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*
Surgery Branch, Division of Clinical Sciences, National Cancer Institute, and
Department of Transfusion Medicine, Clinical Center, National Institutes of Health, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Vaccines are aimed at enhancing the systemic immune competence of the host. However, achievement of substantial antitumor effector functions is complex and includes localization and persistence of active Ag-specific T cells at the tumor site (8, 9). Studies with 111In-labeled TIL have shown that their localization is necessary, although not sufficient, for the observation of tumor regression (10). Thus, it is reasonable to propose that tumor-host interactions could be better considered in the organ targeted by treatment.
Tumor-host interactions have traditionally been studied in excised surgical specimens by morphological or molecular methods to estimate protein or gene expression or to identify genetic abnormalities (9). These methods, however, do not allow the assessment of functional interactions between various cell populations infiltrating the tumors. The results of functional studies with freshly isolated tumor cells or lymphocytes can be confused by the extensive contamination with various cell types and the altered conditions of cells recently subjected to enzymatic or mechanical treatment. The expansion of TIL-tumor cell pairs from excised tumors has provided elegant models for the in vitro characterization of CTL-tumor interaction (11). Although it is not clear whether cultured cells are representatives of in vivo conditions, experiments performed with cell lines establish important principles of T cell-epitope interaction, which may allow formulation of useful hypotheses. Analysis of reagents obtained from excised tumor specimens, however, yields static information about a disease characterized by extreme genetic instability (12). The natural course of the removed tumor cannot be followed prospectively; therefore, to take the excised lesion as representative of other lesions left in vivo, homogeneity among metastases must be assumed. However, synchronous metastases are quite heterogeneous in expression of MA and HLA class I molecules (13), and this heterogeneity affects the T cell population of a given lesion (14). Furthermore, MA and HLA class I molecule expression gradually decrease with time either in reflection of the progressive dedifferentiation of tumor cells or as the result of immune selection (13, 15). Thus, tumor-T cell interactions could be better studied by following the progression of events occurring within the same lesion. This could be achieved by serial analysis of identical tumor samples through fine needle aspiration (FNA) biopsies, which do not require excision of the tumor and therefore provide the opportunity to evaluate dynamically the expression of relevant markers on tumor or T cells (16, 17). The serial sampling of tumor metastases may yield important information about the adaptation in time of the host’s immune response to changing tumor cell phenotypes and at the same time allow correlation with size measurements of the same lesion collected throughout a relevant observation period. Because of the limited amount of material obtainable, however, FNA suffers its own limitations. The primary purpose of this study was, therefore, to evaluate the feasibility of developing tumor/T cell pairs from limited FNA material, which could be eventually used for serial analysis of the same tumor lesions.
An example of the dynamic nature of the tumor-host interaction is the adaptation of the tumor environment to vaccination with epitopes derived from MA. We previously noted a functional dissociation between systemic and local immune response in a patient whose disease progressed in spite of the maintenance of a systemic vaccine-specific T cell reactivity (17). Fading treatment effectiveness at the tumor site could be best explained, in that case, by loss in time of the target MA. Other observations suggest that MA and/or HLA class I molecule down-regulation is a common event in response to systemic therapies intended to enhance antitumor immune responses (13, 18, 19, 20, 21, 22, 23, 24). The secondary goal of this study was, therefore, to sequentially analyze tumor and T cell adaptation over time in a small cohort of patients treated with an epitope-specific vaccination.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The melanoma cell lines 624.38 (HLA-A*0201/0301, B*1402/-, Cw*0702/0802) was generated by limiting dilution from a metastatic lesion (25). The A375 Mel (HLA-A*01/0201, B*17/-, Cw*06/-) melanoma cell line was purchased from the American Type Culture Collection (ATCC) (Manassas, VA). 586-Mel (HLA-A*31, B8/-), 537-Mel (HLA-A*1, 26 B44,70), 1362 Mel (HLA-A*A-1, 25 B8,61), 1379 Mel (HLA-A-A*-11,0 B-35,60), and 888-Mel (HLA-A*01/2402, B*52/55, Cw*0102/1201) cell lines were derived from surgically removed metastatic lesions of patients treated at the Surgery Branch, National Cancer Institute (Bethesda, MD), and have been previously extensively characterized with regard to HLA and MA expression (26). The 583-EBV lymphocytic cell line (HLA-A3/30, -B7/44) was also developed from a melanoma patient and was used as an MA-negative control. All cell lines were maintained in complete medium consisting of RPMI 1640 (Biofluids, Rockville, MD) supplemented with 10 mM HEPES buffer, 100 U/ml penicillin-streptomycin (Biofluids), 10 µg/ml ciprofloxacin (Bayer, West Haven, CT), 0.03% L-glutamine (Biofluids), and 0.5 mg/ml amphotericin B (Biofluids) and 10% heat-inactivated FBS (Biofluids). T2 (ATCC), a cell line defective for endogenous processing and expressing HLA-A*0201 (27), was used to test CTL specificity toward HLA-A*0201-restricted epitopes.
Peptides
Four different peptides, MART-1:27–35 (MART-1, AAGIGILTV) and the modified gp100:209–217 (210M) (g209-2M, IMDQVPFSV), gp100:280–288(288V) (g280-9V, YLEPGPVTV), and tyrosinase:368–376(370D) (tyrosinase, YMDGTMSQV), were used for vaccination and in vitro testing as later specified. Furthermore, the following peptides were used for in vitro testing: the native gp100:209–217 (g209, ITDQVPFSV,); and gp100:280–288 (g280, YLEPGPVTA). The Flu-M1:58–66 peptide (Flu, GILGFVFTL by Multiple Peptide Systems, San Diego, CA) was also used as control in sensitization assays. All peptides, with the exception of Flu, were provided by the Cancer Therapy Evaluation program (National Cancer Institute). The peptide were produced to GMP (good manufacturing procedure) grade by solid phase synthesis techniques and solubilized in either sterile water or DMSO (Sigma, St. Louis, MO) according to their biochemical characteristics.
Patient selection and treatment
FNA biopsies were obtained from patients with metastatic melanoma undergoing various immunotherapy protocols at the Surgery Branch, National Cancer Institute. To demonstrate the utility of repeated sampling of the same lesion, we characterized FNA material from five HLA-A*0201 patients vaccinated with the MA epitopes MART-1, tyrosinase, g280-9V, and g209-2M. Patients received four separate s.c. injections of each peptide emulsified in IFA at 3-wk intervals. HLA typing and subtyping for HLA class I and II were determined on patients’ PBMC or tumor cell lines using sequence specific primer-PCR (28, 29). When necessary, the identity of some HLA alleles was conclusively determined by sequencing of cDNA (30).
Expansion of TIL from FNA
With a 23-gauge needle, cells were aspirated from metastases and immediately suspended in Iscove’s medium (IM) (Biofluids) supplemented with 10 mM HEPES buffer, 100 U/ml penicillin-streptomycin (Biofluids), 10 µg/ml ciprofloxacin (Bayer), 0.03% L-glutamine (Biofluids), 0.5 mg/ml amphotericin B (Biofluids) and 10% heat-inactivated human AB serum (Gemini Bioproducts, Calabasas, CA). For TIL expansion, total cells, including tumor cells, RBC, and TIL, were counted and cultured at a concentration of 5 x 106 FNA cells/well of 24-well plates (Falcon, Franklin Lakes, NJ) with IM plus 6000 IU/ml (Chiron, Emeryville, CA). Epitope-specific TIL were expanded from serial FNA of metastases obtained before and after two and four vaccinations. The FNA material was cultured in 24-well plates at a concentration of 2 x 106 cells/well with 4–5 x 106 autologous irradiated (50 Gy) PBMC pulsed with 1 µM peptide. The in vitro sensitization was performed by separately stimulating FNA aliquots with each of the peptides MART-1, g209-2M, g280-9V, tyrosinase, and Flu. After 24 h and every 2 days thereafter, 300 IU/ml IL-2 were added to the cultures. Simultaneously with the FNA, leukaphereses were obtained and parallel cultures of Ficoll-Hypaque gradient-separated PBMC were performed. PBMC (4 x 106/well) were cultured in a 24-well plates with 1 µM peptide as described for the TIL or with no peptide. The addition of irradiated APC in TIL was not deemed necessary for PBMC cultures, which already contained a large monocytic population (3). To test whether results equivalent to TIL cultures could be obtained by the addition of irradiated APC to PBMC cultures, PBMC were also stimulated with 5 x 106 autologous peptide-pulsed, irradiated PBMC. These treatments were equivalent to the in vitro sensitization with peptide alone. After 10–12 days in culture, T cells were harvested and tested for Ag recognition.
Development of tumor cell lines from FNA
Tumor cell lines were developed in culture conditions identical with those adopted to expand TIL but without IL-2. The autologous cell lines were HLA class I and II matched to the phenotype of patients screened, and when equivocal (loss of MA expression) their neoplastic phenotype was proved by electron microscopy and karyotyping.
Assessment of Ag recognition by T cells derived from PBMC and TIL
Ag recognition by T cells derived from PBMC and TIL was assessed
by IFN-
release assay. Effector cells (1 x
105) were plated with 5 x
104 stimulator cells (tumor cells or T2 cells
pulsed with natural and modified MA epitopes or Flu peptide) in 96-well
round-bottom plates in 200 µl IM. After a 24-h incubation at 37°C,
the plates were centrifuged, and the supernatant was harvested for
analysis by ELISA (Endogen, Cambridge, MA). IFN- was reported as
picograms of IFN- per milliliter secreted by 5 x
104 effector cells in 24 h, and values
double the background and >100 pg/ml were considered positive.
Phenotypic characterization of tumor cell lines and T cells
Cell surface expression of HLA, MA, and other surface Ags was determined by flow cytometry. MA expression was assessed by intracellular staining by fixing cells in 200 µl acetone for 10 min at room temperature and staining with the primary mAb (26). The following mAbs were used for detection of HLA surface expression: W6/32 (Sera Labs, Westbury, NY) specific for a monomorphic determinant of the HLA class I heavy chain (31); IVA-12 (ATCC) for HLA class II; KS-I (32) for HLA-A2. Anti-MART-1/MelanA murine IgG2b (M2-7C10) (16, 33) and anti-Pmel17/gp100 mAbHMB45 (Enzo Diagnostics, Farmingdale, NY) were used for MA detection. Abs used to characterize T cells included: anti-human CD8-FITC, CD4-PE, TCR-FITC, CD45RA-FITC, CD45RO-PE, cataneous lymphocyte Ag (CLA)-purified, CD95 ligand (L) (FASL)-purified, CD11b (MAC-1)-PE, CD44(pgp-1)-FITC, CD152(CTLA-4)-PE (PharMingen, San Diego, CA), CD56-PE, CD28-FITC, CD62L-PE, CD154-PE (Becton Dickinson, San Jose, CA); and FITC-anti-human CD49a, CD49b, CD49d, CD49e, CD49f (Serotec, Raleigh, NC). Primary staining with the purified Ab CD95L was followed by secondary staining with FITC-goat anti-mouse IgG. Staining with anti-human CLA was followed by secondary staining with FITC-anti rat IgM. Tetrameric peptide-HLA-A2 complexes were synthesized as described previously (34). The final concentration of tetramer was adjusted to 1 mg/ml for g209, g209-2M, MART-1 tetramer HLA (tHLA), and to 0.5 mg/ml for Flu tHLA. For FACS staining, TIL and PBMC cultures were washed and suspended at 2 x 105 cell/50 µl cold FACS buffer (PBS, 5% inactivated FCS (Biofluids). Cells were incubated on ice with 1 µg tHLA for 15 min and then with 20 µl anti-CD8-FITC (Becton Dickinson) for 30 min. Cells were washed twice in 2 ml cold FACS buffer before analysis by FACS (Becton Dickinson); 50,000 events were acquired for PBMC and TIL cultures.
For immunocytochemistry (IHC) staining, cytospin preparations of sequentially obtained FNA material were fixed in acetone and stained with the same mAbs used for the FACS analysis with the exception of HMB45 (Biogenex, San Ramon, CA). For secondary staining, biotinylated goat anti-mouse IgG (Kirkegaard & Perry Laboratories, Gaithersburg, MD) was used followed by avidin-biotin-peroxidase (Vectasin Elite Kit, Vector Laboratories, Burlingame, CA) (16).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We expanded tumor cell and TIL lines from 18 FNA of melanoma
metastases in 12 patients undergoing immunological treatment at our
institution (Table I
). TIL could be
derived in 14 cultures whereas tumor cell lines could be derived in
only 8 FNA from 6 patients. TIL-autologous tumor cell pairs could be
obtained in five patients. In four patients, TIL-tumor cell pairs could
be derived from the same lesion. TIL were most commonly CD4 or CD8
expressing T cells (Table II) and had a
phenotype consistent with Ag-experienced T cells (CD45RO positive,
CD45RA negative). Activation markers demonstrated occasional expression
of CD154 (CD40L) in cultures with a high percentage of
CD4+ T cells, low expression of CD28, and no
expression of CD152 (CTLA4) and CD95L (FasL) (data not shown). Analysis
of markers related to trafficking to target organs identified high
levels of CD44 and CD49d. Other homing receptors were less frequently
(CD11b, CD49b, CD49f), rarely (CLA, CD62L), or not (CD49a, CD49e)
expressed. Tumor cell lines were characterized according to the
expression of HLA class I and II molecules, MA and ICAM-1 (CD54) (Table III). All cell lines expressed HLA class
I molecules and cell lines obtained from HLA-A*0201 patients stained
with KS-1 mAb specific for this allele. Not uncommonly for melanoma
(35), several cell lines also expressed HLA class II
molecules. As noted in cell lines derived from excised surgical
specimens (26), gp100 was expressed less frequently than
MART-1. The less frequent detection of gp100 in the cell lines derived
from FNA was also in accordance with the most frequent loss of gp100
observed by IHC of the same FNA (Table III). A comparison of the
expression of MA in cytospins of the original FNA compared with the
cell lines suggested that the latter do not always accurately reflect
the expression of MA in vivo, possibly because of overgrowth of
MA-negative tumor cells during prolonged culture conditions. An example
is cell line KA, R Th. This cell line stained positive for MART-1 at an
early passage (Table III, FACS*, passage 5) but decreased the
expression of this MA in subsequent passages (FACS**).
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The 14 TIL cultures were analyzed for recognition of autologous
and/or HLA-matched tumors (Table IV).
Five of six TIL cultures could recognize available autologous tumor
cells. In addition two TIL cultures for which no autologous tumor was
available could recognize HLA-matched tumor cell lines. One TIL culture
(CM, L Ax) developed from a lesion that had lost expression of common
differentiation Ags such as MART-1 and gp100 could recognize a new MA
expressed by autologous or allogeneic melanoma cell lines in
association with HLA-Cw*0702 (Panelli et al., manuscript in
preparation). Two TIL cultures (KA, R Th and KA, R Hip) highly reactive
to autologous tumor cells and HLA-A*0201 allogeneic melanoma cells
included a significant proportion of MART-1 specific CTL according to
tHLA staining (Fig. 1). Calculation of
epitope-specific CTL indicated that MART-1-specific T cells represented
4 and 19% of KA, R Th and KA, R Hip CD8+ cells.
Although the expression of MART-1 in the autologous cell line was
reduced (Table III
), analysis of MA expression in cytospins of the FNA
from which the tumor and TIL were obtained had shown expression of
MART-1 in vivo. These data suggest that it is possible to expand tumor
and T cell lines, occasionally in pairs, from FNA that can be utilized
in studies aimed at their functional characterization.
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To assess the feasibility and usefulness of TIL expansion from
sequential FNA biopsies of the same lesion, we adopted an
epitope-specific model. Patients were vaccinated by s.c. administration
of g209-2M, g280-9V, MART-1, and tyrosinase peptides in IFA at 3-wk
intervals. FNA were obtained before vaccination and 3 wk after the
second and fourth vaccinations. Simultaneously, patients were
leukapheresed and PBMC were induced in vitro. To exclude the
possibility that blood contaminating the FNA could be accountable for
the expansion of epitope-specific T cells by direct stimulation of
PBMC (36), we tested differences in ability to induce
Flu-specific CTL in cultures derived from FNA material and PBMC.
Flu-specific T cell could be induced significantly more often in PBMC
(p < 0.001, Fisher’s; Fig. 2), suggesting that blood contamination
of the FNA was either absent or minimal.
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). No MA-specific TIL could be derived
from prevaccination FNA. After vaccination, we could generate MART-1-,
g209-, and g280-specific TIL in two, three, and three patients
respectively (Fig. 3). In no case could
tyrosinase-specific T cells be induced from FNA or PBMC. The
combination of results obtained with various attempts in the same
patient gave a reasonable success rate in expanding vaccine-specific
TIL and suggested localization of vaccine-induced T cells at the tumor
site. The success rate in expanding vaccine-specific TIL was not as
high when each culture attempted was separately considered. Overall, 21
attempts were made to induce TIL specific for MART-1, g209-2M, or
g280-9V (7 for each peptide) in prevaccination FNA and 36 in
postvaccination FNA (12 for each peptide) (Table V). Vaccine-specific T
cells could not be raised in prevaccination FNA cultures, whereas 12 of
36 attempts performed in postvaccination FNA generated MA-specific TIL
(33%, Fisher’s p between pre- and postvaccination
cultures, <0.005). Thus, repeated attempts of expansion from FNA
material may enhance the sensitivity of the method.
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). TIL elicited with the modified g209
and g280 peptides could recognize the epitope used for the vaccination,
autologous tumor (when available) or HLA-A*0201-matched melanoma cells.
Recognition of autologous tumor could not be easily explained for
gp100-specific TIL because in some instances phenotypic
characterization of the autologous cell lines revealed good expression
of HLA class I alleles but severely decreased expression gp100 (Table VII). It is possible that the limited
number of cells expressing gp100 could still induce release of IFN-
by the TIL. Alternatively, T cells specific for other MA might have
been included in the TIL populations. "Concomitant expansion" of T
cells nonspecific for the stimulus applied in culture was noted in
KAe-2 g209-2M TIL. This TIL, although induced by g209-2M stimulation,
demonstrated a predominance of MART-1-specific CD8+ T cells (Fig. 4). Evidently, T cells of various
specificity present in high frequency in the original FNA material
might expand independently from the specific stimulus applied in
culture because of the general stimulatory conditions provided ex
vivo.
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and Fig. 1). Epitope-specific reactivity in TIL is demonstrable only in association with enhanced systemic T cell responses to vaccination
Analysis of the local response to vaccination was compared with
the systemic response in simultaneously obtained PBMC. Four of five
PBMC cultures could generate 209-2M or 280-9V-specific T cells after
two vaccinations and three of five were reactive to MART-1 (Fig. 5). Postvaccination, MA-specific TIL
could be elicited only in patients that also demonstrated enhanced
systemic reactivity toward the same epitope. Detection of
vaccine-specific immune reactivity in peripheral blood lymphocytes did
not correspond always to the presence of vaccine-reactive TIL. These
data suggest that development of systemic immunoreactivity toward the
vaccine is necessary but not sufficient for the localization of immune
responses at the tumor site. Flu reactivity was present in PBMC
cultures from five of five patients with no significant pre- and
postvaccination differences. In patient MK, no vaccine-specific T cell
could be induced from PBMC or from FNA material, but it was possible to
induce Flu-reactive T cells from PBMC, suggesting that this patient was
not immunocompromised.
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). This once again suggests that
variables other than those evaluable by direct confrontation of
autologous tumor-T cell pairs should be considered to explain their
coexistence in vivo. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The original expectation of these studies was to identify lesions characterized by different clinical behavior and correlate such behaviors with data obtained from the functional characterization of tumor cells and/or TIL expanded from the same lesions. Lack of dramatic responses in the lesions studied and the complexity of the information obtained limited the ability to make definitive conclusions about conditions determining treatment outcome in this study. Furthermore, this study underlined the complexity of the dynamic analysis of tumor-host interactions. Loss of MA expression occurring in cell lines in correlation with culture passages does not have a definite explanation and it is not clear whether, during in vitro culture, more anaplastic cells with a dedifferentiated phenotype have a selective growth advantage. In general, however, MA expression and in particular gp100 expression was lower even in the original FNA materials compared with previous experiences (13), perhaps as a consequence of the previous Ag-specific treatments received by the patients.
An interesting observation was the identification of TIL capable of
recognizing autologous and HLA class I-matched tumors. These TIL have a
memory phenotype characterized by predominant expression of the CD45RO
marker (Table II). However, this cannot in itself prove their true
origin from cancer-populating T cells inasmuch as this phenotype have
been identified also in PBMC of melanoma patients (40).
However, the expansion of T cell cultures from FNA with the addition of
IL-2 alone that could recognize tumor targets in an HLA-restricted
fashion suggests that their origin was likely from TIL rather than
blood contaminating the FNA material. It was also possible to induce
more easily vaccine-specific TIL after vaccination than before. This,
of course, does not exclude that more sensitive methods of induction
could have detected TIL specific for these MA also in prevaccination
specimens as previously observed (41, 42).
Vaccine-specific TIL could be expanded at the tumor site only when
systemic responses could be simultaneously noted. Thus, the observation
of vaccine-specific TIL could in theory represent only an artifact due
to the in vitro expansion of passenger T cells in peripheral blood
contaminating the FNA aspirate material. Such a possibility cannot be
totally excluded. On the other hand, two observations suggest that the
T cells expanded represented genuine TIL residing in the tumor: 1) it
was difficult to expand Flu CTL from the FNA, whereas we have
previously shown that it is extremely easy to expand Flu-specific CTL
from PBMC cultures in patients with melanoma (36); 2) the
concomitant expansion of T cells recognizing other MA in response to
stimulation with one epitope suggests a precursor frequency of
MA-specific TIL superior to the one expected in PBMC (Table VI). Indeed
we have never noted concomitant expansion of non-epitope-driven
MA-specific CTL in PBMC cultures (data not shown).
This study proposes an alternative view of tumor monitoring in which events are analyzed at the tumor site where they are most relevant. The major limitation of this approach is the limited amount of material obtainable and the necessity for expansion of TIL and tumor cells for functional studies to be compared with information collected by IHC of remaining aliquots of the same FNA. It can be foreseen that in the future new technologies may be applied to allow extensive analyses of materials from small samples. Distinct populations of cells could be sorted by microdissection (43, 44) or epitope-HLA tetramers (14, 34), and their status of activation could be directly tested by sensitive methodologies such as Taqman-based real time RT-PCR (45) or intracellular FACS analysis (46). Collection of cDNA libraries from FNA of metastases could profile patterns of expression of thousands of genes in a single experiment (47). More recently, the expansion of tHLA-sorted T cell lines after a primary expansion has been proposed (48), which may lead to a higher yield of epitope-specific TIL from FNA. This information, combined with knowledge of the natural history of the lesion left in situ, might yield clinical material for correlation of laboratory findings with clinical outcome and identification of the algorithm necessary for tumor regression.
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Footnotes |
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2 Abbreviations used in this paper: MA, melanoma Ag; FNA, fine needle aspirate; IHC, immunocytochemistry; TIL, tumor-infiltrating lymphocytes; tHLA, soluble MHC-peptide tetramer; MART-1, MART-1:27–35(AAGIGILTV); g209-2M, gp100:209–217 (IMDQVPFSV); g280-9V, gp100:280–288(YLEPGPVTV); tyrosinase, tyrosinase:368–376 (YMDGTMSQV); IM, Iscove’s medium; L, ligand.
Received for publication May 6, 1999. Accepted for publication October 8, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-2. J. Immunother. 19:375.[Medline]
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, patient WW;
, patient MC; 
, patient MK; 
, patient CD.