Flt3-Ligand Rhesus Macaque
Description
The Flt3 Ligand Rhesus Macaque is purified by proprietary chromatographic techniques.
Product Specs
Q&A
What is Flt3-Ligand and how does it function in rhesus macaques?
Fms-like tyrosine kinase 3 ligand (Flt3L) is a growth factor critical for the differentiation and self-renewal of CD34+ progenitor cells in primates, including rhesus macaques. In research contexts, it has been utilized to expand dendritic cell (DC) subsets, treat radiation-induced myelosuppression, and serve as an adjuvant for various vaccines . The ligand interacts with the Flt3 receptor expressed primarily on hematopoietic progenitor cells and certain immune cell populations. When administered to rhesus macaques, Flt3L significantly increases circulating CD34+ progenitor cells (up to 30-fold) and expands dendritic cell populations .
How do researchers identify and characterize Flt3L-mobilized cells in rhesus macaques?
Circulating dendritic cells in rhesus macaques are identified within lineage-negative, HLA-DR-positive cell populations. These include:
CD34+ progenitor cells are typically identified by flow cytometry, with baseline frequencies significantly higher in bone marrow (median 6.51%) compared to peripheral blood (median 0.135%) . The activation status of DCs can be determined by measuring surface expression of CD80 and CD86 co-stimulatory molecules, which show slight increases after Flt3L mobilization but typically remain at levels characteristic of immature DCs unless further stimulated .
How does the timing of Flt3L administration affect experimental outcomes?
The timing of Flt3L administration relative to experimental interventions is critical. Research shows that peak expansion of CD34+ cells occurs on day 12, which is 5 days after discontinuing a 7-day Flt3L treatment . For dendritic cells, peak mobilization occurs approximately 4 days post-treatment completion . Interestingly, these peaks do not correlate with serum Flt3L concentrations, which typically peak around day 4 of administration .
When using Flt3L as a vaccine adjuvant, optimal results have been observed when administering Flt3L-Fc fusion protein 7 days before the arenaviral vaccine, allowing for proper expansion of innate immune cells before vaccination . This specific timing enhances T-cell activation and leads to improved vaccine-induced immune responses.
What differential effects does Flt3L have on various immune cell populations?
Flt3L administration has distinct effects on different cell populations:
This differential response is important when designing experiments targeting specific cell populations.
How does Flt3L administration affect proliferation kinetics of progenitor cells?
This reduction in Ki67+ CD34+ bone marrow cells correlates inversely with the number of CD34+ cells per ml of bone marrow aspirate (R = -0.645, P < 0.0038) . These findings suggest a negative regulatory mechanism in the bone marrow, possibly involving an autocrine/paracrine feedback loop at the Flt3/Flt3L axis. Conversely, Ki67 is significantly upregulated in the bulk bone marrow mononuclear cell population during Flt3L administration, likely reflecting proliferation of more committed precursors such as dendritic cells or monocyte precursors .
How effective is Flt3L in mobilizing cells in SIV/SHIV-infected macaques compared to naive animals?
This research demonstrates that Flt3L can be safely and effectively used in infected animals without increasing viral load, which has important implications for immunotherapeutic approaches in HIV/SIV research.
What are the mechanisms behind the differential mobilization patterns observed with Flt3L?
The observation that Flt3L mobilizes CD34+ cells in peripheral blood without expanding these cells in the bone marrow has led to two competing hypotheses:
-
Emigration Hypothesis: Mobilized CD34+ cells in peripheral blood represent emigrants from the bone marrow pool, which explains why no expansion is observed in the bone marrow itself .
-
In Situ Proliferation Hypothesis: The CD34+ progenitor cell population already in circulation proliferates in response to Flt3L, independent of bone marrow emigration .
The downregulation of Ki67 in bone marrow CD34+ cells during Flt3L administration suggests strong regulatory pressure to prevent hyperproliferation in this tissue. This regulatory mechanism may involve autocrine/paracrine feedback loops at the Flt3/Flt3L axis, as recently described for human progenitor cells in vitro .
How do researchers utilize Flt3L as a vaccine adjuvant in rhesus macaque models?
Flt3L has shown significant potential as a vaccine adjuvant in rhesus macaque models, with administration strategies focusing on proper timing and dosing:
-
Recent research demonstrated that administering a rhesus-specific engineered Flt3L-Fc fusion protein 7 days before arenaviral vaccine dramatically enhanced vaccine immunogenicity .
-
This pre-treatment approach resulted in significant enhancement in the magnitude, breadth, and polyfunctionality of vaccine-induced T-cell responses .
-
Mechanistically, Flt3L administration increases the frequency and activation of innate immune cells, particularly dendritic cells, which are critical for initiating adaptive immune responses .
-
No treatment-related adverse events or antidrug antibody generation was observed after repeated dosing, indicating a favorable safety profile .
This approach has particular relevance for developing more effective vaccines against challenging pathogens such as HIV/SIV, where robust cellular immune responses are desirable.
What methodological approaches can optimize Flt3L-mobilized cell collection for downstream applications?
For researchers seeking to collect and utilize Flt3L-mobilized cells:
-
Timing is critical: Collection of peripheral blood for CD34+ progenitor cells should be performed around day 12 (5 days post-treatment), while dendritic cell collection is optimal approximately 4 days after completing a 7-day treatment regimen .
-
Cryopreservation is viable: Flt3L-mobilized dendritic cells have been shown to be resilient to freeze-thawing, maintaining their functional capacity .
-
In vitro activation: Overnight culture activates Flt3L-mobilized dendritic cells, up-regulating CD80/CD86 expression and interleukin-12 release, with CD40L providing additional stimulation . This activation is even more pronounced in enriched DC cultures.
-
Cell enrichment: For applications requiring higher purity, enrichment methods for specific DC subsets can enhance the activation potential observed during subsequent culture .
How do Flt3L effects in rhesus macaques compare to those in humans and other species?
The expression of Toll-like receptors (TLRs) within different subsets of macaque dendritic cells exactly mimics expression in human DCs but differs significantly from murine DCs . This provides important justification for using non-human primates to evaluate Flt3L and TLR ligands as adjuvants in vivo when modeling human responses.
Specific similarities between rhesus and human systems include:
-
Plasmacytoid DCs from rhesus macaques express TLR-7 and TLR-9 and respond to viral infection by producing large amounts of IFNα and upregulating CD86 expression .
-
Rhesus macaque myeloid DCs express TLR-3, -4, -7, and -8, and respond to agonists for each TLR by upregulating surface expression of CD40 and CD86 .
-
Rhesus macaque pDCs constitutively express high levels of interferon regulatory factor-7 (IRF-7), which is necessary for driving transcription of IFN-α, similar to human pDCs .
These similarities make rhesus macaques an excellent model for studying Flt3L effects relevant to human applications.
What are the limitations and constraints when extrapolating Flt3L research from rhesus macaques to humans?
While rhesus macaques provide an excellent model for human immunology, researchers should consider several key differences:
-
MHC differences: Rhesus macaques have two MHC class I loci (A and B) and three MHC class II loci (DP, DQ, and DR), and unlike humans, rhesus can have several MHC class I alleles per haplotype .
-
Dosing considerations: The standard dose of 100 μg/kg/day used in macaques may require adjustment when translated to human applications based on differences in body mass, metabolism, and receptor expression.
-
Temporal dynamics: While the general patterns of cell mobilization are likely similar between species, the precise timing and magnitude may vary and should be validated in human studies.
-
Age-related considerations: T cell repertoire undergoes significant shrinkage in aged rhesus macaques with the appearance of T cell clonal expansions, similar to elderly humans . This may affect the response to Flt3L in aged individuals.
Product Science Overview
The recombinant Flt3-Ligand for rhesus macaques is produced using E. coli expression systems. This recombinant protein is a single, non-glycosylated polypeptide chain containing 159 amino acids and has a molecular mass of approximately 18.0 kDa . The recombinant protein is purified using proprietary chromatographic techniques to ensure high purity and activity .
The biological activity of the recombinant Flt3-Ligand is measured by its ability to stimulate the proliferation of human AML5 cells. The effective dose (ED50) for this activity is less than 1.0 ng/ml, corresponding to a specific activity of 1,000,000 IU/mg . This high level of activity makes it a valuable tool for research in hematopoiesis and immunology.
Flt3-Ligand has been extensively studied for its role in enhancing the immunogenicity of vaccines. For instance, administration of Flt3-Ligand has been shown to promote the expansion of dendritic cell populations in the spleen and blood, which are critical for initiating immune responses . In rhesus macaques, the use of a rhesus-specific engineered Flt3L-Fc fusion protein has demonstrated significant enhancement in vaccine-induced T-cell responses .
The lyophilized form of recombinant Flt3-Ligand is stable at room temperature for up to three weeks. However, for long-term storage, it should be kept desiccated below -18°C. Upon reconstitution, the protein should be stored at 4°C for short-term use (2-7 days) and below -18°C for future use . It is important to avoid repeated freeze-thaw cycles to maintain the protein’s stability and activity .
