CD40-activated B cells express full lymph node homing triad and induce T-cell chemotaxis: potential as cellular adjuvants

Numerous potential tumor-rejection antigens have been identified in the past.¹  The majority of vaccination strategies use cellular adjuvants to direct T-cell responses against these target structures. Dendritic cells (DCs) as “nature's adjuvant” are a major focus due to their natural capacity to take up and present antigen and home to secondary lymphoid organs, where they effectively induce T-cell responses.²  However, difficulties to generate large amounts of DCs necessary for vaccination and to identify the optimal DC subtype have sparked an interest in alternative antigen-presenting cells (APCs).

B cells have been demonstrated to present antigen to T cells in vivo.³  CD40 activation dramatically improves antigen presentation by normal and malignant B cells and has therefore been studied as an approach to generate autologous nonartificial APCs for immunotherapy.^4,5  CD40-activated B cells (CD40-B cells) not only expand antigen-specific CD4⁺ and CD8⁺ T cells, but also prime naive T cells in vitro.^6-9  Most importantly, CD40-B cells can be generated in large amounts from small samples of peripheral blood from healthy donors and cancer patients under good manufacturing practice (GMP) conditions.⁶  CD40-B cells are therefore used by a growing number of investigators as APCs in vitro and potentially represent a complementary cellular adjuvant in DC-based immunotherapy in particular in pediatric patients¹⁰  and patients with low marrow reserve.^8,9,11 

Unlike dendritic cells, it is not the B cells' physiologic role to survey peripheral tissues and home to secondary lymphoid tissue upon antigen uptake. It therefore remains unclear whether they express the respective factors and receptors that are necessary for homing to draining lymph nodes and/or to attract T cells (eg, in the T-cell areas of secondary lymphatic organs).¹²  The requirements necessary for successful migration of antigen-presenting cells to secondary lymphoid organs have most extensively been studied for DCs. These cells mature upon encounter of antigen and certain inflammatory stimuli, down-regulate CCR1, CCR2, CCR5, CCR6, and CXCR-1, and up-regulate surface CXCR-4 and CCR-7 expression within 6 to 8 hours.^13,14  CCR1, CCR2, CCR5, CCR6, and CXCR-1 are receptors necessary for homing to inflammatory sites, while CXCR4 and CCR7 have an antagonistic function and mediate chemotaxis to secondary lymphoid organs.^13,15,16  Of interest, monocyte-derived DCs, as currently applied in cancer vaccine trials, lack l-selectin (CD62L), a key molecule implicated in the initial phase of migration through high endothelial venules. As shown in animal and human studies, homing of these cells can be very limited.^12,17,18  Accordingly, transfection of CD62L into mature DCs has been shown to improve homing properties.¹⁹ 

Here, we determined the expression of these important homing receptors on CD40-B cells and investigated whether CD40-B cells are equipped with the necessary receptors and ligands for migration to secondary lymphoid organs. To successfully interact with T cells, APCs not only need to locate to T-cell areas of lymph nodes but also must actively attract T cells. To determine the potential of CD40-B cells to actually physically interact with T cells and not only relocate to their vicinity, we assessed the expression of important T-cell attractants and determined the capacity of CD40-B cells to induce T-cell migration.²⁰ 

CD40-activated B cells and monocyte-derived DCs were generated from peripheral-blood mononuclear cells (PBMCs) as previously described.^5,6,21  Anti-CD40mab (α-CD40) and TNFα were added to the DC cultures for the last 48 hours.

Chemokine receptor expression was determined by flow cytometry using standard procedures. Antibodies were purchased from R&D Systems (Minneapolis, MN), with the exception of CCR5 (Pharmingen, San Jose, CA) and CD20 (Dako, Carpinteria, CA).

CD40-activated B cells were harvested on day 14 of culture and stored at –80°C. Target preparation and array hybridization were performed on the HG-U133A platform (Affymetrix, Santa Clara, CA) as described previously.²² 

Detection of chemokines was carried out using the TaqMan Master technology (Roche Applied Science, Mannheim, Germany) according to the manufacturer's recommendations.

For B-cell migration, 5 × 10⁵ CD40-B cells were transferred into the upper chamber of 5-μm pore size transwell plates (Costar, Cambridge, MA). Varying amounts of chemokines (R&D Systems) were added to the lower chamber. Migration of cells was assessed after 2 and 3 hours using a Coulter Z2 counter (Beckman Coulter, Fullerton, CA) and a FACS Canto (Becton Dickinson, Heidelberg, Germany). For analysis of T-cell migration, magnetic-activated cell sorter (MACS)–purified CD4⁺ or CD8⁺ T cells were used.

We assessed whether CD40-B cells express chemokine receptors up-regulated on mature DCs and associated with homing of APCs to secondary lymphoid organs (CXCR4 and CCR7) or homing to inflammatory sites.^13,23,24  To better understand the overall homing properties, we performed comprehensive gene-expression profiling of B cells before (n = 4) and after (n = 4) 14 days of CD40L/IL-4 culture using the Affymetrix technology as well as flow cytometric analysis. In contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18) on transcriptional and protein level (Figure 1A; also Table S1, available on the Blood website; see the Supplemental Tables link at the top of the online article). These receptors are crucial for homing to secondary lymphoid organs.¹²  They are implicated in rolling, activation, and adhesion to high endothelial venules via addressin (PNAD), SLC/CCL21, and ICAM-1, respectively, in order to enable extravasation.²⁵  While CD62L, CXCR4, and CCR7 were moderately down-regulated on CD40-B cells after the 14-day culture period, surface expression of CD11a/CD18 had increased.²⁶  This expression pattern was stable over at least 6 weeks of B-cell culture (data not shown). Resting and CD40-B cells lacked significant expression of CCR1, CCR2, CCR3, CCR4, CXCR1, CXCR2, CXCR3, CXCR5, and CXCR6. Upon activation, CCR5, CCR6, and CXCR3 were down-regulated (data not shown). The expression pattern of the homing molecules CD62L, CCR7/CXCR4, and LFA-1 on CD40-B cells is comparable with naive T cells and B cells.^13,26,27  Expression of these molecules in the absence of CXCR5 suggests that CD40-B cells might be able to enter the T-cell areas, but not germinal centers,²⁸  and therefore could colocalize with T cells in the T-cell–rich areas of secondary lymphoid organs.

Using a standard migration assay, we next determined whether CCR7 and CXCR4 are functional and mediate chemotaxis. In the absence of chemokines, CD40-B cells showed very little spontaneous migration. However, in the presence of increasing concentrations of CXCL12, the ligand for CXCR4, migration of CD40-B cells was significantly increased by 3.2-fold over background at a concentration of 1000 ng/mL (Figure 1B). CXCL12 is expressed in secondary lymphoid organs and has been demonstrated to mediate migration of mature DCs.²⁹  Similarly, increasing concentrations of CCL19 and CCL21, which are expressed in T-cell areas of lymph nodes and by lymphatic endothelial cells, increased CCR7-mediated migration of CD40-B cells 5.9-fold (CCL19) and 6.2-fold (CCL21) (Figure 1C).^13,30  Migration was shown to be directed only toward increasing chemokine concentrations excluding random, activation-induced locomotion (data not shown). Taken together, these data suggest that ex vivo–generated CD40-B cells have the capacity to migrate in response to chemokines important for homing to secondary lymphoid organs.

To induce immunity, antigen-presenting cells need to physically encounter T cells in a coordinated migration, which is particularly regulated by chemokine gradients in the T-cell areas of secondary lymphoid organs.¹²  The induction of T-cell chemotaxis by APCs has been studied extensively for DCs. Maturation stimuli such as LPS, dsDNA, and IL-1β induce significant alterations of chemokine profiles and ensure efficient antigen presentation to T cells. In order to determine the profile of chemoattractants secreted by CD40-B cells, we determined the expression of known T-cell attractants and chemokines produced by mature DCs. Expression of chemokines CCL17 (TARC), CCL5 (RANTES), CCL22 (MDC), and CXCL10 (IP-10) was detected using comprehensive gene-expression analysis (Table S2) and RT-PCR (data not shown).

To assess whether this array of chemokines expressed by CD40-B cells actually induces T-cell migration, we performed standard transwell migration assays. Highly purified CD4⁺ or CD8⁺ T cells were isolated from peripheral blood, and chemotaxis was determined in response to supernatant from CD40-B cells. T cells were transferred into the upper chamber containing fresh B-cell medium and allowed to migrate for 2 hours. When supernatant from CD40-B cells was added to the lower chamber, T-cell migration increased 3.1-fold for CD4⁺ and 2.5-fold for CD8⁺ T cells, clearly demonstrating the chemotactic potential of CD40-B cells. As controls, CXCL12 and CCL21 as well as supernatant from monocyte-derived immature and mature DCs were used (Figure 2A). Migration was shown to be directed toward increasing concentrations of supernatant, thus excluding random, activation-induced locomotion (data not shown).

Blocking experiments using single monoclonal antibodies against the chemokine receptors of interest did not lead to significant blockade of migration (data not shown), supporting the notion that numerous chemokine-chemokine receptor interactions are responsible for strong T/B-cell interactions and/or that chemokine-independent mechanisms play a role (data not shown).

Phenotypic analysis of migrated T cells demonstrated that SDF-1α and SLC did not induce migration of a particular subgroup, while supernatants from CD40-B as well as immature and mature monocyte-derived DCs promoted migration of effector memory T cells. Correspondingly, chemotaxis of naive T cells was reduced under these conditions, in particular when supernatant from CD40-activated B cells was used (Figure 2B). Based on these findings, a combined approach using DCs for priming and CD40-B cells for frequent, high-dose expansion of antigen-experienced cells might be envisioned.

These data strongly suggest that in vitro–generated CD40-activated B cells are equipped with the respective chemokines and receptors, allowing them to home to secondary lymphoid organs to attract T cells.