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From the Institute of Pathology,*
University of
Würzburg, Würzburg; and the Medizinische Klinik und
Poliklinik V,
University of Heidelberg,
Heidelberg, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, interleukin-2). By
interphase fluorescence in situ hybridization analysis
the translocation t(11;18)(q21;q21) was found in four of nine (44%)
cases studied. Interestingly, there was a four times higher
proliferation and survival rate of purified t(11;18)-positive tumor B
cells in vitro, although there were no
significant profile differences from the TITLs in vivo.
The finding of essential costimulating molecules in low-grade MALT-type
lymphomas in vivo indicates a locally directed cognate
T-/B-cell interaction. Consequently, a potentially equipped
inflammatory background may not only determine the fate of autoreactive
B-cells, but is also crucial to lymphoma maintenance and
progression.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently, in vitro and molecular studies suggested that
MALT-type lymphoma B cells are antigen sensitive but
autoreactive6-8
and thus have escaped a tolerance
mechanism that is normally checked by various T-cell subsets through
interactions of cell surface molecules and cytokines. Experimental
observations suggest that antigen and Fas-ligand/CD40-ligand expressed
on activated T-cells act during cognate T-/B-cell interaction and are
crucial checkpoints for germinal center B-cell maturation, generating
marginal zone B cells, but act to delete harmful B
cells9,10
. The maturation of B cells to effective
producers of high-affinity and high-specificity antibodies is
controlled by defined T-cell subsets through cell surface molecules and
soluble cytokines.11-13
In particular, whereas Th1-type
cytokines (ie, interleukin-2 (IL2), interferon- (INF-)) are
important in cell-mediated immune responses and the eradication of
microbial pathogens,14
Th2- and Th3-type cytokines (IL4,
IL6, IL10, and transforming growth factor ß1
(TGF-ß1)) have been demonstrated to be crucial for B-cell
maturation15,16
but may also support the growth of
otherwise uncontrolled B cells in autoimmune disorders17
and suppress cell-mediated autoimmunity (ie, TGF-ß1).
Therefore, the discovery of functionally heterogeneous CD4+
T-cell subsets that secrete different cytokines may determine not only
the type of B-cell immune response but may also provide key molecules
in B-cell lymphoma initiation and progression.
Recently, in a larger series a t(11;18)(q21;q21) chromosome translocation was found to be the characteristic aberration in low-grade MALT-type lymphoma18 involving a novel gene MLT and the HIAP1 gene, encoding an inhibitor of apoptosis.19 Although its biological and clinical significance is so far unknown, it is suggested that the rearranged genes may support increased B-cell survival and decrease T-cell-dependent help.20
We present herein a detailed characterization of the microenvironment of low-grade MALT-type lymphomas, which is different from chronic gastritis, and show that essential costimulatory molecules are present in all cases irrespective of the t(11;18) alteration, which may provide an important background for tumor progression.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Fresh tissues were received within 2–6 hours after surgery for
the preparation of cell suspensions, snap-freezing in liquid nitrogen,
and routine fixation for histological examination. Nonneoplastic
tonsillar tissues with reactive tonsillitis and resection samples from
patients with primary low-grade MALT-type lymphoma of the stomach were
selected from the lymph node registry in Würzburg. The tumors
were always confined to the stomach and regional lymph nodes without
clinical evidence of generalized disease (Table 1)
. Additionally, one low-grade MALT-type
lymphoma of the thyroid gland and one case of gastritis (no. 11; as a
control) were investigated (Table 1)
. For diagnostic purposes, the
morphological and immunophenotypical analyses of paraffin-embedded and
freshly frozen sections were performed by standard methods as recently
described.1
In all cases the diagnosis was confirmed
according to the criteria of the Revised European American Lymphoma
(REAL) Classification.21
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Immunohistochemical staining was performed on 4-µm cryostat sections of fresh frozen surgical specimens. Biopsy tissues were kept at -70°C as snap-frozen blocks until sections were prepared at the time of the experiments. The immunoperoxidase method was applied by a three-step incubation procedure with diluted affinity-purified anti-rabbit, anti-rat, anti-mouse antibodies (all from Dako, Hamburg, Germany) and serum from each species as a control, as described in detail elsewhere.22 APAAP staining was used to visualize mouse anti-human CD40-ligand (kind gift from RA Krozcek, Berlin, Germany) as described.23 The following primary antibodies were used in this study: rabbit anti-human Fas-ligand (SC-834-G; Santa Cruz Biotechnology, Santa Cruz, CA), rat anti-human IL-2 (MQ1–17H12; Pharmingen, Heidelberg, Germany), mouse anti-human IL-10 (mAb 2,17; R&D Systems, Wiesbaden, Germany), rat anti-human IL-13 (JES10-5A2; Pharmingen), and TGF-ß1 (BMS4435; Boehringer Mannheim, Mannheim, Germany). CD40-ligand and Fas-ligand served in immunostained cytospins as positive controls for cytokines from different cell lines (Jukat, Raji, THP-1) stimulated with phorbol myristic acetate/ionomycin or lipopolysaccharide over 24 hours.24,25
Flow Cytometry
Flow cytometric analysis was performed on a FACScan (Becton
Dickinson) with an argon ion laser tuned at 488 nm, using LYSIS II for
data acquisition and analysis and triple immunostaining with directly
conjugated antibodies (anti-CD19 (HD 37; Sigma); anti-
(KP-53;
Sigma), anti-
(HP6054; Sigma), anti-CD3 (UCHT-1; Sigma), anti-CD14
(Leu-M3; Becton Dickinson), anti-CD40 (mAb89),26
anti-CD4
(R-8886; Sigma), anti-CD8 (R-806; Dako), anti-CD38
(MHCD3804; MEDAC), anti-IgD (I-2768; Sigma), anti-IgM (P-9295; Sigma),
anti-CD25 (R-811; Dako), anti-CD69 (BD 347823; Becton Dickinson)). Each
measurement contained 20,000 cells. As a specificity control and
for instrument setup isotype controls were used in parallel for each
directly conjugated antibody regarding species, isotype, and type of
fluorochrome (all from Sigma, Deisenhofen, Germany).
B-Cell Purification, Cell Culture, Stimulation, and Cytogenetic Analysis
Viable lymphocyte single-cell suspensions were isolated by density-gradient centrifugation and negative depletion using magnetic beads coupled with anti-CD2 and anti-CD14 (Dynal, Hamburg, Germany) to remove macrophages and T cells. Thereafter, as a control, nonneoplastic memory B cells from tonsils were depleted of CD38+ (clone ACT 13.5; Serotec, Wiesbaden, Germany) and IgD+ (clone HJ9; Sigma) cells to remove germinal center cells, plasma cells, and mantle zone cells as described elsewhere.27,28 Lymphoma cells were further purified by negative depletion of nonneoplastic bystander B cells, using beads coated with antibodies to immunoglobulin heavy and light chains not expressed by the lymphoma, including IgD. Tumor-infiltrating T cells were isolated from primary cell suspensions, using anti-CD4+- and anti-CD8+-coupled magnetic beads and Detach-a-beads (all from Dynal). If the purity of cell isolates was >95%, CD19+/IgD-/CD38-/sIg+ cell suspensions were subjected to further analyses. Purified B cells were stimulated for reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analyses with 500 ng/ml ionomycin and 10 ng/ml 12-O-tetradecanoylphorbol-13-acetate for 6 or 24 hours. To measure the proliferation the B cells were stimulated in the CD40-system as described previously.6
Cytogenetic analyses were performed as described.18 The presence of the t(11;18)(q21;q21) was determined either by conventional karyotyping or by applying a fluorescence in situ hybridization (FISH) assay that was recently developed to detect the chromosomal breakage in 11q21 in interphase cells (A. Rosenwald, manuscript submitted for publication). Karyotypes were described according to the International System for human cytogenetic nomenclature.
Western Blot
For each sample total protein extraction was prepared from 1 x 106 cells from cell culture. The cells were resuspended in 100 µl of Laemmli buffer with 1 µmol/L leupeptin (Sigma), 1 µmol/L pepstatin (Sigma), and 1 mmol/L pefabloc (Boehringer Mannheim)29 and boiled for 5 minutes, and the DNA was shared with a cannula. The solution was spun down, and the protein supernatant was stored at -20°C. The protein extractions were monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.29 In each lane 30 µl of protein extract mixed with bromphenol blue was applied. Proteins were transferred to a nitrocellulose membrane, which was probed with anti-CD40-ligand (TRAP cell culture supernatant; dilution 1:5) or anti-Fas-ligand (clone G247-4; Pharmingen) as the primary antibody, respectively. For detection the ECL system (Amersham, Freiburg, Germany) was used according to the manufacturer’s instructions.
RNA Extraction
All tumor tissue blocks were snap-frozen in liquid nitrogen and stored at -70°C until extraction of RNA. Total RNA was prepared (TRIzol reagent; Life Technology, Paisley, UK) from 20 sections of about 10 µm from the frozen tumor tissue blocks. The integrity of the RNA was checked by electrophoresis on a 2% formaldehyde-agarose gel, and the yield of RNA was quantitated by measuring the optical density. To check for carry-over material during the isolation step, extraction buffer without tissue was used as a negative control.
cDNA Synthesis
First-strand cDNA was synthesized with 1 µg of total cellular
RNA. RNA, 2 µg of dT-15 primer, and diethylpyro-carbonate
(DEPC)-treated water at a final volume of 8 µl were incubated
for 10 minutes at 65°C. After chilling on ice, a master mix
consisting of deoxynucleoside triphosphates and dithioreitol (final
concentrations of 1 mmol/L each and 10 mmol/L, respectively), 25 U of
recombinant RNase inhibitor (Promega, Heidelberg, Germany), RT-buffer,
and 200 U of Moloney-murine reverse transcriptase (GIBCO BRL,
Eggenstein, Germany) were added to a final reaction volume of 25 µl.
After 70 minutes of incubation at 37°C, the samples were heated to
98°C for 4 minutes. The efficiency of cDNA synthesis was assessed by
PCR with glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-specific
primers (Table 2)
. The GAPDH-PCR was
performed in a final volume of 25 µl, using 2.5 µl 10x PCR-buffer
(USB, Cleveland, OH), 2.0 µl 25 mmol/L MgCl2, 0.5 µl 10
mmol/L dNTPs, 0.5 µl 20 pmol of each primer, 0.5 U
Taq-polymerase (USB), 18.4 µl PCR-water, and 1 µl cDNA.
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For gross quantitation, we used expression of GAPDH as an external
standard, which was amplified in separate reactions. Twenty-five cycles
of amplification for each sample were carried out in a DNA thermal
cycler 2600 (Perkin-Elmer Centus, Emeryville, CA), and aliquots were
taken after 21, 23, and 25 cycles. Each cycle of amplification
consisted of 30 seconds of denaturation at 94°C, 30 seconds of
annealing at 60°C, and 1 minute of elongation at 72°C. The aliquots
were subjected to electrophoresis, and the amount of amplification was
assessed by comparing signal intensites. After the cDNA amount in each
sample was adjusted to generate an equal result in GAPDH amplification,
specific RT-PCRs for CD40-ligand, Fas-ligand, and cytokines were
carried out. Specific oligonucleotides were selected in such a manner
that they were only able to hybridize with cDNA and not with genomic
DNA (Table 2)
. The master-mix conditions for the specific PCRs were the
same as already described for GAPDH, except for the MgCl2
concentration (Table 2)
. PCR fragments were sequenced to ascertain
specificity. Ionomycin-stimulated peripheral blood lymphocyte (PBL)
cDNA was used as a positive control. The specificity and sensitivity
for each primer pair of the cytokine PCRs were seperately tested as
described.30
Two microliters of cDNA from stimulated PBLs
was taken as the control cDNA in each test reaction. The primer pairs
were tested with different MgCl2 concentrations (1.0, 1.5,
2.0, 2.5, 3.0, 3.5 mmol/L) and different annealing temperatures. Under
consideration of the intensity of the PCR product and the purity of the
backround analyzed on an agarose gel, we selected the most
sensitive combination of MgCl2 concentration and annealing
temperature. The control PCRs in Figure 5
showed different intensities
due to the different concentrations of cytokine mRNAs in the control
PBLs.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As determined by FACScan analysis, low-grade lymphomas contained
high numbers of tumor-infiltrating T-lymphocytes (TITLs) (for details
see Figure 1A
). The CD4/CD8 ratio was 3.3
in t(11;18)-positive cases and 3.3 in t(11;18)-negative cases. To
investigate whether TITLs were activated (CD69+) and
immunocompetent (CD28+), three-color fluorescence-activated
cell sorter (FACS) analysis was used. As shown in Figure 1, B and C
,
low-grade TITLs expressed significantly more CD69 than the T cells from
PBLs of the same patient. Moreover, in contrast to PBL T cells, all
lymphoma TITLs expressed CD28, with an even higher average mean
fluorescence intensity (MFI). Although CD69 and CD28 MFI expression was
higher in t(11;18)-negative low-grade lymphoma TITLs (MFI CD69/28:
12/33) as compared with t(11;18)-positive TITLs (MFI CD69/28: 9/31),
the differences were statistically insignificant.
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To investigate whether CD40-ligand and Fas-ligand mRNA were
present in lymphoma tissues in vivo, a semiquantitative
RT-PCR was used. Within the low-grade lymphomas 80% were positive for
CD40-ligand and Fas-ligand mRNA, whereas immunohistochemistry found
protein expression that paralleled mRNA expression in all cases.
Notably, CD40-ligand-positive as well as Fas-ligand-positive cells were
found in considerable numbers outside the reactive residual germinal
centers within the tumor mass (Figure 2,B and C)
.
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. Because B cells were recently found
to be able to express low basic levels of CD40-ligand
mRNA,31
we also measured the protein expression by Western
blot assay and could not detect any CD40-ligand protein in normal
memory B cells and tumor B cells as compared with the positive control
(Figure 4A)
. In addition, neither
Fas-ligand mRNA nor protein was found in MALT tumor B cells (Figure 4B)
.
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The most prominent cytokine found in all lymphoma tissues by
RT-PCR and immunohistochemistry was TGF-ß1, which was
expressed in the cytoplasm of tumor cells and by endothelial cells of
small vessels (Figure 5C)
. IL10 mRNA- and
protein-expressing cells were present in 60% of low-grade lymphomas.
IL13 mRNA/protein was detected in 40% of the cases.
Cytokine-expressing cells were found to be loosely distributed within
the tumor tissue (Figure 5, D and E)
.
IL2 was found in three low-grade cases (nos. 1, 6, and 8 (weak)), with
some scattered positive cells surrounding small blood vessels (Figure 5B)
. IFN- was found at a very low level in one low-grade case (no.
1), whereas no protein expression was found by immunohistochemistry
(data not shown). IFN- receptor mRNA (as a control) was always
detecable.
t(11;18) May Affect Tumor B-Cell Proliferation and Survival in Vivo
In one t(11;18)-negative and t(11;18)-positive case, cell culture
material was compared with normal memory B cells in vitro.
Strikingly, the basal proliferation (without any stimulation) and the
survival rate were significantly higher in the t(11;18)-positive case
as compared with the t(11;18)-negative case or the control memory B
cells (Figure 6A)
. Notably, the
differences in survival could be reversed by triggering the
lymphocytes in the CD40 system (Figure 6B)
.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Beyond the ability of CD4+ T cells to provide help in the priming of tumor-specific CD8+ CTLs, evidence from mouse models and cell lines indicates that tumor-specific CD4+ T cells can orchestrate additional effector functions in tumor immunity.34-37 Therefore, the observation that CD40-ligand was expressed within the MALT tumor tissues of at least low-grade lymphomas is important in three ways. First, as shown recently,2,6 CD40-ligand is the most potent stimulus for proliferation of MALT-type lymphoma B cells in vitro and may also promote tumor growth in vivo. Second, CD40-ligand is able to inhibit drug-induced apoptosis in lymphoma cell lines in vitro,38,39 suggesting that it may induce the resistance of tumor cells to proapoptotic and antiproliferative effects of cytotoxic drugs in vivo. Third, CD40-ligand is known to be expressed on T cells after triggering by specific antigens.40 Its detection in tumor tissues thus favors the idea of antigen-mediated tumor promotion by activated T cells. This notion is further strengthened by the observation that the TITLs but not the tumor B cells expressed CD40-ligand, in contrast to the recently described aberrant CD40-ligand expression of tumor-B cells in some low-grade non-Hodgkin’s lymphoma (eg, chronic lymphocytic leukemia41,42 ). Therefore, the presence of CD40-ligand in tumor tissues points to the possibility of nonautonomous tumor growth supported by activated CD40-ligand-positive T cells and is consistent with tumor regression after antibiotic treatment.
In addition to CD40-ligand, we also found Fas-ligand to be expressed in vivo. During normal T-/B-cell interaction Fas-ligand expression is commonly detectable on highly activated but not on resting T cells and is important to the deletion of harmful autoreactive B cells.
Given that most MALT-type B-cell lymphomas were found to express antigen receptors with specificity for autoantigens,7,8,29,43,44 the presence of Fas-ligand in the tumor tissues was surprising. A possible explanation for this result could be either an aberrant expression of Fas-ligand by the tumor cells as described recently for some carcinomas45 or a so far unknown escape mechanism in which the tumor B cells become resistant to Fas triggering. Our results favor the latter idea, because we could not detect any Fas-ligand mRNA or protein expression in normal or tumor B cells, even under rigorous stimulation in vitro. Furthermore, it is possible that impaired Fas-mediated apoptosis or Fas mutations as described recently in some B-cell lymphomas46 are operative in MALT-type lymphomas as well and may lead to a functional Fas resistance.20,47
The finding of Th2- and Th3-type cytokines but only low levels of
Th1-type cytokines in MALT tumor tissues in vivo is in
contrast to a recent report on four cases in which TITLs secreted a Th1
cytokine (INF-) after H. pylori stimulation in
vitro.48
This discrepancy may be explained either by
TGF-ß1 expression in the tumor cells in vivo,
which may cause a deviation of the T-cell response to a Th2 phenotype,
as shown recently in mice,49
or by in vitro
selection (due to culture conditions) of an otherwise (eg, major
histocompatibility complex-restricted) controlled Th1 population,
although it cannot be ruled out that a few cells in the tumor
microenvironment may be functionally significant. However, the
existence of Th1 cytokines would argue against tumor promotion and
favor antitumor immunity,37
whereas Th2-type (IL10, IL13)
and Th3-type (TGF-ß1) cytokines were shown to be potent
promoters for B-cell proliferation and
differentiation.40,50,51
The observation that activated T cells secrete Th2-type cytokines in MALT-type lymphomas in vivo may attribute to them a function that goes beyond solely translating H. pylori-specific signals into growth signals for MALT lymphoma B cells. Thus Blanckenstein et al52 recently proposed that presentation of tumor antigens by B cells to CD4+ T cells diverts antitumor immunity toward a "nonproductive" Th2-type humoral immune response and away from a "productive" Th1-type cellular response. Therefore, it is possible that a Th1-weighted immunity established during H. pylori-associated chronic inflammation as described recently53,54 may support antitumor immunity, whereas a Th2-weighted profile in vivo favors progression of lymphoproliferative disease and aberrant B-cell clones. If this indeed turns out to be a general phenomenon it will be important to design strategies that will break Th2-mediated T-cell tolerance and/or polarization to promote the development of an effective tumor immunotherapy, even in low-grade MALT-type lymphomas progressively growing after H. pylori eradication. In this context, it may be important that the negative and the positive t(11;18) cases did not differ regarding their expression profiles and appear to be dependent on the same environment. A reason for this could be that the t(11;18) translocation, which represents the most frequent structural abnormality in MALT-type lymphomas,18 involves genes that do not impair the T-/B-cell interaction. This may explain why purified low-grade tumor B cells respond uniformly in the context of CD40 triggering and cytokine stimulation2 but behave differently without exogenous stimuli in vitro. Therefore, it will be interesting to compare tumor regression in vivo with respect to the presence of t(11;18) because tumor B cells may still benefit from the immunological setting but have developed sophisticated survival strategies, even without a need for continuous T-cell collaboration.
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Acknowledgements |
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Footnotes |
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Supported by the Wilhelm-Sander Stiftung (grant 94.025.2, SFB 172 (B4) and grant 98.086.1 to S. S.) and the 1ZKF2000 (B3 to A. G.)
Accepted for publication August 1, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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