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CUTTING EDGE |
Departments of
*
Pathology and Laboratory Medicine and
Medicine, University of British Columbia and Vancouver Hospital and Health Sciences Centre, Vancouver, British Columbia, Canada;
Department of Pediatrics, University of Alberta, Edmonton, Alberta, Canada; and
§
Department of Pathology and Laboratory Medicine, British Columbia’s Children’s Hospital, Vancouver, British Columbia, Canada
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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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The genetic basis of XLP has been attributed to mutations in signaling
lymphocytic activation molecule (SLAM) associated protein (SAP)
(10, 11, 12), an intracellular molecule expressed in T cells
and consisting almost entirely of a single, noncatalytic, Src-homology
2 domain. SAP has been shown to block recruitment of SHP-2 phosphatase
by competitively binding to tyrosine recognition motifs of SLAM and
2B4, costimulatory molecules expressed on the surface of T/B cells and
T/NK cells, respectively (10, 13). The engagement of 2B4
on NK cells has previously been demonstrated to promote spontaneous
cytotoxicity and augment secretion of IFN-
(14, 15, 16, 17).
Because SAP has been shown to interact with 2B4 in vitro
(13), we sought to determine whether mutations in SAP
might affect normal signaling mediated through ligation of 2B4 in
patients with XLP.
We have sequenced a novel mutation in SAP from autopsy specimens obtained from two maternally related cousins clinically diagnosed with XLP. Based on the molecular structure of the SAP-SLAM interaction (18, 19), this mutation (Arg55Leu in exon 2) is predicted to disrupt binding between the Src-homology 2 domain of SAP and the cytoplasmic domain of SLAM. It is likely that this mutation also interferes with SAP/2B4 binding because 2B4 and SLAM share strong amino acid homology, including careful conservation of the tyrosine recognition motifs contained within their cytoplasmic domains (16, 20). We have genotypically identified two additional related males from the extended family who carry the same mutation. Both affected males were healthy, and neither had clinical or serologic (EBV viral capsid Ag IgG negative) evidence of prior EBV infection. In this report, we describe the abnormal function of NK and lymphokine-activated killer (LAK) cells obtained from these boys. We further show that, in contrast to cells obtained from healthy controls, the cytotoxic activity of NK cells obtained from XLP patients cannot be enhanced through 2B4 ligation. These findings suggest that the interaction of SAP with 2B4 is required for optimal NK/LAK cell immunity, that SAP Arg55 is essential for MHC-unrestricted cytotoxic function, and that alterations to this pathway contribute to the defective immunity described in XLP.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PBMC were isolated by Ficoll density gradient centrifugation. Cells were resuspended in complete medium and incubated in culture flasks at 37°C for 4 h to deplete monocytes. The nonadherent fraction was used as effector cells in cytotoxicity assays. The cell lines used were K562, Raji, NK-92ci, and MV4;11 (Dr. K. Schultz, University of British Columbia, Vancouver British Columbia, Canada). Primary lymphocyte subsets were purified with immunomagnetic cell separation kits (Miltenyi Biotec, Auburn, CA) according to the manufacturer’s specifications. The study was approved by the University of British Columbia Clinical Research Ethics Board, and informed consent was obtained from all subjects before the collection of blood.
Flow cytometry and Abs
Samples were analyzed on a FACScalibur flow cytometer (Becton Dickinson, San Jose, CA) using CellQuest software (Becton Dickinson). Briefly, 200,000 cells were analyzed after staining with appropriate Abs: anti-CD3-FITC, anti-CD56-PE, anti-CD48, or isotype controls (PharMingen, Mississauga, Ontario, Canada). The C1.7 Ab was purchased from Biodesign International (Kennebunkport, ME).
Chromium release assays (CRA)
CRA were performed using standard protocols. Effector cells were
either nonadherent PBMC or LAK cells, and target cells were
51Cr-labeled K562, MV4;11, or Raji. Plates were
incubated for 6–8 h at 37°C. Supernatants (100 µl/well) were
collected and radiation was measured. Spontaneous release was
determined by incubation of target cells in medium alone, and maximum
release was determined by the addition of 100 µl of 5% Triton X-100
detergent to target cell suspension. Specific lysis (%) was calculated
according to the formula: 100 (experimental release - spontaneous
release)/(maximum release - spontaneous release). For 2B4
augmentation assays, mAb (isotype control or C1.7) was added directly
to the plated samples at a final concentration of 0.2 µg/ml.
LAK cell generation
Nonadherent PBMCs were plated at 2 x 106/ml in six-well plates in complete medium with 1000 IU/ml recombinant human IL-2 (PharMingen). The supernatant (nonadherent cells and media) was removed on day 3 and replaced with 2 ml of media, 2 ml of medium, and 500 IU/ml of IL-2. Every 3 days, for 12 days, media was replaced accordingly. On day 14, adherent cells were harvested for use in cytotoxicity assays.
Expression analysis
Cells were lysed in Trizol (Life Technologies, Grand Island, NY) and RNA was isolated according to the manufacturer’s instructions. SAP was amplified by PCR from oligo(dT) cDNA using the forward primer 5'-GAG GAA TTC AGG CCA TGG ACG CAG TGG C-3' and reverse primer 5'-AAG CCGCTC GAG TTT TAT TTT TCT TCA TG-3'. The PCR parameters were 95°C x 30 s, 60°C x 30 s, and 72°C x 1 min for 30 cycles. DNA from PBMC was purified using the PureGene DNA isolation Kit (Gentra Systems, Minneapolis, MN). Amplification of DNA from exon 2 of SAP was performed using SAP exon 2 forward primer sequence 5'-CAA TGA CAC CAT ATA CGT GT-3' and SAP exon 2 reverse primer sequence 5'-GCT TCC TTA ATG ATC CAT GA-3'.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Nonadherent PBMC derived from healthy controls and two XLP
patients were assessed for their ability to lyse K562 cells in standard
CRA. In accordance with previous reports (5, 9), NK cells
obtained from XLP patients effected significantly less cytotoxicity
against target cells than NK cells obtained from healthy controls (Fig. 1
; p < 0.005 at all E:T
ratios, Student’s t test). To determine whether
equivalent numbers of NK cells were present in each assay, we
immunophenotyped the effector cells to determine the proportion of
CD3-CD56+ cells. The numbers of NK cells present
in the effector cell fraction from healthy control and XLP patients
ranged from 7 to 19 and 5 to 7%, respectively (data not shown).
However, the vast majority of healthy controls had between 7 and 10%
NK cells in their effector PBMC, suggesting that XLP patients have only
a modest decrease in numbers of NK cells and that this decrease in
numbers alone cannot account for the large decrease in cytotoxicity.
Nevertheless, because the proportion of NK cells present within the
effector cell population varied considerably among normal controls, we
could not conclude with certainty that the reduced cytotoxicity was due
to an intrinsic NK defect and not to fewer absolute numbers of effector
cells. Therefore, we sought to characterize the cytotoxic function of
LAK cells generated from PBMC, reasoning that this lymphocyte subset
would represent a more homogenous population of effector cells.
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We generated long-term cultured LAK cells by incubating
nonadherent PBMC from two XLP patients and healthy controls (including
an unaffected sibling) with recombinant IL-2. LAK cultures generated
from XLP patients and controls were assessed for their capacity to
mediate lysis of the NK-resistant cell line, Raji (Fig. 2a). Equivalent numbers of
cultured LAK cells from the XLP patients effected significantly lower
killing than those generated from healthy controls, indicating that LAK
cells generated from XLP patients possess a profound defect in
cytotoxic function. To determine whether the effector cells established
from XLP patients and controls were phenotypically similar, LAK cells
were stained for surface expression of CD3 and CD56 (Fig. 2, b and c). Interestingly, LAK cells obtained from
XLP patients yielded substantially fewer
CD3-CD56+ cells than
healthy controls, suggesting a defect in generating this particular
subset of LAK effector cells. Furthermore, because the proportion of
CD3+CD56+ cytokine-induced
killer cells was similar in both groups, and because this population of
cells normally exhibits potent cytotoxicity (21, 22), the
inability of these effectors to kill Raji targets implies that these
cells may be similarly affected by mutations in SAP.
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Because the cytotoxic function of both NK and LAK cells was
deficient in the XLP patients, we sought to establish whether SAP is
normally expressed in these lymphocyte subsets to correlate aberrant
function with the Arg55Leu mutation. Expression
of SAP mRNA was detected by RT-PCR in an NK-derived cell line, NK-92ci
(23), IL-2 established LAK cells from XLP patients or
healthy controls and primary NK cells (Fig. 3). This observation suggests that the
mutant form of SAP, which is expressed in NK and LAK cells, might
account for their defective cytotoxic function.
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Most clinically defined cases of XLP studied so far have been
associated with mutations of SAP (24). Because SAP has
been shown to associate with the intracellular domain of human 2B4
(13), an activating molecule expressed on human NK cells
(16, 17, 20), we assessed the consequence of 2B4 ligation
on NK-mediated cytotoxicity. Using C1.7, a mAb with specificity for the
human 2B4 Ag (25), we first determined the effect of 2B4
ligation on NK cytotoxicity using nonadherent PBMC derived from healthy
volunteers as effector cells. Although 2B4 is expressed by several
leukocyte populations, including CD8+ T cells,
the ligation of 2B4 induces effector functions and biochemical changes
solely in NK cells (26). Therefore, augmentation of
cytotoxicity by the addition of mAbC1.7 reflects the specific activity
of NK cells and not other cytotoxic lymphocytes. Fig. 4a demonstrates the effect of
adding mAbC1.7 to a standard NK cell CRA. Addition of mAbC1.7 (to a
final concentration of 0.2 µg/ml) significantly increased specific
lysis by healthy control-derived NK cells, whereas the addition of an
isotype-matched Ab (directed against CD57) failed to influence
cytotoxicity. The augmentation effect reflects enhanced spontaneous
cytotoxicity and not Ab-dependent cellular cytotoxicity (ADCC) as K562
cells do not express 2B4 (Fig. 4d). To further confirm this
result, we reassessed this phenomenon using an NK effector cell line,
NK-92ci (23), which also expresses the 2B4 molecule (Fig. 4c). Addition of mAbC1.7 reproducibly augmented
NK-92ci-mediated cytotoxicity against the myeloid leukemic cell line,
MV4;11 (27), whereas addition of an isotype-matched Ab
directed against another highly expressed surface molecule on NK-92ci
(CD28) failed to enhance killing (Fig. 4b). Again, the
possibility that ADCC may have confounded the enhancing effects of
mAbC1.7 was ruled out as NK-92ci does not express CD16 (Fig. 6d). These results, taken
together, indicate that ligation of 2B4 by mAbC1.7 specifically
augments killing by non-HLA restricted effector cells via a spontaneous
cytotoxic mechanism.
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We subsequently determined the ability of 2B4 ligation to augment
NK cytotoxicity in PBMC obtained from XLP patients. Nonadherent PBMCs
from healthy controls (n = 7) and XLP
(n = 2) patients were repeatedly tested for their
ability to lyse 51Cr-labeled K562 target cells in
the presence or absence of mAbC1.7. The addition of mAbC1.7
significantly augmented the specific lysis of NK cells obtained from
healthy controls. However, mAbC1.7 ligation of 2B4 failed to augment
the lytic activity of NK cells obtained from XLP patients (Fig. 6
). Therefore, aside from exhibiting a
reduced basal level of cytotoxicity, NK cells from XLP patients are
unresponsive to 2B4-mediated enhancement of cytotoxicity.
Augmentation of NK lysis by 2B4 ligation is dependent on target cell expression of CD48
Because the natural ligand for 2B4 is CD48 (16, 17, 20), we next investigated whether the augmentation of NK
cytotoxic function by ligation of 2B4 is influenced by the use of
target cells that express CD48. We repeated the cytotoxicity assays
using as effector cells NK-92ci but, rather than using CD48-negative
target cells such as MV4:11 or K562, we used the CD48-positive cell
line, Raji (Fig. 5, a–c). We reasoned that physiological
ligation of 2B4 by endogenously expressed CD48 might conceal the
effects of exogenous ligation by mAbC1.7. As predicted, ligation of 2B4
failed to augment NK-92ci cytotoxicity against Raji cells (Fig. 5e).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In addition, we have demonstrated that SAP is expressed in NK and LAK cells derived from healthy controls as well as in LAK cells derived from XLP patients with an Arg55Leu SAP mutation. Therefore, the altered NK/LAK cell function observed in XLP patients correlates with the expression of the Arg55Leu mutant form of the SAP gene and may explain why XLP patients are unable to control infection with a common viral pathogen and maintain immune surveillance against lymphoid malignancies.
To examine the role of 2B4 in a uniform population of effector cells, we assessed the ability of the NK-derived cell line, NK-92ci, to lyse different target cell lines. NK-92ci effected enhanced lysis only of those targets that lacked expression of the 2B4 ligand, CD48, implying that target cell expression of CD48 conceals the effect of mAbC1.7. In support of this observation, transfection of K562 cells with CD48 reduced their susceptibility to anti-2B4-induced augmentation while enhancing their overall susceptibility to lysis (26). Interestingly, the susceptibility of CD48-transfected K562 cells to lysis could not be entirely accounted for by the transfection, implying that additional factor(s) must contribute to the augmenting effects of mAbC1.7. Moreover, addition of Fc fragments to NK cytotoxicity assays failed to entirely block the effect of 2B4 ligation (26). Although many of the CD48-negative cell lines express Fc receptors on the surface, this phenomenon was not due to ADCC because NK-92ci does not express CD16, and K562 does not express 2B4. An alternative explanation is that myeloid cells, which are primarily CD48 negative (28), may express an additional surface molecule that provides the primary stimulus needed to initiate costimulation through 2B4. The role of ADCC in the clinical phenotype of XLP is unclear. Some investigators report normal ADCC function in XLP patients (8), whereas others suggest that ADCC may be abnormal (6). We are currently investigating the ADCC function of NK cells obtained from these affected boys to determine whether the defect in 2B4 augmentation is one of several global defects in NK cell signaling or whether perhaps SAP is involved in ADCC.
Although there is considerable evidence supporting the role of CTL in controlling EBV infection (29, 30, 31), less is known about the specific function of NK or LAK cells in controlling either acute or latent EBV infection. NK cells are known both to proliferate during acute EBV infection (32) and to regulate the outgrowth of EBV-transformed B cells in vitro (33, 34), and LAK cells have been useful in the treatment of posttransplantation EBV-associated malignancies (35, 36). Because the major defect in XLP patients is an inability to control EBV infection, our findings provide evidence that NK and/or LAK cells may be vital for protective immunity to EBV.
How might defective NK or LAK cells account for the EBV-selective immunodeficiency seen in XLP patients? CD48, the natural ligand for human 2B4, has been shown to be dramatically up-regulated on B lymphocytes following EBV infection (37). Subsequent interaction of CD48 with 2B4 may potentiate the cytotoxic effects of normal NK or LAK cells on EBV-infected cells, and this interaction may be an essential element in the control of viral replication. Alternatively, NK and/or LAK cells may play a role in regulating the cellular response to viral infection, and functional expression of 2B4 may be critical for maintaining CTL homeostasis following EBV infection. This latter mechanism would account for the uncontrolled lymphoproliferation seen in XLP patients following exposure to EBV.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Rusung Tan, Department of Pathology and Laboratory Medicine, British Columbia’s Children’s Hospital, 4480 Oak Street, Room 2G5, Vancouver, British Columbia V6H 3V4.
3 Abbreviations used in this paper: XLP, X-linked lymphoproliferative disease; SLAM, signaling lymphocytic activation molecule; SAP, SLAM-associated protein; LAK, lymphokine-activated killer; CRA, chromium release assays; ADCC, Ab-dependent cellular cytotoxicity.
Received for publication June 27, 2000. Accepted for publication August 2, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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X 174. Birth Defects Orig. Artic. Ser. 19:321.[Medline]
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A. Aoukaty and R. Tan Association of the X-linked Lymphoproliferative Disease Gene Product SAP/SH2D1A with 2B4, a Natural Killer Cel |
) were assayed for lytic activity against
51Cr-labeled K562 cells. All assays were performed in
triplicate and on three separate occasions. Each data point is the mean
value of the repeated experiments and the error bars refer to the SD
generated from the three independent assays.
). b, NK-92ci were tested in three independent
assays for cytotoxic function against 51Cr-labeled MV4;11
target cells without Ab (
) or XLP patients
(n = 2, 
) were tested in three independent
assays for cytotoxicity against 51Cr -labeled K562 cells in
the presence (•,