The applicant has demonstrated that primitive hematopoietic progenitors expressing CD34, but lineage negative and HLA-DR-(Lin-34+DR-) can give rise not only to myeloid progenitors in long-term bone marrow culture (LTBMC), but to natural killer cells (NK) after coculture in direct contact with bone marrow stroma. In contrast, CD3 positive T lymphocytes are not identified in these long term cultures presumably because of the absence of thymic-derived differentiation signals. He hypothesizes that external specific microenvironment signals provided by soluble factors, cellular ligands, and extracellular matrix (ECM) components are responsible for inducing differentiation along the NK or T-cell lineage. Acquisition of CD7 on adult human CD34+ marrow cells correlates with CD3z transcription, a pattern observed in mature NK. Additional expression of CD2 on CD34+/CD7+ cells correlates with not only CD3z but also CD3g and CD3d transcription, components present in T-cells but not NK cells. Coculture of CD34+/CD7+/CD2+/CD3- progenitors with BM stroma results in NK progeny only. However, coculture of the same progenitors with TE feeders results in mature T-cell progeny suggesting that the starting population may contain common NK/T progenitors.
In Specific Aim 1, he will adapt an in vitro culture of human thymic epithelial (TE) cells monolayers to induce CD34+ adult marrow progenitors to differentiate along the T-cell lineage. PCR will be employed to evaluate discrete molecular events to precisely dissect the microenvironment specific differentiation into the NK (transcription of CD3z, NK-tr and NKG2 mRNA) and T-cell lineage (CD3gdz mRNA and TCR rearrangement).
In Specific Aim 2, these molecular markers will be used to identify signals (soluble, ECM, cell ligands) provided by BM stroma or TE feeders to induce NK and T-cell differentiation.
In Specific Aim 3, in vitro mechanisms which promote maintenance of single cells in the CD34+/Lin-/DR- populations capable of myeloid, NK and T-cell differentiation will be examined. The persistence of lymphoid progenitors (NK and T) will be evaluated by utilizing sequential microenvironment switch culture assays. The applicant hypothesizes that maintenance of multilineage progenitors in this population is influenced by a balance of myeloid and lymphoid differentiation factors. He will determine the effect of inducers (IL3, IL7) and inhibitors (MIP-1a) of differentiation on NK and T-cell development throughout this process. Single cell analysis and retroviral marking studies will be employed to document the common origin of myeloid and NK/T progenitors in sequential microenvironment specific switch cultures.
