This study demonstrates that anti-third party CTLs can efficiently kill B cell lymphomas through a novel TCR-independent mechanism that requires engagement of MHC class I molecules on the lymphoma cells with CD8 molecules on the CTLs. Using mutant cell lines and blocking antibodies, the researchers showed that lymphoma cell killing begins with conjugate formation between CTLs and tumor cells mediated by ICAM-1 and LFA-1 binding, followed by slower MHC-I dependent induction of apoptosis upon binding of MHC-I on tumor cells to CD8 on CTLs. This novel finding suggests MHC-I molecules can signal tumor cell death independent of antigen presentation when engaged by CD8, representing a new role for MHC-I in

1. The Journal of Immunology
TCR-Independent Killing of B Cell Malignancies by
Anti–Third-Party CTLs: The Critical Role of MHC–CD8
Engagement
Assaf Lask,*,1
Polina Goichberg,*,1
Adva Cohen,* Rinat Goren-Arbel,* Oren Milstein,*
Shraga Aviner,* Ilan Feine,* Eran Ophir,* Shlomit Reich-Zeliger,* David Hagin,*
Tirza Klein,†
Arnon Nagler,‡
Alain Berrebi,x
and Yair Reisner*
We previously demonstrated that anti–third-party CTLs (stimulated under IL-2 deprivation against cells with an MHC class I
[MHC-I] background different from that of the host and the donor) are depleted of graft-versus-host reactivity and can eradicate
B cell chronic lymphocytic leukemia cells in vitro or in an HU/SCID mouse model. We demonstrated in the current study that
human allogeneic or autologous anti–third-party CTLs can also efficiently eradicate primary non-Hodgkin B cell lymphoma by
inducing slow apoptosis of the pathological cells. Using MHC-I mutant cell line as target cells, which are unrecognizable by the
CTL TCR, we demonstrated directly that this killing is TCR independent. Strikingly, this unique TCR-independent killing is
induced through lymphoma MHC-I engagement. We further showed that this killing mechanism begins with durable conjugate
formation between the CTLs and the tumor cells, through rapid binding of tumor ICAM-1 to the CTL LFA-1 molecule. This
conjugation is followed by a slower second step of MHC-I–dependent apoptosis, requiring the binding of the MHC-I a2/3 C region
on tumor cells to the CTL CD8 molecule for killing to ensue. By comparing CTL-mediated killing of Daudi lymphoma cells
(lacking surface MHC-I expression) to Daudi cells with reconstituted surface MHC-I, we demonstrated directly for the first time
to our knowledge, in vitro and in vivo, a novel role for MHC-I in the induction of lymphoma cell apoptosis by CTLs. Additionally,
by using different knockout and transgenic strains, we further showed that mouse anti–third-party CTLs also kill lymphoma cells
using similar unique TCR-independence mechanism as human CTLs, while sparing normal naive B cells. The Journal of
Immunology, 2011, 187: 2006–2014.
D
evelopment of strategies to generate ex vivo immune
cells selectively endowed with graft-versus-leukemia
(GVL), while depleted of graft-versus-host (GVH) re-
activity, potentially represents a major challenge in bone marrow
(BM) transplantation and in cancer immunotherapy. Our previous
studies showed that ex vivo stimulation of mouse CD8 T cells
against third-party stimulators (carrying an MHC class I [MHC-I]
background different from that of the host and the donor, hence,
termed “third party”) under IL-2 deprivation led to the selective
growth of third-party–restricted CTL clones concomitantly with
a loss of other clones, including anti-host clones that are unable to
produce their own IL-2 (1–3). Such anti–third-party CTLs were
found to be markedly depleted of GVH reactivity upon trans-
plantation into lethally irradiated mice (2) and exhibited strong
veto activity in vitro (4) and in vivo (2). Thus, their potential role
in tolerance induction, especially in the context of BM allografting
under reduced-intensity conditioning (RIC), was demonstrated in
mouse models (2). This ability of donor anti–third-party CTLs to
kill cognate alloreactive host CTL precursors, when the donor
anti–third-party CTLs are recognized by the naive host CTL
precursors, is initiated upon immune-synapse formation through
TCR recognition of the donor cells by the host naive T cells.
Perhaps somewhat confusing is that the anti–third-party CTLs
serving as target are capable of killing the recognizing naive
T cells. Early studies using Fas and Fas ligand (FasL) knockout
(KO) T cells in long-term MLR cultures indicated a role for Fas–
FasL killing (4), whereas a more recent study using microscopy
imaging indicated that short-term killing through a perforin-
mediated mechanism can also occur (5). In both instances, the
interaction of CD8 on the veto anti–third-party CTL with a3
domain of MHC on the recognizing host CTL precursor is critical
for induction of the killing process.
Based on the insights from the mouse model, a new procedure for
the generation of human anti–third-party CTLs was developed (3).
When studied in a human–mouse chimeric model, these newly
generated human anti–third-party CTLs did not negatively affect
the engraftment of fully allogeneic normal B and T cells (6).
Surprisingly, we found that such allogeneic or autologous anti–
third-party CTLs exhibited potent killing of B cell chronic lym-
phocytic leukemia (B-CLL) (6) cells. Thus, these host nonreactive
anti–third-party CTLs could potentially be used to facilitate en-
graftment of allogeneic hematopoietic stem cells, as well as pro-
vide GVL reactivity.
*Department of Immunology, Weizmann Institute of Science, Rehovot 76100, Israel;
†
Tissue Typing, Rabin Medical Center, Petach Tikva 49100, Israel; ‡
Division of
Hematology, Sheba Medical Center, Tel-Hashomer 52621, Israel; and x
Hematology
Institute, Kaplan Medical Center, Rehovot 76100, Israel
1
A.L. and P.G. contributed equally to this study.
Received for publication January 24, 2011. Accepted for publication June 6, 2011.
This work was supported by the Gabriella Rich Center for Transplantation Biology
Research, Mrs. E. Drake, and by a research grant from Roberto and Renata Ruhman.
Address correspondence and reprint requests to Prof. Yair Reisner, Department of
Immunology, Weizmann Institute of Science, Rehovot 76100, Israel. E-mail address:
yair.reisner@weizmann.ac.il
The online version of this article contains supplemental material.
Abbreviations used in this article: B-CLL, B cell chronic lymphocytic leukemia;
B2m, b2-microglobulin–reconstituted Daudi; BM, bone marrow; B-NHL, B cell
non-Hodgkin’s lymphoma; DHE, dihydroethidium; FasL, Fas ligand; GVH, graft-
versus-host; GVL, graft-versus-leukemia; KO, knockout; b2m, b2-microglobulin;
MHC-I, MHC class I; RIC, reduced-intensity conditioning; wt, wild-type.
Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00
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2. In the current study, we demonstrated that anti–third-party CTLs
also exhibited marked reactivity against different types of B cell
lymphoma while sparing normal naive B cells; therefore, their use
could be extended to treat B cell malignancies other than B-CLL.
Furthermore, the availability of different B cell lymphoma lines
enabled us to investigate the mechanism by which anti–third-party
CTLs can induce apoptosis in lymphoma cells in the absence of
TCR recognition.
MHC-I molecules are highly expressed on immune cells, and
their major role is to present antigenic peptide fragments to CD8+
T cells (7) through their TCR and its coreceptor CD8 (8, 9). Our
novel finding is in agreement with a growing body of evidence
highlighting another role for MHC-I molecules in eliciting an
intracellular signaling event upon their engagement. This event
results in either positive or negative regulation of immune cell
function and viability (10–15). Using different blocking Abs and
mutated lymphoma cell lines, we showed a novel role for the
MHC-I molecule in mediating induction of apoptosis of malig-
nant B cells by anti–third-party CTLs. The initial step in the de-
scribed killing process is a rapid conjugate formation mediated
via an ICAM-1:LFA-1 interaction. Interestingly, this is followed
by unique TCR-independent killing, which is dependent on the
interaction of CD8 molecules on the CTL and MHC-I molecules
on the affected lymphoma cells.
Materials and Methods
Cells
Whole blood was collected from lymphoma patients in the leukemic phase
and from healthy volunteers. The lymphoma samples included stage IV
follicular lymphoma (one case), lymphoplasmacytic lymphoma (two cases),
and splenic lymphoma with villous lymphocytes (one case). The study was
approved by the National Ethics Committee, Israel, and informed consent
was obtained from patients and healthy donors.
PBMCs were obtained by Ficoll density-gradient centrifugation. HLA
typing was performed by molecular methods for patient samples. Briefly,
DNA was purified from PBMCs derived from EDTA-treated blood samples
using the High Pure PCR Template Preparation Kit (Roche Diagnostics,
Hilden, Germany), and HLA-A and -B typing was performed by PCR using
RELI SSO HLA-A and HLA-B Typing kits (Invitrogen, Carlsbad, CA). For
healthy samples, HLA typing was performed by serology methods.
BL-44 (Burkitt’s lymphoma) and Granta519 (mantle cell lymphoma)
lines were cultured in RPMI 1640 supplemented with 10% FCS and
antibiotics. Daudi and b2-microglobulin (b2m)-reconstituted Daudi (B2m)
Burkitt’s lymphoma cell lines were kindly provided by Dr. R.D. Salter
(University of Pittsburgh School of Medicine, Pittsburgh, PA) (16). The
H.My2 C1R HLA-A2 wild-type (wt) transfectant B cell lines were also
kindly provided by Dr. R.D. Salter (17); the H.My2 C1R HLA-A2 K66A
transfectant was kindly provided by Dr. B.M. Baker (University of Notre
Dame, Notre Dame, IN) (18). Daudi and H.My2 cell lines were cultured in
IMDM with 10% FCS and antibiotics.
Animals
Female 6–12-wk-old BALB/c, CB6, FVB, and C57BL/6 mice were ob-
tained from Harlan Laboratories (Rehovot, Israel). Breeding pairs of Prf2/2
,
Gld2/2
, Tnf2/2
, and Trail2/2
KO mice of H-2b
background and transgenic
OT1/rag2/2
mice of C57BL/6 background expressing a TCR specific for
OVA 257–264 aa in the context of H2Kb
on CD8 T cells were obtained
from The Jackson Laboratory (Bar Harbor, ME). NOD-SCID mice were
bred and housed at the Weizmann Institute. All mice were kept in small
cages (five animals in each cage) and fed sterile food and acid water.
Generation of nonalloreactive anti–third-party CTLs
As described previously (6), PBMCs were obtained by Ficoll density-
gradient centrifugation of buffy coats from healthy donors (allogeneic)
or from whole-blood samples of lymphoma patients (autologous). The
PBMCs were then stimulated with an allogeneic EBV-transformed B cell
line of an HLA background different from that of the host HLA. Cells were
cultured at a 40:1 PBMC/stimulator ratio for 10 d. The cultured cells were
then mounted on Ficoll to discard dead cells and were restimulated with
EBV stimulators at a 4:1 ratio. Four days later, the subpopulation of CD8+
T cells was isolated by one cycle of negative selection with anti-CD56–
coated magnetic beads (CD56 MACS; Miltenyi Biotec), followed by
positive selection of CD8+
cells with anti-CD8–coated magnetic beads
(CD8 MACS; Miltenyi Biotec). CD8+
T cells were then cultured in
complete RPMI 1640 containing 300 IU/ml recombinant human IL-2
(EuroCentus, Amsterdam, The Netherlands) and EBV stimulators at a
4:1 ratio. Culture was replenished with IL-2, every 2–3 d, for 7 d post-
initial IL-2 addition. Using FACS analysis, CD8 purity of CTLs was shown
to be .90% CD8+
on day 14 and on the day of the experiment (day 21).
Mouse anti–third-party CTLs were prepared as previously described (2).
Briefly, splenocytes of the donor mice were cultured against irradiated
third-party splenocytes for 6 d under cytokine deprivation. Subsequently,
cells were subjected to positive selection of CD8+
cells using Magnetic
Particles (BD Pharmingen). The isolated cells were restimulated with ir-
radiated third-party splenocytes, and recombinant human IL-2 (40 U/ml)
was added every second day.
Flow cytometry
The following labeled anti-human Abs were used: CD19-PE (Miltenyi
Biotec); CD20-FITC, CD56-PE, and CD8-allophycocyanin (Biosciences
Pharmingen, San Diego, CA); and Pan gd TCR-PE (Beckman Coulter,
Krefeld, Germany). Indirect immunolabeling by anti–HLA-ABC (W6/32;
Serotec, Oxford, U.K.) was performed using secondary PE-conjugated
IgG (Jackson ImmunoResearch Laboratories, West Grove, PA). The fol-
lowing labeled anti-mouse Abs were used: CD8 (allophycocyanin/PE),
CD-19 (allophycocyanin/FITC), and B-220 (PE) (BD Bioscience). For the
in vitro killing assays, lymphoma cells were prelabeled with 0.15 mg/ml
calcein AM (Molecular Probes, Eugene, OR). The MLR was followed by
Annexin V labeling (Biosciences Pharmingen).
MLR-killing assay
Anti–third-party CTLs and lymphoma cells were obtained by Ficoll
density-gradient centrifugation, after which the lymphoma cells were la-
beled with 0.15 mg/ml calcein AM, a vital dye that is released upon cell
death, according to the manufacturer’s instructions, and brought to a con-
centration of 1 3 106
cells/ml in the appropriate media. Next, 2.5 3 105
calcein-labeled lymphoma cells were incubated with or without anti–third-
party CTLs at the indicated ratio and time intervals in 24-well plates. No
exogenous cytokines were added to the MLR. Cells were recovered and
analyzed for survival using surface markers or by measuring the number of
surviving calcein-stained lymphoma cells by FACS. To obtain absolute
values of cells, samples were suspended in constant volume, and flow
cytometric counts for each sample were obtained during a constant pre-
determined period of time and compared with flow cytometric counts
obtained with a fixed volume and fixed numbers of input cells (19). Sur-
vival rates are presented relative to the survival of lymphoma cells alone.
Preparation of naive normal B cells
Splenocytes of BALB/c mice were harvested, and single-cell suspensions
were prepared. The cell suspensions were fractionated on Ficoll-Paque Plus
(Amersham Biosciences, Uppsala, Sweden) to remove RBCs. The isolated
mononuclear cells were then subjected to positive selection of B cells using
magnetically labeled anti-B220 Abs using the IMAG magnetic separation
system (BD Bioscience, San Diego, CA). Purity and efficiency of cleaning
were tested by FACS analysis.
Inhibition of B cell lymphoma killing by blocking Abs
CTLs or lymphoma cells were preincubated for 30 min at a minimal vol-
ume with the indicated neutralizing Ab at the indicated concentrations.
Blocked cells were then incubated with the other nonblocked component
of the MLR for 36 h at a 1:5 ratio in favor of the anti–third-party CTLs.
Cell survival was analyzed by FACS. The following neutralizing Abs were
used: hNKG2D-blocking mAb (clone 149810; R&D Systems, Minneap-
olis, MN); LFA-1 (CD11a)-blocking Ab (clones TSI.18.1.1.2 or 2F12),
ICAM-1 (CD54)-blocking Ab (clone MC1615XZ), and HLA-ABC
(W6/32)-blocking Ab (AbD (Serotec); ICAM-2 (CD102)-blocking Ab
(clone CBR-IC2/2) and CD50 (ICAM-3) (clone CBR-IC3/1)-blocking Ab
(Bender MedSystems, Vienna, Austria); and anti-CD8 (LT8)-blocking
Ab (GeneTex, San Antonio, TX).
Conjugation assay
Efficiency of cell–cell binding was assayed by conjugate-formation anal-
ysis, as previously described (20). Briefly, following incubation with the
indicated Ab, calcein AM (0.15 mg/ml)-prelabeled anti–third-party CTLs
(1 3 106
cells/ml) were combined with equal numbers of dihydroethid-
ium (DHE) (3 mg/ml; Sigma-Aldrich, Rehovot, Israel)-prelabeled target
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3. B cells, pelleted for 2 min at 500 rpm, and further incubated for 5–10 min
at 37˚C. Following a 5-s vortex, the percentage of conjugates was deter-
mined by FACS.
Detection of apoptosis by Annexin V staining
Samples from in vitro cultures were incubated with a mixture of selected
mAbs labeled with different fluorochromes for 20 min at 4˚C. After washing
off the unbound free Ab, samples were incubated with 5 ml Annexin V-
FITC or Annexin V-allophycocyanin (Biosciences Pharmingen) for 10 min
at room temperature. Subsequently, unbound Annexin V was washed out,
and samples were analyzed by FACS (21).
In vivo analysis of anti–third-party CTL-mediated killing
Target lymphoma cells (20 3 106
/mice) were labeled with 5 mM CFSE and
injected i.p. into NOD-SCID mice in the presence or absence of a 4-fold
excess of anti–third-party CTLs. Peritoneal exudates were collected
5 d postinjection, and the numbers of CFSE+
cells per volume were de-
termined by FACS. Labeling with anti-human CD8-allophycocyanin Ab
(Biosciences Pharmingen) was performed to assay the presence of the
anti–third-party CTLs in the peritoneal fluids of the coinjected mice.
Statistical analysis
Statistical significance was established using the Student t test.
Results
Allogeneic and autologous anti–third-party CTLs induce
apoptotic death of B cell non-Hodgkin’s lymphoma cells
To determine whether anti–third-party CTLs are endowed with
antilymphoma cell reactivity, we initially tested their ability to
kill the BL-44 and Granta519 cell lines, both of which represent
highly aggressive malignancies (22, 23). As can be seen in Fig.
1A, marked killing of 73.5 6 3% of lymphoma cells was found
following incubation with a 5-fold excess of anti–third-party
CTLs. The killing kinetics of lymphoma cells by anti–third-
party CTLs was much slower than was the killing of cognate
third-party stimulators by the same CTLs. Thus, when tested 24 h
after the initiation of culture, 41.1 6 1.3% of noncognate lym-
phoma cells were killed compared with 92 6 0.7% of cognate
target cells (Fig. 1A).
The killing of lymphoma cells by anti–third-party CTLs was
dose dependent. As can be seen in Fig. 1B, 58% of lymphoma
cells were viable after incubation at a 1:1 lymphoma cell/CTL
ratio, whereas only 23% of lymphoma cells were viable after in-
cubation with a 10-fold excess of anti–third-party CTLs. Like-
wise, the level of Annexin V+
lymphoma cells increased from 42
to 77% upon escalation of the lymphoma cell/CTL ratio from 1:1
to 1:10 (Fig. 1B). When the broad-range caspase inhibitor ZVAD-
fmk was added to the cell culture, killing was blocked (Fig. 1B).
These data are consistent with our previous results on B-CLL
eradication (6) and indicated that killing of lymphoma cells by
anti–third-party CTLs is mediated by apoptosis.
We next examined the ability of the anti–third-party CTLs to kill
primary tumor cells obtained from peripheral blood of stage IV
lymphoma patients. We used four patients with HLA-A and -B
types that were not cross-reactive with the stimulator cells used for
the generation of anti–third-party CTLs. As can be seen in Fig.
1C, following incubation of the primary lymphoma cells with
FIGURE 1. Anti–third-party CTLs are endowed with reactivity against lymphoma cell lines and primary PBLs from lymphoma patients. A, Calcein AM-
prelabeled lymphoma cells (BL-44 or Granta519 cell lines) or calcein AM-prelabeled cognate target cells (third-party B cell stimulators) were incubated for
the indicated time intervals with or without 5-fold excess of anti–third-party CTLs. Numbers of viable calcein AM+
Annexin V2
cells were determined by
FACS, as demonstrated in the representative scatograms (right panels). Data are shown as the percentage of killing compared with target cells incubated in
the absence of CTLs (0%). Data shown are mean 6 SD of at least three independent experiments, each in triplicate. *p , 0.01, changes in the number of
live cells versus no CTL and versus target-restricted CTLs. B, BL-44 or Granta519 lymphoma cell line cells were incubated for 36 h with or without anti–
third-party CTLs at the indicated ratios in the absence or presence of ZVAD-fmk and examined by FACS for viability and Annexin V labeling (as described
in Materials and Methods). A representative experiment with BL-44 is shown. C, Killing of primary tumor cells obtained from PBLs of lymphoma patients
or lymphoma cell lines following 36 h of incubation in the presence or absence of allogeneic anti–third-party CTLs. Data represent mean 6 SD of five
independent experiments, performed in triplicates (n = 4 patients). *p , 0.01, changes in the number of live cells versus values obtained in the absence of
CTLs. D, Killing of primary lymphoma cells following 36 h of incubation in the presence or absence of autologous (Auto) or allogeneic (Allo) anti–third-
party CTLs. Data represent mean 6 SD of five independent experiments, performed in triplicates (n = 4 patients). *p , 0.01, decrease in the number of live
cells versus values obtained in the absence of CTLs.
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4. allogeneic anti–third-party CTLs for 36 h, primary lymphoma
cells were killed with a similar efficiency to that found for the
lymphoma cell lines described above.
To test whether the killing of lymphoma cells is mediated by
residual alloreactivity, we generated anti–third-party CTLs from
the patients’ own T cells. As can be seen in Fig. 1D, a similar level
of killing of primary lymphoma cells was induced by autologous
or allogeneic anti–third-party CTLs. Considering that autologous
T cells are markedly depleted of self-reactive clones, the similar
killing of lymphoma cells by autologous and allogeneic anti–
third-party CTLs strongly indicated that this form of killing can-
not be attributed to contamination of the anti–third-party CTLs
with residual alloreactive clones.
The killing of lymphoma cells by anti–third-party CTLs is TCR
independent and does not use other killing mechanisms
commonly used by CTLs
Although the similarity in lymphoma killing exhibited by autol-
ogous and allogeneic anti–third-party CTLs ruled out the potential
involvement of residual alloreactive clones, it also suggested that
the TCR of these CTLs directed against a third party likely does
not mediate this killing. The role of the TCR in this killing was
more definitively addressed by using mutated B cell lympho-
blastoid target cells previously shown to express an MHC-I mutant
not recognizable by the TCR. In this experiment, we made use of
the previous finding that the K66A mutation in the a1 helix of the
HLA-A2 allele, in which the lysine residue is replaced by alanine,
disrupts recognition by the TCR (24). These findings were con-
sistent with an earlier study showing that this mutation disrupts
recognition of HLA-A2 by a series of alloreactive CTLs (25).
Thus, we compared the ability of anti–third-party CTLs to kill the
B cell lymphoblastoid cell line H.My C1R, expressing a wt HLA-
A2, with the killing of the B cell lymphoblastoid H.My C1R cell
line expressing mutated HLA-A2 (K66A) by anti–third-party CTLs.
As shown in Fig. 2A, this comparison did not reveal a significant
difference in the cytotoxic efficacy. Thus, the killing by anti–third-
party CTLs is not disrupted by a mutation in MHC-I, which is
known to block TCR recognition of peptide–MHC-I complexes.
Interestingly, other mechanisms generally used by CTLs were also
excluded. The potential induction of apoptosis by death molecules,
such as TNF, FasL, or TRAIL, was ruled out by using specific
blocking Abs that did not affect the killing (data not shown). To test
the efficacy of anti-FasL Ab, a positive control study was performed
using supernatant containing soluble FasL. Although the soluble
FasL induced apoptosis in BL-44 and Jurkat cells, apoptosis was
prevented by the addition of the blocking Ab (data not shown).
However, to further define the possible involvement of such
death molecules that were previously found to play a role in
generating the veto activity by anti–third-party CTLs, we carried
out mouse experiments using different KO mice. To that end, we
used a mouse model based on the A20 B cell lymphoma/leukemia
line (26) spontaneously derived from BALB/c (H-2d
) mice.
Initially, we tested the ability of mouse anti–third-party CTLs to
kill A20 cells. As can be seen in Fig. 2B, allogeneic anti–third-
party CTLs exhibited dose-dependent killing of A20 target cells.
Similarly to the human setting, this killing is not mediated by
alloreactivity, because anti–third-party CTLs derived from F1
mice (CB6 H2bd
), which are not alloreactive against BALB/c
parental cells, displayed a comparable level of killing (Fig. 2B).
In addition, as for the human counterparts, the death of the mouse
lymphoma cells was due to apoptosis, as indicated by Annexin V
staining (Fig. 2C).
Importantly, as shown in Fig. 2D, examination of anti–third-
party CTLs from FasL-mutated (gld), TNF KO, or Trail KO mice
did not reveal a significant reduction in their ability to kill A20
cells compared with their wt counterparts. Thus, in accordance
with our blocking studies in the human setting, the killing of
B cell lymphoma cells is likely not mediated by death molecules
belonging to the TNF family.
Likewise, a perforin-based mechanism was ruled out by an assay
for BLT esterase 24 h after the initiation of MLR with human anti–
third-party CTLs (Supplemental Fig. 1A) or by using mouse CTLs
from perforin KO mice (Fig. 2D). Furthermore, in addition to the
experiments described above indicating that the killing of B cell
lymphoma by human anti–third-party CTLs is TCR independent,
the mouse model enabled us to directly examine this important
question. Thus, CTLs generated from OT-1–RAG2/2
transgenic
mice, in which the CD8+
T cells express only a TCR specific for
the SIINFEKL peptide of OVA in the context of H2-Kb
(not
expressed by the A20 lymphoma cells), exhibited similar killing to
that found when using nonalloreactive F1-derived (BALB 3 B6)
anti–third-party CTLs (Fig. 2E).
Thus, again, as for human anti–third-party CTLs, the killing of
lymphoma cells is TCR independent.
In addition, a potential involvement of gd T cells in the observed
killing is highly unlikely, considering that gd T cells are markedly
depleted in the process of anti–third-party CTL generation and
could barely be detected at the end of the culture (Supplemental Fig.
1B). Furthermore, we also showed, by using blocking Abs, that this
killing does not involve the NKG2D ligand (Supplemental Fig. 1C).
Mouse anti–third-party CTLs spare normal naive B cells
We previously suggested that human anti–third-party CTLs are
unable to affect the engraftment of normal B cells, because the
human IgG secretion by normal B cells was not significantly af-
fected upon infusion of anti–third-party CTLs into a human/mouse
chimera (6). Yet, it could be argued that this indirect evidence
based on measuring Abs does not rule out possible elimination
of different types of normal B cells. Although it is difficult to
maintain human B cells through the time required for HLA typing
(needed in our routine assay to avoid cross-reactivity) in the ab-
sence of antigenic stimulation (27), this problem can be circum-
vented by using well-defined mouse strains. Thus, we attempted to
directly assess in vitro the reactivity of mouse-derived anti–third-
party CTLs against normal naive B cells. Interestingly, in contrast
to A20 lymphoma cell eradication, the anti–third-party CTLs did
not affect naive B cells (Fig. 2F).
LFA-1– and ICAM-1–mediated interactions are required for
lymphoma cell killing by anti–third-party CTLs
Initial experiments using Transwell filters suggested that the killing
of lymphoma cells is contact dependent (data not shown). As can
be seen in Supplemental Video 1, live-imaging experiments revealed
that anti–third-party CTLs bind to target lymphoma cells and rap-
idly form conjugates (on a time scale between seconds and a few
minutes). These conjugates remained stable and long lasting during
the follow-up period of 1 h. The conjugates were also monitored by
FACS in the presence of blocking Abs. Preincubation of the lym-
phoma cells with anti–ICAM-1 or preincubation of the CTLs with
anti–LFA-1 reduced CTL–lymphoma conjugate formation by two
thirds (Fig. 3A). In contrast, neutralization of the interactions of TCR
(CD3), CD8, MHC-I, CD102 (ICAM-2), or CD50 (ICAM-3), using
blocking Abs, did not affect the percentage of cells in lymphoma–
CTL conjugates (Fig. 3A). Importantly, LFA-1 neutralization on
CTLs, as well as ICAM-1, but not ICAM-3 (CD50), blockade on
lymphoma cell lines, prevented the killing of lymphoma cells fol-
lowing coculture with anti–third-party CTLs (Fig. 3B). Thus, in
the absence of TCR recognition, the LFA-1:ICAM-1 interaction is
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5. crucial for the killing of the tumor target cells. This critical role for
the LFA-1–ICAM-1 interaction was also verified in the mouse
model, using blocking Abs for LFA1 and ICAM1 (Supplemental
Fig. 2).
MHC-I expression is essential for lymphoma cell eradication
by anti–third-party CTLs
Given the importance of the CD8–MHC-I interaction demonstrated
in mice (4, 28, 29), we sought to evaluate whether a role for this
interaction also exists in the killing of lymphoma cells by human
anti–third-party CTLs. Masking of a2/a3 domains of MHC-I on
target B cells by neutralizing Ab significantly impaired the anti–
third-party CTL-mediated killing. Thus, killing of target lym-
phoma cells was decreased from 50 6 4.9% to 18 6 1.0% when
anti HLA-ABC Ab was added to the incubation with anti–third-
party CTLs (Fig. 4A, HLA-ABC). A similar decrease in killing
was observed upon the addition of anti-CD8 Ab, suggesting that
CD8 binding to the MHC-I C region is required for lymphoma cell
killing. Inhibition of CD8 binding to the MHC-I C region simi-
larly abrogated the killing of primary PBLs obtained from lym-
phoma patients (Fig. 4B). Blocking of CD8 molecules using
blocking Abs inhibited the killing in the mouse setting as well
(Supplemental Fig. 2).
To further validate the role of MHC-I signaling in lymphoma
cell killing, we used the Daudi (Burkitt’s lymphoma) cell line,
which exhibits markedly reduced surface MHC-I expression due
to a deficiency in b2m expression (16). The cytotoxic activity of
anti–third-party CTLs against Daudi cells was compared with
Daudi cells with reconstituted MHC-I surface expression, ach-
ieved by overexpression of transfected b2m. As depicted in Fig.
4C, the efficiency of B2m and BL-44 cell killing was nearly
identical, with killing of 56 6 0.5 and 62.7 6 3.2% of the lym-
phoma cells, respectively, at a 1:5 ratio of lymphoma cells/anti–
third-party CTLs. In contrast, killing of MHC-I–deficient Daudi
cells did not exceed 20% (Fig. 4C). These data implicated func-
tional MHC-I expression on lymphoma cells in the mechanism of
anti–third-party CTL-mediated killing.
Despite the impaired sensitivity to killing displayed by the
MHC-I–deficient Daudi cells, conjugate formation was not af-
fected, because MHC-I–expressing B2m and MHC-I2
Daudi cells
displayed comparable ability to form conjugates (18.8 6 3.5% and
14.2 6 4.0%, respectively; Fig. 4D). This finding, which is con-
sistent with our demonstration that anti-HLA class I Ab did not
block conjugate formation (Fig. 3A), strongly suggested that
MHC-I is not required for the initial interactions between B lym-
phoma and anti–third-party CTL cells, but it is critical for the in-
duction of apoptosis.
Finally, to further evaluate the importance of MHC-I expression
for the eradication of lymphoma target cells by anti–third-party
CTLs in vivo, we used adoptive-transfer experiments in NOD-
SCID mice. As can be seen in Fig. 5, following transplantation
into recipient mice, CTLs efficiently killed B2m cells. Thus, al-
though only 10.83% of the B2m cells survived in the presence
of a 4-fold excess of anti–third-party CTLs, Daudi cells lacking
functional MHC-I were resistant to killing by the same CTL lines,
and 86.87% of the Daudi cells survived under the same conditions.
FIGURE 2. The killing of tumor B cell lines by anti–third-party CTLs is
TCR independent and does not use typical death molecules involved in
alloreactivity. A, H.My2 C1R HLA-A2 wt transfectant and H.My2 C1R
HLA-A2 K66A mutant transfectant cell lines were incubated for 48 h with
or without anti–third-party CTLs at a 1:5 ratio in favor of anti–third-party
CTLs. The killing of the cells was analyzed by FACS, as described in
Materials and Methods. The percentage of killing was calculated as de-
scribed in Materials and Methods (mean 6 SD of four independent
experiments in triplicates). B, The killing of lymphoma cells in the murine
setting is dose dependent and not mediated by alloreactivity; calcein AM-
prelabeled murine A20 lymphoma cells (BALB/c origin) were incubated
with or without allogeneic (C57BL/6) or F1 (CB6-H2bd
)-derived anti–
third-party CTLs for 24 h in the indicated CTL/lymphoma cell ratios.
Numbers of viable calcein AM+
Annexin V2
lymphoma cells were de-
termined by flow cytometry (mean 6 SD of at least three independent
experiments, each in triplicate). C, The killing of murine lymphoma cells
by anti–third-party CTLs is mediated by apoptosis. Calcein AM-prelabeled
A20 lymphoma cells were incubated for 16 h with or without a 5-fold
excess of allogeneic (C57BL/6), F1 (CB6), or syngeneic (BALB/c) derived
anti–third-party CTLs and were then examined using flow cytometry for
calcein AM+
Annexin V+
expression (mean 6 SD of at least three in-
dependent experiments, each in triplicate). D, The killing of lymphoma
cells by anti–third-party CTLs is not mediated by the typical killing
molecules of CTLs. Calcein AM-prelabeled murine A20 lymphoma cells
were incubated for 16 h with or without C57BL/6 wt-, Prf2/2
-, Gld2/2
-
(FasL KO), Trail2/2
-, or TNF2/2
-derived anti–third-party CTLs at a 5:1
CTL/lymphoma cell ratio. Numbers of viable cells were determined by
flow cytometry (mean 6 SD of at least three independent experiments,
each in triplicate). E, The killing of A20 lymphoma cells by anti–third-
party CTLs is TCR independent. Calcein AM-prelabeled murine A20
lymphoma cells were incubated with or without OT-1/Rag2/2
(transgenic
TCR)- or F1 (CB6)-derived anti–third-party CTLs for 16 h (37˚C in 5%
CO2) at a 5:1 CTL/lymphoma cell ratio. Numbers of viable cells were
determined by flow cytometry (mean 6 SD of two experiments each in
triplicate). F, Anti–third-party CTLs kill lymphoma cells but spare normal
naive B cells. Naive B cells harvested from BALB/c mice by magnetic
beads or A20 cells were incubated for 16 h with syngeneic anti–third-party
CTLs at a 5:1 CTL/B cell ratio. At the end of the culture, the number of
viable CD19+
cells (B cells) was determined by flow cytometry. Results
shown represent average 6 SD of triplicates in one representative exper-
iment out of three performed, each in triplicates. *p , 0.05, changes in the
percentage of Annexin V+
live lymphoma cells versus A20-only group;
**p , 0.01, Student t test.
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6. Collectively, these data demonstrated that MHC-I expression is
required for lymphoma cell eradication by anti–third-party CTLs.
Discussion
We recently demonstrated that autologous or allogeneic anti–third-
party CTLs can be used to eradicate pathological cells from B-
CLL patients (6). In the current study, we demonstrated that anti–
third-party CTLs also exhibited significant killing of B cell non-
Hodgkin’s lymphoma (B-NHL) cells. By using blocking Abs and
mutated cell lines, we elucidated the initial molecular events that
trigger this cytotoxic activity. Our data strongly suggested that
unlike the classical killing of cognate targets by CTLs, the de-
scribed killing of B cell malignant cells by anti–third-party CTLs
is TCR independent.
This intriguing attribute of anti–third-party CTLs is supported
by several findings. First, the lymphoma cells are killed efficiently
both by allogeneic and autologous anti–third-party CTLs. Second,
TCR-independent killing is directly demonstrated by using ma-
lignant cells (H.My C1R) expressing mutated HLA-A2 at a posi-
tion critical for TCR recognition (position K66) (24). Although
this mutation disrupts recognition of peptide–MHC-I complexes
by the TCR, these HLA-A2–mutated cells were depleted at a
similar rate to that of H.My C1R cells expressing wt HLA-A2,
when cocultured with anti–third-party CTLs. Finally, the most
definitive demonstration that the mechanism is TCR independent
is afforded by the mouse studies in which anti–third-party CTLs
derived from OT1/Rag2/2
mice exhibited comparable killing of
A20 lymphoma cells to that found for anti–third-party CTLs of
(host 3 donor)F1 origin. Thus, CTLs expressing a transgenic TCR
directed against an irrelevant Ag still demonstrated significant
killing of lymphoma cells.
In previous studies investigating the veto mechanism and the
killing of CTL precursors by anti–third-party CTLs, both FasL–Fas
interaction (4) and perforin (5) secretion were shown to play
a role. Interestingly, in contrast to these veto mechanisms, the
killing modality of malignant B cells is mediated through a dif-
ferent mechanism. Granule-independent killing is indicated in the
human setting by testing for BLT esterase and further supported in
the mouse model by using anti–third-party CTLs from perforin
KO mice. FasL-independent killing is shown in human CTLs by
using blocking Abs and in the mouse model by demonstrating that
CTLs from FasL-mutated gld mice do not exhibit significantly
reduced killing of A20 lymphoma cells.
FIGURE 3. LFA-1:ICAM-1–dependent interactions are essential for B lymphoma target cell killing by anti–third-party CTLs. A, DHE-prelabeled BL-44
or Granta519 cells were incubated (+Ab) or not (no Ab) for 15 min with 10 or 20 mg/ml of anti-CD54 or 20 mg/ml of anti-CD102, CD50, or HLA-ABC Ab.
Calcein AM-prelabeled anti–third-party CTLs were incubated or not with 10 or 20 mg/ml of anti-CD11a, anti-CD8, or anti-CD3 Ab. Lymphoma cells and
CTLs were combined at a 1:1 ratio, and conjugate formation was analyzed by FACS, as described in Materials and Methods. An allogeneic line of DHE-
labeled anti–third-party CTLs was used as a nonrelevant target (T cells) to exclude the possibility of nonconjugate-coincident events. Data are presented as
fold change relative to the percentage of cells in double-positive clusters formed in the absence of Ab (mean 6 SD of three to six independent experiments,
each in triplicate). *p , 0.001, compared with no Ab. Representative FACS scatter graphs of conjugates formed in the absence (no Ab) or presence of LFA-
1–neutralizing Ab (+Ab) are shown (right panels). B, BL-44 or Granta519 cells (2 3 105
) were incubated for 36 h in the absence or presence of a 5-fold
excess of anti–third-party CTLs with or without 10 mg/ml of anti-CD11a, CD54, or CD50. Data are shown as mean 6 SD (n = 10 for anti–third-party CTL
lines). *p , 0.05, **p , 0.01, changes in the killing percentage versus samples cultured in the absence of Ab.
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7. Interestingly, the involvement of other killing mechanisms
generally used by CTLs in inducing apoptosis, such as TNF,
TRAIL, or NKG2D, were ruled out indirectly in the human setting
by using blocking Abs and more directly in the mouse studies by
using CTLs derived from TNF or Trail KO mice or by neutralizing
Ab for NKG2D. Furthermore, we showed that the level of gd T cell
depletion in our CTL preparation ruled out the potential in-
volvement of these cells.
Indeed, our results suggested that anti–third-party CTLs induce
apoptosis in B cell lymphoma through a novel mechanism. Ini-
tially, using Transwell filters, we found that the killing of lym-
phoma cells by anti–third-party CTLs is contact mediated. More-
over, evaluation of conjugate formation by FACS, as well as by
live video microscopy, revealed that these interactions resulted in
rapid conjugate formation. Ab blockade showed that conjugates
are largely mediated by nonspecific adhesion molecules of the
integrin family, namely, LFA-1 molecules on the anti–third-party
CTLs and ICAM-1 molecules on the lymphoma cells. Our results
are consistent with earlier studies indicating that adhesion ring
junctions, composed of high surface densities of ICAM-1 and
LFA-1 or peripheral supramolecular activation complexes, can be
formed between CD8 T cells and target cells in the absence of
TCR engagement (30, 31). ICAM-1 is the most potent ligand of
LFA-1 in cell-adhesion assays (32), and activation of lymphocytes
leads to an increase in ICAM-1 expression (33). However, despite
the fact that ICAM-1–LFA-1 interactions are essential for long-
lasting (∼1 h) conjugate formation between CTLs and lymphoma
cells, these interactions alone are not sufficient to induce caspase-
mediated apoptosis of lymphoma cells. Thus, further engagement
of MHC-I on the lymphoma cells by the CD8 molecules on the
CTLs is required to achieve killing following the initial binding.
This was first demonstrated using blocking Abs directed against
the a3/a2 domain of MHC-I or against CD8, both of which
inhibited the killing, but not the formation, of cell conjugates.
However, more definitive evidence for the central role of MHC-I
was provided by comparing Daudi cells lacking MHC-I surface
expression and a Daudi transfectant re-expressing MHC-I. In the
absence of TCR-mediated signaling, the engagement of the MHC-I
C region by the CTL CD8 molecules led to the initiation of ap-
optosis. However, considering that ICAM-1 is expressed on many
cell types, it is unlikely that it could afford, together with HLA
expression, sufficient conditions for apoptosis induction. Thus,
although we showed that, in the absence of these interactions
apoptosis will not ensue, other internal-signaling pathways, es-
pecially those regulating apoptosis inhibitors, probably underlie the
FIGURE 6. Proposed model for the killing of lymphoma cells by anti–
third-party CTLs. 1, Initially, fast-forming adhesion interactions are me-
diated by ICAM-1 molecules on the lymphoma cell and by LFA-1 mole-
cules on the CTL. 2, In the absence of TCR engagement, following the
initial binding, the a3 domain in the MHC-I on the lymphoma cell
becomes engaged by the CD8 molecule on the CTL. 3, Apoptotic signaling
is induced and mediated through caspases 3 and 8, leading to cell death.
FIGURE 4. Anti–third-party CTLs reactivity against lymphoma cells
in vitro is impaired in the absence of functional MHC-I. A, BL-44 or
Granta519 line cells were incubated for 36 h with or without a 5-fold excess
of anti–third-party CTLs in the presence or absence of 10–20 mg/ml anti
MHC-I (HLA-ABC) or anti-CD8 Ab. Data are shown as mean 6 SD of 10
independent experiments in triplicates. *p , 0.05, **p , 0.01, changes in
the killing percentage compared with samples cultured in the absence of Ab.
B, Primary lymphoma cells were incubated as in A, with or without anti–
third-party CTLs from the same healthy donors in the presence or absence of
anti–MHC-I Ab in two regimens; in the 20 mg 3 2, the second dose was
added 18 h after the initiation of culture. Data are from two individual
experiments using cells from different patients. *p , 0.01, changes in the
average killing percentage of cells in the presence of anti–MHC-I versus
values obtained in the absence of Ab. C, Daudi, BL-44, or B2m cells were
incubated for 36 h in the absence or presence of anti–third-party CTLs at
the indicated ratios. The number of live cells was determined as in Fig. 1
(mean 6 SD of four to six independent experiments, each in multiplicates).
*p , 0.01, differences, at each time point, in the killing percentage of Daudi
cells versus B2m cells. D, Conjugate-formation capacity of Daudi and B2m
cells with anti–third-party CTLs was assayed as in Fig. 3A. Data are shown
as mean 6 SD (n = 5 CTL lines).
FIGURE 5. Anti–third-party CTLs reactivity against lymphoma cells is
impaired in the absence of functional MHC-I in vivo. Detection of B2m or
Daudi cells in the peritoneum of NOD/SCID mice 5 d postinjection in the
absence (2) or presence (+) of a 4-fold excess of anti–third-party CTLs.
Data from individual mice (circles) and the means of three independent
experiments (anti–third-party CTL lines) are shown as the percentage of
live cells compared with target cells incubated in the absence of CTLs
(100%). *p , 0.01, changes in the percentage of live cells versus samples
in the absence of anti–third-party CTLs. ns, nonsignificant.
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8. predisposition of a B cell tumor to induction of apoptosis upon
engagement of its MHC with the CD8 on the CTL.
MHC-I clustering was previously implicated in apoptosis in-
duction in several cell types (10, 34). Several early studies sug-
gested that engagement of the a3 domain of the MHC-I by Abs
induces apoptosis in resting or in activated B cells (35, 36). In
addition, Pedersen et al. (12) observed that cross-linking of MHC-
I molecules on B cell lymphoma cells led to mobilization of in-
tracellular Ca2+
ions. Hence, it was suggested that apoptosis in-
duction via MHC-I could be mediated by protein tyrosine kinases.
Similarly, Yang et al. (37, 38) discovered that recruiting the
MHC-I molecules to the lipid rafts by specific b2M mAbs induced
apoptosis of hematological malignant cells while sparing normal
cells. This apoptosis was shown to be mediated via activation of
JNK signaling. Thus, further studies are warranted to evaluate
the possibility that the CTL CD8 molecules induce clustering of
MHC-I molecules on the malignant cell, resulting in their re-
cruitment into lipid rafts. Likewise, the signal-transduction path-
way that follows the MHC-I engagement by the CTLs requires
further elucidation. Such signaling might not be mediated directly
through the cytoplasmic tail of MHC-I, because it was shown that
this tail lacks phosphorylation sites (14, 15, 39). Furthermore, it
was demonstrated that Ab-induced cross-linking of MHC-I on
Jurkat cells expressing a truncated MHC-I, lacking the cytoplas-
mic tail, evoked the same increase in the intracellular Ca2+
ions
concentration as did that in Jurkat cells expressing native MHC-I
molecules (14).
Collectively, our data suggested the following sequence of events
for apoptosis induction in B cell lymphoma by anti–third-party
CTLs (Fig. 6). First, nonspecific, yet long-lasting, adhesive in-
teractions are formed between anti–third-party CTLs and target
cells. These interactions are mediated by LFA-1 molecules on
the CTLs and ICAM-1 molecules on the lymphoma cells. Next,
CD8 molecules on the CTLs engage the a3 domain of the MHC-I
on the lymphoma cells. Upon MHC-I–CD8 engagement, a signal
transduction is initiated, which is likely the result of recruitment
of signal molecules adjacent to the intracellular tail of the MHC-I
molecule and not the tail itself (14, 15, 34). These, as-yet-
undefined signals lead to cell death by apoptosis.
In addition to the novel role of MHC–CD8 interaction in the
mechanism of action underlying the killing by anti–third-party
CTLs, our results extend the potential therapeutic application of
CTLs, previously shown to eradicate B-CLL (6), to the treatment
of other forms of B-NHL. In particular, the use of these cells is
attractive in the context of hematopoietic stem cell transplantation.
The use of hematopoietic stem cell transplantation for tolerance
induction as a prelude to cell therapy with donor T cells is being
investigated by several groups, and major efforts are currently
directed at reducing transplant-related mortality by eliminating the
risk for GVH disease and by reducing the toxicity of the condi-
tioning protocol. T cell-depleted BM transplants were shown to
adequately eliminate GVH disease. However, even in leukemia
patients undergoing supralethal conditioning, mismatched T cell-
depleted BM transplants are vigorously rejected. This barrier can
be overcome through the modulatory activity of CD34+
cells,
which are endowed with veto activity, by the use of megadose
stem cell transplants (40, 41). Nevertheless, the number of human
CD34 cells that can be harvested is not likely to be sufficient to
overcome rejection under RIC. To address this challenge, the use
of anti–third-party veto CTLs was initially proposed to provide
additional veto activity and to enhance engraftment of purified
CD34+
stem cells in patients treated with RIC (2). We previously
suggested, based on indirect evidence, that these anti–third-party
CTLs might not negatively affect normal allogeneic B and T cells
(5). Sparing of normal B cells is now directly demonstrated by
showing that anti–third-party CTLs do not adversely affect naive
B cells in vitro, whereas they kill malignant B-NHL cells both
in vitro and in vivo. This selective killing is of great clinical sig-
nificance, because it will spare the naive B cell population and will
not delay humoral immune reconstitution against new Ags (42,
43). Thus, anti–third-party CTLs could be highly attractive for use
in B-NHL patients, because, in addition to their prominent toler-
izing activity demonstrated in mouse models, they might further
improve the clinical outcome by virtue of their GVL reactivity.
Alternatively, our finding that autologous anti–third-party CTLs
are as effective as their allogeneic counterparts offers another
option for cell therapy in the context of autologous transplan-
tation. Although autologous transplants have lower transplant-
related mortality (44), allogeneic transplants exhibit increased
overall survival due to the enhanced GVL reactivity mediated by
adoptively transferred, as well as newly formed, T cells (45–47).
Further studies to evaluate whether the autologous anti–third-party
CTLs could effectively eradicate residual disease and, thereby,
enhance overall survival compared with allogeneic anti–third-
party CTLs in the context of “megadose” purified CD34 cell
transplants is warranted.
Disclosures
The authors have no financial conflicts of interest.
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2014 TCR-INDEPENDENT KILLING OF LYMPHOMA CELLS BY CTLs
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