T Cell Hybridoma Technology and Its Application

About T Cell Hybridoma

In developing T lymphocytes, TCR variable region exons ale assembled from germline variable (V), diversity (D) and joining (J) segments. V (D) J recombination is initiated by the lymphocyte—specific recombination activating gene (RAG)I and 2 which cooperate to direct site-specific cleavage after recognizing and synapsing the target recombination signal sequences (RSS). The cleaved gene segments are then joined by constitutively expressed DNA-repair enzymes, such as the terminal deoxynucleotidyl transferase (TdT) that is a template-independent DNA polymerase that catalyzes non-homologous end-joining reactions. A large number of different V, D, J recombination with nucleotide insertion and deletion at junctional sites consist of the consequence of diversity of the T cell repertoire, thereby increasing diversity for recognition of a vast array of antigens．

Through activation of recombination activating gene (RAG)1 and 2 early in T cell development, TCR β locus rearrangement proceeds stepwise: first Dβ to Jβ recombination on both alleles, followed by Vβ recombination to all assembled DJβ complex on a single allele. Transcription through a functional Vβ-Dβ rearrangement generates TCR β chains which was further combined with pre. Ta molecules to form pre-TCRs. Signaling via the pre．TCRs result in：temporarily down—regulate RAG genes expression; thymocyte expansion and maturation to the double positive stage (CD4⁺CD8⁺); subsequent feedback signal that blocks further Vβ rearrangement to enforce TCR β allelic exclusion, result in the “one lymphocyte-one antigen receptor” the general principle of TCR expression; initiation of the rearrangements at the a locus; Finally, mature TCRaβ were expressed on the cell surface, pTa and RAGs expression were shut-off．

Immunological dogma holds that once T cells exit the thymus, RAG 1 and RAG2 are permanently inactivated, which suggested that functional antigen receptor gene rearrangement that leads to expression of a cell surface receptor ensures that the receptor will not be further altered at the level of V(D)J recombination.T cells will experience rearrangement of functional antigen receptor genes, in which the encounter of autoreactive T cells with antigen (in the context of APC) will result in deletion of those T cells by apoptosis before leaving into periphery. However, functional aβ TCR alone is not sufficient to silence RAG 1 and RAG2 expression, mature T cells modify their receptors through additional RAG-mediated V(D)J recombination events to change the specificity of antigen recognition, generate a new receptor in replacement of the initial one, which not only protect cells from apoptisis, but also boost the recognition specificities of TCR repertoire.

T cell hybridoma technology

It is well known that normal T cells are difficult to grow in vitro for a long time; whereas T lymphoma cells can proliferate indefinitely under in vitro conditions. After merging the two cells, the traits we expect to obtain from the two parental cells can be expressed by the T hybridoma progeny cells. The T hybridoma not only has the ability of the parental normal T cells to perform immune function, but also has the characteristics that the parental tumor cells proliferate in vitro or as a transplanted tumor in the animal. By cloning these cells, monoclonal T cell hybridomas with various immune functions can be obtained; and a large number of highly active T cell antibodies production are obtained.

The mechanism of T-T cell fusion, the required conditions and specific procedures are basically the same as those of B-B fusion. The tumor cells used for fusion are various deficient strains derived from T lymphoma cells. The T cells used for fusion should be activated first. When fused with unseparated lymphocytes, cell fusion occurs randomly, T lymph Tumor cells are not selective for T cells, and they have the same chance of fusion with various types of cells. Therefore, we should try to select T lymphoblasts as pure as possible as parental T cells in order to improve the fusion rate.

   The preferred fusion agent is polyethylene glycol (PEG). The molecular weight is from 1000 to 6000. The more commonly used concentration is 40--45%. Usually HAT is used as the selection culture solution. It is reported in the literature that the screening effect of HAT is not good. Hybridomas grow slowly and chromosomes are easy to lose. The reason may be due to thymidine. It is recommended to replace HAT with diazoacetylserine (A) and hypoxanthine (H) selection solution (AH). The former and the latter in comparison, the positive rate of hybridomas was increased by 3 times, the growth rate of fused cells was increased by twice over, and the number of chromosomes was stable, and the success rate of recloning was also increased.

  Hybrid cells were screened using a fluorescence activated cell separator (FACS) and this method was used to allow HGPRT+ T lymphoma cells to be used for fusion. The cells to be tested are first stained with a fluorescent antibody against the normal T cell surface antigen, and the hybrid cells and T cells are isolated, but due to the low frequency of hybridization and the non-specific staining of the tumor cells, the method of analyzing the DNA content is required to perform the second method. The step was screened and stained with dibenzimivir 33258. The dye had no effect on cell viability. After staining, the fluorescence density was linear with the number of chromosomes. To avoid the interference of G2 tumor cells, only the DNA content was higher than four ploid cells.

    After the fused cells are seeded into the culture plate, trophoblast cells are generally added, including mouse peritoneal cells, thymocytes, adherent cells in human PBL, and irradiated mouse spleen cells. Adding trophoblast cells can double the fusion rate. After the hybridoma cells grow stably, the HT medium is changed, and then the ordinary medium is gradually transferred, and it is generally necessary to clone again to obtain monoclonal cells.

Application of T cell hybridoma

In theory, cell hybridization technology enables people to obtain T cell hybridomas with any immune function, and has successfully established various T cell hybridomas with auxiliary, inhibitory and killing functions. The function of the T cell subset is significant. The regulation of immune responses by T cells can be roughly divided into two categories, sub-helper and inhibition, which are all performed by different T cell subsets.

There are many reports on suppressive hybridomas. A T cell hybridoma cell is injected into the irradiated mouse with the spleen cells of the sensitized mouse and the corresponding antigen, and the function of producing antibodies in the spleen cells is inhibited. Clearing the sensitized lymphocytes to kill the corresponding tumor cells in vitro and in vivo, the injection of the supernatant can promote the growth of the tumor in the mouse, but if the tumor cells are first absorbed in vitro, the effect disappears. Unrelated tumor cells have no effect. It is indicated that the factor in the supernatant specifically binds to the tumor cells, thereby demonstrating that the inhibitory factor produced by the T cell is a blocking factor that inhibits the mechanism immune response and promotes tumor growth.

The specificity of hybridoma cell killing is generally closely related to the specificity of parental CTL. The two functions of killing function and growth-dependent IL-2 in vitro are closely related. There is no clear answer to explain this phenomenon, probably due to these two. The characteristics are controlled by a common gene or by several closely linked genes located on the same chromosome. These genes are open in cells with killing function, and these chromosomes may be fused in hybridoma cells without killing function. It was lost soon. In short, it is possible to obtain a hybridoma with killing function by fusion, but it is difficult to maintain its function stably for a long time.

In addition to the above, studies on T cell receptors have been made and substantial progress has been made. The antigen receptor of T cells isolated on the surface of T hybridoma cells was analyzed by structural analysis. It was found that the T cell receptor is a macromolecule with a molecular weight of 85000-95000, and has two chains of a and β, which are linked by a disulfide chain. In addition, it has been found that the receptors on the surface of different hybridomas are different, indicating that the amino acid arrangement order also has a constant region and a variable region.

Reference

[1] Sleckman B P, Peterson A, Jones W K, et al. Expression and function of CD4 in a murine T-cell hybridoma[J]. Nature, 1987, 328(6128):351-353.

[2] Schreiber R D, Pace J L, Russell S W, et al. Macrophage-activating factor produced by a T cell hybridoma: physiochemical and biosynthetic resemblance to gamma-interferon.[J]. Journal of Immunology, 1983, 131(2):826.

[3] Ratnofsky S E, Peterson A, Greenstein J L, et al. Expression and function of CD8 in a murine T cell hybridoma[J]. Journal of Experimental Medicine, 1987, 166(6):1747-1757.

[4] Sarin A, Adams D H, Henkart P A. Protease inhibitors selectively block T cell receptor-triggered programmed cell death in a murine T cell hybridoma and activated peripheral T cells.[J]. Journal of Experimental Medicine, 1993, 178(5):1693-700.