Experimental methods in immunology

Experimental Methods in Immunology

Stem Cells

The growth of stem cells in the bone marrow is the basis for cellular immunity. In vitro mimicry of this growth can be achieved by filling a semisolid medium with stromal cells. By adding different growth factors and cytokines, as well as stem cells of various differentiation level, the influence of chemical mediators of hematopoiesis can be understood. HSCs can be taken from a donor and injected into a person who has a defective or absent hematopoietic system. As little as 10% of the donor's bone marrow is removed and injected into the recipient, and the HSCs will find their way to the bone without direction, replenishing the hematopoietic system of the recipient. There are several types of stem cell grafts:

• autologous--donor is the recipient themself; the recipient can freeze HSCs prior to chemotherapy or radiation therapy for cancer; additionally, genetically engineered autologous HSCs can be injected back into the patient
• syngeneic--donor is genetically identical to recipient (identical twins)
• allogeneic--donor is genetically different from recipient
  • this can lead to graft-vs.-host disease (GVHD)
  • GVHD is based on MHC/HLA types, and so a bone marrow "match" occurs when an allogeneic member of the population is found to have relatively similar MHC/HLA types relative to the recipient

Knock-Out Mice

Genetic factors that produce certain cytokines (or certain cell types themselves) can be studied throught the use of knock-out mice. In these mice, a certain gene is inactivated and the animal is allowed to grow. The resulting phenotype is compared against other knock-out mice, in an attempt to piece together the genetic basis of different cellular functions and phenotypes. The process of gene knockout has become fine-tuned in recent years. Basically, a gene library is searched for a candidate gene to be knocked-out. A full mouse genome with the candidate gene knocked-out is developed and grown in a preliminary stem cell, and it is injected into a mouse stem cell via electroporation. Besides making the candidate gene inoperable, the altered genome will usually have two factors:

• A gene to make the cultured preliminary stem cell resistant to a certain antibiotic. Because not all of the stem cells will incorporate the altered genome, an antibiotic is applied to the treated cells, and only those that survive have therefore been injected with the altered genome (including both the antibiotic resistance gene and the knocked-out gene sequence)
• A gene to make the phenotype of a fully-altered knockout mouse obvious. For example, the altered mouse genome might include a gene to make the knockout mice pure white in color. The stem cells, before being treated, are collected from a black mouse. Thus, when the black fur mother mouse is injected with the treated stem cells, she will give birth to baby mice with varied coat colors, from black to white and grey in between. The nearly-white progeny are then cross-bred with each other, and the process is repeated until mice with pure white coats are created. These mice are most likely to have both the pure white genotype and the knocked-out gene. Thus, linked phenotypes are a marker for linked genotypes.

Several genes effect hematopoiesis, and have been developed via gene knockout. These include:

• GATA-2--regulates lymphoid and myeloid cell production, as well as RBC (erythrocyte) production
• Ikaros--regulates lymphoid cell production
• Oct-2--regulates differentiation of naive B cells into plasma cells

Hematopoietic Stem Cell Concentration

Irradiation of a mouse can wipe out the HSCs in the bone marrow, destroying the mouse's ability to produce red blood cells and leukocytes. These cells can be replaced with the bone marrow cells of an immunologically identical mouse, although in the injected replacement cells very few cells are actually HSCs. In order to concentrate the amount of actual HSCs in a sample, mice can be irradiated and replacement bone marrow cells are injected with fluorescent immunoglobulins that bind to mature RBCs and WBCs. A flow cytometry method can now be used to weed out these labelled cells, and the process can be repeated until only those that are most likely undifferentiated remain. The HSCs can be found in a subset of cells that contain the CD34 receptor on their surface; the CD34+ subpopulation is almost totally HSC with few differentiated cells.

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