Author response for "Non invasive in vivo monitoring of dimethyl fumarate treatment in EAE by assessing the glucose metabolism in secondary lymphoid organs"

Abstract CD4+CD25+ regulatory T cells (Treg) mediate alloresponses in murine models of bone marrow transplantation (BMT), leading to protection from graft-versus-host disease (GvHD). However, in vivo migration and tissue localization of Treg during this inflammatory response remain unclear. We previously demonstrated co-localization of Treg with effector T cells (Tcon) with initial expansion in secondary lymphoid organs prior to migration into inflamed tissues in a major MHC-mismatched BMT model. To explore the stimuli for Treg proliferation, we evaluated the role of the allogeneic environment by transferring FVB donor luciferase-expressing (luc+) Treg into lethally-irradiated syngeneic recipients. Unlike the allogeneic irradiated setting where Treg expand in the presence or absence of Tcon, adoptively transferred luc+ Treg were not detected in secondary lymphoid organs of syngeneic lethally-irradiated BMT recipients by in vivo bioluminescence imaging (BLI). Syngeneic luc+ Tcon also had significantly different in vivo dynamics, with a 4 day delay and only moderate expansion in lymph nodes. Proliferation was not detected in the spleen, unlike their allogeneic Tcon counterparts, nor in the bone marrow compartments, as seen in lymphopenic models. To assess whether irradiation induced the observed in vivo dynamics of Treg in the allogeneic setting, we transferred FVB luc+ Treg or luc+ Tcon into unirradiated Balb/c Rag2−/−gamma chain (γC) −/− recipients, which lack T, B, and NK cells. After adoptive transfer into Rag2−/−γC−/− recipients, robust Tcon proliferation was observed in secondary lymphoid organs and the bone marrow compartments; however, Treg expansion was weak, and specific localization to lymphoid or nonlymphoid tissues was not observed. Treg were stimulated to localize to and expand in secondary lymphoid organs by the co-transfer of Tcon in unirradiated Rag2−/− (γC) −/− or by conditioning Rag2−/− (γC) −/− recipients with irradiation. Exogenous IL2 administration two weeks following luc+ Treg transfer into unirradiated Rag2−/− (γC) −/− recipients similarly led to localization and expansion of Treg in secondary lymphoid organs. These studies indicate the critical role of proinflammatory cytokines, such as IL2, generated either by irradiation-induced tissue damage or donor Tcon, in the expansion and localization of Treg. Differences between Tcon and Treg expansion in syngeneic or unconditioned allogeneic Rag2−/− γC−/− hosts suggest an important role of conditioning with irradiation alone or in concert with the allogeneic environment, in providing distinct signals for Tcon versus Treg activation, proliferation, and localization.

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CD40 Ligand Activation Alters B Cell Migration to Secondary Lymphoid Organs.

Blood ◽

10.1182/blood.v108.11.935.935 ◽

2006 ◽

Vol 108 (11) ◽

pp. 935-935

Author(s):

Yvonne A. Efebera ◽

Tahamtan Ahmadi ◽

Amanda Flies ◽

David H. Sherr

Keyword(s):

B Cells ◽

Lymph Nodes ◽

B Cell ◽

Cd40 Ligand ◽

Lymphoid Organs ◽

Cell Populations ◽

Secondary Lymphoid Organs ◽

Activated B Cells

Abstract Background: An increased understanding of the requirements for antigen presentation has encouraged development of cell-based cancer vaccines. Trials using dendritic cells (DC) as antigen presenting cells (APC) for immunotherapy of several malignancies have shown considerable success. However, the difficulty in generating large numbers of DC required for these immunizations has led to the search for alternative APC. One such candidate is the CD40 ligand (CD40L)-activated B cell, populations of which can readily be expanded in vitro. To be an effective vehicle for antigen presentation to T cells, CD40L-activated B cells must be capable of migrating to secondary lymphoid organs. Therefore, CD40L-activated B cell migration following subcutaneous or intravenous injection was evaluated. Methods: Splenic B cells from GFP transgenic mice were activated with CD40L + IL-4 and expanded in vitro prior to i.v. or s.c. injection of 3–4 x 107 into C57BL/6 mice. Recipient mice were sacrificed 2 hrs or 1–14 days thereafter and the percentage of GFP+/B220+ B cells quantified in spleens and lymph nodes by flow cytometry. Localization of these cells within lymphoid organs was determined by immunohistochemistry. In some experiments, activated C57BL/6 B cells were labeled with carboxy fluorescein succinimidyl ester (CFSE) to evaluate cell growth in vivo. Results: Murine B cell populations were readily expanded by culture on CD40L-transfected L cells in the presence of IL-4. CD40L-activated B cells expressed high levels of CD80, CD86, and LFA-1 but decreased levels of L-selectin relative to naive cells. Following i.v. injection, activated B cells were detected in spleens and lymph nodes within 1 day. Peak concentrations of activated B cells were noted in spleens and lymph nodes on days 7 (4.8% of injected cells) and 10 (1.25% of injected cells) respectively, suggesting expansion of the activated B cell population in vivo. Naive B cells injected i.v. were detected within 1 day but their number declined precipitously thereafter. Following s.c. injection, peak levels of CD40L-activated B cells were noted on day 5 (spleens) and day 7 (lymph nodes). As determined by immunohistochemistry, both CD40L-activated and naïve B cells injected i.v. appeared in B cell regions of spleens and lymph nodes. While the kinetics of accumulation of CD40L-activated B cells injected s.c. or i.v. were similar, s.c. injected CD40L-activated B cells homed to the T cell regions of spleens and lymph nodes. CFSE experiments indicated that these activated B cells continue to grow in vivo. In contrast, naïve B cells injected s.c. only appeared in B cell regions. Conclusion: CD40L-activated B cell populations can readily be expanded in vitro, CD40L-activated B cells migrate to secondary lymphoid organs even when injected s.c., activated B cell populations expand in vivo, and s.c. injected, CD40L-activated B cells preferentially home to T cell regions of secondary lymphoid organs. These results suggest that this effective APC may serve as an important vehicle for delivery and presentation of exogenous (e.g. tumor) antigens to T cells in vivo.

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