Restoration of CCL21 Accelerates Lymph Node (LN) and Spleen Regeneration Post-Transplant and Improves Anti-Pathogen and Anti-Tumor Effector Responses
Authors: Stefanski Heather E , Goren Emily , Jonert Leslie , Mule James J. , Blazar Bruce R. 
Published: October 2019

Abstract
Abstract 212

Introduction:
BMT conditioning causes substantial stromal cell injury, which may hinder lymphopoiesis and immune recovery post-transplant. The C-C chemokine CCL21, produced by LN fibroblastic reticular cells (FRCs), is critical for the trafficking of CCR7 expressing T cells into secondary lymphoid organs (i.e. spleen, LN). We first reported that radiation conditioning markedly reduces FRCs, CCL21 expression, and lymphoid tissue inducer cells that are critical for lymphoid organogenesis. As a consequence of these events, mice have substantial T cell lymphopenia and poor responses to pathogens and neoantigens (Kelly et al, Blood, 2010). We hypothesized that CCL21 deficiency would preclude the recruitment of recent thymic emigrants to secondary lymphoid organs where they would receive necessary signals for their survival. Moreover, within the secondary lymphoid organs, T cells would be unable to properly migrate to B cell zones. Thus, we tested whether restoring CCL21 protein could improve LN stromal cell architecture, T cell trafficking and consequently, T cell effector responses.

Methods/Results:
C57BL/6 mice were lethally irradiated, rescued with congenic T cell depleted bone marrow, and given CCL21 cDNA containing or null adenovirus vector transduced dendritic cells (DCs) that were pulsed with lysates from either vesicular stomatitis or attenuated Listeria monocytogenes expressing the nominal antigen ovalbumin (VSV-OVA; Listeria-OVA). The DC vaccines were given intramuscularly beginning 21 days post-transplant and then weekly for three total doses. On day 42, mice were challenged with either VSV-OVA or Listeria-OVA and euthanized on day 43 or 50, respectively. Compared to DC/null, DC/CCL21 vaccine substantially improved spleen and LN architecture and markedly increased ER-TR7+ LN stromal cell density. These architectural changes were associated with significantly increased CCL21 expression (p<0.05 for both CCL21 density per B cell area and normalization to non-BMT controls determined by ELISA), resulting in a significant increase in total LN cells, CD8+ T cells and lymphoid tissue inducer cells (all p<0.05). Expression of the B cell chemoattractant, CXCL13, and B cell follicle number were significantly increased (p<0.05) in the spleen and LN of DC/CCL21 vaccinated mice and T cells were found juxtaposed to B cells within B cell zones. Compared to DC/null, DC/CCL21 LN CD8+ T cell number normalized vs non-BMT controls, and clearance of Listeria-OVA by the liver was significantly (p<0.05) improved and similar to non-BMT controls (3.5 vs 1.8 vs 1.2 × 105 colony-forming units, respectively). Similarly, VSV-OVA clearance was significantly improved in DC/CCL21 vs DC/null vaccines, both less than non-BMT controls (13.5 vs 26 vs 8 splenic plaque-forming units, respectively; all group-wise comparisons, p<0.05). In order to determine whether improved immune function could enhance the endogenous CD8+ T cell response to acute myeloid leukemia (AML) challenge post-BMT, congeneic BMT recipients were vaccinated with DC/CCL21 or DC/null pre-loaded with AML lysates. All mice received a lethal dose (106) of C57BL/6 AML cells expressing firefly luciferase (C1498) on d.42 post-BMT (see Figure). Compared to the uniform lethality in both DC/null vaccines and non-vaccinated, non-BMT controls, mice receiving DC/CCL21 had a significant increase (p=.0241 compared to DC/null) in survival with 50% of mice surviving long-term.

Conclusions:
Taken together, these data indicate that DC/CCL21 vaccines can potently restore LN and splenic architecture as well as improve endogenous T cell responses to pathogens and AML challenge. Because DC/CCL21 vaccines are in clinical trial for patients with lung cancer or melanoma, our studies may provide the foundation for future clinical trials of DC/CCL21 vaccination in patients receiving pre-transplant conditioning regimens. If our preclinical data prove translatable, DC/CCL21 vaccination could increase immune reconstitution in BMT recipients. This could lead to decreased relapse and increased pathogenic responses to life-threatening infections and decreasing morbidity and mortality post transplant.

Disclosures:
No relevant conflicts of interest to declare.