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TRANSPLANTATION
From ITERT-INSERM U437, Nantes; INSERM U119, Marseille;
and Sangstat Europe, Lyon, France.
Immunosuppression with B7 antagonists might have 2 opposite
effects: reducing T-cell costimulation through CD28 but also preventing CTLA-4 from transmitting its negative regulatory signal. We therefore hypothesized that a selective blockade of CD28 might be qualitatively different from blocking B7. It was previously reported that CD28 modulation prolongs allograft survival in the rat and reverses induction of experimental autoimmune encephalomyelitis in mice. However, whether CD28 or B7 blockade results in similar
immunosuppression on alloimmune and self-restricted responses to
soluble antigens has not yet been investigated. Here, we addressed this
issue in vitro with antagonist anti-CD28 Fab fragments and in vivo
using the modulating anti-rat JJ319 monoclonal antibody. As in the
inhibition of B7 with CTLA4 immunoglobulin, anti-CD28 Fab fragments
inhibited allogenic T-cell proliferation in mixed cultures. In vivo
modulation of CD28 blocked the expansion of alloreactive T cells and
promoted their apoptosis. In contrast, selective blockade of CD28 did
not modify T-cell proliferative responses and antibody production to
soluble antigens, whereas blocking B7 with CTLA4 immunoglobulin did.
Our data show that blocking CD28, while leaving CTLA4-B7 interactions
undisturbed, inhibits alloreactive CD4+ T-cell expansion
but does not modify the response to nominal antigens presented in
the context of a self-major histocompatibility complex. That B7
engagement is needed for self-restricted responses whereas
engagement of CD28 is not essential adds to the suggestion that another
unidentified ligand of B7 might deliver a costimulatory signal in the
absence of CD28.
(Blood. 2002;99:2228-2234) Complete activation of T cells requires the
delivery of at least 2 signals by antigen-presenting cells (APCs). The
first is provided through T-cell receptor (TCR) engagement of
the peptide-major histocompatibility complex (MHC) on the APC, and the
second is provided by the interaction of accessory receptors on T cells with their ligands on APCs and is referred to as costimulation. The
CD28 molecule on T cells binds to B7-1 or B7-2 on APCs, thereby providing signal 2 for the initiation of naive T-cell responses. In
conjunction with a TCR stimulus, it allows high-level interleukin-2 (IL-2) production and provides an essential survival signal for T
cells,1 thereby preventing apoptosis or the induction of anergy that may occur in response to signal 1 alone. CD28 regulates T-cell cycle entry and progression through the G1 phase in an IL-2-independent manner, resulting in the activation of
cyclins.2 After activation, T cells up-regulate the
surface expression of CTLA-4, a homologue of CD28, that binds the same
ligands with higher affinity and serves as a negative regulator of
T-cell activation. In addition to its competition with CD28 for binding
to B7 molecules, CTLA-4 recruits phosphatases that dephosphorylate
activated molecules in the CD3 complex, and it reduces nuclear
translocation of RelA, which leads to a suppression of the production
of multiple cytokines produced by Th1 and Th2 cells, including IL-2,
interferon (IFN)- Despite evidence that CTLA-4 is a regulator of T-cell responses, some
observations indicate that CTLA-4 may be of relatively minor importance
for the down-regulation of primary immune responses.5 In
addition, though lymphoproliferative disease is observed in CTLA-4-deficient mice, arguing for a role of CTLA-4 in inhibiting T-cell expansion, it is not observed in chimeric recipients when wild-type and CTLA-4 In vitro studies with murine and human T-cell clones have indicated
that antigenic stimulation in the absence of costimulation signaling
through B7/CD28 causes the T cells to enter a state of long-term
hyporesponsiveness, known as anergy, connected to the failure to induce
IL-2 gene transcription.16 Immunosuppression with B7
antagonists, however, might have 2 opposite effects Here, we added to these studies a comparison between the effects of
antagonizing B7 versus CD28 on immune responses to nominal antigens and
to direct allorecognition. Using either Fab fragments from an
anti-human CD28 antibody25 in vitro or the modulating anti-rat CD28 JJ319 antibody21 in vivo, we demonstrated
that though, as expected, B7 inhibition reduces direct pathway
activation of the growth of T cells subjected to allostimulation and
immune responses to soluble antigens, selective inhibition of CD28 does not modify the responses of T cells activated against
self-MHC-restricted determinants. No modification of antibody
responses, antigen-triggered cell proliferation, or cytokine induction
was noted. In contrast, it was effective in reducing the proliferation
of T cells stimulated through the direct allorecognition pathway in
vitro and in vivo. These data indicate that blocking CD28 results in a
qualitatively different effect from blocking B7. In addition, our
results match recent data suggesting additional yet unidentified
ligands for B7 on T cells.
Animals and cells
Antibodies and reagents
Proliferation assays Human peripheral blood mononuclear cells (PBMCs) or CD4+ T cells, or rat T cells were seeded in triplicate at a final concentration of 105 cells/well in RPMI 1640 (Sigma), glutamine, nonessential amino acids, sodium pyruvate, and antibiotics, and 10% heat-inactivated autologous serum (plus 5 × 10 5 M 2- ME for rat cells) and were cultured for
5 days with 2 × 104 allogeneic dendritic cells (for rat)
or 105 PBMCs (human) irradiated at 30 Gy.
Proliferation was measured by 1 µCi (37 Bq) 3H
incorporation after 16-hour incubation. Results
expressed are cpm per triplicate or proliferation indexes: (cpm
MLR cpm stimulating cells alone)/(cpm responding cells alone).
Immunizations and self-restricted responses LEW.1A rats were immunized in the footpad with 50 µg KLH in complete Freund adjuvant. Serum and draining lymph nodes were harvested 11 days after injection. Triplicate 0.2-mL cultures of draining lymph node cells (3 × 105/well) were cultured in RPMI 1640 (Sigma) supplemented with 2-ME, glutamine, nonessential amino acids, sodium pyruvate, antibiotics, and 0 to 50 µg/mL KLH. Cultures were incubated for 72 hours and were pulsed with [3H] thymidine (1.0 µCi (37 Bq)/10µL per well) for the last 16 hours. Data are presented as stimulation indexes (average counts per minute in cultures with stimulus divided by those in control cultures without stimulus) or cpm (cpm = mean cpm in cultures with
stimulus mean cpm in control cultures without stimulus).
Assay for DNP- and KLH-specific antibody production Serum levels of anti-dinytrophenol (DNP) and anti-KLH IgG1, IgG2a, IgG2b, IgG2c, and IgM subclasses were determined by enzyme-linked immunosorbent assay, as described previously.26 Briefly, 96-well microtiter plates (Immulon, Chantilly, VA) were coated with bovine globulin-DNP or with KLH at
5 µg/mL. After blocking the plates with gelatin (Sigma) and
incubating serum serial dilutions in 0.1% phosphate-buffered
saline-Tween 20 for 2 hours at 37°C, plates were developed using
horseradish peroxidase-conjugated mouse monoclonal anti-rat
individual isotypes (IgG1, MARG1-2; IgG2a, MARG2a-1; IgG2b, MARG2b-3;
IgG2c, MARG2c-5; IgM, MARM-4; generous gift from H. Bazin, University
of Louvain, Belgium). The concentration of anti-DNP antibody was
estimated using standard curves generated by incubating the DNP-coated
plates with anti-DNP rat monoclonal antibody (IgG1, LODNP-1; IgG2a,
LODNP-16; IgG2b, LODNP11; IgM, LODNP34; generous gift from H. Bazin).
Titer comparisons were performed in the linear range of the assay.
Titers of anti-DNP IgG2c responses and anti-KLH antibody responses are
represented as the serum dilution needed to reach an OD405
of 0.5 in the assay.
Graft-versus-host disease induction and monitoring of cell proliferation in vivo A 1:1 mixture of mononuclear cells from rat spleen and mesenteric lymph nodes was resuspended at 5 × 107 cells/mL in RPMI containing 5 µM 5-6-carboxy fluorescein diacetate succinimidyl ester (CFDASE; Molecular Probes, Eugene, OR) and was incubated at 37°C for 20 minutes, as previously described.27 Unbound CFDASE or the deacetylated form, CFSE, was quenched by 3 washes in complete medium. CFSE-labeled cells (2 × 108) were injected intravenously in congeneic or allogeneic rats treated 24 hours earlier with whole-body sublethal irradiation of 8 Gy using a cobalt Co 60 source. Recipients were killed after 3 days for analysis of spleen and mesenteric lymph node cells. Sensitivity of FL1 detection was adjusted so that residual recipient T cells could be distinguished from CFSE-labeled cells that divided up to 9 times.Flow cytometry At the time of harvest, spleen and mesenteric lymph node cells were washed in cold phosphate-buffered saline containing 1% bovine serum albumin and 0.1% sodium azide. At least 2 × 105 cells per sample were stained with biotin-conjugated monoclonal antibody specific for CD4 (W3/25), CD8 (ox8), CD25 (ox39), and CD62L (ox85) or with biotin-conjugated Annexin-V (Immunotech, Marseille, France) followed by PC5-conjugated streptavidin (Immunotech). Cytometry was performed on a Becton Dickinson FACSCalibur single-laser cytometer using standard Cell Quest acquisition analysis software, and fluorescence compensation was achieved using the appropriate single-fluorochrome-labeled samples. Twenty thousand events in the lymphocyte acquisition gate were collected. Analysis of cell division (CFSE-fluorescence profile) was restricted to the CD4+ subset of CFSE-labeled cells.Quantitation of cytokine mRNA Cells (4 × 106) from mixed lymphocyte reactions were collected after 5 days of culture. Cells were lysed in guanidinium isothiocyanate, and total RNA was extracted as previously described.28 Total RNA (10 µg) was treated with DNAse (Promega, Charbonnières, France) and was retro-transcribed using 100 µM oligo dT (Gibco BRL), 10 mM dithiothreitol (Promega), 0.5 mM each of 4 dNTPs, 40 U RNAse OUT, and 200 U M-MLV reverse transcriptase. Quantitative polymerase chain reaction was carried out in a 7700 Sequence Detector TaqMan (Perkin Elmer) using the following primers : IL-2, 5'-AAACACAGCTACAACTGGAGCA-3' and 5'-GCTGATTAAGTCCCTGGGTCTT-3'; IFN-, 5'-TGTCCAACGCAAAGCAATACA-3' and 5'-TTCGCTTCCCTGTTTTAGCTG-3';
IL-4, 5'-CACCGAGTTGACCGTAACAGAC-3' and 5'-TACTCTGGTTGGCTTCCTTCAC-3';
IL-5, 5'-TGTATGCCATCCCCACAGAA-3' and 5'-TTTCCACAGTACCCCCTTGC-3'; HPRT,
5'-ATTGACACTGGCAAAACAATGCA-3' and 5'-TCCAACACTTCGTGGGGTCC-3'.
Selective blockade of CD28 reduces T-cell proliferation in primary and secondary mixed lymphocyte reaction To compare a selective blockade of CD28 with a blockade of B7, we first assayed the effect of monovalent Fab fragments from a blocking anti-CD28 antibody with that of CTLA4 immunoglobulin on mixed lymphocyte reactions (MLRs) using pure T cells as proliferating responders. The direct presentation pathway accounts for 90% of the measured proliferation in this setting.29 Allogenic proliferation of PBMCs or CD4+ T cells was typically reduced by 60% in the presence of optimal doses of anti-CD28 Fab fragments (10 µg/mL), whereas CTLA4 immunoglobulin (5 µg/mL) reduced proliferation to 75% in human MLR and nearly completely in rat MLR (Figure 1A-B). Proliferation was reduced to the same extent with anti-CD86 antibodies (data not shown). A similar reduction of proliferation was obtained ex vivo using CD4+CD28 T cells from rats treated with the
modulating JJ319 anti-CD28 antibody, stimulated with allogenic APCs
(Figure 1C-D). The observed reduction of T-cell proliferation with CD28
or B7 blockade was paralleled by a reduction in the production of
IFN- and IL-2 in human MLR (Figure 2).
Up-regulation of messenger RNA for IL-4 and IL-5 was also inhibited by
anti-CD28 Fab and CTLA4 immunoglobulin. In secondary stimulation in
vitro with donor APC, the addition of anti-CD28 Fab or CTLA4
immunoglobulin reduced proliferation by 40% to 50% (Figure
3), showing that the inhibition of CD28 or B7 also blocked the proliferation of primed alloimmune cells to a
similar extent.
Down-modulation of CD28 in vivo inhibits donor T-cell expansion and development of graft-versus-host disease To investigate whether the reduced proliferation of allogeneic T cells by selective CD28 blockade we observed in vitro could also be evidenced in vivo, we followed up the fate and function of T cells that recognize recipient alloantigens after in vivo transfer. We used a model in which CFSE-labeled spleen cells from LEW.1A rats were transplanted into sublethally irradiated allogenic LEW.1W recipients. In this model, donor CD4+ T cells engraft, expand, and represent more than 95% of proliferating cells, resulting in lethal GVHD within 13 days. Sublethally irradiated recipients were treated with modulating anti-CD28 mAb, CTLA4 immunoglobulin, or mouse IgG. On day 3, CFSE+ donor cells in recipient spleen and mesenteric lymph nodes were analyzed for CFSE fluorescence intensity, expression of CD25, and binding of Annexin V on donor CD4+ T cells. Fifty percent of CFSE-labeled CD4+ T cells recovered after 3 days either had undergone no division (ie, their fluorescence was maximal) or had undertaken a single mitosis. Because halving of CFSE intensity was also found in syngenic controls on 50% of the cells, we considered one division as not related to allogenic stimulation. Homeostatic growth (more than one division) of syngenic CFSE-labeled CD4+ T cells in irradiated recipients was significant (10% or 15% of transplanted cells in mesenteric lymph nodes or spleen, respectively) but was weaker than the proliferating allogenic CD4+ T cells, which represented 35% or 40% of mononuclear cells in MLN (not shown) and spleen (Figure 4A), respectively. Most (80%) of proliferating allogenic T cells underwent between 6 and 9 mitoses after 3 days.
Treatment of recipients with CTLA4 immunoglobulin reduced recovery of
proliferating cells by 60% (10% of allospecific growth instead of
25% in control IgG-treated animals). Treatment of recipients with
modulating anti-CD28 mAb was even more efficient and reduced recovery
of proliferating cells by more than 80% (5% of allospecific growth
instead of 25% in control IgG-treated animals) (Figure 4B). CD25 was
expressed on 12% of host (CFSE In contrast to alloreactive responses, antibody responses to nominal antigens were not modified by selective down-modulation of CD28 To compare the effect obtained using B7 antagonists with CD28 antagonists on antibody responses, we immunized rats intraperitoneally with 10 µg DNP-OVA without adjuvant or with 50 µg KLH subcutaneously in CFA and treated them with control IgG, CTLA4 immunoglobulin, or modulating anti-CD28 mAb every 2 days from day 0 until day 6 after immunization. Blood sampling at day 6 of animals treated with anti-CD28 mAb confirmed that CD28 had been fully modulated on PBMCs. Animals treated in parallel and killed on day 6 confirmed that a full modulation of CD28 expression was also obtained on spleen and draining lymph node T cells. Treatment with CTLA4 immunoglobulin inhibited antibody responses of all subclasses measured after 2 weeks, regardless of whether adjuvant was used in immunizations. In sharp contrast, no reduction, in any subclass, could be observed after treatment with anti-CD28 mAb, even when antigen was given without adjuvant (Figure 5A-B). Thus, the antibody response in the rat with fully modulated CD28 was normal.
Proliferative responses to KLH immunization are unmodified by specific CD28 down-modulation Draining lymph node cells from rats immunized with KLH in Complete Freund Adjuvant (CFA) were restimulated in vitro with KLH, and we measured proliferation after 3 days. Cells from rats treated with control IgG or with anti-CD28 mAb fully responded to this secondary stimulation, whereas proliferation was reduced by 75% if CTLA4 immunoglobulin was infused (Figure 6A). Moreover, the addition of anti-CD28 Fab fragments in vitro in KLH-restimulated cells from immunized untreated animals did not reduce proliferation, whereas CTLA4 immunoglobulin was effective in this setting (Figure 6B). Thus, proliferative responses to soluble antigens can be inhibited by B7 blockade but not by CD28 blockade.
In this study, we investigated the role of CD28 in T-cell responses to alloantigens elicited by the "direct" presentation pathway and to nominal antigens presented in the context of self-MHC molecules. We compared the blockade of CD28 (using Fab fragments from antagonist antibodies or modulating mAb) with that of B7 (using CTLA4 immunoglobulin). Fab fragments do not induce signal transduction, yet they remain capable of efficient blockade of B7 binding.19 We show first that anti-CD28 Fab reduced the proliferation of CD4+ T cells in vitro elicited by the direct pathway of allorecognition. CD28 and B7 blockade reduced the production of Th1 and Th2 cytokines in primary MLR. A reduction in TH2 development after CD28 blockade is consistent with the role of CD28 in increasing IL-4 receptor sensitivity, which drastically promotes Th2 generation through the IL-4-mediated pathway.30 CD28 and B7 blockade were also effective in secondary stimulation (Figure 3), indicating that naive and primed alloreactive cells equally necessitate costimulation through CD28. In vivo, in a model of GVHD in which mitosis of alloreactive cells can be monitored, the administration of modulating anti-CD28 mAb is effective in the inhibition of allogenic T-cell proliferation. Proliferating cells had an activated phenotype: they expressed high levels of CD25 (and low levels of CD62L, not shown), regardless of whether modulating anti-CD28 mAb was given. Administration of CTLA4 immunoglobulin consistently reduced by 40% the percentage of CD25+ cells in proliferating cells (and increased in proportion the percentage of CD62L+ cells, not shown). That B7 blockade and not CD28 blockade inhibits activation of alloreactive T cells, whereas CD28 blockade inhibits proliferation more strongly, suggests that B7 delivers an activation signal through a molecule other than CD28. Reports showing that CTLA-4 can promote costimulation in vivo7,8 suggest that a signal through CTLA-4 might promote activation in GVHD. Alternatively, a B7 ligand other than CTLA-4 and CD28, the existence of which was functionally demonstrated recently, might promote activation. In contrast with data previously reported in mice,31 proliferating rat T cells did not commit to apoptotic cell death in untreated, allogenic, irradiated recipients (Figure 4D). In anti-CD28-treated rats, however, it was clear that T cells became increasingly susceptible to apoptosis with each cycle of cell division. Above 8 divisions this susceptibility to apoptosis decreased and was correlated with a higher recovery of alloreactive cells. This commitment to apoptotic cell death with proliferation was not present with B7 blockade (Figure 4), suggesting that the difference lies in the unaltered CTLA-4-B7 interaction when anti-CD28 mAb, but not CTLA4 immunoglobulin, is used, which might promote the inhibition of cell division and cell death. Maximal apoptotic events were measured only after 7 divisions, which refers to reports showing that cross-linking of CTLA-4 on resting CD4+ T cells blocks transition from G0 to G1 and induces the antiapoptotic factor Bcl-xL,17 whereas cross-linking CTLA-4 on activated CD4+ T cells induces Fas-independent cell death.18 However because of the complex and yet not fully clarified mechanisms of action of CTLA-4 in vivo that have been reported so far, ranging from costimulation7,8 to inhibition,32 a role for a free CTLA-4-B7 interaction that may inhibit allogeneic T-cell growth in our GVHD model remains speculative. Collectively, these data show that blocking costimulation through CD28 or B7 results in a reduction of alloreactive T-cell proliferation but that the mechanisms are different. Soluble antigens are processed by self-APCs that expose antigenic peptides in association with self-MHC class 2 molecules. APCs then signal CD4+ T cells that proliferate and provide help for effector T-cell responses and antibody responses. Molecules stimulated by B7 appear essential for the development of the immune response to self-restricted presentation because CTLA4 immunoglobulin completely blocks the induction of T-dependent alloantibodies.15 Here, we found that a selective blockade of CD28 in the rat has no effect on self-restricted responses measured in vivo or ex vivo, whereas B7 blockade with CTLA4 immunoglobulin clearly inhibits this type of response in primary or secondary stimulation. The strength of immunization does not appear to determine whether CD28 is required as similar results were obtained in a weak (low dose without adjuvant) and in a strong (high dose in CFA) immunization protocol. This suggests at least that B7 molecules are instrumental in self-restricted responses to soluble antigens, independent of CD28, and it would mean that either B7-CTLA-4 interaction is paradoxically required for self-restricted responses to occur or that another, yet unidentified, ligand for B7 on T cells is necessary. The first hypothesis is refuted by numerous studies evidencing suppression of the production of multiple cytokines3 and cyclins33 after engagement of CTLA-4, which is clearly identified as a CD28 antagonist. However, other reports have shown that CTLA-4 can actually costimulate T-cell clonal expansion and the production of cytolytic T cells.7,8 The second hypothesis pertains to the data by Mandelbrot et al34 showing that B7-dependent costimulation can be evidenced in CD28-CTLA4 double-knockout mice. Autoreactive T cells that serve as a pool of potentially
pathogenic cells are positively selected in the thymus for their low
affinity for peptides bound to self-MHC molecules expressed on cortical
epithelial cells. Their low affinity is likely caused by the
physico-chemical characteristics of the TCR- In conclusion, our data demonstrate that CD28 costimulation is required for full responses of CD4+ T cells to stimulation by allogenic APCs but that it is not required for responses to soluble antigens, whereas both types of responses require B7 molecules. Selectively blocking CD28 might be relevant for clinical transplantation because the direct presentation pathway of alloantigens can lead to acute rejection, but the induction of immune tolerance is thought to require a self-restricted response and a free CTLA-4-B7 interaction.
We thank C. Usal and S. Iyer for excellent technical assistance, G. Boulday and J. M. Heslan for technical advice, and R. Peach for purified CTLA4 immunoglobulin. We also thank P. Vusiau for biosensor analysis, F. Nisol and H. Bazin for anti-rat immunoglobulin mAb and DNP-OVA, and M. Brunet for irradiations.
Submitted July 6, 2001; accepted October 25, 2001.
Supported in part by the Fondation Progreffe and by the postgenome program, grant 109, of the French government (MENRT).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Jean-Paul Soulillou, ITERT, INSERM U437, CHU Hotel Dieu, 30 Bld Jean Monnet, 44093 Nantes, France; e-mail address: bvanhove{at}nantes.inserm.fr.
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  A.-S. Dugast, T. Haudebourg, F. Coulon, M. Heslan, F. Haspot, N. Poirier, R. Vuillefroy de Silly, C. Usal, H. Smit, B. Martinet, et al. Myeloid-Derived Suppressor Cells Accumulate in Kidney Allograft Tolerance and Specifically Suppress Effector T Cell Expansion J. Immunol., June 15, 2008; 180(12): 7898 - 7906. [Abstract] [Full Text] [PDF]
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C. Guillonneau, C. Seveno, A.-S. Dugast, X.-L. Li, K. Renaudin, F. Haspot, C. Usal, J. Veziers, I. Anegon, and B. Vanhove Anti-CD28 Antibodies Modify Regulatory Mechanisms and Reinforce Tolerance in CD40Ig-Treated Heart Allograft Recipients J. Immunol., December 15, 2007; 179(12): 8164 - 8171. [Abstract] [Full Text] [PDF]
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 B. Vanhove, G. Laflamme, F. Coulon, M. Mougin, P. Vusio, F. Haspot, J. Tiollier, and J.-P. Soulillou Selective blockade of CD28 and not CTLA-4 with a single-chain Fv-{alpha}1-antitrypsin fusion antibody Blood, July 15, 2003; 102(2): 564 - 570. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
reducing T-cell
costimulation through CD28 and preventing CTLA-4 from transmitting its
negative regulatory signal. Therefore, we hypothesized that selective
blockade of CD28 could be more effective at inducing immunosuppression
and anergy than B7 blockade. Expected molecular consequences of the
selective blockade of CD28 with an unmodified CTLA-4-B7 interaction
would be a block in the transition from G0 to G1 and an induction of
the antiapoptotic factor Bcl-xL in resting CD4+ T
cells,
) Recipients treated with control IgG.
(
) Recipients treated with CTLA4 immunoglobulin. (
) Recipients
treated with anti-CD28. Nb indicates the number of mitosis according to
fluorescence intensity halving.
CDR3 region, which
tends to be cross-reactive and "sticky."