Cytotoxic T Lymphocyte Trafficking and Survival in an Augmented Fibrin Matrix Carrier
et al. (2012) Cytotoxic T Lymphocyte Trafficking and Survival in an Augmented Fibrin Matrix Carrier. PLoS
ONE 7(4): e34652. doi:10.1371/journal.pone.0034652
Cytotoxic T Lymphocyte Trafficking and Survival in an Augmented Fibrin Matrix Carrier
Zhaoxia Zou 0
Erin Denny 0
Christine E. Brown 0
Michael C. Jensen 0
Gang Li 0
Tatsuhiro Fujii 0
Josh Neman 0
Rahul Jandial 0
Mike Chen 0
Paul Proost, University of Leuven, Rega Institute, Belgium
0 1 Division of Neurosurgery, City of Hope National Medical Center, Duarte, California, United States of America, 2 Department of Cancer Immunotherapeutics & Tumor Immunology, City of Hope National Medical Center , Duarte, California , United States of America
Cell-based therapies have intriguing potential for the treatment of a variety of neurological disorders. One such example is genetically engineered cytotoxic T lymphocytes (CTLs) that are being investigated in brain tumor clinical trials. The development of methods for CTL delivery is critical to their use in the laboratory and clinical setting. In our study, we determined whether CTLs can migrate through fibrin matrices and if their migration, survival, and function could be modulated by adding chemokines to the matrix. Our results indicated that CTLs can freely migrate through fibrin matrices. As expected, the addition of the monocyte chemotactic protein-1 (MCP-1), also known as chemokine C-C motif ligand 2 (CCL2), to the surrounding media increased egress of the CTLs out of the fibrin clot. Interleukin (IL) -2 and/or IL-15 embedded in the matrix enhanced T cell survival and further promoted T cell migration. The interleukin-13 receptor alpha 2 specific (IL-13R alpha2) T cells that traveled out of the fibrin clot retained the capacity to kill U251 glioma cells. In summary, CTLs can survive and migrate robustly in fibrin matrices. These processes can be influenced by modification of matrix constituents. We conclude that fibrin matrices may be suitable T cell carriers and can be used to facilitate understanding of T cell interaction with the surrounding microenvironment.
Adoptive immunotherapy using genetically engineered
cytotoxic T lymphocytes (CTLs) is being investigated as a potential
treatment for cancer in the central nervous system and elsewhere
[1,2]. Pilot clinical trials have been initiated to evaluate the
feasibility and safety of local-regional delivery of autologous
IL13zetakine redirected CTL clones in patients with recurrent
glioblastoma (GBM). These results indicate the adoptive transfer
of tumor-specific cytotoxic T cells to the tumor bed is a promising
strategy, though its clinical application may be limited due to the
shortcomings of previous and current delivery methods.
While systemic delivery is appealing for its convenience,
nonspecific targeting, high cell dosage requirements, and the presence
of the blood-brain barrier are formidable obstacles. In clinical
trials local delivery can be achieved using catheter-based delivery
systems. Catheter-based delivery systems, however, are associated
with risks such as infection and hemorrhage. Additionally,
catheter-based delivery systems used at our institution present
significant logistical challenges. First, injection results in the
formation of a pocket of T cells at the catheter tip because cell
sized particles cannot flow through the extracellular space. With
point source distribution, CTLs may have to seek and find distant
targets in the brain diminishing the chances of successful therapy.
Second, to attain an adequate dose, multiple injections are needed.
These multiple injections lengthen hospital stay creating more
cost, risk, and patient inconvenience. We hypothesized that T cells
could be delivered more efficiently by embedded them in a fibrin
matrix placed in the resection cavity during surgery. The fibrin
matrix is a commercially available (Baxter, Tisseel) biodegradable
adhesive obtained by a combination of human-derived fibrinogen
and thrombin, duplicating the last step of the coagulation cascade.
Tisseel is normally used in neurosurgery as a dural sealant and a
hemostatic agent. Studies have shown that the application of fibrin
glue to the brain is safe and well tolerated [3,4,5].
Our approach of using fibrin glue as a vehicle for CTLs has not
been previously described, though fibrin glue has been widely used
for delivery of a variety of cells [6,7,8,9,10,11,12,13,14,15]. Fibrin
matrices are employed not only as a cell carrier, but also as
suitable vectors for drugs and exogenous growth factors.
Therefore, there exists the potential to combine cell-based
therapies with modulating biological factors in the fibrin glue.
A prerequisite for use of fibrin matrices as a T cell delivery
vehicle is that the cells must be able to migrate out of the fibrin
clot. Additionally, these cells must survive the passage and remain
efficacious tumor killers. In this study, we demonstrated the ability
of T cells to robustly migrate through a bio-enhanced matrix while
maintaining potency against glioma cells.
Materials and Methods
Recombinant human interleukin (IL) -2 was purchased from
Cell signaling. Recombinant human IL-15 and monocyte
chemotactic protein-1 (MCP-1) were purchased from R&D
Systems (Minneapolis, MN). Each cytokine and chemokine was
reconstituted in PBS with 0.1% human serum albumin (PBS
HSA) and stored at 220uC until time of use. On the day of
experimentation, aliquots were diluted in RPMI media to the final
concentrations as indicated.
Cell lines and Cultures
In our experiments, firefly luciferase (ffl+) expressing
interleukin-13 receptor alpha 2 specific (IL-13R alpha2) CTLs cells were
cultured in RPMI 1640 (Invitrogen) with 10% heat-inactivated
fetal calf serum (FCS), 25 mmol/L HEPES, and 2 mmol/L
Lglutamine supplemented with 50 U/mL rhIL-2 (Chiron). U251
glioblastoma cells were grown in Dulbeccos modified Eagles
medium supplemented with 10% heat-inactivated FCS, 25 mmol/
L HEPES, and 2 mmol/L L-glutamine.
For conditioned medium, U251 cell lines were grown to 60
90% confluency and cells were then transferred to serum-free
medium. 24 and 48 hours after the transfer, the cell-free
supernatants were collected.
Fibrin Matrix (Tisseel) Formulation and Dissolution
The sealer protein component (fibrinogen-containing
component) was reconstituted in aprotinin solution (Baxter Healthcare),
and the thrombin component was reconstituted in 30 mM CaCl2.
Both solutions were diluted to the appropriate concentration using
Trisbuffered saline (TBS) and 30 mM CaCl2 in TBS for the sealer
protein and thrombin components, respectively. Formulation 1
was composed of 2.5 mg/ml fibrinogen and 2.5 U/ml thrombin.
Formulation 2 was composed of 5 mg/ml fibrinogen and 5 U/ml
thrombin. Finally, Formulation 3 was composed of 5 mg/ml
fibrinogen and 1 U/ml thrombin. In select experiments tissue
plasminogen activator (tPA) was used to dissolve the fibrin clots to
determine the number of remaining T cells. The CellTiter-Glo
Luminescent Cell Viability Assay (Promega) was performed as a
surrogate for cell count.
Incorporation of T Cells in Fibrin Matrix and Chemotaxis
T cell/fibrin clot constructs were prepared in the following
manner. Following harvest with trypsinEDTA, 100 ml of ffl+
IL13 zetakine CTLs (7.5*106 cells/mL) were mixed with 150 ml
sealer protein solution. 150 ml of thrombin solution was
subsequently added to the sealer proteincell solution and mixed by
tapping and tilting the plates. Then 400 ml of the combined
mixture was placed in 24-well 5 mm QCM Chemotaxis Cell
Migration Assay plates (Millipore). Plates were incubated in a 5%
CO2 incubator for 8, 24, or 48 hours. At each time point, the
number of T cells that migrated out of the clots from the upper
chamber to the lower chamber was assayed using the
CellTiterGlo Luminescent Cell Viability kit and measured using a
luminescence plate reader. The number of T cells that had
migrated out of the fibrin matrix was calculated according to the
standard curve; then the percent migration was calculated by
dividing the number of collected T cells by number of initial T
cells placed in the clot and multiplying the result by 100.
Cytotoxicity of IL-13 zetakine CTLs passage through the fibrin
matrix may adversely affect the ability of CTLs to kill tumor cells.
The potency of the CTLs was assayed in the following manner.
Effector cells were used 13 to 15 days after stimulation with
muromonab-CD3 (OKT3) and irradiated feeder cells. 7.56105
IL-13 zetakine CTLs per fibrin clot were plated into the upper
chamber of 24 well chemotaxis assay plates. The CTLs that had
migrated out were collected after 24 hours. U251 ffl+ target cells
(200,000 cells/ml) were co-cultured with increasing numbers of
CD8+ effector zetakine CTLs in a 5% CO2 incubator. After 4 or
24 hours of incubation, Dual-Glo Luciferase Assay Reagent was
added to each well of the 96-well plate. The plate was incubated
for 10 minutes at room temperature, and luminescence was read
from 8 mm above the bottom of the plate on the luminescence
Human Chemokine Levels in Glioma-Conditioned
Chemokine levels of MCP-1, C-C motif ligand 5 (CCL5), and
C-X-C motif ligand 10 (CXCL10) were determined using their
respective Human ELISA Kits (Invitrogen) as per the
manufacturers instructions. Briefly, samples, controls, MCP-1, CCL5 (data
not shown), or CXCL10 standards were incubated for 2 hours at
room temperature with a biotinylated Hu MCP-1, CCL5, or
CXCL10 Biotin Conjugate solution, and then incubated for
30 minutes at room temperature with Streptavidin-HRP Working
Solution. Absorbance detection of captured cytokines was
quantified by Optical Density using a Microplate Reader.
Stimulation of T Cells in the Fibrin Clots and Effect on
Survival and Migration
IL-2 and/or IL-15 were added to the fibrin matrix to investigate
whether T cell survival and migration could be enhanced. T cell
fibrin clots were prepared as described above. IL-2 (500 u/ml),
IL15 (100 ng/ml), or IL-2 + Il-15 were mixed into the fibrin clots.
The IL-2 and IL-15 concentrations were chosen based on previous
studies and our preliminary experiments . The CellTiter-Glo
ATP assay was performed to quantify CTL migration after
The means and standard deviations of the obtained data were
calculated. One-way analysis of variance (ANOVA) with post-hoc
testing was used for comparing means of three or more variables.
Statistical analysis was carried out using SAS software (SPSS
version 12.0.1) on a Microsoft Windows XP system. The threshold
for statistical significance was set at 0.05.
T Cell Migration Through Low Viscosity Fibrin Matrices
It is essential that CTLs are able to travel through the fibrin clot
to reach their target. We examined the effect of viscosity on T cell
migration using 6 formulations of varying thrombin and
fibrinogen concentrations. T cells that had exited to the
surrounding media were collected and quantified using an ATP
assay as a surrogate of cell count. The CellTiter-Glo ATP assay
showed a highly linear relationship between cell count and
luminescence (R2 = .98; data not shown).
Formulation 1 of lower fibrinogen and thrombin concentrations
was not only less viscous but also allowed the most T cells (25%) to
move out of the fibrin glue (p,0.01) (Figure 1). Formulation 2
(5.0 mg/ml fibrinogen and 5.0 U/ml thrombin) and Formulation
3 (5.0 mg/ml and 1.0 U/ml) allowed only 5% of T cells to migrate
through the clot. T cells were not capable of passaging through the
fibrin matrix when concentrations of fibrinogen and thrombin
exceeded 5.0 mg/ml and 1.0 U/ml respectively (data not shown).
At concentrations lower than those reported in Formulations 1,
the fibrin glue did not form a clot, and therefore these formulations
were not further analyzed.
Chemoattractants in the Surrounding Media Increase CTL
Migration Out of the Fibrin Clot
MCP-1 is a known T cell chemokine; its ability to penetrate into
fibrin matrices, however, was unknown. We indirectly examined if
MCP-1 could diffuse into the fibrin clot to promote CTL
trafficking. As shown in Figure 1, the addition of MCP-1 to the
media outside of the clot significantly increased CTL migration in
the three formulations shown (p,0.01).
In addition to secreting other CTL chemokines such as IL-8,
tumors can regulate activation of normal T cell expression and
secretion . The collective effect of these chemokines on T cell
trafficking from the fibrin clot was assessed (Figure 2). CTLs in the
fibrin matrices were exposed to standard culture media or
conditioned media obtained from culturing U251 glioblastoma
cells. There was significantly (p,0.05) more migration out of the
clot when glioma-conditioned media was used.
The fate of T cells remaining in the clot was also assessed
because it was unclear if those cells were trapped or had perished.
The clots were dissolved with tPA, and the cells were quantified
using an ATP assay (Figure 2). Regardless of the type of media, the
total number of cellsCTLs in or out of the clotwas slightly
over 30% of the initial cell count. This indicated that one limiting
factor was T cell demise within the fibrin matrix.
Chemokine Level in Glioma-Conditioned Medium
We demonstrated that there was significantly more migration
out of the clot when glioma-conditioned media was used
compared to standard culture media. To further investigate the
mechanism for the observed trafficking of T cells in
gliomaconditioned media, we measured chemokine levels. MCP-1 and
CXCL10 were detected at high levels in glioma-conditioned
supernatants compared to standard culture media (p,0.01)
(Figure 3). Thus, the observed migrational response in Figure 2
may be a consequence of chemokine-dependent directional
IL-2 and IL-15 Alone or in Combination Enhances T Cell
Survival and Promotes Trafficking
Cell survival after implantation is a major concern. To enhance
survival and potentiate trafficking, the cytokines IL-2 and IL-15,
alone or in combination, were added to the fibrin matrix. Prior to
this experiment, we confirmed that IL-2 and IL-15 caused
expansion and an increase in the diameter of CTLs, the later
reflective of T cell health (data not shown). Addition of IL-2
(500 u/ml), IL-15 (100 ng/ml), or IL-2+Il-15 into the fibrin clot
dramatically (p,0.01) increased T cell egression at multiple time
points (Figure 4). In contrast, the CTL migration in the untreated
group decreased to almost 0% after 96 hours. The most trafficking
was seen when IL-2 and IL-15 were combined; 65% of CTLs had
migrated out. These findings suggest the proliferative effect of the
cytokines is preserved in the fibrin matrix.
IL-13R alpha2 CTLs That Migrate Out of the Matrix
Remain Potent Against U251 Glioma Cells
Whether CTLs are still potent after migrating out of the fibrin
clot is unknown. U251 glioma cells expressing firefly luciferase
(U251 ffl+) were co-cultured with increasing numbers of
gliomaspecific primary IL-13 zetakine CTLs that had migrated out of the
fibrin matrix (Figure 5). Although U251 cells died in a time
dependent manner, glioma cell death was dose-dependent (in
terms of CTL to U251 ratio) only at a shorter treatment time
(4 hours). 24 hours after incubation U251 cells were almost
completely eradicated when cultured with a five fold greater
number of CTLs.
Adoptive immunotherapy is a theoretically elegant approach for
the treatment of brain tumors; however, the optimal method to
deliver T cells has not been developed. We hypothesized that in
lieu of the catheter-based delivery system, T cells can be
distributed by using a fibrin matrix carrier that is placed into the
resection cavity at the time of surgery. This method would confer
potential advantages. One is that the viscous nature of the
substance improves localization of the T cells to the tumor bed,
preventing washout into the subarachnoid space. Another
potential advantage is that catheter implantation is not required
and therefore certain risks are avoided. The most important
potential benefits relate to dose and distribution. A much larger
dose can be delivered. Additionally, bioactive reagents can be
added to the fibrin matrix to enhance the survival and function of
the T cells.
Our finding that CTLs can passage through low viscosity fibrin
matrices is consistent with studies reporting migration through
Tisseel of fibroblasts, chondrocytes, neutrophils and monocytes
[18,19,20,21]. The concentration of fibrinogen and thrombin that
we determined as optimal (2.5 mg/ml fibrinogen and 2.5 U/ml
thrombin) is similar to those reported in Cox et al.s study which
examined fibroblast migration through fibrin clots (517 mg/mL
fibrinogen and 1167 U/mL thrombin).
Though it was obvious that high concentrations of fibrinogen
were inhibitory, the active or passive mechanism(s) responsible for
CTL egress out of Tisseel have not been defined. In vivo, tumor
cells secrete chemoattractants such as MCP-1 which play an
Figure 3. Cytokine concentrations in glioma-conditioned media (GC-Media) and standard culture media (Media). Cytokine
concentrations were measured using Human ELISA Kits. (A) Increased MCP-1 concentrations in GC-Media (* p,0.0001, 24 h or 48 h vs. control). (B)
Increased CXCL10 concentrations in GC-Media (* p,0.0001, 24 h or 48 h vs. control). The data represents the means 6 SD of three independent
important role in the host antitumor immune response by
recruiting CD4+ and CD8+ cells [22,23,24]. It was uncertain if
CTL chemotactic factors could diffuse from the surrounding
media into the clot to promote migration. We have found that
glioma cell line U251 produces sufficient levels of MCP-1 to
chemoattract T cell migration through fibrin clots in vitro. Our
data indicated that MCP-1 containing or glioma-conditioned
mediathe later theoretically full of MCP-1 and other
chemokinessignificantly enhanced T cell migration suggesting that
targeting of remote tumor cells is possible.
We allow that factors other than MCP-1 can enhance T cell
migration. Although no other study has examined CTL migration
using a fibrin clot, Nishimura et al. have shown that chemokines
such as CXCL10, also referred to as interferon gamma-induced
protein 10 (IP-10), can play a critical role in the recruitment of Tc1
effector cells to the brain tumor site . Our present study
showed that CXCL10, in fact, was present in high concentrations
in glioma-conditioned media and could potentially promote CTL
migration out of the fibrin clot. In addition to the identity of
chemokines, other variables such as the amount or type of
chemokine-secreting residual tumor may be important.
Even under optimal circumstances, without additional aid only
about one-fourth of the CTLs were able to leave the fibrin matrix.
We observed that most had perished by days 36. Survival varied
depending on the initial health of the T cells which corresponded
to their status in the stimulation cycle. Of the T cells remaining in
the matrix, over two thirds of the initial cells were no longer viable.
Improving viability would be expected to increase migration due
to an enlarged pool of available cells.
Adoptively transferred antigen-specific CTLs are highly
dependent on exogenous cytokines such as IL-2, IL-7, IL-15 and IL-21
for their continued growth and survival . Systemic
administration of IL-2 has been used to enhance T cell expansion and
persistence in vivo . Administration of lowdose IL-15 has been
shown to promote the persistence of adoptively transferred
tumorspecific T cells in murine tumor models; however, the systemic
toxicity and the expansion of unwanted cells, including regulatory
T cells, limit the clinical value of this strategy [28,29]. We
demonstrated that IL-2 and IL-15 added to the fibrin clot
improved survival, possibly caused proliferation, and subsequently
increased T cell migration by 23 fold.
Migration to the target is insufficient if the T cells lose their
capacity to kill the tumor cells during passage. IL-13 zetakine
CTLs have been previously shown to recognize and kill
glioblastomas and medulloblastomas expressing IL-13R alpha2
[30,31]. In fact, IL-13 zetakine CTLs have been shown to
recognize and eliminate brain tumor stem-like initiating cells .
Our data indicated that IL-13 zetakine CTLs that had moved out
of the fibrin matrix killed U251 glioma cells in a time and dose
dependent fashion. The optimal dose required in vivo has yet to be
determined. However, the observed dose dependency suggests that
larger doses possible with fibrin matrix delivery would confer a
We have shown that robust migration can be achieved with IL-2
and IL-15, chemotaxis mechanisms are not impaired by the
matrix, and that CTLs that have passaged through the matrix
remain potent. The significant logistical and biological advantages
associated with this technique are promising but will need to be
confirmed in vivo and in future clinical trials.
We are indebted to Araceli Naranjo who provided invaluable assistance
with procurement of reagents for this study.
Conceived and designed the experiments: ZZ ED MC. Performed the
experiments: ZZ ED. Analyzed the data: GL JN RJ. Contributed reagents/
materials/analysis tools: CEB MCJ. Wrote the paper: ZZ TF RJ MC.
1. Kruse CA , Cepeda L , Owens B , Johnson SD , Stears J , et al. ( 1997 ) Treatment of recurrent glioma with intracavitary alloreactive cytotoxic T lymphocytes and interleukin-2 . Cancer Immunol Immunother 45 : 77 - 87 .
2. Rosenberg SA , Dudley ME ( 2009 ) Adoptive cell therapy for the treatment of patients with metastatic melanoma . Curr Opin Immunol 21 : 233 - 240 .
3. Ghulam Muhammad AK , Yoshimine T , Maruno M , Takemoto O , Hayakawa T ( 1997 ) Topical application of fibrin adhesive in the rat brain: effects on different cellular elements of the wound . Neurol Res 19 : 84 - 88 .
4. Kassam A , Nemoto E , Balzer J , Rao G , Welch WC , et al. ( 2004 ) Effects of Tisseel fibrin glue on the central nervous system of nonhuman primates . Ear Nose Throat J 83 : 246 - 248 , 250, 252 passim.
5. Lee KC , Park SK , Lee KS ( 1991 ) Neurosurgical application of fibrin adhesive . Yonsei Med J 32 : 53 - 57 .
6. Andrades P , Asiedu C , Rodriguez C , Goodwin J , Deckard LA , et al. ( 2007 ) Insulin secretion from pancreatic islets in fibrin glue clots at different fibrinogen and thrombin concentrations . Transplant Proc 39 : 1607 - 1608 .
7. Chen SJ , Chang CM , Tsai SK , Chang YL , Chou SJ , et al. ( 2010 ) Functional improvement of focal cerebral ischemia injury by subdural transplantation of induced pluripotent stem cells with fibrin glue . Stem Cells Dev 19 : 1757 - 1767 .
8. Christman KL , Vardanian AJ , Fang Q , Sievers RE , Fok HH , et al. ( 2004 ) Injectable fibrin scaffold improves cell transplant survival, reduces infarct expansion, and induces neovasculature formation in ischemic myocardium . J Am Coll Cardiol 44 : 654 - 660 .
9. Currie LJ , Sharpe JR , Martin R ( 2001 ) The use of fibrin glue in skin grafts and tissue-engineered skin replacements: a review . Plast Reconstr Surg 108 : 1713 - 1726 .
10. Falanga V , Iwamoto S , Chartier M , Yufit T , Butmarc J , et al. ( 2007 ) Autologous bone marrow-derived cultured mesenchymal stem cells delivered in a fibrin spray accelerate healing in murine and human cutaneous wounds . Tissue Eng 13 : 1299 - 1312 .
11. Hafez AT , Afshar K , Bagli DJ , Bahoric A , Aitken K , et al. ( 2005 ) Aerosol transfer of bladder urothelial and smooth muscle cells onto demucosalized colonic segments for porcine bladder augmentation in vivo: a 6-week experimental study . J Urol 174 : 1663 - 1667 ; discussion 1667 - 1668 .
12. Jung SN , Rhie JW , Kwon H , Jun YJ , Seo JW , et al. ( 2010 ) In vivo cartilage formation using chondrogenic-differentiated human adipose-derived mesenchymal stem cells mixed with fibrin glue . J Craniofac Surg 21 : 468 - 472 .
13. Rainer C , Wechselberger G , Bauer T , Neumeister MW , Lille S , et al. ( 2001 ) Transplantation of tracheal epithelial cells onto a prefabricated capsule pouch with fibrin glue as a delivery vehicle . J Thorac Cardiovasc Surg 121 : 1187 - 1193 .
14. Wechselberger G , Russell RC , Neumeister MW , Schoeller T , Piza-Katzer H , et al. ( 2002 ) Successful transplantation of three tissue-engineered cell types using capsule induction technique and fibrin glue as a delivery vehicle . Plast Reconstr Surg 110 : 123 - 129 .
15. Wechselberger G , Schoeller T , Stenzl A , Ninkovic M , Lille S , et al. ( 1998 ) Fibrin glue as a delivery vehicle for autologous urothelial cell transplantation onto a prefabricated pouch . J Urol 160 : 583 - 586 .
16. Kinter AL , Godbout EJ , McNally JP , Sereti I , Roby GA , et al. ( 2008 ) The common gamma-chain cytokines IL-2, IL-7, IL-15, and IL-21 induce the expression of programmed death-1 and its ligands . J Immunol 181 : 6738 - 6746 .
17. Brown CE , Vishwanath RP , Aguilar B , Starr R , Najbauer J , et al. ( 2007 ) Tumor-derived chemokine MCP-1/CCL2 is sufficient for mediating tumor tropism of adoptively transferred T cells . J Immunol 179 : 3332 - 3341 .
18. Cox S , Cole M , Tawil B ( 2004 ) Behavior of human dermal fibroblasts in threedimensional fibrin clots: dependence on fibrinogen and thrombin concentration . Tissue Eng 10 : 942 - 954 .
19. Gille J , Meisner U , Ehlers EM , Muller A , Russlies M , et al. ( 2005 ) Migration pattern, morphology and viability of cells suspended in or sealed with fibrin glue: a histomorphologic study . Tissue Cell 37 : 339 - 348 .
20. Hanson AJ , Quinn MT ( 2002 ) Effect of fibrin sealant composition on human neutrophil chemotaxis . J Biomed Mater Res 61 : 474 - 481 .
21. Mana M , Cole M , Cox S , Tawil B ( 2006 ) Human U937 monocyte behavior and protein expression on various formulations of three-dimensional fibrin clots . Wound Repair Regen 14 : 72 - 80 .
22. Carr MW , Roth SJ , Luther E , Rose SS , Springer TA ( 1994 ) Monocyte chemoattractant protein 1 acts as a T-lymphocyte chemoattractant . Proc Natl Acad Sci U S A 91 : 3652 - 3656 .
23. Roth SJ , Carr MW , Springer TA ( 1995 ) C-C chemokines, but not the C-X-C chemokines interleukin-8 and interferon-gamma inducible protein-10, stimulate transendothelial chemotaxis of T lymphocytes . Eur J Immunol 25 : 3482 - 3488 .
24. Zheng M , Sun G , Cai S , Mueller R , Mrowietz U ( 1999 ) Significant reduction of T-cell chemotaxis to MCP-1 in patients with primary and metastatic melanoma . Chin Med J (Engl) 112 : 493 - 496 .
25. Nishimura F , Dusak JE , Eguchi J , Zhu X , Gambotto A , et al. ( 2006 ) Adoptive transfer of type 1 CTL mediates effective anti-central nervous system tumor response: critical roles of IFN-inducible protein-10 . Cancer Res 66 : 4478 - 4487 .
26. Sallusto F , Lenig D , Forster R , Lipp M , Lanzavecchia A ( 1999 ) Two subsets of memory T lymphocytes with distinct homing potentials and effector functions . Nature 401 : 708 - 712 .
27. Dudley ME , Wunderlich J , Nishimura MI , Yu D , Yang JC , et al. ( 2001 ) Adoptive transfer of cloned melanoma-reactive T lymphocytes for the treatment of patients with metastatic melanoma . J Immunother 24 : 363 - 373 .
28. Blanco-Jerez C , Plaza JF , Masjuan J , Orensanz LM , Alvarez-Cermeno JC ( 2002 ) Increased levels of IL-15 mRNA in relapsing-remitting multiple sclerosis attacks . J Neuroimmunol 128 : 90 - 94 .
29. Roychowdhury S , May KF , Jr., Tzou KS , Lin T , Bhatt D , et al. ( 2004 ) Failed adoptive immunotherapy with tumor-specific T cells: reversal with low-dose interleukin 15 but not low-dose interleukin 2 . Cancer Res 64 : 8062 - 8067 .
30. Kahlon KS , Brown C , Cooper LJ , Raubitschek A , Forman SJ , et al. ( 2004 ) Specific recognition and killing of glioblastoma multiforme by interleukin 13- zetakine redirected cytolytic T cells . Cancer Res 64 : 9160 - 9166 .
31. Stastny MJ , Brown CE , Ruel C , Jensen MC ( 2007 ) Medulloblastomas expressing IL13Ralpha2 are targets for IL13-zetakine+ cytolytic T cells . J Pediatr Hematol Oncol 29 : 669 - 677 .
32. Brown CE , Starr R , Martinez C , Aguilar B , D'Apuzzo M , et al. ( 2009 ) Recognition and killing of brain tumor stem-like initiating cells by CD8+ cytolytic T cells . Cancer Res 69 : 8886 - 8893 .