## 2 Materials and Methods  
### Materials  
Blood samples from healthy volunteers were collected by technicians at the National Hospital for Tropical Diseases under the protocol approved in ethics approval number IRB-VN01.001/IRB00003121/FWA 00004148 from the Ethics Committee at the Hanoi Medical University. The samples were collected between June and December 2020, stocked in K2 EDTA tubes, and processed within 24 h. Lymphocyte Separating Medium, Pancoll human (PAN Biotech, Aidenbach, Germany) was used for isolating PBMCs from total blood cells. Tritest reagent (BD Biosciences, Becton, San Jose, CA, USA) containing fluorescence-labeled antibodies including CD45-PerCP/CD4-FITC/CD3-PE, was used to label cells in the FACS method using the FACCalibur system and Multiset software (BD Biosciences). FACS Lysis Solution 10X Concentrate (BD Biosciences) was diluted to 1X concentration by adding 9 mL of distilled water to 1 mL of 10X solution, and used to lyse red blood cells in the FACS method. Trucount tubes (BD Biosciences) with a predefined number of fluorescence beads were used as a reference to determine the absolute number of cells in FACS analysis. Anti CD4 Abs labeled with FITC and anti CD3 Abs labeled with PE (CD4-FITC, CD3-PE, Thermo Fisher Scientific, Waltham, MA, USA) were used for immunofluorescence staining. Anti-Human CD4 Particles--DM (NP-CD4) from BD Biosciences were used for cell capture and separation using a Dynamag(tm-2 magnet (Thermo Scientific), and were then counted using Countess\({}^{\text{TM}}\), an automated cell-counting machine. A hemocytometer ([PERSON], Lauda-Konigshofen, Germany) was used to estimate the cell concentration in the sample after PBMC isolation under a Primo Star (2) Microscope ([PERSON], Oberkochen, Germany). Phosphate buffered saline (PBS, pH 7.4) supplemented with 0.5% BSA and 2 mM EDTA (PBS\({}^{\star}\)) was used as a buffer for the incubating and washing steps. Isolation of Peripheral Blood Mononuclear Cells (PBMC) from Whole Blood by Sucrose Gradient Centrifugation  
PBMCs were isolated from blood by sucrose gradient centrifugation using the Pancoll Human Kit (PAN-Biotech). Briefly, approximately 4 mL of blood was collected in K2 EDTA tubes and diluted 2-fold with PBS. Diluted blood was then carefully layered on 8 mL Pancoll solution such that the two phases were separated. After centrifugation at 800\(\times\)on an Eppendorf 5810R for 20 min, brake = 0, and at room temperature, four fractions were separated from top to bottom: yellow serum, opaque white PBMCs, clear white Pancoll solution, and red blood cells. The PBMC phase was harvested into a new centrifuge tube using a pipette and was then filled with two- to three-fold volume of PBS, followed by centrifugation at 300\(\times\)\(g\) for 10 min. After centrifugation, the cell pellet was precipitated at the bottom and the supernatant was discarded. The washing step was repeated once again for purified PBMCs. Isolation of CD4\({}^{+}\) T Cells from PBMCs and Whole Blood Using Anti-CD4 Antibodies Conjugated with Magnetic Nanoparticles  
As a conventional method, approximately 10\({}^{7}\) PBMCs previously isolated from whole blood were incubated in 1 mL PBS with 50 \(\mu\)L NP-CD4 for 30 min. The sample suspension was then placed on a DynamagTM-2 magnet for 10 min, following which the supernatant was discarded. The washing steps were performed twice by repeating washes with 1 mL PBS, placing the tube on the magnet for 7 min, and discarding the supernatant. The cell sample was finally resuspended in 1 mL PBS*. Separation efficiency (SE) was calculated as the ratio of the number of CD4\({}^{+}\) T cells separated by the number of CD4\({}^{+}\) T cells in the sample before separation. MACS separation of CD4\({}^{+}\) T cells from whole blood was performed using the same procedure as the conventional method, but with various ratios of blood input and NP-CD4.