10 min at room temperature with 5 µl Annexin V-FITC antibody. Cells were then resuspended in 200 µl binding buffer and added 5 µl propidium iodide (PI; 1 µg/ml). Samples were analyzed by flow cytometry. In selected experiments, soluble rhCD95 (Fas:Fc; R&D Systems, Minneapolis, MN) was added to the culture at various concentrations (8, 4, 2, and 1 µg/ml) using PHA-stimulated blasts in the presence of anti-CD95 (500 ng/ml; clone CH11, Upstate Biotechnology, Lake Placid, NY) as a positive control of apoptosis for the measurement of the inhibitory effect of Fas:Fc.

Flow cytometry
Cells from APB and UCB were stained with CD45-FITC/CD4-RD1/CD8-ECD/CD3-PC5 (Coulter, Miami, FL) or CD4-FITC (Caltag), CD45RA-phycoerythrin (PE; Diaclone, Cedex, France), and CD3-PC5 (Coulter) to determine the percentage of different subsets of cells.

Resting and activated cells were stained with anti-CD25 mAb labeled with PE (Caltag), and the expression of CD25 (IL-2 receptor {alpha}) was analyzed by flow cytometry (EPICS-XL, Coulter). For down-regulation follow-up, purified cells were adjusted at 0.4 x 106 cells/ml and incubated for the indicated times in wells previously coated with anti-CD3 (0.7 µg/ml; OKT3). Cells were resuspended and transferred to ice-cold PBS, washed twice, and stained with the specific mAb directly labeled: CD3-PC5 (Coulter), CD4-FITC (Caltag), and CD45RA-PE (Diaclone). Cell-surface expression of TCR-CD3 was assessed by flow cytometry, and the mean fluorescence intensity (MFI) was measured at each point. The positive proportion of each subset and the MFI of CD3 expression were calculated from these gated subsets using EXPOTM 32 MultiCOMP software (Coulter). CD95 (Fas) expression was determined by cytometry using anti-CD95-FITC (Becton Dickinson).

RNase protection assay
Assays were performed following the RiboQuant multiprobe RNase protection assay system instructions with slight modifications [30 ]. We used two sets of multiprobe (hAPO 2 and hAPO 3; Becton Dickinson) to determine the expression of genes related to the bcl-2 family and tumor necrosis factor receptor (TNFR) family, respectively, including templates for the analysis of L32 and glyceraldehyde 3-phosphate dehydrogenase housekeeping genes. Antisense RNA probes were performed using [{alpha}-32P]-UTP and T7 RNA polymerase. Labeled probes were dissolved in hybridization buffer and adjusted to 2.5 x 105 cpm/µl and 4.3 x 105 cpm/µl for hAPO 2 and hAPO 3, respectively. Total RNA (2 µg) isolated from purified CD4+CD45RA+ cells was dissolved in 8 µl hybridization buffer. Diluted probe (2 µl) was added to each RNA sample and incubated for 12–16 h. Unhybridized probes and target RNAs were digested with RNase A, and the samples were treated with proteinase K. The remaining "RNase-protected" probes were purified, dissolved in 1x loading buffer, and resolved on 5% denaturing polyacrylamide gel according to their size. Dried gels were exposed to X-ray film at -70°C, and semiquantitative analysis was measured using the Fluor-S system. The identity and quantity of each mRNA species sample were then determined based on the signal intensities given by the appropriately sized protected probe-fragment bands.

Western blot
After culturing, cells were harvested and lysed in 10 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, and 1% Triton X-100. Lysates were cleared with a 10,000 rpm spin at 4°C for 15 min. Volumes of lysate containing 1.5 µg protein were fractionated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis by using Bio-Rad minigel apparatus (Bio-Rad, Hercules, CA). The fractionated proteins were transferred to immobilon polyvinylidene difluoride membranes (Millipore, Bedford, MA) using a semidry transblot apparatus. The blots were blocked overnight at 4°C with 10 mM Tris pH 7.5, 0.1 M NaCl containing 0.1% Tween-20 (Sigma Chemical Co., St. Louis, MO) and 1% bovine serum albumin. The blots were probed with anti-CD95L mAb (clone G247-4, Becton Dickinson) followed by horseradish peroxidase mouse anti-human IgG (Pierce, Rockford, IL). Finally, membranes were developed by chemiluminiscence using enhanced chemiluminescence (Pierce).

Statistics
Data are presented as mean ± SD. A nonparametric Mann-Whitney test was used to compare UCB and APB values. P values <0.05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Immunophenotyping resting lymphocytes from UCB and APB
The distribution of lymphocyte subsets in blood samples from UCB and APB was evaluated. Although UCB and APB presented a similar proportion of CD4+CD3+ (44.7±14.1% in UCB vs. 40.2±15.2% in APB), UCB showed a lower percentage of CD3+CD8+ (14.0±7% in UCB vs. 26.5±10% in APB). Consequently, the ratio between CD3+CD4+/CD3+CD8+ was significantly higher in UCB (3.19 vs. 1.51). As expected, CD4+ cells from UCB contained significantly increased numbers of CD45RA+ (89.4±14% vs. 49±15%) but reduced numbers of the CD45RO+ cell than APB. Naive CD4+ cells from UCB and APB express similar amounts of CD28, CD62L, CCR7, and CD27, without any of those markers defining a distinct population among UCB– and APB–CD4+CD45RA+ cells. When analyzing the expression of CD3/TCR on the cell surface of T cells (Fig. 1 ), the MFI of CD3 on each of the different subsets of T cells (CD3, CD4, CD45RA) was slightly but consistently higher in UCB in more than 20 independent experiments (MFI of CD4+CD45RA+ cells was 6.6±0.50 in APB vs. 7.89±0.53 in UCB, P<0.001). This result demonstrated that UCB–T subsets display a higher number of CD3 molecules on the surface than APB cells.



View larger version (16K):
[in this window]
[in a new window]
  		Figure 1. CD3 expression levels on CD4+CD45RA+cells from APB and UCB. Expression of CD3 on the cell surface was measured by flow cytometry on resting CD4+ and CD4+CD45RA+ cells from APB and UCB. Cells were labeled with anti-CD3-PC5, anti-CD4-ECD, and anti-CD45RA-FITC, as described in Materials and Methods. After gating the indicated populations, the CD3 expression level was determined and compared between UCB (black line) and APB (gray line). A representative histogram of 20 experiments is shown (MFI of CD4+CD45RA+ cells was 6.6±0.50 in APB vs. 7.89±0.53 in UCB, P<0.001).

 
Activation and expansion of naive CD4+ UCB cells after primary stimulation
Anti-CD3-induced DNA synthesis by UCB–T cells was not significantly different from that exhibited by adult T cells (our previous unpublished results and ref. [31 ]). To compare the functional capacity of naive CD4+ from UCB with naive CD4+ cells from APB, equivalent numbers of highly purified CD4+CD45RA+ cells were used to reduce the contribution of uncontrolled elements present in various proportions in UCB and APB samples. CD4+CD45RA+ cells from UCB or APB were incubated on plates with anti-CD3, anti-CD3 + anti-CD28 or anti-CD3 + rIL-2. The concentration of anti-CD3 used (0.7 µg/ml) had provided consistent activation of T cells from APB in previous mononuclear cell cultures. [3H]-Thymidine incorporation revealed that naive CD4+ cells from UCB, but not from APB, were able to synthesize DNA with anti-CD3 stimulation alone (Fig. 2A ). With the addition of a costimulatory signal (anti-CD28), the two populations reached similar high levels of DNA synthesis. Although neither APB nor UCB cells respond to IL-2 alone (2245±1338 in APB vs. 6532±1911 in UCB), immobilized anti-CD3 mAb induced an IL-2 responsive state in APB cells, as the addition of exogenous rIL-2 to wells with immobilized anti-CD3 reduced the difference between CD4+CD45RA+ cells from UCB and APB.



View larger version (15K):
[in this window]
[in a new window]
  		Figure 2. Proliferation and expansion of UCB naïve CD4 cells after stimulation with anti-CD3. (A) Comparison of proliferative response of naïve CD4+ cells from UCB and APB. CD4+CD45RA+ cells (2x105) were cultured in plates with immobilized anti-CD3 (0.7 µg/ml; OKT3), immobilized anti-CD3 + anti-CD28 (a-CD28; 0.5 µg/ml), or rIL-2 (50 IU/ml). Cells were pulsed with [3H]-thymidine and collected 72 h after initial stimulation. Data are the mean of five independent experiments. Statistical analysis (*) demonstrated that there was a significant incorporation of [3H]-thymidine in the UCB cells when cultured with OKT3 (P<0.02). Although there was a small [3H]-thymidine incorporation in APB cells with OKT3 (P<0.04), UCB and APB reached different levels of DNA synthesis (P<0.03). (B) Comparison of the expansion of naïve CD4+ cells from UCB and APB in the presence of gradual concentrations of immobilized anti-CD3 ranging from 0.5 to 2.5 µg/ml. After 72 h in culture, UCB (solid line) and APB (dotted line) cells from individual wells were harvested and counted manually, and the number of cells is shown. Data are the mean of five independent experiments. The number of cells was significantly different in the presence of OKT3 concentrations ranging from 0.5 to 2.5 µg/ml (P<0.05).

 
As [3H]-thymidine incorporation represents DNA labeling in S-phase, the definitive cell division was quantified by counting the number of cells in the well after the stimulation with anti-CD3 alone. Gradual concentrations of anti-CD3 mAb ranging from 0.5 to 2.5 µg/ml were used to coat 96-well plates, and at daily intervals, cells were harvested and counted. As seen in Figure 2B , the number of cells in anti-CD3-coated wells was compared with the initial number of cells put in culture (2.5x105 cells). In agreement with the [3H]-thymidine incorporation experiment, CD4+CD45RA+ cells from UCB were able to proliferate with an average expansion of 1.5 to twofold at day 3. Anti-CD3 mAb alone induced CD4+ cells to enter the cell cycle to achieve expansion without the requirement of exogenous rIL-2 or costimulatory signals. Activated CD4+CD45RA+ cells proliferated in clusters during anti-CD3 activation.

On the contrary, CD4+CD45RA+ APB cells were not able to proliferate, regardless of the concentration of anti-CD3 in studies on days 3 and 5. This suggests that the presence of anti-CD3 mAb under the titration range shown is insufficient to activate CD4+CD45RA+ APB cells. It is unlikely that proliferation of UCB cells induced by immobilized anti-CD3 was a result of the presence of a minor population of contaminating accessory cells, as proliferation was not observed after culturing highly purified UCB–CD4+CD45RA+ cells with a mitogenic dose of PHA (data not shown) [28 ]. Furthermore, the <2% of contaminating cells are in the lymphoid region of the flow cytometry analysis, and they are CD45-, suggesting the presence of few nucleated red blood cells abundant in cord blood samples [5 ].

After 18 h of stimulation, the MFI values of CD3 showed that CD4+CD45RA+ UCB and APB cells expressed less than 20% of the TCR/CD3 surface expression (data not shown). Cell stimulation conditions did not influence membrane expression of other surface molecules (CD4 and CD45), concluding that down-regulation was specific for TCR/CD3 components. Moreover, TCR/CD3 complexes were in fact internalized and not simply masked by down-regulating antibodies, as control experiments with FITC-labeled anti-Ig detected no Igs adhering to the cell surface (data not shown).

Phenotype of activated CD4+CD45RA+ UCB cells
Forty-eight hours after stimulation with anti-CD3, CD4+CD45RA+ UCB cells showed a characteristic enlargement (Fig. 3 ), and CD4+CD45RA+ APB cells showed small morphology, representative of resting lymphocytes. The blastic transformation of naïve CD4+ UCB cells, after stimulation with anti-CD3, may be attributed to their higher sensitivity when compared with APB cells. After stimulation with anti-CD3 in the presence of exogenous rIL-2 or costimulatory signals, naïve CD4+ cells from UCB as well as from APB showed significant enlargement. These results suggest that naïve CD4+ APB cells are more dependent on costimulatory signals than naïve CD4+ UCB cells to undergo activation.



View larger version (41K):
[in this window]
[in a new window]
  		Figure 3. Phenotypic analysis of primary activation of naïve CD4+ cells from APB and UCB. (A) Changes in morphology after 48 h of activation were monitored by flow cytometry analysis of cellular forward-scatter (FS; y-axis) and side-scatter (SS; x-axis; lanes 1 and 3). Lanes 2 and 4 represent staining cells with anti-CD25-PE mAb. Naïve CD4+ cells were isolated as described in Materials and Methods and were stimulated for 48 h in the presence of medium, anti-CD3 (OKT3), anti-CD3 plus anti-CD28 (OKT3 + a-CD28), rIL-2 (50 IU/ml), and anti-CD3 plus rIL-2 (OKT3 + rIL-2). This figure represents a minimum of five experiments. (B) Blastic formation in UCB cells, but not in APB cells, after 48 h in culture with OKT3. After the culture, cells were harvested, cytospinned at 400 rpm for 1 min, and stained with methylene blue solution (x400).

 
At different time points (24 h, 48 h, and 72 h) after activation, UCB and APB naïve CD4+ cells were stained with specific anti-CD25 mAb and analyzed by cytometry. CD25 was up-regulated on UCB cells 24–48 h after anti-CD3 activation, whereas CD25 expression remained low on APB up to 72 h after anti-CD3 activation (67.55±3.6% CD25+ cells in UCB vs. 31.7±5.3% in APB, P<0.03). This observation was consistent with the higher proliferation rate and the blastic morphology of the naïve CD4+ cells from UCB after primary activation with anti-CD3.

In spite of the significant proliferation and expansion of UCB naïve CD4+ cells following anti-CD3 stimulation, the levels of IL-2 in the supernatants after 24 h in culture were low in UCB and undetectable in APB cultures (140±20 pg/ml in UCB vs. <40 pg/ml in APB supernatants). At 48 h and 72 h after the initiation of the culture, IL-2 levels in the supernatants of anti-CD3-activated naive UCB cells were undetectable by ELISA. There was no detection of IL-2 on supernatants of APB cells at 24, 48, or 72 h. The experimental conditions did not allow discarding IL-2 consumption as the major cause of not detecting IL-2 in the supernatants. The kinetics of IL-2 production by naive CD4 cells from UCB and APB after activation with anti-CD3 + anti-CD28 revealed that the peak was at 24 h with both populations, producing similar high IL-2 levels (>10,000 pg/ml). This result is consistent with the comparably high proliferation observed in both cultured populations and demonstrates that naïve CD4+ cells from UCB and APB are able to produce IL-2 under optimal conditions of activation.

Naive CD4 UCB cells undergo cell death in response to primary stimulation in vitro
Considering that thymocytes and naive murine neonatal T cells are sensitive to cell death following TCR ligation [32 33 34 ] and that naïve CD4+ UCB cells have been characterized as recent thymic emigrants, we tested whether T cells from UCB may also undergo apoptosis in response to primary stimulation. Naive CD4+ cells were activated on plates with immobilized anti-CD3, and at 24, 48, and 72 h after activation, cells were stained with Annexin V and PI and were analyzed by cytometry (Fig. 4A and 4B ). By 72 h, the spontaneous apoptosis of UCB–CD4+CD45RA+ cells was remarkably high, and after activation with anti-CD3, naive CD4+ UCB cells showed an even more extensive apoptosis. On the contrary, naive CD4+ APB showed similar low levels of apoptosis up to 5 days following culture in anti-CD3-coated wells (data not shown). The induced apoptosis in UCB cells was prevented by the addition of rIL-2 or by costimulatory signals (anti-CD28). As naive CD4+ UCB underwent a significant expansion after anti-CD3 activation, in absolute numbers, the number of apoptotic UCB naive CD4+ cells was remarkably high. It is interesting to note that during the cytometry analysis of apoptosis, a high fraction of dead cells was located in the blastic morphology region, suggesting that these dead cells had been previously activated.



View larger version (31K):
[in this window]
[in a new window]
  		Figure 4. UCB–CD4+CD45RA+ cells undergo apoptosis after anti-CD3 stimulation. (A) Neonatal and adult CD4+CD45RA+ cells were cultured with medium, plate-bound anti-CD3 (OKT3), anti-CD3 + anti-CD28 (OKT3+ a-CD28), and anti-CD3 + rIL-2 (OKT3+IL-2). At 48 h after the initiation of the culture, the cells were harvested and stained with PI and Annexin V-FITC. The percentage of cells falling within each region is indicated. The figure is representative of five independent experiments. (B) Percentage of apoptotic cells at 48 h under the indicated conditions. Statistical differences were indicated: **, P < 0.01.

 
To determine possible mechanisms involved in the apoptosis induced after primary activation on UCB cells, we compared the expression of apoptotic genes at mRNA level from purified, naive UCB and APB–CD4+ cells, using multiprobe RNase protection assay (hAPO 3; Fig. 5A ). The profile of mRNAs in resting naive CD4+ UCB cells was similar to APB cells. Upon activation with anti-CD3, anti-CD3 + anti-CD28, or anti-CD3 + rIL-2, there was a similar induction of the mRNAs encoding FADD, FAF, and TNFR1 in UCB and APB cells. TRAIL expression showed high variability among different UCB samples. CD95 and CD95L levels were up-regulated in both populations after activation. However, CD95 mRNA levels were lower in activated naive CD4+ cells from UCB than in APB. This result was confirmed at protein level by flow cytometry (Fig. 5B) . In contrast, CD95L mRNA levels were significantly higher in naive CD4+ cells from UCB than from APB. By Western blot, the higher levels of CD95L protein in anti-CD3-activated UCB cells than APB cells were confirmed (Fig. 5C) .



View larger version (36K):
[in this window]
[in a new window]
  		Figure 5. Analysis of possible genes involved in the apoptosis after primary activation of UCB and APB naïve CD4+ cells. (A) A representative, radiographic profile of several genes related to apoptosis. UCB and APB–CD4+CD45RA+ cells were cultured for 48 h in the presence of medium, immobilized anti-CD3 (OKT3), anti-CD3 + anti-CD28 (OKT3+a-CD28), and anti-CD3 + rIL-2 (OKT3+rIL-2). Total RNA from resting and stimulated cells was analyzed for distinct mRNA species using RiboQuant multiprobe RNase protection assay system with the hAPO 3 multiprobe template set. Free probe and other single-stranded RNA molecules were digested with RNases. The RNase-protected probes were purified, resolved on denaturing polyacrylamide gels, and imaged by autoradiography. Bands of housekeeping genes (L32) were included for normalizing signals, and probe bands were quantified with Fluor-S image system (Bio-Rad). FADD, Fas-associated death domain; FAF, Fas-associated protein factor; TRAIL, TNF-related apoptosis-inducing ligand; RIP, receptor-interacting protein. (B) The expression of CD95 protein was determined by flow cytometry on the surface of UCB and APB cells after 48 h of culture in the presence of medium, anti-CD3 (OKT3; P<0.03), anti-CD3 + anti-CD28 (OKT3+a-CD28; P<0.03), and anti-CD3 + rIL-2 (OKT3+rIL-2; 50 IU/ml; P<0.04). Cells were gated on FS and SS to exclude debris. The values, shown in the flow cytometric profiles, represent the proportion of cells that fall in the window of positive cells ± SD from five separate experiments. (C) Western blot analyzed CD95L protein levels. After 48 h in culture in the presence of medium (lanes 1), anti-CD3 (lanes 2), anti-CD3 + anti-CD28 (lanes 3), and anti-CD3 + IL-2 (lanes 4), cells were harvested. Extracted proteins corresponding to 1 x 106 cells were blotted on polyvinylidene difluoride membranes and were probed for anti-CD95L as described in Materials and Methods. Blots were stripped and reprobed with anti-ß actin to verify the same protein levels per lane.

 
The neutralizing effect of a soluble form of CD95 that binds to the CD95L on the cell surface was used to determine whether CD95 was participating in the apoptosis induced in UCB-naive CD4+ cells by primary activation with anti-CD3, or alternatively, high CD95L expression was a reflection of the higher level of activation achieved by these cells. Cells were cultured in the presence of a chimeric molecule consisting of the extracellular region of human CD95 and the Fc portion of human IgG (Fas:Fc) as described previously [35 ]. Whereas the Fas:Fc molecule at 2–4 µg/ml was able to inhibit 50–60% of the CD95L-induced apoptosis of PHA blasts [36 ], it had no effect on the anti-CD3-induced death of UCB–CD4+CD45RA+ cells (data not shown). This result therefore suggested that anti-CD3-induced apoptosis in UCB was via a CD95-independent mechanism.

The expression of bcl-2, bax, and bcl-xL mRNA was examined in UCB and APB naive CD4+ cells by RNase protection assay (hAPO 2; Fig. 6 ) after the normalization against mRNA of housekeeping gene L32. After culturing CD4+CD45RA+ cells for 48 h in media, UCB and APB cells expressed comparable low levels of bcl-2 and bax, and both failed to express bcl-xL. Although upon activation with anti-CD3, almost half of the CD4+CD45RA+ cell from UCB but not from APB undergoes cell death, both populations expressed similar levels of bcl-2. After activation with anti-CD3, bax expression was induced in UCB cells, and the average in UCB did not differ significantly from APB (5.9±2 in UCB vs. 8±3 in APB). Bcl-xL was also induced in APB and UCB cells, and with costimulatory signals, the expression increased in both populations. It is interesting to note that apoptosis was mostly inhibited with the addition of rIL-2; however, bcl-xL levels were equivalent in UCB cells after OKT3 and OKT3 + IL-2 activation.



View larger version (16K):
[in this window]
[in a new window]
  		Figure 6. Analysis of bcl-2, bax, and bcl-xL gene expression after primary activation of UCB and APB naïve CD4+ cells. UCB– and APB–CD4+CD45RA+ cells were cultured for 48 h under the indicated conditions. Total RNA from cells was analyzed for bcl-2, bax, and bcl-xL mRNA species using RiboQuant multiprobe RNase protection assay system with the hAPO 2 template set. Bands of housekeeping gene (L32) were included for normalizing signals. Each histogram shows the mean ± SD of the normalized values of bcl-2, bax, and bcl-xL gene expression from six different samples analyzed in two separated experiments. Statistical differences in bcl-xL expression under OKT3 culture were observed (P<0.03).

 
The results indicate that while rescuing UCB–CD4+CD45RA+ cells from TCR-mediated apoptosis with anti-CD28 or rIL-2, there was no change in the ratio of bcl-2 and bax. Thus, TCR-mediated apoptosis in naive UCB cells was not directly related to alterations in the balance of the expression of pro- and antiapoptotic members of the bcl-2 family.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we have examined the functional potential of UCB–T cells. To this end, highly purified CD4+CD45RA+ cells were used to determine the activation level achieved after TCR/CD3 engagement. Anti-CD3 mAb alone stimulated UCB–CD4+CD45RA+ cells to proliferate and express CD25 in the absence of accessory cells, costimulatory signals, or exogenous rIL-2. In contrast, an additional contribution of costimulatory signals or exogenous IL-2 was required for inducing APB–CD4+CD45RA+ cells to proliferate.

It has previously been shown that the proliferation of UCB–T cells to mitogens does not differ from the response of ABP T cells [14 15 16 ], but UCB–T cells are significantly less robust than APB–T cells in their responses to anti-CD3 or anti-CD2 stimulation [17 , 18 ]. As there is no indicator of the activated cell type in these T cell cultures, the mixture of naive and memory cells in APB–T cells might have complicated the interpretation of these results. Using purified CD4+CD45RA+ cells, UCB and APB do not acquire similar activation status following PHA, anti-CD2, or PMA plus ionomycin stimulation [26 ], but these experimental conditions are unable to identically reproduce those mechanisms initiated by TCR engagement.

The triggering of the TCR/CD3 complex is proposed to be insufficient to induce an effective T cell activation [37 , 38 ]. However, the activation of UCB naïve CD4+ cells is not under so restrictive control as in adult naive cells [39 ]. The activation signal delivered by anti-CD3 antibodies to resting CD4+CD45RA+ UCB cells was enhanced along with the density of anti-CD3 bound to the plastic. In comparison with reports showing similar CD3 expression levels on UCB and APB T cells [18 ], our analysis revealed a slight but consistently higher expression of CD3 on the surface of UCB–T cells. In our experiments, the expression of CD3 was determined separately on each subset of UCB and APB T cells (CD4+, CD45RA+) to avoid a masking effect from different CD3 expression on naive and memory or on CD4 and CD8 cells. Therefore, the sensitivity of UCB cells to activation via TCR may be related to their higher CD3 expression.

As the TCR down-regulation extension on UCB–CD4+CD45RA+ cells was similar to APB cells, the efficient T cellactivation in UCB cells correlated strictly with a higher number of internalized TCRs [40 ]. Probably, there are different signaling mechanisms initiated by internalized TCRs that can be responsible for the different requirements of UCB and APB cells to enter in the cell cycle and proliferate (manuscript in preparation).

Proliferation relies on a number of factors involving the capacity of T cell subsets to enter the cell cycle, production of the primary T cell growth factor IL-2, and up-regulation of CD25. In our experiments, there is an apparent discrepancy between the effective activation of UCB naive CD4+ cells after TCR engagement alone and the low amount of IL-2 secreted at 24 h. A significant earlier IL-2 production may not have been detected in our experiments, but this is improbable, as the peak of IL-2 gene expression in most activatory systems is ~24 h. Presumably, the low concentration of IL-2 detected at 24 h by anti-CD3-stimulated UCB–CD4+CD45RA+ cells rapidly disappeared as a result of autocrine consumption during posterior proliferation [41 ].

IL-2 gene transcription in UCB naive CD4+ cells is under relatively more stringent control compared with other genes involved in activation-marker modulation or cell-cycle entry. Thus, silencer activity mediated through the Pu element of the IL-2 promoter was exclusively found in resting naive CD4 cells [42 ]. An IL-2-independent UCB cell proliferation depending on other unidentified soluble factors as described in other scenarios [43 , 44 ], would provide an alternative explanation for the activation of UCB naïve cells in the absence of IL-2 production.

Death of TCR-activated UCB cells may be a result of a premature exit from the cell cycle with the consequent impairment of IL-2 production. Alternatively, the maintenance of CD25 expression after consuming most of IL-2 could have been negative for the survival of UCB cells as long as they remain activated, engaging UCB–T cells to death. The IL-2 production at 24 h in culture with OKT3 could be sufficient for driving proliferation of UCB cells after signaling through CD25 on the surface of activated UCB cells. The consumption without production could result in an apoptotic regulatory mechanism to control those cells that have been able to initiate the proliferation in the absence of a costimulatory signal. The preventive effect of exogenous rIL-2 on the TCR-mediated apoptosis in UCB cells is consistent with the latter hypothesis [45 ].

The results did not formally prove that cell proliferation was indeed necessary for negative signaling, but more than 40% of dead cells displayed blast morphology (data not shown). It has been postulated that when TCR/CD3 triggering induces cell death, it requires a preactivated state of the cell associated with proliferation [46 ]. This phenomenon of activation and apoptosis in UCB naive CD4+ cells is not unique. It has also been reported that high doses of soluble antigen or superantigens cause a rapid increase in T cells bearing target Vß followed by a rapid decline of reactive cells [47 , 48 ]. Similarly to UCB naïve CD4 cells with anti-CD3, superantigens deliver an overpowering stimulus that drives proliferation and death [49 ]. However, the massive proliferation is not an essential prerequisite for superantigen-mediated T cell deletion [50 ]. Further experiments will be necessary to determine whether UCB cell proliferation is a requirement for the TCR-mediated apoptosis.

There are two major mechanisms of T cell death involving a requirement for cell activation and a central role for IL-2. Our observations suggest that a generalized mechanism known as activation-induced cell death cannot account for TCR-mediated UCB apoptosis, as high levels of CD95L were not mediators of the killing. Conversely, CD95Lhigh naive UCB cells may have acquired an increased ability to induce apoptosis in other CD95-sensitive targets [51 ]. CD95L expression was high in TCR-activated UCB cultures, suggesting that increased levels rather correspond to the up-regulation that occurs after activation. Our observations also indicated that TCR-mediated apoptosis in naïve UCB cells did not directly alter the balance of the pro- and antiapoptotic members of the bcl-2 family associated with passive cell death after cytokine deprivation [52 ]. The possibility of a heterogenous protein expression of bcl family members in these cultures was not addressed by this study and remains undefined. Recently, BH3-only proteins have been described as a novel neutralization mechanism of the antiapoptotic properties of bcl-2 family members [53 ]. Transforming growth factor-ß is another mechanism that promotes the elimination of post-activated T cells once the initial stimulus has been resolved without directly inducing apoptosis via CD95 or influencing bcl proteins [54 ].

In UCB naive cells, combined activation of antigen and costimulatory receptors leads to full activation of TCR-coupled signaling pathways and reduced the percentage of cell death probably by increasing IL-2 production. However, with the present experiments, we cannot exclude that CD28 is also capable of costimulating independently of IL-2 and nuclear factor of activated T cells [43 ] and significantly up-regulating bcl-xL in UCB cells [55 ].

Engaging TCR with anti-CD3 on thymocytes and naive CD4+ cells from UCB produces apoptosis [33 ]. The similar response between cells from both sources could be a reflection of the progression from neonatal to adult immunocompetence in vivo, as these cells have been considered recent thymic emigrants [9 ].

From the TCR engagement, we cannot conclude about the response of T cells to alloantigens, but it is still tempting to speculate how UCB T cells will be allostimulated. After UCB transplantation, the first wave of T cell reconstitution is originated from the graft-derived mature donor T cells infused [56 ]. UCB-cell stimulation through antigen receptors without engagement of costimulatory receptors could induce a strong, tolerizing stimulus by generating suboptimal activation before clearing many activated cells [57 ]. With their death, the overall T cell numbers decrease, and the relative number of specific T cells to particular antigens or that bear certain TCRs also changes. Although we do not know whether naive phenomena in vitro may be bypassed in vivo by unforeseen factors, and there are no data available on the role of peripheral expansion of UCB cells after transplant, it is interesting to note that UCB-transplanted patients have a greater proportion of apoptotic T cells in the peripheral blood than patients who have received bone marrow or mobilized peripheral blood stem cells [58 ]. In this case, apoptosis is likely to be selective for donor-reactive cells, as only activated cells are susceptible to death. The deletion of donor-reactive clones has been contemplated as the basis of transplantation tolerance [59 ] to explain the apparently reduced GvHD in UCB-transplanted individuals. Although graft-versus-leukemia potential of UCB cells could have been reduced by the apoptosis of donor-reactive cells, our findings on CD4+ naive cells might not necessarily be translated into the lytic activity of cord blood cells. Recently, some reports have demonstrated the induction of graft-versus-leukemia effect and the complete remission of acute leukemia following unrelated umbilical cord blood transplantation [60 ].


   ACKNOWLEDGEMENTS
 
This work was supported by FIS 00/0583 grant. E. C. is a research fellow, and S. V. has a Research Scientist contract from Fondo Investigaciones Sanitarias. We thank Cord Blood Bank of Barcelona for kindly providing cord blood units.

Received March 10, 2003; accepted July 29, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Gluckman, E., Rocha, V., Boyer-Chammard, A., Locatelli, F., Arcese, W., Pasquini, R., Ortega, J., Souillet, G., Ferreira, E., Laporte, J. P., Fernandez, M., Chastang, C. (1997) Outcome of cord-blood transplantation from related and unrelated donors. Eurocord Transplant Group and the European Blood and Marrow Transplantation Group N. Engl. J. Med. 337,373-381[Abstract/Free Full Text]
  2. Rubinstein, P., Carrier, C., Scaradavou, A., Kurtzberg, J., Adamson, J., Migliaccio, A. R., Berkowitz, R. L., Cabbad, M., Dobrila, N. L., Taylor, P. E., Rosenfield, R. E., Stevens, C. E. (1998) Outcomes among 562 recipients of placental-blood transplants from unrelated donors N. Engl. J. Med. 339,1565-1577[Abstract/Free Full Text]
  3. Clerici, M., DePalma, L., Roilides, E., Baker, R., Shearer, G. M. (1993) Analysis of T helper and antigen-presenting cell functions in cord blood and peripheral blood leukocytes from healthy children of different ages J. Clin. Invest. 91,2829-2836
  4. Harris, D. T., LoCascio, J., Besencon, F. J. (1994) Analysis of the alloreactive capacity of human umbilical cord blood: implications for graft-versus-host disease Bone Marrow Transplant. 14,545-553[Medline]
  5. Han, P., Hodge, G., Story, C., Xu, X. (1995) Phenotypic analysis of functional T-lymphocyte subtypes and natural killer cells in human cord blood: relevance to umbilical cord blood transplantation Br. J. Haematol. 89,733-740[Medline]
  6. D’Arena, G., Musto, P., Cascavilla, N., Di Giorgio, G., Fusilli, S., Zendoli, F., Carotenuto, M. (1998) Flow cytometric characterization of human umbilical cord blood lymphocytes: immunophenotypic features Haematologica 83,197-203[Abstract/Free Full Text]
  7. Cairo, M. S., Wagner, J. E. (1997) Placental and/or umbilical cord blood: an alternative source of hematopoietic stem cells for transplantation Blood 90,4665-4678[Free Full Text]
  8. Delespesse, G., Yang, L. P., Ohshima, Y., Demeure, C., Shu, U., Byun, D. G., Sarfati, M. (1998) Maturation of human neonatal CD4+ and CD8+ T lymphocytes into Th1/Th2 effectors Vaccine 16,1415-1419[CrossRef][Medline]
  9. Hassan, J., Reen, D. J. (2001) Human recent thymic emigrants–identification, expansion, and survival characteristics J. Immunol. 167,1970-1976[Abstract/Free Full Text]
  10. Risdon, G., Gaddy, J., Horie, M., Broxmeyer, H. E. (1995) Alloantigen priming induces a state of unresponsiveness in human umbilical cord blood T cells Proc. Natl. Acad. Sci. USA 92,2413-2417[Abstract/Free Full Text]
  11. Paiva, A., Freitas, A., Loureiro, A., Couceiro, A., Martinho, A., Simoes, O., Santos, P., Tomaz, J., Pais, M. L., Breda Coimbra, H. (1998) Functional aspects of cord blood lymphocytes response to polyclonal and allogeneic activation Bone Marrow Transplant. 22(Suppl. 1),S31-S34
  12. Slavcev, A., Striz, I., Ivaskova, E., Breur-Vriesendorp, B. S. (2002) Alloresponses of cord blood cells in primary mixed lymphocyte cultures Hum. Immunol. 63,155-163[CrossRef][Medline]
  13. Porcu, P., Gaddy, J., Broxmeyer, H. E. (1998) Alloantigen-induced unresponsiveness in cord blood T lymphocytes is associated with defective activation of Ras Proc. Natl. Acad. Sci. USA 95,4538-4543[Abstract/Free Full Text]
  14. Ruiz-Requena, R., Garrido, F., Martin-Andres, A., Osorio, C. (1977) Studies on T cells in newborns. Higher reactivity of umbilical cord blood lymphocytes to PHA as measured by whole blood microtechnique Rev. Esp. Fisiol. 33,181-186[Medline]
  15. Griffin, J. F., Bulmer, R., Wilson, E. W. (1979) Cord blood lymphocyte subpopulations and mitogenic activity in whole blood microculture J. Reprod. Immunol. 1,219-227[CrossRef][Medline]
  16. D’Arena, G., Cascavilla, N., Carotenuto, M. (1998) Blastogenic response of activated human umbilical cord blood T-lymphocytes Haematologica 83,1048-1050[Abstract/Free Full Text]
  17. Pirenne-Ansart, H., Paillard, F., De Groote, D., Eljaafari, A., Le Gac, S., Blot, P., Franchimont, P., Vaquero, C., Sterkers, G. (1995) Defective cytokine expression but adult-type T-cell receptor, CD8, and p56lck modulation in CD3- or CD2-activated T cells from neonates Pediatr. Res. 37,64-69[Medline]
  18. Hassan, J., Reen, D. J. (1997) Cord blood CD4+ CD45RA+ T cells achieve a lower magnitude of activation when compared with their adult counterparts Immunology 90,397-401[CrossRef][Medline]
  19. Miscia, S., Di Baldassarre, A., Sabatino, G., Bonvini, E., Rana, R. A., Vitale, M., Di Valerio, V., Manzoli, F. A. (1999) Inefficient phospholipase C activation and reduced Lck expression characterize the signaling defect of umbilical cord T lymphocytes J. Immunol. 163,2416-2424[Abstract/Free Full Text]
  20. Wilson, C. B., Westall, J., Johnston, L., Lewis, D. B., Dower, S. K., Alpert, A. R. (1986) Decreased production of interferon-gamma by human neonatal cells. Intrinsic and regulatory deficiencies J. Clin. Invest. 77,860-867
  21. Cohen, S. B., Dominiguez, E., Lowdell, M., Madrigal, J. A. (1998) The immunological properties of cord blood: overview of current research presented at the 2nd EUROCORD workshop Bone Marrow Transplant. 22(Suppl. 1),S22-S25
  22. Lee, S. M., Suen, Y., Chang, L., Bruner, V., Qian, J., Indes, J., Knoppel, E., van de Ven, C., Cairo, M. S. (1996) Decreased interleukin-12 (IL-12) from activated cord versus adult peripheral blood mononuclear cells and upregulation of interferon-gamma, natural killer, and lymphokine-activated killer activity by IL-12 in cord blood mononuclear cells Blood 88,945-954[Abstract/Free Full Text]
  23. Qian, J. X., Lee, S. M., Suen, Y., Knoppel, E., van de Ven, C., Cairo, M. S. (1997) Decreased interleukin-15 from activated cord versus adult peripheral blood mononuclear cells and the effect of interleukin-15 in upregulating antitumor immune activity and cytokine production in cord blood Blood 90,3106-3117[Abstract/Free Full Text]
  24. Reen, D. J. (1998) Activation and functional capacity of human neonatal CD4 T-cells Vaccine 16,1401-1408[CrossRef][Medline]
  25. D’Arena, G., Musto, P., Cascavilla, N., Minervini, M. M., Di Giorgio, G., Maglione, A., Carotenuto, M. (1999) Inability of activated cord blood T lymphocytes to perform Th1-like and Th2-like responses: implications for transplantation J. Hematother. Stem Cell Res. 8,381-385[CrossRef][Medline]
  26. Gerli, R., Bertotto, A., Crupi, S., Arcangeli, C., Marinelli, I., Spinozzi, F., Cernetti, C., Angelella, P., Rambotti, P. (1989) Activation of cord T lymphocytes. I. Evidence for a defective T cell mitogenesis induced through the CD2 molecule J. Immunol. 142,2583-2589[Abstract]
  27. Bell, E. B., Sparshott, S. M. (1990) Interconversion of CD45R subsets of CD4 T cells in vivo Nature 348,163-166[CrossRef][Medline]
  28. Katzen, D., Chu, E., Terhost, C., Leung, D. Y., Gesner, M., Miller, R. A., Geha, R. S. (1985) Mechanisms of human T cell response to mitogens: IL 2 induces IL 2 receptor expression and proliferation but not IL 2 synthesis in PHA-stimulated T cells J. Immunol. 135,1840-1845[Abstract]
  29. Koopman, G., Reutelingsperger, C. P., Kuijten, G. A., Keehnen, R. M., Pals, S. T., van Oers, M. H. (1994) Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis Blood 84,1415-1420[Abstract/Free Full Text]
  30. Sabzevari, H., Propp, S., Kono, D. H., Theofilopoulos, A. N. (1997) G1 arrest and high expression of cyclin kinase and apoptosis inhibitors in accumulated activated/memory phenotype CD4+ cells of older lupus mice Eur. J. Immunol. 27,1901-1910[Medline]
  31. Splawski, J. B., Jelinek, D. F., Lipsky, P. E. (1991) Delineation of the functional capacity of human neonatal lymphocytes J. Clin. Invest. 87,545-553
  32. Smith, C. A., Williams, G. T., Kingston, R., Jenkinson, E. J., Owen, J. J. (1989) Antibodies to CD3/T-cell receptor complex induce death by apoptosis in immature T cells in thymic cultures Nature 337,181-184[CrossRef][Medline]
  33. Shi, Y. F., Bissonnette, R. P., Parfrey, N., Szalay, M., Kubo, R. T., Green, D. R. (1991) In vivo administration of monoclonal antibodies to the CD3 T cell receptor complex induces cell death (apoptosis) in immature thymocytes J. Immunol. 146,3340-3346[Abstract]
  34. Adkins, B., Chun, K., Hamilton, K., Nassiri, M. (1996) Naive murine neonatal T cells undergo apoptosis in response to primary stimulation J. Immunol. 157,1343-1349[Abstract]
  35. Brunner, T., Mogil, R. J., LaFace, D., Yoo, N. J., Mahboubi, A., Echeverri, F., Martin, S. J., Force, W. R., Lynch, D. H., Ware, C. F., et al (1995) Cell-autonomous Fas (CD95)/Fas-ligand interaction mediates activation-induced apoptosis in T-cell hybridomas Nature 373,441-444[CrossRef][Medline]
  36. Cheng, J., Zhou, T., Liu, C., Shapiro, J. P., Brauer, M. J., Kiefer, M. C., Barr, P. J., Mountz, J. D. (1994) Protection from Fas-mediated apoptosis by a soluble form of the Fas molecule Science 263,1759-1762[Abstract/Free Full Text]
  37. Bretscher, P. (1992) The two-signal model of lymphocyte activation twenty-one years later Immunol. Today 13,74-76[CrossRef][Medline]
  38. Jenkins, M. K. (1992) The role of cell division in the induction of clonal anergy Immunol. Today 13,69-73[CrossRef][Medline]
  39. Mueller, D. L., Jenkins, M. K., Schwartz, R. H. (1989) Clonal expansion versus functional clonal inactivation: a costimulatory signalling pathway determines the outcome of T cell antigen receptor occupancy Annu. Rev. Immunol. 7,445-480[Medline]
  40. Viola, A., Lanzavecchia, A. (1996) T cell activation determined by T cell receptor number and tunable thresholds Science 273,104-106[Abstract]
  41. Swoboda, R., Bommhardt, U., Schimpl, A. (1991) Regulation of lymphokine expression in T cell activation. I. Rapid loss of interleukin-specific RNA after removal of the stimulating signal Eur. J. Immunol. 21,1691-1695[Medline]
  42. Mouzaki, A., Rungger, D., Tucci, A., Doucet, A., Zubler, R. H. (1993) Occurrence of a silencer of the interleukin-2 gene in naive but not in memory resting T helper lymphocytes Eur. J. Immunol. 23,1469-1474[Medline]
  43. Boulougouris, G., McLeod, J. D., Patel, Y. I., Ellwood, C. N., Walker, L. S., Sansom, D. M. (1999) IL-2-independent activation and proliferation in human T cells induced by CD28 J. Immunol. 163,1809-1816[Abstract/Free Full Text]
  44. Razi-Wolf, Z., Hollander, G. A., Reiser, H. (1996) Activation of CD4+ T lymphocytes form interleukin 2-deficient mice by costimulatory B7 molecules Proc. Natl. Acad. Sci. USA 93,2903-2908[Abstract/Free Full Text]
  45. Mueller, D. L., Seiffert, S., Fang, W., Behrens, T. W. (1996) Differential regulation of bcl-2 and bcl-x by CD3, CD28, and the IL-2 receptor in cloned CD4+ helper T cells. A model for the long-term survival of memory cells J. Immunol. 156,1764-1771[Abstract]
  46. Wesselborg, S., Janssen, O., Kabelitz, D. (1993) Induction of activation-driven death (apoptosis) in activated but not resting peripheral blood T cells J. Immunol. 150,4338-4345[Abstract]
  47. White, J., Herman, A., Pullen, A. M., Kubo, R., Kappler, J. W., Marrack, P. (1989) The V beta-specific superantigen staphylococcal enterotoxin B: stimulation of mature T cells and clonal deletion in neonatal mice Cell 56,27-35[CrossRef][Medline]
  48. Kawabe, Y., Ochi, A. (1991) Programmed cell death and extrathymic reduction of Vbeta8+ CD4+ T cells in mice tolerant to Staphylococcus aureus enterotoxin B Nature 349,245-248[CrossRef][Medline]
  49. Webb, S., Morris, C., Sprent, J. (1990) Extrathymic tolerance of mature T cells: clonal elimination as a consequence of immunity Cell 63,1249-1256[CrossRef][Medline]
  50. McCormack, J. E., Callahan, J. E., Kappler, J., Marrack, P. C. (1993) Profound deletion of mature T cells in vivo by chronic exposure to exogenous superantigen J. Immunol. 150,3785-3792[Abstract]
  51. Drenou, B., Choqueux, C., El Ghalbzouri, A., Blancheteau, V., Toubert, A., Charron, D., Mooney, N. (1998) Characterisation of the roles of CD95 and CD95 ligand in cord blood Bone Marrow Transplant. 22(Suppl. 1),S44-S47
  52. Akbar, A. N., Borthwick, N. J., Wickremasinghe, R. G., Panayoitidis, P., Pilling, D., Bofill, M., Krajewski, S., Reed, J. C., Salmon, M. (1996) Interleukin-2 receptor common gamma-chain signaling cytokines regulate activated T cell apoptosis in response to growth factor withdrawal: selective induction of anti-apoptotic (bcl-2, bcl-xL) but not pro-apoptotic (bax, bcl-xS) gene expression Eur. J. Immunol. 26,294-299[Medline]
  53. Hildeman, D. A., Zhu, Y., Mitchell, T. C., Kappler, J., Marrack, P. (2002) Molecular mechanisms of activated T cell death in vivo Curr. Opin. Immunol. 14,354-359[CrossRef][Medline]
  54. Sillett, H. K., Cruickshank, S. M., Southgate, J., Trejdosiewicz, L. K. (2001) Transforming growth factor-beta promotes ‘death by neglect’ in post-activated human T cells Immunology 102,310-316[CrossRef][Medline]
  55. Boise, L. H., Minn, A. J., Noel, P. J., June, C. H., Accavitti, M. A., Lindsten, T., Thompson, C. B. (1995) CD28 costimulation can promote T cell survival by enhancing the expression of Bcl-XL Immunity 3,87-98[CrossRef][Medline]
  56. Storek, J., Dawson, M. A., Storer, B., Stevens-Ayers, T., Maloney, D. G., Marr, K. A., Witherspoon, R. P., Bensinger, W., Flowers, M. E., Martin, P., Storb, R., Appelbaum, F. R., Boeckh, M. (2001) Immune reconstitution after allogeneic marrow transplantation compared with blood stem cell transplantation Blood 97,3380-3389[Abstract/Free Full Text]
  57. Schwartz, R. H. (1996) Models of T cell anergy: is there a common molecular mechanism? J. Exp. Med. 184,1-8[Free Full Text]
  58. Lin, M. T., Tseng, L-H., Pei, J., Garcia, M., Barsoukow, A., Akatsuka, Y., Hansen, J. (1997) Cell death among peripheral blood T cells following allogeneic marrow transplantation: correlation with markers of activation Blood 90(Suppl. 1),541a
  59. Li, X. C., Strom, T. B., Turka, L. A., Wells, A. D. (2001) T cell death and transplantation tolerance Immunity 14,407-416[CrossRef][Medline]
  60. Haut, P. R., Gonzalez-Ryan, L., Wang, L-J., Olszewski, M., Morgan, E., Kletzel, M. (2002) Induction of a transient graft vs. leukemia effect following unrelated cord blood transplantation Pediatr. Transplant. 6,348-351[CrossRef][Medline]



This article has been cited by other articles:


Home page
 NeoReviewsHome page
D. A. Randolph
The Neonatal Adaptive Immune System
NeoReviews, October 1, 2005; 6(10): e454 - e462.
[Full Text] [PDF]

Home page
 J. Immunol.Home page
C. A. Thornton, J. W. Upham, M. E. Wikstrom, B. J. Holt, G. P. White, M. J. Sharp, P. D. Sly, and P. G. Holt
Functional Maturation of CD4+CD25+CTLA4+CD45RA+ T Regulatory Cells in Human Neonatal T Cell Responses to Environmental Antigens/Allergens
J. Immunol., September 1, 2004; 173(5): 3084 - 3092.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.0303098v1
74/6/998    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cantó, E.
Right arrow Articles by Vidal, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cantó, E.