Role Of CD44 And Telomeres In Generation Of Tumor-Reactive Memory T Lymphocytes

Project summary:Recent clinical trials have shown that adoptive cell transfer immunotherapy with autologous anti-tumor tumor-infiltrating lymphocytes(TILs)with high-dose interleukin-2 injection following lymphodepletion chemotherapy can cause tumor regression in approximately 50%of treated patients with refractory metastatic melanoma.It has also been indicated that transfer of cells with memory properties may be superior mediators of an anti-tumor response.Nevertheless,memory T cell populations in human tumor-reactive T cells for adoptive cell transfer have not fully characterized.Considering that high expression of CD44 seems to be the most reliable marker for memory cells in mice,we hypothesize that CD44 also plays an important role in the generation and maintenance of memory T cells in the administered TIL samples for adoptive cell transfer therapy.In this proposed project,we aim to(1)determine the expression of CD44 isoforms on human memory T cells from tumor-reactive tumor-infiltrating lymphocytes by means of fluorescence activated cell sorter(FACS)analysis and reverse transcription-polymerase chain reaction(RT-PCR)cloning methods to know if CD44 can be used in the identification of tumor-reactive memory T cells in tumor-infiltrating lymphocytes,(2)determine the regulation of CD44 expression and its cellular signaling pathways upon stimulation by CD44 ligand(such as hyaluronan,fibronectin,chondroitin and collagen)binding and rapid expansion protocol in the maintenance of tumor-reactive memory T cells in tumor-infiltrating lymphocytes,and(3)determine the association of CD44 and telomere length in the generation and maintenance of tumor-reactive memory T cells in tumor-infiltrating lymphocytes.Manipulations of memory T cell generation and maintenance in vitro may improve adoptive immunotherapy.Understanding the role of CD44 and telomere length in generating and maintaining tumor-reactive memory T cells for adoptive cell transfer could lead to improved immunotherapy for the patients with cancer.Our studies demonstrated that CD62L,CD27 and CD28 positive effector memory T cells were present in the TIL samples initiated from the tumor tissues of melanoma patients and T cell expansion led to the significant loss of memory T cells.CD27 and CD28 positive T cells had high levels of CD44 expression.T cell expansion resulted in significant down-regulation of CD44 expression.IL-2 and anti-CD3 antibody stimulation may be responsible for CD44 down-regulation on CD8+ T cells during expansion.Furthermore,CD44 down-regulation using siRNA on TILs dramatically decreased IFN-y and IL-2 release upon tumor stimulation.These results suggested that the regulation of CD44 expression in TILs may play an important role in memory T cell maintenance and anti-tumor immune response.