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*
Laboratory of Experimental Immunology, Division of Basic Sciences, National Cancer Institute, Frederick, MD 21702; and
Instituto de Inmunologia, Facultad de Medicina, Universidad Central de Venezuela, Caracas, Venezuela
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In addition to their cytotoxic activity, NK cells participate either directly or indirectly in multiple developmental and regulatory aspects of the immune system characterized by rapid response to exogenous and endogenous signals by producing a variety of cytokines and chemokines (2, 3, 4).
The ability of NK cells to produce hemopoietic cell growth factors,
IFN, IL, TNF 
and 
, TGF, and other growth factors, combined with
their ability to respond rapidly to exogenous signals by
up-regulating mRNA expression for various cytokines within
minutes demonstrates the importance of NK cells as mediators or
effectors of the intercellular communication network (reviewed in Ref.
2).
Under activated conditions, NK cells express and up-regulate the receptors for a variety of chemotactic factors, cytokines, growth factors, and hormones including the expression of the ligand of the CD40 receptor (CD154) thus enhancing NK cell interactions within the immune system. For example, it has been shown that NK cells are involved in the regulation of B lymphocyte functions (5, 6, 7, 8). Donor type-activated NK cells promote marrow engraftment and B cell development during allogeneic bone marrow transplantation in mice (9) and in humans (10). Also, human NK cells can enhance the B cell proliferative responses to the surface Ig cross-linking agents anti-IgM or Staphylococcus aureus Cowan strain (6).
By direct interaction between B cells and NK cells, it has been shown
that B cells are able to stimulate the production of IFN-
by NK
cells and activation of the NK cells (11). Thus, both B
cells and NK cells are capable of interacting in a spontaneous manner
leading to a costimulatory effect.
The evidence described above suggests that NK cells are involved in B cell maturation, Ig secretion, and isotype switching, pathways well-known to be regulated by CD40-CD154 interaction (12). The absence, or blockade, of the CD40-CD154 interaction results in gross impairment of the B cell physiological and molecular pathways that seem to be unique to the T-dependent Ab response.
Whether the CD40-CD154 engagement plays a role in B cell regulation by NK cells remains to be clarified. The expression of mRNA for CD40 ligand (CD40L)2 on purified NK cells has been reported previously (13); more recently, a role for CD40-CD154 in NK cell interaction with other cells has been described (14). NK cell clones expressing CD154 were able to kill target cells expressing CD40 receptors. Although freshly isolated human NK cells were unable to lyse CD40+ targets, when activated with rIL-2 they were able to kill the CD40-transfected cells. This study also reported that coexpression of CD40 and MHC class I Ag on the target cell inhibited the lysis by the NK cells, suggesting a regulatory role of MHC class I in the CD40-triggered killing.
Direct interaction between B and NK cells under activated conditions are supported by several studies, particularly in mouse models (15, 16, 17, 18). Evidence from human studies suggests that NK cells and B cells can spontaneously interact in vitro, as demonstrated by conjugate formation and activation of the interacting cells (11). However, this activating function is poorly understood.
NK cells are potent regulatory cells in the innate immune system, characterized by their spontaneous interaction with immune and nonimmune cells (11, 19). In addition, growing evidence suggests that NK cells might be involved in the development of autoimmune diseases (20). However, the mechanisms by which NK cells modulate these responses are not entirely clear. In this study, we tested the hypothesis that spontaneous interaction between human B cells and autologous NK cells might be important in initiating the innate B cell response which, under the appropriate conditions, might provide an early protective advantage to the host or contribute to the development of autoimmunity.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RPMI 1640 culture medium and Dulbecco’s PBS,
L-glutamine, and penicillin-streptomycin were purchased
from BioWhittaker (Walkersville, MD); FBS' was purchased from Biofluids
(Rockville, MD). Lymphocyte separation medium and BSA were obtained
from ICN Pharmaceuticals (Aurora, OH). Percoll was obtained from
Amersham Pharmacia Biotech (Upsala, Sweden); polyoxyethylene-sorbitan
monolaurate (Tween 20) was obtained from Sigma-Aldrich (St. Louis, MO).
ELISA kits for human TNF, IL-6, IL-4, IFN-, and IL-10
quantification were obtained from R&D Systems (Minneapolis,
MN).
Monoclonal and polyclonal Abs
Anti-CD3 FITC, anti-CD19 PE, anti-CD14 FITC, anti-CD56 PE, anti-CD5 FITC, anti-CD40 PE, anti-CD40L (anti-CD154) PE, anti-CD27 FITC, anti-IgM FITC, anti-IgG, IgD, and anti-CD27 FITC mAbs were purchased from BD Biosciences (San Jose, CA). Anti-CD56 microbeads (CD56 beads) and streptavidin microbeads were obtained from Miltenyi Biotec (Oslo, Sweden). CD40-muIg fusion protein was obtained from Ancell (Bayport, MN).
Lymphocyte preparation
PBMC were isolated from the buffy coats of healthy donors (obtained from the National Institutes of Health blood bank) after centrifugation on a lymphocyte separation medium. Cells were washed twice with Dulbecco’s PBS and suspended in RPMI 1640 medium supplemented with 2 mM of L-glutamine, 100 IU/ml penicillin, 50 µg/ml streptomycin, and 10% FCS. Adherent cells were removed by incubation in plastic flasks for 1 h at 37°C and the nonadherent cells were recovered by being gently washed with warmed medium and incubated on nylon wool columns for 1 h at 37°C. The nylon nonadherent cells (mostly T cells and NK cells) were eluted with prewarmed RPMI 1640 medium and the nylon adherent cells (enriched B cell fraction) were recovered by washing and soaking the nylon wool with cold PBS plus 1 mM of EDTA.
Cell fractionation by Percoll gradient
Both nylon adherent (B enriched fraction) and nonadherent cells (T cells plus NK cells) were fractionated separately on a seven-step Percoll gradient as previously described (21). High-density fractions (F4 to F6) from the nylon adherent cell gradients containing 60–80% resting B cells were used for further purification of B cells and the low density fraction 2 from the nonadherent cell gradients (40–60% NK cells) was used to purify NK cells. Fraction 5 from the NK cell gradient (96 ± 2% CD3+ T cells) was used as the source of T cells.
Purification of B cells and NK cells by magnetic columns
B cells and NK cells obtained by Percoll gradient centrifugation were further depleted from the remaining T lymphocytes and monocytes by negative selection with anti-CD3 and anti-CD14 mAbs. The cells were labeled for 30 min on ice with biotinylated anti-CD3 and anti-CD14. After removing the unbound Abs by washing with cold PBS plus 1% BSA, the cells were incubated for 15 min with streptavidin microbeads (Miltenyi Biotec) and the positive cells (CD3+ and CD14+) were removed with a magnetic column (MACS; Miltenyi Biotec). Final purity was 98 ± 1% CD19-positive cells for B cells and 97 ± 2% CD56+/CD5- cells for NK cells as determined by flow cytometry analysis (FacsSort, BD Biosciences) with anti-CD19 and anti-CD56/anti-CD5, respectively.
Phenotypically, the B cell population was composed of 85 ± 3% IgM+IgD+; 2 ± 0.5% IgM alone, 3 ± 0.6% IgD alone, 6 ± 2% IgM-IgD-IgG+; 13 ± 3% CD5+ (B1), 21 ± 3% IgM+IgD+, CD27+, and <3 ± 2% IgA+ cells as determined by flow cytometry.
In vitro assay for B cell activation with NK cells
Highly purified B lymphocytes (1 x 106 cells/ml) were mixed at different ratios with autologous NK cells or T lymphocytes in 1.5-ml polystyrene microcentrifuge tubes (Fisher Scientific, Pittsburgh, PA). The cell suspension was centrifuged at 200 x g for 10 s in a microcentrifuge (Capsule; Tomy, Tokyo, Japan) and incubated for 5 min at 37°C in a water bath. Then, cell mixtures were immediately transferred to an ice bath. These conditions were previously determined as the minimum time necessary to activate the B cells with the NK cells as evaluated by tyrosine-phosphorylation studies (data not shown). In some experiments, the cell mixture was only desegregated and the B cell concentration was adjusted to 5 x 105 cells/ml in RPMI 1640 plus 10% FCS (complete medium) and cultured for 6 days at 37°C in an atmosphere of 5% CO2. In other experiments, the stimulating T cells or NK cells were removed from the mixture by magnetic columns (as described above) after labeling at 4°C with biotinylated anti-CD3 plus streptavidin microbeads or anti-CD56 microbeads, respectively. The remaining B cells stimulated with T cells (BsT cells) or with NK cells (BsNK cells) contained <2% of T cells or NK cells as determined by flow cytometry using double-labeling with anti-CD5 FITC/anti-CD19 PE and anti-CD19 FITC/CD16 PE, respectively. Both BsT cells and BsNK cells were adjusted to 5 x 105 cells/ml and cultured for 6 days in the same conditions as above. Each culture was performed in triplicate in 1 ml of medium using 12 x 75 round-bottom sterile culture tubes (BD Biosciences). Finally, the cell cultures were centrifuged at 1200 x g for 10 min and the cell-free supernatants were harvested, filtered through a 0.22 µm Millipore filter (Bedford, MA), and tested for secreted Ig.
Fixation of NK cells
In some experiments, the NK cells were fixed for 5 min with 1% glutaraldehyde, washed three times with PBS, rested for 1 h at room temperature (RT) to achieve polymerization of the fixative, and suspended in RPMI 1640 plus 10% FCS before use for B cell stimulation.
Ig analysis
Quantitation of IgM, IgG, and IgA in cell-free supernatants was performed by an ELISA specific for human IgM, IgG, and IgA (Bethyl Laboratories, Montgomery, TX). Nun Maxisorp C bottom-well plates (Nunc, Naperville, IL) were coated with isotype-specific capture Abs at 1 µg/well in 0.1 ml of 0.5 M sodium carbonate (pH 9.6) for 1 h at RT. The plates were washed twice with a wash solution containing 50 mM of Tris (pH 8.0), 0.1 M of NaCl, and 0.05% Tween 20 and incubated for 30 min at RT with a postcoat solution (1% BSA in 50 mM of Tris (pH 8.0), 0.15 M of NaCl) to block nonspecific binding. Subsequently, cell-free supernatants and standards (reference serum or calibrator) were added in duplicate to the plate wells (100 µl/well). Dilution of the standards was made in a blocking solution to avoid binding of serum components to the wells. All plates were incubated for 1 h at RT, washed three times with wash solution, and incubated 1 h at RT with 100 µl/well optimal concentration of isotype-specific (anti-human IgM, IgG, and IgA) Abs conjugated with HRP. After washing three times, the enzyme substrate hydrogen peroxide plus 3,3',5,5'-tetramethylbenzidine was added for 20 min at RT in the dark and the reaction was stopped with 50 µl of 2 M of H2SO4. Plates were read in a MRX microplate reader (Dynatech Laboratories, Chantilly, VA) and isotype concentrations were extrapolated from a reference curve (range: 500–7.5 ng/ml of the corresponding isotype). A revelation program incorporated with the microplate reader (revelation program, Dynatech) calculated the Ig concentration.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous reports (11, 22) have hypothesized that
contact interaction between B cells and NK cells induces two-way
activation signals. These may be important in the regulation of B cell
function by the NK cells, especially if occurring in the absence of
exogenous stimuli. To test this hypothesis, we evaluated Ig synthesis
by highly purified human resting B cells cocultured with autologous NK
cells. A fixed number of 5 x 105 B cells were
cocultured for the indicated days at 37°C with NK cells, using
different B-NK cell ratios and the B cell function was monitored by Ig
production in cell-free supernatants as described in Materials
and Methods. As shown in Fig. 1
, cocultures of B cells with NK cells at
1:1 and 1:2 B-NK ratios induced consistent activation and
differentiation of the B cells into Ab-producing cells. The peak for
each Ig isotype varied from a 1:1 B-NK cell ratio for IgG to a 1:4 B-NK
cell ratio for IgA. In general, B cell and NK cell cocultures at B-NK
ratios higher than 1:4 were not effective in inducing the activation
and differentiation of the B cells, suggesting that this NK cell
function is regulated for a restricted range of interaction with the B
cells.
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). The data demonstrate that the
response of the B cells to stimulation with NK+
PWM is over 40-fold for IgM and IgG in both unfractionated B cells and
high-density B cells. However, when we compare the Ig synthesis induced
by NK cells alone with respect to B cells plus medium, PWM, and IL-2
alone, the NK-induced Ig is over 10-fold higher. Although the isotype
distribution is similar between unfractionated B cells and high-density
B cells, the background level of Ig showed a higher variability in
comparison to the high-density fraction. The NK-induced Ig response by
unstimulated NK cells is similar to the B cell response reported by
Cocks et al. (13) that was achieved by stimulation of
purified B cells with CD40L-transfected cells. Thus, in contrast with
Gray and Horwitz (22) who study the amplification by IL-2
of the NK-induced Ig synthesis, we have analyzed in greater detail this
consistent B cell activation induced by unstimulated autologous NK
cells to understand its possible role in the immune response.
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) follows kinetics similar to that
reported for T cell-dependent human B cell differentiation into IgM-,
IgG-, and IgA-producing cells (23). This Ig response
reaches its maximum between 7 and 8 days with a plateau between 8 and
10 days of culture. Cocultures for 12 days or more showed a decrease in
the accumulated Ig levels in comparison with those with 8–10 days of
incubation, due probably to an increase in the protease activity
released by dead cells or generation of some other factors interfering
with the ELISA cocultures of B cells with unstimulated T cells
that failed to activate the B cells (data not shown). This
stimulatory activity of the NK cells on the B cells was selective and
specific because T cells failed to induce the B cell activation.
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To evaluate whether activation of B cells by NK cells was
contact-dependent or contact-independent, we tested the activation of B
cells by NK cells using transwell plates containing two chambers
separated by a semipermeable membrane (polycarbonate, 0.4-um pore
membrane). This membrane permits the circulation of soluble factors
between both chambers but prevents B cell and NK cell contact. A fixed
number of 2.5 x 105 B cells were seeded in the lower
chamber and cocultured with 5 x 105 NK cells seeded
in the upper chamber (1:2, B-NK cell ratio) for 7 days at 37°C in
RPMI 1640 medium plus 10% FCS. As shown in Fig. 3, NK cells failed to activate the B
cells when cocultured in the transwell plate in comparison to those
cocultured in the same plate at an identical B-NK cell ratio. These
results show that activation of resting B cells by unstimulated NK
cells is a cell contact-dependent process and, in contrast to
mitogen-activated B cells which are induced to Ab production by NK
soluble factor(s) (7), resting B cells are not inducible
by NK cell soluble factors alone.
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To evaluate whether the B cell activation required the continuous
presence of the NK cells to become activated, we designed experiments
of 5-min interaction between B cells and NK cells at 37°C as
described in Materials and Methods. B cells were mixed
at a 1:1 ratio with autologous NK cells or T cells inducing a close
cell-cell contact for 5 min at 37°C. Then, the mixture was
desegregated by vortexing in 1 mM of EDTA and suspended in RPMI 1640
plus 10% FCS or T cells and NK cells were depleted by magnetic columns
using anti-CD3 and anti-CD56 microbeads, respectively. The
number of B cells in each experimental condition (BsNK, BsT) and those
cocultured together (B + NK) and (B + T), was adjusted to 5 x
105 B cells/ml and cultured for 6 days at 37°C in RPMI
1640 medium containing 10% FCS. Cell-free supernatants were tested for
Ig production. As shown in Fig. 4, the B
cells cocultured with NK cells (B + NK mixed) and B cells stimulated
for only 5 min with NK cells (BsNK) underwent activation and
differentiation into Ig-producing cells in a similar way. This data
indicates that a 5-min interaction with NK cells is sufficient to
induce B cell differentiation into Ab-producing cells. We also
evaluated the synthesis of IL-4, IL-10, IL-6, TNF, and IFN- in
these cultures. As shown in Fig. 5, both
B + NK and BsNK cocultures produced TNF and IL-6, while IFN- was
present only in B + NK cocultures, suggesting that this factor
(produced by the NK cells) was not critical for the differentiation of
the B cells after stimulation with the NK cells. IL-10 and IL-4 were
not detected in these system (data not shown).
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The contact-dependent cognate recognition involved in B cell activation suggested that it might require an active membrane function. Cognate interaction is an active process that requires cell membrane polarization, a phenomenon involved in many other processes, such as cell differentiation, induction of immune response, and target cell recognition and killing (24, 25). Cell polarization is required for the conjugate formation between NK cells and their target cells (26, 27).
To further evaluate whether the activation of B cells by NK cells
requires an intact NK cell membrane mobility, we performed experiments
in which the NK cell membrane was fixed with 1% glutaraldehyde before
testing its capacity to stimulate B cells. Glutaraldehyde-fixed NK
cells failed to activate the B cells after a 5-min interaction (Fig. 6) or when cocultured for 6 days. These
results indicate that this contact-dependent activation phenomenon is
also dependent upon an active cellular membrane. Longer interaction (6
days coculture) failed to activate the B cells (data not shown). The
possibility that the fixed NK cells can be deleterious for the B cell
activity is unlikely because the B cells were still able to respond to
fresh NK cells when cocultured for 6 days or when cocultured with fresh
NK cells plus PWM (IgM = 626 ng/ml; IgG = 130 ng/ml; and
IgA = 36 ng/ml).
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Numerous studies have dealt with the molecular mechanisms
regulating B cell and T cell interactions, suggesting that the ligand
pair CD40/CD154 plays an important role (12). To determine
whether the capacity of the NK cells to induce B cell activation was
dependent on the CD40-CD154 interaction, we first cocultured both B and
NK cells in the presence of different doses of anti-CD154 mAb
(range: 0.1–2 µg/ml). As a control for nonspecific binding via
FcR, we included an identical concentration of irrelevant mouse
IgG1 that matched the anti-CD154 isotype. As shown in Fig. 7, anti-CD154 inhibited the
activation of the B cells in a dose-dependent manner as demonstrated by
the inhibition of IgM, IgG, and IgA synthesis.
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A, the
presence of anti-CD154 or CD40-mIg during the B-NK interaction
blocked the activation of the B cells, suggesting a critical role of
the CD40-CD40L(CD154) interaction in the activation of the B cells by
NK cells.
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B, pretreatment of NK cells with
anti-CD154 abolished their capacity to activate the B cells. Depletion of CD27+ B cells and CD5+ B cell subpopulations modifies the B cell response to NK cell activation
As described above, the Ig response in our system is reminiscent of a secondary response in which the IgG was the main isotype in most of the donors. In addition, phenotypic analysis of our resting B cell population obtained from the high-density Percoll gradient revealed a population of 21 ± 6% CD27+ cells and 13 ± 3% CD5+ (B1) cells. From these, only 2.5 ± 1% were CD27+/CD5+.
To determine whether CD27+ B cells and CD5+ B cell subpopulations were participating in the B cell response induced by NK cells, we proceeded to deplete these B cell subsets from the tested B cell populations before their interaction with the NK cells. This approach was used due to the difficulty in isolating these B cell subsets by negative selection, avoiding the presence of Ab on the responder B cells. Purified B cells were labeled with biotin-conjugated anti-CD27+, anti-CD5+, or anti-CD27+ anti-CD5 mAbs and the positive cells were removed by magnetic columns using streptavidin microbeads as described in Materials and Methods. The unfractionated B cell population (total), CD27- B cells, CD5- B cells, and the double-negative (CD27-CD5-) B cells were cocultured at 5 x 105 cells/ml with autologous NK cells at a 1:2 B-NK cell ratio for 7 days at 37°C. Cell-free supernatants were tested for Ig synthesis.
As shown in Fig. 9, depletion of
CD27+ B cells abrogated IgM synthesis induced by
the NK stimulation and reduced the levels of IgG and IgA. A similar
effect was also observed when the CD5+ B cell
subset was removed from the responder B cells. B cells depleted in both
B cell subsets (CD27+ and
CD5+) did not respond to activation with
autologous NK cells, suggesting a critical role of these B cell
subpopulations in the B cell response induced by NK cells. Although
depletion of CD27+ includes switched cells
(IgG+ B cells), the depletion of
IgG+ memory B cells alone (performed in separated
experiments, data not shown) did not induce significant changes in the
IgG synthesis, suggesting that other subsets of memory B cells may be
responsible for IgG switching by activation with the NK cells.
Recently, studies have shown that human
IgM+IgD+CD27+
B cells switch to all IgG subclasses (28). These B cell
subsets in our B cell population accounted for 18 ± 5% of the
total B cells, while the IgG+ subset was 6
± 2% of the total B cells. Although the
IgM+IgD+CD27+
B cell subset represents nonactivated resting B cells, it has been
shown to express higher levels of Ig mRNA than naive
(IgM+IgD+) B cells
(29). Thus, our results suggest that in addition to
IgG+ B celIs, IgM+
IgD+CD27+ B cells may be
responsible for the switch to an IgG isotype in our experimental
system.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1:1. We also demonstrate that 5 min of close contact at 37°C
between both cells, followed by removal of the NK cells, was sufficient
to induce the differentiation of the B cells into Ig-producing cells.
This activating function is contact-dependent and requires an active NK
cell membrane because coculturing both cells in transwell plates or
fixing the NK cell with glutaraldehyde failed to activate the B cells.
Indeed, cognate interaction as a result of cell-cell conjugation has
been shown to be an important feature of the interaction of B cells and
NK cells (11), a process that requires the redistribution
of adhesion molecules and cell membrane polarization as demonstrated in
conjugate formation of NK cells with target cells (27). It
has been shown that many membrane proteins are freely mobile and thus
present an array of independent binding sites (30). Thus,
very low affinity interactions can effectively generate organized areas
of synapses. Moreover, the capacity to activate resting B cells, in the
absence of exogenous stimuli, is specific for NK cells, because
autologous resting T cells failed to induce B cell activation. It is
well known that helper T cells may activate and cooperate with B cells
only after their activation using Ag receptors or by Ag-unlinked
(cognate) interaction (31). Thus, the lack of B cell
activation by unstimulated T cells was expected.
Although B cell and NK cell interaction has been supported by
several studies, particularly in rodent models, the experimental
designs required the preactivation of the NK cells (22) or
the B cells (17). In human studies, the evidence for
direct interaction of unstimulated NK cells with B cells is more
limited. In a previous report, Wyatt and Dawson (11)
showed that human tonsil B cells are able to conjugate to NK cells and
to induce their activation and IFN- production, which was suggested
as a possible mediator of the Ig synthesis inhibition reported for NK
cells (5, 32, 33). However, in this study, the
ability of B cells to function as Ab-producing cells was not reported.
In our system, the production of IFN- was detected only in B cells
cocultured with the NK cells (B + NK), but not in the supernatant of B
cells stimulated for 5 min with NK cells. This result suggests that
IFN- may not play a critical role.
Although in our system the B cell activation is contact dependent, as
demonstrated by transwell plate cultures, we cannot exclude the
possibility that during the interaction (11), the NK cells
produce soluble factors in the intercellular space capable of driving
the B cells into Ab-producing cells. The synthesis of a late-acting
soluble factor by the NK cell, which has an enhancing effect on Ig
synthesis by B cells, has been hypothesized (34). It is
possible that B-NK cell interaction may reduce the B cell threshold
necessary to respond to activating signals (35). Indeed,
it has been shown that activated B cells are able to synthesize and
express CD40L13 and to produce cytokines with B
cell costimulatory activity. Among these cytokines, IL-6, IL-10, and
TNF have been shown to function as autocrine loops
(36, 37, 38). Although in our system IL-10 was not detected in
the cultures supernatant of BsNK cells, IL-6 and TNF were always
present.
The role of CD40-CD40L (CD154) interaction on B cell activation and differentiation has been well established for T-dependent Ab responses. However, the role of this interaction in the T-independent B cell response remains to be elucidated. In our system, B cell activation by the NK cell was inhibited by the presence of either anti-CD154 mAb or CD40-mIg fusion protein during the B-NK cell interaction or by pretreatment of the NK cells with anti-CD154 before their interaction with the B cells. This data suggests that the CD40-CD154 interaction plays an important role in this T cell-independent NK-mediated activation of B lymphocytes. The expression of CD154 on NK cells has been well-documented. Purified NK cells express mRNA for CD40L (13) and CD40L surface expression and its regulatory role on NK cell cytotoxicity have been demonstrated in IL-2-activated NK cells and NK cell lines (39). Thus, very low levels of CD154 may be expressed constitutively on the NK cells or during the interaction with the B cells.
The possibility that activated B cells may express CD40L as suggested by Grammer et al. (40) is of functional importance in our system due to its possible contribution in homotypic B cell costimulation which, in combination with autocrine B cell factors, might drive the B cell into an Ab-producing cell. However, the possibility that CD40L acts as initiator of the B cell activation is very unlikely because purified B cells under the same interacting conditions as the B-NK cell mixture never become activated. This data suggests that contact-dependent NK cell activating signals are required.
This direct contact activation of the B cells by NK cells which, in addition to the CD40-CD40L interaction, includes the activation of cytokine production by the B cell capable of promoting its differentiation into an Ab-producing cell, is extremely important in autoimmunity and oncogenesis. In addition, increasing evidence, both in human and mouse models, has suggested an important role of NK cells in autoimmunity (20, 41). Nevertheless, the link between the NK cell and the autoreactive B cell or transformed B cell is still missing.
In our study, the Ig response elicited by NK-activated B cells is reminiscent of a secondary Ab response expressing most of the Ig isotype (IgM, IgG, and IgA). This result prompted us to hypothesize that the responder B cells might belong to a certain subset of memory B cells or autoreactive B cells. One interesting B cell subgroup is CD27+ B cells, which include, in addition to classical isotype-switched IgG+, IgA+, and IgE memory B cells, the IgM+IgD+ and IgM-only B cells (42). In contrast, CD5+ B cells are long-lived cells that appear to be derived from precursor cells present in infant, but not in adult, bone marrow and classified as B1 cells (43). As they arise soon after birth, they are the major source of Ab production in young individuals and might be important for T cell-independent Ab response. As they tend to produce autoreactive Igs, they have been linked to autoimmune diseases. Remarkably, the malignant cells in nearly all cases of human chronic lymphocytic leukemia carry the CD5 marker, suggesting that this malignancy arises from the CD5+ B cell subset. Both, the CD27+ B cells and CD5+ B cell subpopulation belong to the resting B cell pool in human peripheral blood and have been implicated in the generation of natural Abs and participation in the pathophysiology of certain autoimmune diseases (20).
This population, once activated by the NK cells, is capable of driving its own differentiation by autocrine synthesis of cytokines. To test this hypothesis, we performed depletion studies of CD27+ memory B cells and CD5 + B-1 autoreactive B cell subsets, which, in our B cell populations, represented 21 ± 5% and 15 ± 3% of the total population, respectively. Removal of CD27+ memory B cells or CD5+ cells significantly reduced the Ig response, while depletion of both B cell subsets completely abolished the Ig response induced by NK cell activation. This data suggests that direct interaction of the NK cells with these two B cell subsets may play an important role in the memory Ab response and B cell-mediated autoimmunity. The substantial reduction of IgM synthesis almost to background levels caused by the removal of CD27+ B cells or CD5+ B cells suggests that the IgM are CD27+/CD5+. Although 2.5% of the B cells were CD27+/CD5+, further studies are needed to elucidate this point. It is possible that after activation with the NK cells both B cell subsets require homotypic interaction to differentiate into IgM-producing cells.
As depletion of the IgG+ B cell subset does not significantly reduce the IgG synthesis, we postulate that another memory B cell subset with switching capacity to IgG may be involved. Recent studies (29, 42) have shown that the germinal center produces three different types of V(D)J mutated B cells in similar proportion including the Ig-switched, IgD-IgM+(IgM only), and IgD+IgM+ cells which together represent the CD27+ compartment of rested recirculating memory B cells. In human studies, mutated Ig sequences are found exclusively in this CD27+ B cell population and the IgD+IgM+CD27+ cells have been shown to express a high switching capacity into most Ig isotypes and IgG subclasses (28). In our study, this IgD+CD27+ population represented 18 ± 5% of the purified B cells. Thus, these cells represent an important candidate for direct interaction with the NK cells.
Although NK cells provide a survival signal to the B cells as determined by the improvement in viability of the B cells cocultured with NK cells in comparison to B cells cultured alone, this is not sufficient to achieve activation of the B cells because a contact-dependent signal is still required. This is demonstrated by B-NK cell cocultures in transwell plates in which the survival signals provided by the NK cells are still present without activation of the B cells. A similar improvement of B cell survival is observed in B cells cocultured with resting T cells without subsequent activation of the B cells. The addition of a survival signal such as IL-2 to resting B cells in the absence of NK cells prolongs their survival without accumulation and secretion of Ig.
The importance of this mechanism of B cell activation in vivo is supported by different studies. For example, the presence of NK cells in the germinal center of the human tonsil in proximity to B cells suggests a close interaction with the B cells in this lymphoid organ (44). Secondly, NK cells could play a critical role in the initiation of an autoimmune response in combination with host intrinsic or extrinsic factors. Studies by Shi et al. (20) have shown that NK cells can affect the outcome of the adaptive immune response. NK cells, but not NK1.1+ T cells, were found to participate in the development of myasthenia gravis in mice (41). Third, the contribution of NK cells to B cell activation and differentiation in vivo has been suggested from human bone marrow transplantation (BMT). In recipients of T cell-depleted allogeneic bone marrow transplants, large granular lymphocytes which contain mostly NK cells were found responsible for the secretion of B cell differentiation factors capable of maintaining the Ig expression and specific Ab levels in the absence of mature T cells. It appears likely that NK cells play a significant role in maintaining B cell function in vivo upon T cell-depleted BMT (10, 45). Donor type-activated NK cells have also been shown to promote BMT in mice (9).
The establishment of a link between NK cells, autoreactive B cells, and circulating memory B cells in our study also suggests a potential regulation by NK cells of the adaptive immune response through their direct interaction with cells and the production of a number of immunoregulatory factors. NK cells might provide an advantage to the host before the development of specific and potent helper T cells. The extension of our findings into animal models should allow further testing of these hypotheses.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: CD40L, CD40 ligand; RT, room temperature; BsNK, B cells stimulated with NK cells; BsT, B cells stimulated with T cells; BMT, bone marrow transplantation.
Received for publication May 11, 2001. Accepted for publication September 21, 2001.
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References |
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B and rel protein in innate immunity. Adv. Immunol. 58:1.[Medline]
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