Intratumoral rhIL-12 administration in head and neck squamous cell carcinoma patients induces B cell activation

Int. J. Cancer: 123, 2354–2361 (2008) ' 2008 Wiley-Liss, Inc.

Intratumoral rhIL-12 administration in head and neck squamous cell carcinoma patients induces B cell activation Carla M.L. van Herpen1*, Robbert van der Voort2, Jeroen A.W.M. van der Laak3, Ina S. Klasen4, Aniek O. de Graaf5, Leon C.L. van Kempen3, I. Jolanda M. de Vries2, Tjitske Duiveman-de Boer2, Harry Dolstra5, Ruurd Torensma2, Johan H. van Krieken3, Gosse J. Adema2 and Pieter H.M. De Mulder1  1 Department of Medical Oncology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands 2 Department of Tumor Immunology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands 3 Department of Pathology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands 4 Department of Blood Transfusion and Transplantation Immunology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands 5 Central Hematology Laboratory/Department of Hematology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands The objectives of this study were to investigate the effects of intratumorally (i.t.) administered recombinant human interleukin-12 (rhIL-12) on the distribution and function of B cells in the primary tumors, the locoregional lymph nodes and peripheral blood of head and neck squamous cell carcinoma (HNSCC) patients. The initial characterization of the patients participating in the phase Ib and phase II studies has previously been reported. After rhIL-12 treatment, fewer secondary follicles with a broader outer region of the mantle zones and an increase in interfollicular B-blasts were seen in the enlarged lymph nodes compared with control HNSCC patients. The size of the germinal center (GC) was diminished, partly due to a decrease in the number of CD571 GC cells that have been associated with immune suppression. These changes did not correlate with signs of apoptosis or CXCR5 expression by B cells. Strikingly, in 3 out of 4 IL-12 treated patients, increased IFN-c mRNA expression by B cells was detected. In addition, a highly significant IgG subclass switch was seen in the plasma with more IgG1, less IgG2 and more IgG4, indicating a switch to T helper 1 phenotype. Finally, peritumoral B cell infiltration was a positive prognostic sign for overall survival in the 30 HNSCC patients investigated, irrespective of IL-12 treatment. In conclusion, these data indicate that after i.t. IL-12 treatment in HNSCC, significant activation of the B cell and the B cell compartment occurred and that the presence of tumor infiltrating B cells correlated with overall survival of HNSCC patients. ' 2008 Wiley-Liss, Inc. Key words: interleukin-12; B cell; head and neck squamous cell carcinoma; secondary follicles; germinal center

Interleukin-12 (IL-12) is a heterodimeric cytokine that consists of 2 disulfide-linked subunits, i.e., IL-12p40 and IL-12p35.1 IL-12 has a wide range of biological activities1,2 and has effects on both the innate and adaptive immune system. It stimulates the proliferation and activation of cytotoxic T lymphocytes (Tc) and natural killer (NK) cells and induces the production of cytokines, especially IFN-g.3–5 IL-12 is the key cytokine in the induction of T helper 1 (Th1) responses and thereby of cellular immunity. Furthermore, it inhibits angiogenesis.6 Because of these abilities, IL12 was used in preclinical models of antitumor immunotherapy. IL-12 induced regression of established primary tumors, inhibited the formation of tumor metastases and prolonged the survival of tumor-bearing mice.7–10 Several phase I11–16 and phase II studies in various cancer types17–20 have been performed, but only a limited number of clinical responses were observed. In most clinical studies, the immunological effects of IL-12 were studied in peripheral blood. However, after IL-12 administration a transient lymphopenia with a redistribution of activated immune cells from the peripheral blood to the lymph nodes (and primary tumor) occurred.21 Thus, monitoring of the immune effects of IL-12 therapy solely in the peripheral blood can be misleading. Recently, we have performed 2 clinical studies in which we administered rhIL-12 intratumorally (i.t.) in head and neck squamous cell carcinoma (HNSCC) patients. HNSCC is an immunoPublication of the International Union Against Cancer

genic tumor and provides an excellent model to study the effects of locoregional immunotherapy. rhIL-12 was administered i.t. rather than systemically because of the expected higher locoregional concentration in the primary tumor and the draining lymph nodes, which may enhance efficacy with less systemic toxicity. First, we performed a phase Ib study in pretreated HNSCC patients with the objective of determining toxicity and to investigate the pharmacokinetic and pharmacodynamic effects in peripheral blood after IL-12 administration.16 Thereafter, we performed a phase II study in nonpretreated HNSCC patients, in which IL-12 was administered before surgery to evaluate the immunological effects in locoregional lymph nodes, primary tumor and peripheral blood.20,21 The largest effect was seen on NK cells, with a higher number in the primary tumor and a high IFN-g mRNA expression in the lymph nodes. An excessive peritumoral CD201 B cell infiltration was observed in some patients. No differences were seen between IL-12-treated and control patients in the numbers or distribution of CD81 or CD41 T lymphocytes, but induction of IFN-g mRNA expression was seen in these T cells. Interestingly, major differences were apparent in the architecture of the enlarged lymph nodes of IL-12 treated patients. Besides fewer primary and secondary follicles with smaller germinal centers (GCs), a redistribution of B cells was also observed in lymph nodes of IL-12treated patients. In addition, a decrease of DC-LAMP1 cells (dendritic cells) in the paracortex was noticed, resulting in a reduction of paracortical hyperplasia. In lymph nodes primary follicles contain mostly mature, naive B lymphocytes. Germinal centers (GC) develop following antigenic stimulation; these follicles are called secondary follicles. In the GC B cell proliferation, selection of B cells producing high-affinity antibodies and generation of memory B cells occurs. Other cells in the GC are follicular dendritic cells (FDCs), CD571 Th cells and CD681 tingible body macrophages. The GCs are sites of tremendous apoptosis, because B cells that do not express high affinity receptors for antigen will not survive. The anatomic segregation of the T and B cells is dependent on chemokines and cytokines. Naive T cells and dendritic cells (DCs) express a chemokine receptor CCR7 and are attracted to the T cell zones, where CCL19 and CCL21 are produced, which attract the CCR71 cells. Naive B cells and CD571 Th cells express another chemokine receptor, CXCR5, that recognizes CXCL13 produced only in the follicles.22 CXCR4 is involved in the homing of antibody secreting cells.23 In preclinical models, IL-12 is shown to have an effect on humoral immunity. IL-12 stimulates B cell growth by inducing IFN 

—Deceased. *Correspondence to: Department of Medical Oncology, Radboud University Nijmegen Medical Centre, P.O. Box 9101 HB, 6500 HB Nijmegen, The Netherlands. E-mail: [email protected] Received 6 July 2007; Accepted after revision 8 May 2008 DOI 10.1002/ijc.23756 Published online 26 August 2008 in Wiley InterScience (www.interscience. wiley.com).

rhIL-12 ADMINISTRATION INDUCES B CELL ACTIVATION

g production in B cells in vitro, causes Ig isotype selection and triggers a cascade of molecular events in human B cells similar to Th1 commitment.26 The effects of IL-12 on B cells in cancer patients have hardly been studied. The objectives of this study were to study the effects of IL-12 on the presence and distribution of cells in the follicles, apoptosis, chemokine receptor expressions on B and T cells, and B cell functions in patients with HNSCC. 24

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Material and methods Patient selection and treatment schedule phase Ib and phase II study In the phase Ib study, all patients (n 5 6) had histological proof of recurrent HNSCC with either local recurrence or subcutaneous or lymph node metastases, not amenable for curative surgery or radiotherapy.16 In the phase II study, all patients (n 5 10) had histological proof of HNSCC, with the primary tumor in the oral cavity or oropharynx, staged as T1-4, N0-2, M0, for which surgical resection including a supra-omohyoid or radical neck lymph node dissection was planned. Patients were previously untreated, i.e., they did not have prior surgery, radiotherapy, or any systemic therapy.20 In both studies the tumor, with a diameter not exceeding 5 cm, had to be accessible for local injection. The other eligibility criteria have been described previously.16,20 Both studies were single center, open-label, nonrandomized studies. rhIL-12 was supplied by Wyeth (Cambridge, MA) and administered by i.t. injection. rhIL-12 was administered weekly at 2 dose levels of 100 and 300 ng/kg body weight, by single or multiple i.t. injections. In the phase Ib study, patients were treated for a period of minimal 8 and maximal 24 weeks. In the phase II study, patients were treated in the normal waiting procedure prior to surgery and received 2 or 3 injections, of which the last was administered 24 hr before surgery. To allow comparison of the immunological parameters of the rhIL-12-treated HNSCC patients in the phase II study, blood samples, lymph nodes and primary tumor resection material from 20 control patients were also collected; these patients were eligible for the study, but preferred not to receive the rhIL-12 injections. All patients have given written informed consent. The local regulatory committee approved the studies. Handling of the resected material and tissue preparation Material from the primary tumor and lymph nodes of patients in the phase II study was put on ice immediately after resection. The pathologist cut out the lymph node specimen freshly. The neck was divided into 6 lymph node regions (I–VI), from which all lymph nodes were collected. Depending on the size of the lymph node, parts were taken for direct snap freezing or single-cell suspension preparation in order to perform flow cytometry or RTPCR. If the primary tumor was sufficiently large, samples were taken for flow cytometry and a sample was snap frozen. Then the primary tumor was fixated in 4% (v/v) phosphate buffered formalin and cut the following day, or after decalcification. The remaining part of the lymph nodes were fixated in unifix, dehydrated and embedded in paraffin. Tissue sections of 4 lm were cut, mounted onto glass slides pretreated with 2% 3-aminopropyltriethoxysilane (Sigma) and dried overnight. Serial sections were stained with haematoxylin and eosin (H&E) or processed for immunohistochemistry. Immunohistochemical staining and scoring One lymph node of 10 IL-12-treated and 10 control patients was subjected to immunohistochemical analysis. Selection of the control patients was based on patient and tumor characteristics and was comparable with the 10 IL-12 treated patients. The lymph nodes of both groups were selected based on histological features and size. The average size, number of primary and secondary follicles, the degree of paracortical hyperplasia and sinushistiocytosis were identical in H&E stained specimens and immunohistochemi-

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cal stained specimens in each group. All chosen lymph nodes were without metastases. Tissue sections were incubated with the following primary antibodies: anti-CD4 (Clone 1F6, 1:80, Lab Vision-Neomarkers, Immunologic, Duiven, The Netherlands), anti-CD20 (Clone L26, 1:500, Dakocytomation, Heverlee, Belgium), anti-CD21 (Clone 1F8, 1:200, Dakocytomation, Heverlee, Belgium), anti-CD57 (Clone NC1, 1:5, Immunotech/Coulter, Mijdrecht, The Netherlands), anti-CD68 (Clone Kp1, 1:2,000, Dakocytomation, Heverlee, Belgium), MIB (Clone Mib-1, 1:200, Dakocytomation, Heverlee, Belgium) and caspase-3 (Clone 19, BD Pharmingen, Erembodegem, Belgium). In brief, sections were deparaffinized in xylene and rehydrated. Endogenous peroxidase was blocked with 0.3% hydrogen peroxide in methanol for 20 min. Antigen retrieval was performed using 0.1 M citrate buffer pH 6.0 (anti-CD20, anti-CD57 and MIB), microwave cooking in 0.1 M EDTA pH 8.0 (anti-CD4), or fresh pronase 0.1%/PBS (anti-CD21, caspase-3), respectively. Subsequently, sections were preincubated with 1% bovine serum albumin in PBS. Following overnight incubation with the primary antibody, the secondary biotin-conjugated antibody and a tertiary complex of streptavidin-avidin-biotin conjugated to 3-amino-9ethyl-carbazole were applied. Finally, the sections were counterstained with Mayer’s haematoxylin. Incubation with phosphatebuffered saline instead of the primary antibody served as a negative control. The numbers of CD41 GC cells and CD681 GC cells were scored semi-quantitatively, using 3–5 categories: none/few, moderate, many/extensive. Quantitative analysis of immunohistochemical staining of the lymph nodes To determine the number of CD571 GC cells, CD211 cells, MIB1 GC cells and to determine the size of the GCs a quantitative analysis was performed. All image acquisition and processing was performed using custom written macros in KS400 image analysis software (version 3.0, Carl Zeiss, Germany). The method of analysis has been described previously.21 Total IgG and IgG subclasses measurement In the phase Ib study, venous blood was collected in week 1, before the first injection, and in week 8, before the eighth injection. Whole blood samples anticoagulated with citrate were obtained for determination of total IgG and IgG subclasses. Total IgG was measured on the Beckman Coulter (Mijdrecht, The Netherlands) Image Nefelometer using Beckman Coulter reagents. Calibration was on the international standard CRM470. IgG subclasses were measured on a BNII Nefelometer (Dade Behring, Brussels, Belgium) using the reagents of The Binding Site (Birmingham, England). Calibration was performed on a CLB (Amsterdam, The Netherlands) standard H1234. Quantitative real-time PCR of IFNc mRNA expression CD41, CD81, CD191 and CD561 cells were FACS-sorted from lymph node suspensions and the fraction of each cell type in the total suspension (fi) was analyzed. Subsequently, RNA was isolated from 2,000 to 5,000 cells, reverse transcribed and subjected to quantitative real-time PCR analysis (TaqMan, Applied Biosystems) as has been reported previously.20 To quantify IFN-g and PBGD-R2 copy number in a given sample, CT values of 2 independent experiments were extrapolated in standard curves generated by serial dilution of plasmid containing amplicons of IFN-g and PBGD-R2, respectively. Subsequently, the IFN-g/PBGD-R2 ratio (r) in CD41, CD81, CD191 and CD561 cells in a locoregional lymph node of control (n 5 4) and IL-12-treated (n 5 4) patients was determined and multiplied by the fraction of that population in the lymph node from which they were isolated (rfi). The relative contribution of a cell type to the total IFN-g expression in a lymph node is expressed the percentage rfi/Rrfi.

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TABLE I – PRIMER AND PROBE SEQUENCES OF BCL-2 AND FAMILY MEMBERS USED FOR QUANTITATIVE PCR

PBGD forward primer PBGD reverse primer PBGD probe Bcl-2 forward primer Bcl-2 reverse primer Bax forward primer Bax reverse primer Bcl-xL forward primer Bcl-xL reverse primer Bak forward primer Bak reverse primer Bid forward primer Bid reverse primer

50 -ggcaatgcggctgcaa-30 50 -gggtacccacgcgaatcac-30 50 -ctcatctttgggctgttttcttccgcc-30 50 -tcgccctgtggatgactga-30 50 -cagagacagccaggagaaatca-30 50 -gagcggcggtgatgga-30 50 -tggatgaaaccctgaagcaaa-30 50 -ccacttacctgaatgaccacctaga-30 50 -cagcggttgaagcgttcc-30 50 -tgagtacttcaccaagattgcca-30 50 -agtcaggccatgctggtagac-30 50 -ccttgctccgtgatgtctttc-30 50 -tccgttcagtccatcccattt-30

RNA isolation, cDNA reaction and quantitative PCR for determination of Bcl-2 family members RNA was isolated using RNAzol B (Campro Scientific, Veenendaal, The Netherlands). Reverse transcription was performed with 1 lg RNA in a total mixture of 20 ll first strand RT buffer with 10 mM dithiothreitol (DTT), 625 lM dNTPs, 2.5 ng/ll oligo dT (all from Life Technologies, Gaithersburg, MD), 0.1 lg/ll random hexamers (Amersham Pharmacia, Piscataway, NJ), 20 U RNAsin (Promega, Madison, WI) and 200 U M-MLV reverse transcriptase (Life Technologies, Gaithersburg, MD). This reaction mixture was incubated at 20°C for 10 min, at 42°C for 45 min and at 95°C for 10 min. Subsequently, 80 ll of H2O was added and samples were stored at 280°C. Quantitative PCR was performed with the ABI/PRISM 7700 Sequence Detection System (Applied Biosystems, Foster City, CA). Primer and probe sequences are given in Table I. PCR conditions were as follows: 10 min at 95°C followed by 45 cycles of 15 sec at 95°C and 1 min at 60°C, with data collection in the last 30 sec. All PCRs, except those for the reference gene PBGD, were performed in SYBR Green PCR buffer with 125 lM dNTP mix, 1.25 U AmpliTaq Gold and 5 mM MgCl2 (Applied Biosystems, Foster City, CA). The PCR for PBGD was performed in TaqMan Universal PCR MasterMix with 200 nM Vic-labeled (6-carboxyrhodamine) PBGD probe (Applied Biosystems). Primer concentrations for the PCRs were 300 nM. All PCRs were performed in a total volume of 50 ll. PCR products were checked by gel-electrophoresis. Western blot analysis of Bcl-2 Cell pellets were resuspended in lysis buffer, containing 142.5 mM KCl, 5 mM MgCl 2, 10 mM Hepes pH 5 7.3, 1 mM EGTA, 1% Triton X-100, 0.5% Nonidet-P40 and freshly added protease inhibitors (0.3 lM aprotinin, 0.3 lM pepstatin A, 10 lM leupeptin and 0.2 mM phenylmethylsulphonyl fluoride and kept on ice for 45 min. Proteins were separated by gel-electrophoresis on 14% tris-glycine gels and transferred to polyvinylidene difluoride membranes. Bcl-2 was stained using an anti-human Bcl-2 monoclonal antibody (clone 124, Dako Diagnostics, UK). Immunophenotyping with flow cytometry The following moAb were used for immunofluorescence analysis: Cy5-conjugated anti-CD3 (UCHT1), anti-CD4 (13B8.2), antiCD8 (B9.11), anti-CD19 (J4.119) (Beckman Coulter, Mijdrecht, The Netherlands), FITC-conjugated anti-CD57 (Leu7) (BD Biosciences, Erembodegem, Belgium), and PE-conjugated antiCXCR5 (51505), anti-CXCR4 (12G5), CCR7 (150503) (R&D systems, Abingdon, UK). Cells were incubated with the appropriate concentration of moAb in PBS supplemented with 20% pooled human serum and 0.1% NaN3 (4°C, 30 min). Cells were washed in PBS supplemented with 1% BSA and analyzed on an Epics XL flowcytometer (Beckman Coulter, Fullerton, CA). Statistics Nonparametric Mann-Whitney U-tests were performed to compare 2 independent samples. Spearman’s correlation coefficient

was calculated for correlation analysis. Paired student t-test was used for calculating differences in the IgG subclasses between week 1 and 8 in each patient. The paired student t-test was suitable, because the values were normally divided and the standard deviation was not different. Overall survival (OS) was calculated from the date of surgery and estimated using the Kaplan-Meier method.27 The comparison of survival parameters was performed using the log-rank test.28 For all tests, p < 0.05 was considered to be statistically significant. All statistical analysis was performed with 2-tailed tests using SPSS 11.0 for Windows. Results Changed architecture of lymph nodes after IL-12 treatment As described previously, histopathological analysis of lymph nodes derived from IL-12-treated and control patients revealed apparent architectural differences. Although lymph nodes of IL-12 treated patients were enlarged, fewer primary and secondary follicles with smaller GCs were observed without significantly affecting the number of B cells present.21 Routinely H&E-stained histopathological sections showed a broader outer region of the mantle zone of secondary follicles in the IL-12-treated lymph nodes compared to the control lymph nodes (Fig. 1). Furthermore, a high number of CD201 centroblasts, i.e., interfollicular B-blasts, was located outside the GCs in IL-12-treated patients (Fig. 2). Occasionally, CD201 B cells were detected in the sinus around the lymph node (Fig. 2). The IL-12 treated lymph nodes sometimes showed bizarre GCs with the follicular dendritic cells (FDCs) located outside instead of inside the GC (Fig. 2). Because it was not possible to count the CD211 cells due to the shape and the staining pattern of the FDCs, we determined the surface area of the CD211 regions in the GCs in the lymph nodes, which was not different between the IL-12-treated and control patients (Table II). However in some patients the FDCs were less densely positioned inside the GC (Fig. 2). The number of CD571 GC cells was assessed quantitatively. The median number of CD571 cells in the GC was strongly decreased in IL-12-treated patients compared to control patients (10.3 vs. 31.4, respectively (p < 0.05) (Table II and Fig. 1)), whereas the number of CD571 cells outside the GC did not differ (as described earlier21). A semi-quantitative scoring of CD41 cells in the GCs, partly the same cells as the CD571 GC cells, showed a trend to fewer CD41 GC cells (mean scoring of 1.4 and 2.2 in IL-12 treated and control patients, respectively). The number of CD681 tingible body macrophages did not differ. Also, the number of MIB1 cells, a proliferation marker, did not differ outside the GC between IL-12 treated and control patients (data not shown). The number of MIB1 cells in GCs was so high that it was impossible to count these proliferating cells. In conclusion, the changed architecture of the secondary follicles in the lymph nodes of IL-12-treated patients is caused mainly by changes in B cell formation and fewer CD571 GC cells. No signs of altered apoptosis or chemokine receptor expression in lymph node derived B and T cells To investigate whether apoptosis played a role in the changed architecture after IL-12 treatment, mRNA expression of Bcl-2 and other members of the Bcl-2 family in mononuclear cells isolated from the lymph nodes were determined. No differences were found in Bcl-2 or Bcl-xl, known as antiapoptotic molecules, and Bax, Bak or Bid known as proapoptotic members between 5 IL-12 treated and 5 control patients (data not shown). Moreover, the protein expression of Bcl-2 in purified lymph node derived T and B cells did not differ between both groups and thus confirmed the RNA expression data (data not shown). Furthermore, active caspase-3 on immunohistochemical sections of lymph nodes did not differ between IL-12 treated and control patients (data not shown). As the expression of CXCR5 and CXCR4 on T and B cells is important for the distribution and traffic of these cells to and

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FIGURE 1 – Differences in the germinal centers of IL-12 treated (a, c and e) and control patients (b, d and f). Panel a and b show hematoxylin stained tissue sections (350). The outer region of the mantle zone is broadened in IL-12 treated patients. Staining with CD21 (c and d) show the less grouped CD211 cells in germinal centers in IL-12 treated patients (3100). Staining with CD57 (e and f) shows the diminished presence of CD571 cells in germinal centers in IL-12 treated patients (3100).

within the lymph nodes, we also determined their expression in 3 IL-12-treated and 3 control patients. No apparent differences were seen in the mean expression of CXCR4 on B (51.9% vs. 58.8%), Th (21.8% vs. 14.9%) and Tc (5.2% vs. 7.0%) cells, and CXCR5 on B (68.7% vs. 67.9%), Th (8.1% vs. 5.2%) and Tc cells (3.6% vs. 5.0%) in IL-12-treated versus control patients, respectively. Altogether, these results do not support a role for apoptosis or altered chemokine receptor expression in the observed lymph node architecture following IL-12 treatment. B cell activation with IFN-c mRNA expression and IgG subclass switch The altered distribution of B cells in the lymph nodes of IL-12treated patients suggested activation of these cells in response to IL-12. Therefore, the IFN-g mRNA expression in purified lymph node derived B cells and the amount of the different IgG subclasses in plasma were determined.

Previously, we showed that the IFN-g mRNA expression in the mononuclear cells of the lymph nodes was more than 100 times higher in IL-12-treated patients than in control patients.20 To investigate whether in B cells IFN-g mRNA expression was increased after IL-12 treatment, quantitative real-time PCR analysis of IFN-g expression on FACS-sorted cell populations of locoregional lymph node suspensions was performed. In the IL-12 treated patients the relative contribution to IFN-g expression of lymphoid subsets revealed an increase in the relative contribution of CD191 and CD561 cells after i.t. IL-12 injection. The highest expression was seen in the CD561 NK cells, whereas the largest relative change was seen in CD191 B cells (Fig. 3). The B cells of all 4 control patients did not express IFN-g mRNA, whereas in 3 out 4 IL-12 treated patients IFN-g expression was readily detected in their B cells. Next IgG subclasses were analyzed in the plasma of 5 patients treated in the phase Ib study, because in this study patients were treated for a longer period than in the phase II study. In all these

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FIGURE 2 – Lymph nodes of IL-12 treated patients. CD20 staining show interfollicular B-blasts that are lying between follicles ( ) (a) and a vessel full of B cells (b) (3200). FDCs are stained with CD21 and show the bizarre germinal centers with the localization of FDCs outside the germinal center (c) (3100) and (d) (3200).

TABLE II – QUANTITATIVE ANALYSIS OF CD571 AND CD211 CELLS IN GERMINAL CENTERS (GCS) IN LYMPH NODES OF IL-12 TREATED AND CONTROL PATIENTS

2

Total size of lymph node (mm ) Mean number of GCs Sum of surface areas of GCs (mm2) Surface areas of GCs/total size of LN Mean size of GC (mm2) Median size of GC (mm2) Total number CD571 cells in GCs Mean number of CD571 cell per GC Median number of CD571 cell per GC Sum of surface areas of CD211 regions Surface area of CD211 regions/total sizeof LN Mean size of CD211 area Median size of CD211 area Mean number of CD211 areas as GC

IL-12 treated (n 5 10)

Control (n 5 10)

24.6 6.2 0.10 0.39 0.011 0.010 98 12.2 10.3 0.33 1.63 0.019 0.013 6.6

21.1 8.3 0.31 1.38 0.032 0.022 447 45.4 31.4 0.45 2.62 0.026 0.016 7.5

p