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F(ab')2 Prevents Graft-Versus-Host Disease by Selectively Depleting Donor T Cells Activated by Recipient Alloantigens1
*
Fred Hutchinson Cancer Research Center, Seattle, WA 98109; and
Department of Medicine, University of Washington, Seattle, WA 98195
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Abs do not
activate naive T cells but induce apoptosis of Ag-activated, cycling T
cells. Here, we report that treatment with anti-CD3
F(ab')2 can selectively induce apoptosis of donor T cells
that recognize a recipient alloantigen, thereby preventing
graft-vs-host disease initiated by a TCR-transgenic T cell population.
The selective elimination of Ag-activated T cells by non-FcR-binding
anti-CD3
Abs could serve as an ideal strategy to prevent
graft-vs-host disease and allograft rejection or to treat autoimmune
disorders. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6 (B6) and (BALB/c x B6)F1 (CB6F1), and founders of OT-I mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Founders of 2C transgenic mice were provided by Dr. Dennis Y. Loh (Nippon Roche Research Center, Kamakura-shi, Japan). 2C, OT-I, and (2C x B6.Ly5.1)F1 mice were bred at the Fred Hutchinson Cancer Research Center (Seattle, WA).
T cell purification and transplantation
Our protocol for T cell purification and transplantation has been described in detail (3, 4). Briefly, CD8+ T cells were purified by positive selection with a magnetic cell separation system (Miltenyi Biotec, Auburn, CA). The purity of CD8+ cells used for transplantation ranged from 95 to 99%. B6 or CB6F1 recipient mice were exposed to 750 cGy at 20 cGy/min, a dose that is immunosuppressive but not lethal for these strains of mice. Purified CD8+ cells were suspended in PBS and injected via the tail vein into 8- to 10-wk-old irradiated recipients within 24 h after irradiation. The number of injected donor T cells was 6–10 x 106/recipient, but in any given experiment, equal numbers of cells were transplanted into each recipient.
Abs and peptides
Anti-CD3 Fos is a genetically engineered
F(ab')2-like anti-murine CD3 mAb (2, 5). For simplicity, the term "anti-CD3
F(ab')2 " is used in this paper to indicate
anti-CD3 Fos. Anti-human CD3 Fos was used as an irrelevant
control Ab for all the experiments, except one in which PBS was used as
solvent control. The antigenic peptide for 2C cells is SIYRYYGL, and
the antigenic peptide for OT-I cells is SIINFEKL (6, 7).
The control peptide was SIIRFEKL. All three peptides are restricted to
H-2Kb and were synthesized by United Biochemical
Research (Seattle, WA).
Carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling and immunofluorescence analysis
Previously described methods were used for fluorescent labeling
of donor T cells (8). Briefly, purified
CD8+ cells were suspended in PBS at 2 x
107/ml and prewarmed to 37°C. An equal volume
of prewarmed 10 µM CFSE (Molecular Probes, Eugene, OR) in PBS was
added to the cell suspension, and cells were incubated for 10 min at
37°C. The labeled cells were washed twice with cold medium containing
10% FCS. Two- or three-color flow cytometry was performed to measure
the expression of surface molecules and intracellular cytokines
according to standard techniques. Analysis was performed by using a
FACScan and CellQuest software (Becton Dickinson, San Jose, CA).
FITC-labeled anti-TCR V
2, biotin-labeled anti-TCR V
5,
Cy-Chrome-labeled anti-CD8, PE-labeled anti-B220, annexin V,
anti-IFN-
, anti-IL-4, anti-IL-5, and anti-IL-10 were
purchased from PharMingen (San Diego, CA). FITC-labeled 1B2 and
biotin-labeled anti-Ly5.1 mAbs were prepared in our laboratory.
Biotinylated Abs were detected with streptavidin-FITC (Caltag,
Burlingame, CA) or streptavidin-PE (Southern Biotechnologies,
Birmingham, AL).
Cytotoxicity
To measure direct cytotoxic activity, splenocytes freshly isolated from the recipients were used as effectors against peptide-pulsed 51Cr-labeled EL-4 (H2b) cells. For induced cytotoxic activity, splenocytes from the recipients were restimulated with antigenic peptides and then used as effectors. Splenocytes were added to U-bottom 96-well plates with 2 x 103 targets/well to achieve the E:T ratios indicated. The plates were centrifuged at 200 x g for 2–3 min and then incubated at 37°C for 4–5 h. Chromium release was measured with a Topcount (Packard, Meridian, CT), and the percent cytotoxicity was calculated as [(experimental release - spontaneous release)/(maximal release - spontaneous release)] x 100%. A lytic unit was arbitrarily defined as the number of T cells required to yield 15% specific lysis of target cells.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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F(ab')2 in vitro
To test the effect of anti-CD3
F(ab')2 on Ag-stimulated T cells, naive 2C
TCR-transgenic CD8 cells were incubated in vitro with irradiated, T
cell-depleted splenocytes as APCs from syngeneic C57BL/6 (B6,
Ld-) or allogeneic (BALB/c x
B6)F1 (CB6F1,
Ld+) mice. 2C cells proliferated in response to
Ld+ but not Ld- APCs, and
the rate of proliferation peaked on day 3. Anti-CD3
F(ab')2 did not induce T cell proliferation in
the absence of Ag but inhibited Ag-induced T cell proliferation in a
dose-dependent manner (Fig. 1
A). In the culture with
syngeneic APCs, anti-CD3 F(ab')2 did not
affect T cell survival, indicating that this Ab did not deplete
unstimulated T cells. In contrast, increasing concentrations of
anti-CD3 F(ab')2 caused a progressive
decrease in the number of surviving 2C cells stimulated with allogeneic
APCs (Fig. 1B). In the presence of anti-CD3
F(ab')2 at concentrations 
200 ng/ml, the number
of viable 2C cells was lower in the culture with allogeneic APCs than
with syngeneic APCs (p < 0.01, t
test). These results demonstrated that anti-CD3
F(ab')2 depleted Ag-activated CD8 T cells in
vitro.
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F(ab')2 prevents GVHD induced by 2C cells
We reasoned that anti-CD3 F(ab')2
could induce Ag-specific tolerance by depleting alloantigen-reactive T
cells in vivo. To test this hypothesis, we transplanted 2C cells into
sublethally irradiated CB6F1 recipients and
tested for GVHD manifested by depletion of recipient B cells
(3). Recipients were treated with PBS or anti-CD3
F(ab')2 at 5, 20, or 80 µg/dose every other day
for 10 doses or at 80 or 160 µg/dose every other day for 5 doses. In
recipients treated with PBS, 2C cells expanded and prevented recovery
of recipient B cells during the first 6 wk after transplantation.
Treatment with anti-CD3 F(ab')2 at 80
µg/dose inhibited T cell expansion, and the recovery of recipient B
cells was identical with that in irradiation controls (Fig. 2). Treatment with anti-CD3
F(ab')2 at lower doses was less effective in
preventing expansion of donor 2C cells and protecting recipient B cells
(data not shown). Treatment with anti-CD3
F(ab')2 at 80 or 160 µg every other day for
five doses initially inhibited expansion of 2C cells and prevented
destruction of recipient B cells. By day 20 after transplantation,
however, the numbers of 2C cells and B cells were indistinguishable
between the recipients treated with anti-CD3
F(ab')2 or PBS for five doses (data not shown).
These results show that anti-CD3 F(ab')2
was able to prevent GVHD-associated B cell depletion when it was
administered at 80 µg every other day for 10 doses.
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To test the hypothesis that anti-CD3
F(ab')2 might deplete alloreactive T cells while
preserving T cells with other specificities in vivo, we used
OVA-specific CD8+ T cells from OT-I
TCR-transgenic mice as a control for the effects of anti-CD3
treatment on donor cells that do not recognize recipient alloantigens.
We distinguish OT-I cells by staining with mAbs specific for TCR V2
and V5.1/2 which comprise the transgenic TCR (7). In
irradiated mice that did not receive 2C or OT-I cells, splenic B cells
recovered by day 30 (Fig. 3A).
In mice transplanted with 2C and OT-I cells, treatment with
anti-CD3 F(ab')2 decreased the number of
2C cells on day 30 compared with treatment with control Ab
(p < 0.001) but had no effect on the numbers
of OT-I cells (p > 0.05) (Fig. 3B).
Recipients treated with anti-CD3 F(ab')2
and irradiation controls had comparable numbers of B cells
(p > 0.05), 10-fold higher than the number in
recipients treated with control Ab (Fig. 3B). These results
demonstrated that treatment with anti-CD3
F(ab')2 reduced the number of 2C T cells, spared
OT-I T cells, and prevented GVHD-related B lymphopenia. Therefore, the
effects of anti-CD3 F(ab')2 were selective
for alloantigen-activated T cells.
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F(ab')2
Treatment with anti-CD3 F(ab')2 could
reduce the number of 2C cells in CB6F1 recipients
either by increasing cell death or by decreasing cell proliferation. To
distinguish between these two possibilities, we analyzed T cell
proliferation and apoptosis by CFSE labeling and annexin V staining,
respectively. In irradiated B6 recipients in which alloantigen is
absent, 2C cells displayed homeostatic proliferation with an average
doubling time of 52 h (Fig. 4A). Treatment with
anti-CD3 F(ab')2 did not block homeostatic
proliferation and did not induce T cell apoptosis. In
CB6F1 recipients in which alloantigen is present,
2C cells were activated and replicated with an average doubling time of
15 h (Fig. 4A). Treatment with anti-CD3
F(ab')2 did not block 2C cell proliferation in
response to alloantigen but increased apoptotic cell death, especially
after the fifth cell division (Fig. 4, B and C).
In B6 recipients, the spleen contained 1.3 x
105 viable 2C cells after treatment with
anti-CD3 F(ab')2 compared with 1.9 x
105 after treatment with control Ab. In
CB6F1 recipients, the spleen contained 0.07
x 105 viable 2C cells after treatment with
anti-CD3 F(ab')2 compared with 1.1 x
105 after treatment with control Ab. These
results confirmed that treatment with anti-CD3
F(ab')2 selectively decreased the number of donor
cells that recognize recipient alloantigen through induction of T cell
apoptosis and not through inhibition of T cell proliferation.
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F(ab')2 selectively
inhibits CTL activity of donor T cells that recognize recipient
alloantigens
To test the function of donor T cells after treatment, we examined
the CTL activity of 2C cells or OT-I cells generated in vivo.
Splenocytes isolated from each recipient were tested for effector
function by a direct cytotoxicity assay without prior restimulation ex
vivo (Fig. 5, 
, 
). For convenience
in comparing results, we defined 1 LU as the number of effectors needed
to lyse 15% of the targets. The spleen in recipients treated with
anti-CD3 F(ab')2 contained 2 U 2C and 6 U
OT-I lytic activity, respectively, compared with 18 U 2C and 3 U OT-I
in recipients treated with control Ab (Fig. 5). These results confirmed
that the inhibitory effect of anti-CD3
F(ab')2 was selective for 2C CTL. We also
examined the ability of residual donor T cells to generate CTL activity
after ex vivo restimulation by APC loaded with antigenic peptides (Fig. 5, •, 
). In recipients previously treated with anti-CD3
F(ab')2, the spleen contained 50 U
peptide-induced 2C lytic activity, compared with 273 U in recipients
previously treated with control Ab (Fig. 5A). The 5- to
9-fold decrease in 2C LU with prior anti-CD3
F(ab')2 treatment corresponds to the decreased
frequency of 2C cells detected by staining with the clonotype-specific
Ab 1B2 (Fig. 3
). Peptide-induced OT-I lytic activity was not decreased
by prior anti-CD3 F(ab')2 treatment (Fig. 5B). We conclude that treatment with anti-CD3
F(ab')2 selectively depleted the donor CTL that
had been activated by recipient alloantigen.
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F(ab')2 does not change
the cytokine profile of donor T cells that recognize recipient
alloantigen in vivo
To test the cytokine profile of residual 2C cells in
CB6F1 recipients, we stained cells for expression
of IFN-, IL-4, IL-5, and IL-10. The cytokine profile of 2C cells was
similar in recipients treated with anti-CD3
F(ab')2 or PBS (Fig. 6). These results indicate that treatment
with anti-CD3 F(ab')2 did not change the T
cell cytokine profile in vivo.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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F(ab')2. Treatment with
anti-CD3 F(ab')2 was associated with
decreased numbers of donor 2C cells in blood (Fig. 2), spleen (Fig. 3),
and lymph nodes (data not shown). Donor 2C cells replicated at an
equal, if not faster, rate in recipients treated with anti-CD3
F(ab')2 (Fig. 4). Furthermore, anti-CD3
F(ab')2 increased the proportion of apoptotic 2C
cells at each cell division in allogeneic recipients (Fig. 4). These
results show that treatment with anti-CD3
F(ab')2 decreased the number of 2C T cells in
allogeneic recipients by causing apoptosis and not by inhibiting
proliferation. Depletion of T cells by anti-CD3
F(ab')2 has been reported by others (9, 10), but our results emphasize that anti-CD3
F(ab')2 selectively depleted donor T cells that
recognized recipient alloantigen and had little or no effect on donor T
cells that did not recognize alloantigen (Fig. 3). Consistent with this
hypothesis, the Ab inhibited CTL activity triggered by recipient
alloantigen without inhibiting CTL activity triggered by control
Ag.
Treatment with anti-CD3 F(ab')2 caused
90–99% depletion of alloreactive T cells during the first 3–14 days
of treatment (Fig. 5 and data not shown), as compared with 60–70% at
7–10 days after stopping treatment (Fig. 4 and data not shown). We
suspect that the residual alloreactive T cell population was able to
expand in vivo after stopping treatment with anti-CD3
F(ab')2. Because complete depletion of 2C after
treatment with anti-CD3 F(ab')2 has never
been seen in our experiments, it is not obvious how incomplete T cell
depletion can prevent GVHD. GVHD is a dynamic process of injury and
recovery; treatment with anti-CD3 F(ab')2
might decrease the number of alloreactive donor T cells below a
threshold required to cause manifestations of GVHD. It is also possible
that a mechanism of regulatory suppression developed during treatment
with anti-CD3 F(ab')2. We speculate that
apoptotic 2C cells might trigger an immunosuppressive effect (16, 17) or that a subset of residual 2C cells might acquire
regulatory control over the function of 2C cells that survive after
treatment with anti-CD3 F(ab')2
(18).
We have studied whether induction of anergy contributes to
immunosuppression after in vivo administration of anti-CD3
F(ab')2. The lower level of 2C-specific CTL
activity after anti-CD3 F(ab')2 treatment
corresponded to the decreased frequency of 2C cells. Thus, it is clear
that donor 2C T cells surviving after anti-CD3 treatment retained
their cytotoxic function (Fig. 5). These residual 2C cells were unable
to proliferate in response to Ag restimulation ex vivo even in the
presence of IL-2 (data not shown), but we suspect that they were able
to proliferate in response to recipient alloantigen in vivo after
stopping treatment. Our results (Fig. 6) demonstrated that skewing of T
cells toward a Th2-like phenotype was unlikely to explain the
immunosuppressive effect mediated by treatment with anti-CD3
F(ab')2 in vivo.
In summary, we have shown that the in vivo administration of
anti-CD3 F(ab')2 prevents GVHD by
selectively depleting donor T cells that recognize recipient
alloantigen. Our study provides both a rationale and a practical
clinical strategy for Ag-specific therapy of T cell-mediated
diseases.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Claudio Anasetti, Human Immunogenetics Program, Mail Box: D2-100, Fred Hutchinson Cancer Research Center, Seattle, WA 98109.
3 Abbreviation used in this paper: GVHD, graft-vs-host disease.
Received for publication October 18, 2000. Accepted for publication February 22, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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F(ab')2 in antigen receptor transgenic murine T cells activated by specific peptide. J. Immunol. 157:3420.[Abstract]
F(ab')2 fragments inhibit T cell expansion in vivo during graft-versus-host disease or the primary immune response to nominal antigen. J. Immunol. 159:5821.[Abstract]
F(ab')2. Transplantation 69:104.[Medline]
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