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Blood, 1 January 2002, Vol. 99, No. 1, pp. 388-390
CORRESPONDENCE
To the editor:
Terminal plasmocytoid differentiation of malignant B cells
induced by autotumor-reactive CD4+ T cells in one case of
splenic marginal zone B-cell lymphoma
In lymphoid organs invaded by malignant B-cell lymphomas, the
development of reactive CD4+ tumor-infiltrating T cells
(TIL-T) at the contact of tumor cells is now firmly
established.1,2 It has been reported that lymphoma B cells
are capable of proliferating in response to various recombinant signals
usually provided by CD4+ T cells, such as interleukin-4
(IL-4), or CD40 ligand.3 However, the exact nature of the
functional relationships between CD4+ TIL-T and autologous
non-Hodgkin lymphoma (B-NHL) cells remains largely unknown mainly
because this question has not yet been extensively investigated in
autologous CD4+ T/malignant B-cell coculture systems in
vitro. We report here evidence that CD4+ TIL-T have the
potential to drive autologous lymphoma B cells toward a terminal
differentiated state, in one case of splenic low-grade, marginal zone
B-cell lymphoma. In May 1998, a 67-year-old man presented with a low-grade lymphoma
(nonfollicular small-cell lymphoma) with involvement of spleen, blood,
periaortic lymph nodes, liver, and bone marrow. The patient underwent
splenectomy in July 1998. Histologic, cytologic, and immunophenotypic
features were compatible with splenic marginal-zone B-cell
lymphoma.4 All malignant B cells were surface
IgM+, kappa+, CD19+,
CD24+, CD40+, and were negative for IgD, CD23,
and CD5. Less than 1% of malignant cells were
CD38+ or CD138+. The percentage of
CD3+ TIL-T was 9% with 5% CD4+ and 4%
CD8+. A population containing both CD4+ T cells and malignant B
cells was negatively selected from total spleen cells by depleting CD8+ T cells, residual NK cells, monocytes, and normal B
cells by one round of immunomagnetic bead depletion. Purity was
assessed by flow cytometry and CD4+ T/malignant B-cell
preparations usually contained 8% to 10% CD4+ T cells and
88% to 90% CD19+ kappa+ B cells. Residual
cells not stained by CD3, CD4, CD19, or kappa antibodies were always
less than 1%. Cocultures were then performed in the presence of
recombinant IL-2 (rIL-2) at 10 UI/mL. Control cultures consisted of
purified malignant B cells cultured with rIL-2 but without the presence
of CD4+ T cells. A representative experiment of CD4+ T/malignant B-cell
cocultures is given in Figure 1. After 7, 14, and 21 days of coculture, cells were harvested, triple stained with
anti-CD4-PE-Cy5, kappa-PE, and CD138-FITC antibodies (Abs)
and analyzed by flow cytometry. CD138 (Syndecan-1) is a transmembrane
heparan sulfate proteoglycan expressed in Ig-producing, normal and
malignant mature plasma cells.5 At analysis, according to
a multicolor gating/painting strategy, CD138+
cells were gated and colored in orange, and
CD138 cells were colored in gray. At the initiation of
the coculture (day 0), the spontaneous formation of conjugates between
T cells and malignant B cells was negligible. At the contact of
malignant B cells, autotumor-reactive CD4+ T cells became
activated, expanded, and formed stable conjugates with malignant B
cells. Between day 7 and day 21 of the coculture, the percentage of
T/B-cell conjugates gradually increased (5% at day 7, 36% at day 14, and 34% at day 21), and this was accompanied by a progressive increase
in the number of free T cells (from 6% at day 0 to 51% at day 21).
CD138+ cells progressively accumulated in the
CD4+ T/B-cell coculture and reached a maximum value of 18%
of the total number of cells at day 14 (1%, 4%, 18%, 13%, at days
0, 7, 14, 21, respectively). CD138+ cells were almost
exclusively confined within CD4+ T/B-cell conjugates
(Figure 1), suggesting that the acquisition of this ag by
malignant B cells was dependent on a close association with
autotumor-reactive CD4+ T cells. To formally show that the
CD138+ cells were truly plasma B cells, these cells were
positively purified by an immunomagnetic method at day 14 of
the coculture and phenotyped (Figure 2).
This method allowed us to obtain a very pure population of
CD138+ cells (98%). As expected, CD3, CD19, CD20, and CD24
were low or absent; the expression of CD40 was intermediate, and kappa light chain was expressed at a low level (mean value: 198 units of
fluorescence) in comparison to fresh malignant B cells (mean value: 1299). A high proportion (85%) of cells strongly expressed CD38, a relevant marker to plasma cells. These cells exhibited a
morphology of plasma cells and contained large amounts of
intracytoplasmic kappa chain as estimated on cytospin slides stained
with fluorochrome-conjugated anti-kappa Abs. To ensure that these
CD138+ plasma cells belonged to the neoplastic clone, IgH
gene rearrangements of purified CD138+ cells were
investigated by a PCR-based method using Fr3 region primers and
compared with IgH gene rearrangements of purified fresh malignant B
cells. Both cell populations showed the same rearrangement bands,
stressing that the CD138+ cells were B cells and clonally
related to the original lymphoma cells (Figure
3). Cell-division tracking using PKH26
dye labeling indicated that more than 95% of the malignant B cells
remained in a nonproliferative state throughout the coculture, formally excluding that the accumulation of CD138+ cells during the
CD4+ T/malignant B-cell coculture might arise from the
rapid proliferation of a minor component of CD138+ cells
present at the initiation of the coculture. Finally, in control
experiments, purified malignant B cells cultured alone in the sole
presence of rIL-2 retained a phenotypic profile identical to fresh
malignant B cells without any down-regulation of CD19, CD20, CD40, and
surface Ig, or up-regulation of CD38 and CD138. Thus, malignant B cells
did not show any tendency to spontaneous or rIL-2-induced
differentiation in the absence of autologous CD4+ T
cells.6-10
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| Figure 1.
Conjugate formation between
autotumor reactive CD4+ T cells and malignant B cells in
CD4+ T/malignant B-cell cocultures, accumulation of
CD138+ cells, and preferential localization of
CD138+ cells within conjugates.
After various time intervals, cells were harvested, triple stained with
anti-CD4-PE-Cy5, kappa-PE, and CD138-FITC Abs, and analyzed by flow
cytometry. At analysis, CD138+ cells were gated and colored
in orange; CD138 cells were colored in grey.
CD138+ and CD138 cells were then
simultaneously visualized on dot plots with CD4-PE-Cy5/kappa-PE
parameters. 50 000 events were collected for each dot plot. The
quadrant statistics cursors were set using appropriate negative control
antibodies. Results are from 1 representative experiment of the 5 performed.
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| Figure 2.
Purified CD138+ cells exhibit phenotypic
features consistent with plasma cells.
At day 14 of the CD4+ T/malignant B-cell coculture,
CD138+ cells were positively purified with immunomagnetic
beads. The dotted histograms were obtained using the monoclonal
antibody (mAb) under study, and the solid histograms were obtained
using the respective isotype-matched control Ab. Histograms were
obtained from 1 of 2 independent experiments yielding similar results.
2 000 to 5 000 cells were acquired for each histogram.
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| Figure 3.
CD138+ plasma cells belong to the same
neoplastic clone than fresh malignant B cells as evidenced by IgH gene
rearrangements.
Fr3 PCR products of DNA samples were obtained from fresh malignant B
cells and from purified CD138+ cells after 21 days of the
CD4+ T/B-cell coculture. Lane 1, molecular weight markers
(sizes in base pairs); lane 2, fresh malignant B cells; lane 3, purified CD138+ cells. Results are from 1 representative
experiment of the 2 performed.
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In the literature, there are now some convincing observations
supporting the notion that B-lymphoma cells could be released from
their apparent maturation block by external signals. Cerutti et
al6 have described that in Burkitt lymphoma, a monoclonal B-cell line was induced to progress throughout a phenotypic
differentiation program that approximated the stages of early
centroblast, centroblast, centrocyte, and memory B cells, after
engagement of CD40 by CD40 ligand and exposure to IL-4 and IL-10. In
the presence of IL-6, these cells were driven to terminally
differentiated plasma cells. Recently, a second experimental model used
a follicular dendritic cell line, HK, and a lymphoma line, L3055, that
resembles centroblasts. L3055 cells proliferated continuously in the
presence of HK cells, whereas they differentiated into a population
with the phenotype of centrocytes after stimulation with CD40 ligand
and IL-4.7 In follicular lymphomas, it was formerly
reported that allogeneic T cells induced fresh malignant B cells to
secrete large amounts of Ig, and this was accompanied by loss of the
surface Ig and development of abundant intracytoplasmic
Ig.8 In 1995, Kramer et al9 reported a patient
with follicular lymphoma who showed in vivo differentiation of
malignant cells after autologous bone marrow transplantation and
treatment with IL-3. The patient developed a plasmocytosis in blood and
bone marrow accompanied by a paraprotein corresponding to the malignant
clone and high IL-6-serum concentration. It was proven that the plasma
cells were clonally related to the original lymphoma cells. The patient
recovered spontaneously and had long-lasting remission, suggesting that
terminal differentiation of malignant B cells was beneficial to the
patient. More recently, Dogan et al10 described that
follicular lymphomas contain a clonally linked but phenotypically
distinct neoplastic B-cell population in the interfollicular zone. It
was demonstrated that the immunophenotype of the neoplastic
interfollicular cells was similar to that of a subpopulation of
postfollicular B cells observed outside the follicles in normal
lymphoid tissue, with down-regulated expression of follicle-center
activation markers such as CD10, CD38, CD80, CD86, and CD95. These
cells are thought to be malignant memory B cells that have
differentiated from the malignant follicle-center B cells. Thus,
differentiation towards a cell with more mature phenotype should be a
possible occurrence in low-grade B-cell lymphoma. Marginal-zone lymphomas tend to have an indolent clinical
course.4 In the current case, the patient has remained
stable without any treatment after undergoing splenectomy 35 months
ago. The results presented here would provide an explanation for this: the tumor cells may have received signals from autotumor-reactive CD4+ T cells leading to a continuous process of
differentiation that may be beneficial to the patient. As demonstrated
in the current study, in-depth analysis of CD4+ T/malignant
B-cell conjugates in CD4+ T/malignant B-cell cocultures
should form the basis for valid evaluation of the effects of
autotumor-reactive T cells on malignant B cells: induction of
differentiation but also growth regulation/apoptosis. These immunologic
criteria would provide a strong rationale to engage individual cellular
immunotherapy with autologous CD4+ T cells in B-cell NHL.
Thierry Bonnefoix, Jian-Qing Mi, Pascal Perron, Mary Callanan, Cosima Semoun, Mireille Favre, Jean-Charles Renversez, Marie-France Sotto, Dominique Leroux, and Jean-Jacques Sotto
Correspondence: Thierry Bonnefoix, Groupe de Recherche sur les
Lymphomes, Institut Albert Bonniot, Rond-Point de la Chantourne, 38706 La Tronche, France; e-mail: thierry.bonnefoix{at}ujf-grenoble.fr
Acknowledgments
Supported by grants from the Association pour la Recherche sur le Cancer.
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