Steven Hallam and Dr. Bradford Berges, Microbiology and Molecular Biology
Introduction
The human immune system is composed of two main subsets: innate and adaptive immunities. Among the adaptive immune cells, B cells play a vital role in stopping infectious agents through the production of antigen specific antibodies. Human B cells are important targets of infection for many human-specific viruses that are poorly understood. Since the infection and study of human subjects is not possible, animal models have been developed to mimic the human immune environment. Rag2-/-γc-/- mice engrafted with human hematopoietic stem cells produce human B lymphocytes and these cells are capable of mounting antigen-specific human antibody responses. However, there is concern in the field that the frequency of specific antibody responses in humanized mice is somewhat low and inconsistent, and it is unclear why some humanized mice respond to antigenic challenge better than others. We hypothesize that it is necessary to allow full development of the human immune system in this model prior to exposure to antigens if robust and consistent human antibody responses are to be detected.
Methods and Materials
Production of humanized mice:
Rag2–/–γc–/– mice were conditioned for engraftment ( 3.5 Gy) within 5 days of birth and 0.2-0.5×106 human CD34+ cord blood cells were injected intrahepatically. Reconstitution was detected at 8 weeks post-engraftment by FACS staining of peripheral blood to detect human and murine leukocytes by hCD45 and mCD45 specific antibodies, respectively.
FACS staining:
Human-specific antibodies were verified to be human-specific by staining non-humanized mouse cells as a control (not shown). Human blood was used to establish FACS settings. The following antibodies were used to detect various B cells subsets: hCD45-PECy7, hCD19-APC-AF750, hCD20-APC, hCD22-PE, hCD27-FITC, hCD38-PECy7, hCD138-PECy5.5. Samples were analyzed on a BD FACSCanto. % Peripheral blood engraftment (%PBE) was determined by dividing human leukocyte populations by the total mouse and human leukocyte population.
Antibody detection:
70μL of whole blood was taken from each mouse and centrifuged to isolate the serum. This serum was then used with the AlerChek total human IgG, IgA, and IgM ELISA kits according to manufacturer’s instruction. Serum dilution for IgA was 1:20, IgM and IgG were 1:100.
Data Normalization:
Total antibody concentration was divided by %PBE to achieve normalization across different samples.
Results
Conclusions
Rag2–/–γc–/– mice engrafted with human hematopoietic stem cells produce human B cells. These cells are detectable in the blood, spleen, bone marrow, and lymph nodes. Human IgG/IgM/IgA are detectable long term in humanized mice. Human IgG/IgM/IgA normalized concentrations increase over time, but do not exhibit a predictable trend. There appears to be no correlation between %PBE and human antibody levels.
Discussion
Even though we were able to show that humanized antibodies were produced and rose in the mice following engraftment, there was no predictable trend or date of leveling off. The antibody levels also did not correlate with %PBE meaning that we may need to pursue an alternative means of determining mouse fitness for research studies. The develop of human immune cells in the mice appears to be very individualized and it may not be possible to determine a population approach to immune development.