Dear Pedro, excellent question! I think, this is impossible, because real memory cells propagating in vivo are not equal to in vitro stimulated T cells. Moreover, I think that TCR transgenic models widely used now to study memory T cells are misleading, because can not reproduce very important conditions resulting to appearance of real memory cells. Particularly, these are selection processes on periphery. Primarily in vitro stimulated T lymphocytes are continually cycling and can not be resting. They need repeated cycles of restimulation every several days (or 1-2 weeks) and are represented by irregular shape blast cells. In contrast, real memory cells in vitro can be resting and represented by round small lymphocytes. In vitro obtained activated T cells should be restimulated with viable APC plus IL-2. In contrast, real memory T cells can be propagated by restimulation with fixed or heat-killed APC plus IL-2, and even in response to anti-CD3 plus IL-2. Then, in my experiments adoptive transfer of primarily in vitro stimulated T cells to mice never resulted in formation of long lived memory T cells. In contrast, the transfer of real memory cells into secondary recipient allowed to detect them in several months after transplantation. Unfortunately, the term "memory cells" is rather undefined now. Under this term different investigators have in mind 1) T cells that encounter antigen secondary in vivo; 2) T cells acquiring specific set of activation markers after primary response in vivo; 3) T cells carrying this specific set in norm (without immunization); 4) T cells proliferating in secondary MLR (or secondary restimulated with antigen-loaded APC) in vitro; 4) T cells acquiring activation markers under lymphopenia conditions, etc. I think, these are very different essences that require detailed analysis and clear definitions.

Desr Dmitry, thanks a lot for your thoughtful answer.

Dear Pedro, we were able to obtain clonal representatives of memory cells by following approach. Mice were immunized by MHC class I disparate tumor cells (2-3 x 10(7)). As a rule, this immunization induces CD8 CTL response with the peak on 10-12 days and subsequent formation of CD8 memory cells. In two months after immunization, mouse splenocytes can be used as a source of central memory cells not displaying immediate cytotoxic function. These cells can be selectively activated to proliferate in vitro by heat-treated (45oC, 1 h) or paraformaldehyde (0.1%) fixed allogeneic splenocytes carrying immunizing MHC class I molecule (R:S ratio should be 2:1 or 1:1). Then, you have different options: 1) on the next day you can measure IL-2 and IFNg release; 2) in 3-4 days you can directly estimate proliferative CD8 memory response (3H-thymidine incorporation, CFSE); 3) in 3 days you can determine also CTL function (using CFSE stained or 51Cr labeled target cells) ; 4) in 3 days you can obtain T hybridomas by fusion with BW5147 thymoma cells transduced by CD8; 3) in 10 days you can restimulate in vitro enriched memory cells by heat-treated allogeneic stimulators plus IL-2 or anti-CD3 plus IL-2 to obtain cell lines and clones. You can see additional information and references here https://www.researchgate.net/publication/233960011_Direct_Recognition_of_MHC_Class_I_Molecules_in_Memory_Response_to_Allogeneic_Tumor_Cells?ev=prf_pub

Article Direct Recognition of MHC Class I Molecules in Memory Respon...

Dear Dmitry, this is great information ! I will check your manuscript. Thanks a lot

Good luck, Pedro! My sincere greetings to you!

I've read some papers on memory T cell subsets:

1. Klebanoff, C.A., et al. Proceedings of the National Academy of Sciences of the United States of America, 2004. 101(7): p. 1969-1974.

2. Klebanoff, C.A., et al. Proceedings of the National Academy of Sciences of the United States of America, 2005. 102(27): p. 9571-9576.

3. Gattinoni, L., et al. Nature Medicine, 2009. 15(7): p. 808-813.

These papers cultured OVA-specific memory T cells. What's the difference between their memory T cells and yours?(To Dmitry)

Thanks!

The answer for your question is Yes, Pedro. As Dmitry has explained before, repetitive in vitro stimulation does generate surface markers that qualify CD4 and CD8 T cells as memory. I have done studies in human PBMCs and expanded memory CD4 T cell lines from non-immunized individuals (PMID 10825580). The protocol is quite well explained, and works likewise for murine LN or splenic cells.

@Fei Yang. Cited works used Pmel-1 TCR transgenic mice as the source of tumor reactive CD8 T cells. These T cells recognize peptide AA25-33 of gp100 protein in the context of murine Db molecule. Really, these cells are monoclonal and represent highly artificial model for real immune and memory responses. In real immune responses you can see polyclonal immune response and subsequent clonal competition and selection resulting in formation of real memory cells. Certainly, TCR transgenic T cells cannot imitate such responses by any means.