Recombinant Rhesus Macaque Granulocyte-macrophage colony-stimulating factor protein (GM-CSF) (Active)

What is Rhesus Macaque GM-CSF and what are its primary biological functions?

Rhesus macaque GM-CSF, also known as Colony Stimulating Factor 2 (CSF2), is a cytokine originally characterized as a growth factor supporting the in vitro colony formation of granulocyte-macrophage progenitors. This 14.4 kDa protein (127 amino acids) stimulates the growth and differentiation of hematopoietic precursor cells from various lineages . Beyond its hematopoietic functions, rhesus macaque GM-CSF can induce endothelial cell migration and proliferation, and stimulate the proliferation of various tumor cell lines, including osteogenic sarcoma, carcinoma, and adenocarcinoma cell lines .

The protein is produced by multiple cell types, including activated T cells, B cells, macrophages, mast cells, endothelial cells, and fibroblasts in response to cytokine or immune and inflammatory stimulation. It plays a critical role in dendritic cell development and differentiation, establishing GM-CSF as a key immunomodulatory molecule in rhesus macaque models .

How has rhesus macaque GM-CSF been utilized in vaccine development research?

Rhesus macaque GM-CSF has been extensively studied in vaccine development, particularly in SIV (Simian Immunodeficiency Virus) vaccine research. In these studies, recombinant modified vaccinia Ankara (MVA) expressing rhesus GM-CSF (MVA/GM-CSF) has been tested for its immunomodulatory effects when co-administered with MVA/SIV macaque 239 vaccine .

Interestingly, research has demonstrated a dose-dependent effect where high doses of MVA/GM-CSF did not affect systemic envelope (Env)-specific antibody responses but decreased:

• Expression of gut-homing receptor α4β7 on plasmacytoid dendritic cells (p < 0.01)

• Magnitudes of Env-specific IgA (p = 0.01) in rectal secretions

• Magnitudes of Env-specific IgG (p < 0.05) in rectal secretions

These findings suggest that while GM-CSF is often used as an adjuvant to enhance immune responses, high doses may actually inhibit certain mucosal antibody responses, highlighting the importance of dose optimization in vaccine studies.

What role does rhesus macaque GM-CSF play in hematopoietic recovery models?

GM-CSF has demonstrated significant effects on hematopoietic recovery in rhesus macaque models following bone marrow transplantation and radiation exposure. In autologous bone marrow transplantation models, rhesus monkeys treated with recombinant GM-CSF after total body irradiation (9.0 Gy) and bone marrow transplantation showed:

These data demonstrate that GM-CSF infusion accelerates hematopoietic regeneration, significantly enhancing both neutrophil and platelet recovery rates even after discontinuation of treatment .

What are the optimal storage and handling conditions for recombinant rhesus macaque GM-CSF?

Recombinant rhesus macaque GM-CSF is typically supplied as a sterile filtered white lyophilized powder. For optimal stability and activity:

• Store desiccated at -20°C

• Upon reconstitution, prepare single-use aliquots to avoid repeated freeze-thaw cycles

• Reconstitute in 0.2 μm filtered PBS, pH 7.4, for most applications

• For long-term storage of reconstituted protein, add a carrier protein (0.1% BSA or HSA) to prevent adsorption to surfaces

• Handle with caution as this is an active protein that may elicit biological responses in vivo

How is the biological activity of recombinant rhesus macaque GM-CSF measured?

The biological activity of recombinant rhesus macaque GM-CSF is typically determined using cell proliferation assays with human TF-1 cells, which are GM-CSF-dependent erythroleukemia cells. The standard measure of activity is represented as:

• ED₅₀ (effective dose for 50% maximal response) of less than 0.1 ng/ml

• This corresponds to a specific activity of >1.0 × 10⁷ IU/mg

Researchers should verify the activity of each new lot of GM-CSF using appropriate bioassays before incorporating it into critical experiments, as activity can vary between preparations.

What delivery methods are most effective for rhesus macaque GM-CSF in vivo studies?

Various delivery methods have been employed for administering rhesus macaque GM-CSF in vivo, each with distinct pharmacokinetic profiles:

• Continuous infusion: Using subcutaneously implanted miniosmotic pumps (e.g., Alzet pumps) delivering at a rate of 50,400 U/kg/day has shown effective results in bone marrow transplantation studies. This method produces a dramatic leukocytosis and substantial reticulocytosis in healthy monkeys .

• Co-administration with viral vectors: When studying vaccine responses, GM-CSF has been delivered via recombinant viral vectors (e.g., MVA expressing GM-CSF) at various doses ranging from 1 × 10⁵ to 5 × 10⁷ PFU, with dose-dependent effects on immunity .

The choice of delivery method should be determined based on the specific research question, desired pharmacokinetics, and experimental timeline.

How does the dose of rhesus macaque GM-CSF affect immune responses in vaccine studies?

Research has revealed complex, dose-dependent effects of GM-CSF on vaccine-induced immune responses. In studies using MVA expressing rhesus GM-CSF co-administered with SIV vaccines, a 500-fold dose range was tested with the following observations:

• Systemic Env-specific antibody responses were not significantly affected by GM-CSF dose

• Mucosal antibody responses were negatively impacted by high doses of GM-CSF

• Expression of gut-homing receptor α4β7 on plasmacytoid dendritic cells was decreased at higher doses

• Protection against SIV challenge was strongest in animals receiving no or low doses (1 × 10⁵ PFU) of MVA/GM-CSF, particularly in animals with restrictive TRIM5α genotypes

These findings demonstrate that while GM-CSF can modulate immune responses, higher doses may not always be beneficial and could potentially impair certain aspects of protective immunity, particularly at mucosal surfaces.

What is the relationship between GM-CSF administration and protection in SIV challenge models?

In TRIM5α-restrictive rhesus macaques, several correlates of protection against SIV challenge have been associated with GM-CSF-modulated immune responses:

• Env-specific rectal IgG (r = +0.6) and IgA (r = +0.6) correlated with protection

• Avidity of Env-specific serum IgG (r = +0.5) correlated with protection

• Antibody-dependent cell-mediated virus inhibition (r = +0.6) correlated with protection

• Notably, titers of neutralizing antibodies did not correlate with protection

These correlations were observed specifically in TRIM5α-restrictive animals but not in TRIM5α-permissive animals, highlighting the complex interaction between host genetics, GM-CSF-modulated immunity, and protection against viral challenge.

What quality control parameters should be verified for recombinant rhesus macaque GM-CSF?

For research applications requiring high-quality recombinant rhesus macaque GM-CSF, several quality control parameters should be verified:

• Purity: >98% as determined by SDS-PAGE and HPLC analyses

• Endotoxin level: <1.0 EU/μg as determined by LAL method

• Biological activity: ED₅₀ <0.1 ng/ml in TF-1 cell proliferation assay

• Identity confirmation: Mass spectrometry analysis or N-terminal sequencing

• Protein concentration: Verified by standard protein quantification methods (BCA assay or A280 measurement)

Researchers should request and review certificates of analysis for these parameters when obtaining new lots of GM-CSF for critical experiments.

What control groups should be included when studying GM-CSF effects in rhesus macaque models?

When designing experiments to study the effects of rhesus macaque GM-CSF, several control groups should be considered:

• Vehicle control: Animals receiving the same vehicle (e.g., saline, PBS) without GM-CSF

• Dose-ranging groups: Multiple doses of GM-CSF to establish dose-response relationships

• Timing controls: When studying time-dependent effects (e.g., different administration schedules)

• Genetic controls: In SIV challenge studies, stratification by relevant genotypes (e.g., TRIM5α) is crucial

• Irrelevant cytokine control: A control group receiving an irrelevant cytokine to distinguish GM-CSF-specific effects from general cytokine effects

Including appropriate controls is essential for rigorous experimental design and valid interpretation of GM-CSF effects.

How should researchers interpret contradictory findings regarding GM-CSF effects in different experimental systems?

When encountering contradictory findings about GM-CSF effects across different experimental systems, researchers should consider several factors:

• Dose effects: GM-CSF has shown opposing effects at different doses, with high doses potentially inhibiting responses enhanced by lower doses

• Genetic background: Host genetics (e.g., TRIM5α genotype) can dramatically influence GM-CSF effects on protection in challenge studies

• Timing of administration: Effects may vary depending on when GM-CSF is administered relative to other interventions or challenges

• Route of administration: Different delivery methods may affect local versus systemic distribution of GM-CSF

• Experimental readouts: GM-CSF may have different effects on various immune parameters (e.g., enhancing systemic responses while inhibiting mucosal responses)

Careful consideration of these factors can help reconcile apparently contradictory findings and lead to a more nuanced understanding of GM-CSF biology.