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*
Institute of Medical Microbiology, University of Regensburg, Regensburg, Germany;
Department of Medicine III with Institute of Clinical Immunology, University of Erlangen-Nuremberg, Erlangen, Germany;
Partners AIDS Research Center and Infectious Disease Unit, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02129; and
§
AIDS Office, Department of Public Health, San Francisco, CA 94140
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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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Cell-mediated immunity has been shown to play an essential role in limiting the severity and duration of a series of viral infections, and there is cumulative evidence to suggest that this may also be the case in HIV-1-infected individuals. Gag-, Pol-, Env-, and Nef-specific cytolytic T cells have been detected early in the course of HIV-1 infection, before the appearance of humoral responses, and they appear to play an important role in the control of the initial viremia 2, 3, 4, 5, 6 . Moreover, studies of people at high risk of infection 7 , uninfected children born to HIV-1-infected mothers 8, 9 , health care workers exposed to HIV-1-contaminated material 10 , and LTNP individuals 11, 12, 13, 14, 15 strongly suggest that CD8+ HIV-1-specific CTL are associated with delaying the onset of disease in HIV-1-infected subjects.
Although HIV-1-specific CTL have been detected in many infected patients 16, 17, 18, 19 , they are ultimately not able to prevent progression of disease in the majority of infected persons 20 . Single amino acid substitutions within CTL epitopes can abrogate recognition by CTL, and it has been hypothesized that sequence variation leading to immune escape may play a role in progression of HIV-1 infection 21, 22, 23, 24, 25, 26 .
In LTNP, both the quality and the quantity of recognized CTL epitopes have been suggested to be involved in controlling HIV-1 infection 12, 13, 27, 28, 29, 30 . Previously, we had reported a subset of LTNP from the San Francisco City Clinic Cohort (San Francisco, CA), in which we detected a strong CTL response, weak neutralizing Abs, and low viral loads 31 . In one subject of this group (subject 15160), infected now for 20 yr with stable CD4 counts >500/µl and persistently low viral titers <500 molecules RNA/ml of plasma, a detailed analysis of the CTL response and viral quasispecies revealed a vigorous polyclonal HLA-class I-restricted CTL response against at least 6 CTL epitopes in p17, p24, reverse transcriptase (RT), Env, and Nef. Most of the epitopes were highly conserved among all known HIV-1 sequences, and the CTL clones conferred broadly cross-reactive recognition of reported HIV-1 variants 28 .
The dominant CTL response in subject 15160 was directed against the HLA-B14-restricted epitope, DRFYKTLRAE, fitting the predicted peptide binding motif for HLA-B14 32 with an arginine at position 2 and a phenylalanine at position 3, but differing from the motif by containing an alanine at position 9 instead of a predicted leucine. This epitope is partially overlapping with the major homology region (MHR) in the C-terminal domain of the p24 capsid moiety within the HIV-1 group-specific Ag, which is highly conserved not only among human and simian immunodeficiency viruses, but even among most known retroviruses 33, 34, 35 . When CTL clones from subject 15160 were tested for recognition of sequence variation within this epitope, they were able to recognize all HIV-1, HIV-2, and SIV variants published in the Los Alamos Database 36 at the time point of analysis 28 . Despite the selective pressure of this HLA-B14-restricted immunodominant CTL response, sequence analysis of viral quasispecies in plasma and PBMC of subject 15160 showed the absence of mutations in the p24-gag epitope, suggesting an essential function of this region during HIV-1 replication, and that efficient control of viral replication may require the generation of a polyclonal CTL response targeting multiple, highly conserved, and functionally important CTL epitopes.
To study potential limitations on HIV-1 for CTL immune escape in this highly conserved HLA-B14-restricted CTL epitope, we analyzed the influence of sequence variation within this epitope on viral replication and on recognition of the epitope by CTL derived from the LTNP subject 15160. Our results indicate that the analyzed sequence motif plays an essential role in HIV-1 replication and that targeting functionally important epitopes by broadly cross-reactive CTL clones impairs viral immune evasion.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Subject 15160 is an asymptomatic homosexual man who had been enrolled in the San Francisco City Clinic Cohort for the study of incidence and prevalence of hepatitis B virus infection. HIV-1 ELISA analysis of stored serum samples indicated that he has been infected since 1978. During the time of generation of CTL clones in 1993, his CD4 counts ranged between 885 and 1233/µl and his viral load was below the limit of detection of 500 copies/ml measured by quantitative PCR (Amplicor Assay, Roche, Nutley). Informed consent was obtained for the studies. The HLA class I type of subject 15160 is A3, A32, B14, B44,C-.
Cell lines and culture media
EBV-transformed B-lymphoblastoid cell lines (B-LCL) were established and maintained as described previously 37 in R20 (RPMI 1640 medium (Sigma, St. Louis, MO) supplemented with 2 mM L-glutamine, 50 U/ml penicillin, 50 µg/ml streptomycin, and 10 mM HEPES containing 20% (v/v) heat-inactivated FCS (Sigma)). For culture of CTL clones media containing 10% FCS (R10) supplemented with 100 U/ml rIL-2 (R10–100) was used. COS-7 cells were maintained in DMEM supplemented with 10% heat-inactivated FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM glutamine. CEM4 cells were obtained from the American Type Culture Collection (Manassas, VA) and grown in RPMI 1640 medium supplemented as described above.
CTL clones
CTL clones were isolated and maintained as previously described 37 using the CD3-specific mAb 12F6 as stimulus for T cell proliferation. Developing clones were screened for specific CTL activity against vaccinia-infected autologous B-LCL-expressing Gag proteins and then tested for recognition of the peptide DRFYKTLRAE. Clones with specific activity were then restimulated every 10–14 days with anti-CD3 or peptide-pulsed B-LCL together with irradiated allogenic PBMC.
Cytotoxicity assay
B-LCL were sensitized with synthetic peptides (10–100 µg/ml) as described and tested in a 4-h chromium release assay 28 . Supernatant fluid was harvested and counted on a 1216 Rackbeta scintillation Counter (LKB/Wallace, Turko, Finland). Spontaneous release was <30% of maximum release unless otherwise noted. For peptide titrations chromium-labeled target cells were incubated with peptides on a 96-well plate for 1 h before adding effector cells.
Synthetic peptides
Peptide synthesis was performed with a 9050 PepSynthesizer (Milligen, Eschborn, Germany) using Fmoc (9-fluorenylmethyloxycarbonyl)-protected amino acids, as described previously 38 . The peptides were purified by reversed phase HPLC using C2/C18 copolymer column (PepS; Pharmacia, Freiburg, Germany) and a gradient of 0–70% acetonitrile in 0.1% trifluoracetic acid. The fractions containing purified peptides were lyophilized and characterized by amino acid sequencing (Applied Biosystems, Weiterstadt, Germany). Lyophilized peptides were reconstituted at 2 mg/ml in sterile distilled water with 10% DMSO with or without 1 mM DTT.
Recombinant vaccinia viruses
Recombinant vaccinia viruses expressing the full-length p55 gag protein (vAbt141) and the SIV gag protein were provided by Drs. Gail Mazzara and Dennis Panicali (Therion Biologics, Cambridge, MA), and vaccinia viruses expressing the lacZ protein (vLac) were provided by Drs. Bernard Moss and Patricia Earl 37 .
Construction of recombinant proviruses
A pUC8 derivative, plin8Pr55 39 , including the complete gag
gene of HX10 33 was used as a template to generate the p24 capsid
mutations indicated in Fig. 1
.
Mutagenesis was performed by applying the QuikChange site-directed
mutagenesis kit (Stratagene, Heidelberg, Germany) on the dsDNA template
according to the manufacturer’s instructions. Briefly, 19 different
synthetic oligonucleotides corresponding to nucleotides 1670–1736 of
HX10 (with reference to the 5' edge of the 5' long terminal repeat)
have been designed to replace each two nucleotides in one of the
triplets (5'-CTG GCC AAG ATA TTT TGA GAT TCT CGG-3') encoding the
analyzed CTL epitope, respectively. Simultaneous mutation of a flanking
HindIII or AccI restriction site facilitated the
screening of potentially recombinant clones after transformation of the
mutagenized DNA into Escherichia coli. The presence of the
desired mutations was confirmed by sequencing the complete gag gene by
Taq cycle sequencing (Applied Biosystems). A 922-bp
SpeI/BclI fragment of each mutant was
subsequently subcloned into the parental HX10 DNA construct to generate
the recombinant proviruses depicted in Fig. 1.
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Transfection of recombinant provirus plasmids into COS-7 cells was performed by the Ca(PO4)2 procedure as described elsewhere 40 . CEM4 cells were transfected with the mutant provirus constructs by the DEAE transfection procedure as described 41 . Briefly, cells (5 x 106) were washed in 5 ml STBS (25 mM Tris-HCL (pH 7.4), 137 mM NaCl, 5 mM KCl, 0.6 mM Na2HPO4, 0.7 mM CaCl2, and 0.5 mM MgCl2) and resuspended in a mixture of STBS transfection buffer containing 10 µl of sterile DEAE dextran (10 mg/ml) together with 5 µg of the proviral DNA. After 30 min of incubation at 37°C, cells were washed twice in STBS, resuspended in 6 ml of complete RPMI 1640 medium, and seeded in a 25-cm2 flask.
Monitoring of virus release and replication
COS-7 cells and cell culture supernatants containing released virus particles were harvested at days 2 and 4 after transfection. Transfected CEM4 cells were split every 2 days at a ratio of 1:3 to maintain the cells in rapid growth. Aliquots of the supernatants were harvested every 2 days. Release of virus particles from COS-7 cells and replication of the wild-type and mutant viruses after transfection of the proviral DNA into CEM4 cells was monitored using a commercial p24 capture assay (DuPont/NEN, Bad Homburg, Germany) and, for comparison, by a nonradioactive RT assay (Retrotech, Munich, Germany). For that purpose, supernatants were precleared by low speed centrifugation at 200 x g for 10 min and then 30 min at 500 x g. Virus was finally collected from the resuspended pellet by centrifugation through a 30% (w/w) sucrose cushion in PBS at 200,000 x g for 2.5 h, resuspended in a small volume of PBS and processed for quantification (p24 Ag or RT activity) according to the manufacturer’s instructions.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Generation of mutant proviruses
For evaluation of this hypothesis, the occurrence of virus
variants was mimicked in vitro by generating a series of infectious
provirus mutants. Using HX10 as a parental provirus, each of the P1–P9
amino acid residues within the above described HLA-B14-restricted p24
CTL epitope was substituted by site-directed mutagenesis according to
the Dayhoff matrix defining the evolutionary frequency for sequence
variation 42 by 1) a functionally related amino acid (conservative
substitution) or 2) a functionally unrelated residue (nonconservative)
(Fig. 1
). Amino acid variations within this epitope, identified from a
comparison of 141 gag sequences listed in the Los Alamos database 36
at the time of constructing the HX10 provirus mutants were included in
the panel of conservative substitutions. Those variants were
Tyr301 to Phe (Y301
F; 35 of 141 compared gag
sequences), Lys302 to Arg (K302R; 1/141),
Thr303 to Ser (T303S; 33/141) and Thr303 to
Ala (T303A, 8/141).
Influence of sequence variation on virus replication
To test for the ability of gag mutants to assemble and release
virus particles, CD4 negative COS-7 cells were transfected with
wild-type and mutant proviral DNA. Sixty hours after transfection,
culture supernatants were harvested and pelleted through a 30% sucrose
cushion. The majority of the substitutions exhibited no or only a mild
effect on virus particle assembly and release as demonstrated by
quantitation of p24 capsid Ag in a commercial sandwich ELISA (Fig. 2, filled bars). Each of the depicted
values represents the mean determined from four independent
transfection experiments. Only the nonconservative amino acid
substitutions R299Y, F300R and L304H resulted in impaired
assembly and release of virus particles (Fig. 2B). These
results were confirmed in an independent assay format by quantifying
the RT activity from pelleted virions, indicating that the reduced Ag
levels determined in the p24 sandwich assay are due to a defect in
assembly and/or particle release rather than to major conformational
changes that may result in an underestimation of the levels of the
released Ag (Fig. 2, grey bars). In addition, these data demonstrate
that the vast majority of the established mutations affected neither
the release of viral particles nor the incorporation of the gag-pol
precursor into the budding virion. Additional experiments showed no
evidence of defects in processing of the gag and gag-pol precursors,
incorporation of viral RNA and cyclophilin, or association of the viral
gp120/gp41 (our unpublished observations). Sucrose sedimentation
analysis indicated no differences in the biophysical properties of the
released virions, either in size, or in their density (our unpublished
observations).
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). In both
cases, culture medium was harvested every 2–3 days, and the course of
the infections was followed over time by quantifying p24 Ag released
from productively infected cells into the culture medium. The peak of
p24 Ag release at day 8 in the case of the wild-type HIV-1-infected MT4
cells (data not shown) and at day 6 in the CEM4 cells transfected with
the parental HX10 provirus construct was representative of normal
infectivity of virus particles, respectively. In comparison, all of the
nonconservative amino acid substitutions abolished viral infectivity
and only 5 of 10 conservative changes yielded replication competent
virus (Fig. 3, A and B). Noteworthy, among these,
three of four virus variants mimicking sequence variations that were
found in the Los Alamos database (Y301F, T303S, T303A) turned out
to be infectious. However, only mutants R299K and Y301F
replicated to levels and with kinetics that were comparable to
wild-type HIV-1. Although the absolute p24 levels produced over time by
mutants F300Y, T303A, and T303S were in a similar range to
those of the parental virus, maximal Ag production was reproducibly
delayed by 2 days (Fig. 3A). These results are in complete
agreement with the observations obtained using infection of MT4 cells
with the COS-7-derived recombinant viruses (data not shown).
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Influence of sequence variation on CTL recognition
To analyze the effect of these conservative or
nonconservative sequence variations on the recognition by CTL derived
from subject 15160, the capability of various synthetic peptides to
sensitize autologous B-LCLs for CTL-mediated lysis was determined in a
standard chromium release assay. As demonstrated in Fig. 4, all peptides that were altered by a
conservative amino acid substitution were still recognized by the CTL
clone EA3. However, several sequence alterations, including the
substitutions at the positions P2 (R299K), P6 (T303S), P8
(R305K), and P9 (A306G) resulted in decreased recognition by the
CTL clone EA3, as determined using limiting amounts of peptide (Fig. 4A).
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W) and P9 (A306F) did not affect specific
killing of the labeled target cells. The phenylalanine to arginine
substitution at the position P3 (F300R) and the leucine to histidine
substitution at the position P7 (L304H) strongly reduced the
capacity of the peptides for sensitizing autologous targets for lysis
by the CTL. The mutations at the positions P2, P4, P5, P6, and P8
completely abrogated CTL recognition (Fig. 4B).
In addition to CTL clone EA3 (Fig. 4), another epitope-specific CTL
clone (CB5) had been generated at the same time point from subject
15160 that displayed the same fine specificity regarding the
recognition of the variant peptides (data not shown).
When tested with synthetic peptides, all sequence variants that did not
abrogate viral replication were still recognized by CTL, although, in
the case of the threonine to serine substitution at position P6
(T303S) and the arginine to lysine mutation at position P2
(R299K), lysis of peptide-sensitized cells was significantly reduced
(Fig. 4A). The peptide T303S, representing the common
HIV-2 and SIV strains, was only recognized at a peptide concentration
of 100 µg/ml. Loss of recognition was already observed by reducing
the peptide concentration by one order of magnitude down to 10 µg/ml,
suggesting a relatively poor capacity of this peptide to sensitize
target cells. To determine whether this peptide would be recognized on
cells after being processed from a gag protein synthesized in the
cytoplasm, the CTL clones EA3 and CB5 were tested for lysis of B-LCL
infected with a SIV-gag recombinant vaccinia virus. This
SIV-gag, as most known SIV and HIV-2 strains, contains a serine instead
of a threonine at position 6 (DRFYKSLRAE). Although specific lysis was
slightly reduced as compared with the abt141 vaccinia vector expressing
the HIV-1-IIIB-gag protein, the SIV-gag expressing B-LCL were lysed by
the CTL (Fig. 5).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The analyzed p24 CTL epitope (amino acids 298–306) shares 6 residues
with the C-terminal portion of the MHR (amino acids 285–304) (Fig. 1),
a stretch of 20 highly conserved residues, which is found in all known
onco- and lenti- viruses as well as in the yeast retrotransposon Ty-3
33, 35, 43 . Functional studies, primarily targeting the most
conserved residues within various MHR of different origin such as Q287,
G288, E291, and R299 (HIV-1 nomenclature; R299 being part of the
analyzed p24 CTL epitope) suggested an essential role of the MHR for
viral replication. However, its precise function still has to be
elucidated, as different mutations within the MHR block viral
replication at different stages during assembly, maturation and early
steps of infectivity 35, 44, 45, 46 . These findings may be explained by
recently reported structural analysis revealing that the MHR within the
C-terminal domain of the HIV-1 capsid protein forms a compact
strand-turn-helix motif that is stabilized by a complex
hydrogen-bonding network and simultaneously contributes to the
hydrophobic core of the protein 47 .
In agreement with the structural information and considerably extending
recent functional analyses, we found that a stretch of nine amino acids
overlapping the C-terminal portion of the MHR by six residues is
critically involved in early steps of viral replication. Mimicry of
epitope variation by substituting each position in the above described
p24 CTL epitope with a functionally related amino acid or alternatively
with an unrelated residue resulted with few exceptions in relatively
moderate effects on virus assembly and particle release (Fig. 2) 35, 44, 45, 46 . Also, RT-activity measurements of released and purified
virions indicated correct incorporation of the gag-pol precursor into
virions (Fig. 2) 48 . Further biochemical analysis of gag and gag-pol
polyprotein processing, association of the viral gp120/gp41,
incorporation of viral RNA and cellular chaperones (cyclophilin A), as
well as sedimentation analysis suggests that most steps during the
assembly process are not affected by these mutations (our unpublished
data).
By contrast, all nonconservative amino acid substitutions (9/9) and
(5/10) conservative substitutions yielded replication-defective viral
mutants when tested on different CD4-positive T cell lines. In
addition, three mutants showed impaired replication kinetics when
compared with parental wild-type HIV-1. As expected, infectivity was
not affected in the majority of sequence variants (3/4) reported most
frequently in the Los Alamos database 36 . Substitution of
Lys302 to Arg (K302R), a very rare sequence variation
(1/144 listed sequences), induced complete loss of infectivity of our
mutant virus, at least with regard to infection of T lymphotropic tumor
cell lines. Assuming that the listed K302R sequence has been
established from an infectious virus we only can speculate about this
discrepancy. All mutations were introduced into a defined HX10
backbone. The impairment of infectivity by the K302R mutation may be
less striking for other HIV-1 strains or in other target cells than in
the one used here. Further studies are ongoing to elucidate the
molecular mechanisms underlying the replication defects that had been
observed for the majority of the mutants both on the level of capsid
assembly and in the early steps of the viral life cycle.
CTL clones against this epitope, generated from a LTNP individual, were
systematically analyzed for recognition of conservative and
nonconservative amino acid substitutions for each of the amino acid
positions (P1–P9). The TCR of these CTL clones exerted broad
cross-reactivity with recognition of all conservative amino acid
substitutions in the epitope. However, several mutations reduced the
capacity of the peptides to sensitize target cells for specific lysis.
The position P6 (T303) appeared to be particularly critical for the
interaction of TCR with the peptide, as even the conservative threonine
to serine exchange (T303S) had a strong negative effect on
recognition. In contrast, an alanine at the same position (T303A)
did not affect peptide recognition indicating that even minor changes
in the side chain of the amino acid residues induce conformational
changes which affect TCR binding. The reduction of the sensitizing
activity of the peptides due to conservative amino acid substitutions
at positions P2 (R229K), P8 (R305K), and P9 (A306G) probably
results from a reduction of the peptide binding to the HLA-B14
molecule, as the P2 and the P9 residues presumably represent the anchor
positions 32 . Except for an aspartate to tryptophane exchange at
position P1 (D298W) and an alanine to phenylalanine substitution
(A306F) at position P9, all other nonconservative substitutions
strongly reduced or, in the most cases, completely abrogated killing by
the CTL clones. This finding demonstrates that single amino acid
mutations in this epitope can abrogate recognition by CTL and that
there is a potential for HIV-1 to escape CTL by single amino acid
mutations in this epitope.
Although the above experiments demonstrate that mutations within this
epitope can lead to escape, all mutations leading to CTL escape
strongly impaired viral replication. The only two mutations which did
not interfere with virus production, but had a significant negative
effect on CTL recognition, were the arginine to lysine substitution
(R299K) at position P2 and, more importantly, the threonine to
serine substitution (T303S) at position P6. Whereas a R299K
mutation has thus far not been identified in any primary HIV-1 isolate,
a serine at P6 is observed in all HIV-2 and SIV strains, but
interestingly never in HIV-1. Despite the strong effect of the
threonine to serine mutation on the capacity to sensitize target cells,
this mutation was still recognized when expressed by a SIV-gag vaccinia
vector, although to a lesser extent when compared with the HIV-1-gag.
This suggests that even this mutant, at least at high Ag
concentrations, would be recognized in naturally HIV-1-infected cells.
Our data demonstrate that the analyzed stretch of nine amino acids in p24 is critically contributing to proper virus replication to an extent that drastically limits the potential of HIV-1 for CTL escape by emergence of sequence variation within this epitope. However, although the amino acid substitutions have been carefully selected based on the Dayhoff matrix and naturally occurring viral variants have been considered in our analysis, a theoretical potential for immune escape cannot be excluded as all mutations were introduced into a defined HX10 proviral background, and only a limited number of possible mutations could be analyzed. Although also being speculative regarding the complex structure of the MHR, HIV-1 strains could theoretically emerge over time with compensatory mutations within the epitope or in other parts of the sequence that might allow for immune escape.
Even the most active peptides corresponding to this p24 epitope lost the capability to sensitize target cells for lysis by the CTL clones at concentrations of 100–10 ng/ml. In comparison, published peptides corresponding to other epitopes, such as HLA-A2 restricted peptides derived from HIV-1-RT and p17 were still active in picomolar concentrations 49 . It is not yet known, whether this difference in the sensitizing activity of peptides is due to a weaker binding of the p24 peptide to the HLA-B14 molecule or due to a lower binding affinity of the broadly cross-reactive TCR of the CTL clones used in the experiments.
HLA-B14, the restricting allele for this epitope, has been shown to be a protective prognostic factor for HIV-1-infected patients 50, 51 . It remains to be determined whether this is associated with the ability to present such highly conserved epitopes as the p24 epitope. As we have analyzed only a single epitope in a single patient further studies with more patients and more epitopes are necessary to define the role of recognition of specific epitopes by HIV-1-specific CTL for suppression of HIV-1. It has been shown that HLA-B27, which is also a protective HLA allele 52 , can also present a very conserved epitope in which the emergence of sequence variation seems to be inhibited by the negative effects on viral replication.
Based on these studies, we conclude that targeting highly conserved epitopes in which variability of HIV-1 is restricted by the requirements of essential biological functions could be an important factor for a sustained control of HIV-1.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Thomas Harrer, Department of Medicine III with Institute of Clinical Immunology, University of Erlangen-Nürnberg, Krankenhausstrasse 12, 91054 Erlangen, Germany. E-mail address:
3 Abbreviations used in this paper: LTNP, long-term nonprogressors (nonprogressing); MHR, major homology region; B-LCL, B-lymphoblastoid cell line; RT, reverse transcriptase.
Received for publication September 9, 1998. Accepted for publication December 18, 1998.
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C. M. Leutenegger, F. S. Boretti, C. N. Mislin, J. N. Flynn, M. Schroff, A. Habel, C. Junghans, S. A. Koenig-Merediz, B. Sigrist, A. Aubert, et al. Immunization of Cats against Feline Immunodeficiency Virus (FIV) Infection by Using Minimalistic Immunogenic Defined Gene Expression Vector Vaccines Expressing FIV gp140 Alone or with Feline Interleukin-12 (IL-12), IL-16, or a CpG Motif J. Virol., November 15, 2000; 74(22): 10447 - 10457. [Abstract] [Full Text]
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) infectious; (
) noninfectious;
the capability of the mutant peptides to sensitize autologous targets
is given as NET%-specific lysis (% specific lysis of
peptide-sensitized B-LCL - % specific lysis of control B-LCL).