Refined through thousands of years of evolutionary advancement, the immune system has developed elegant strategies to defend the body against foreign pathogens and disease targets. One of these mechanisms involve cytotoxic T lymphocytes (CTLs). These T cells are a subset of white blood cells found in peripheral blood, lymph nodes, thymus and spleen. They are distinguished from other cells types by their unique expression of the surface marker CD8. CTLs are so named for their ability either directly or indirectly through biochemical cascades to produce targeted cell death. Each CTL expresses a slightly different T cell receptor (TCR), which recognizes a particular peptide sequence sequestered within the Major Histocompatibility complex (pMHC) on target cells. All cells constantly present digested protein fragments representative of the cellular proteome as a pMHC. The peptide is 7-10 amino acids long and provides sufficient information for the immune system to differentiate between the health state and identity of cells. For example, cells whose control over their own translational machinery that has been usurped by viruses will display “foreign” pMHC, which the immune system recognizes as a breach and launches a series of responses eventually leading to the elimination of the disease target. In similar fashion, T cells can recognize cancer antigens. These peptide sequences are either unique (derived from oncogenetic mutations) or self antigens (derived from chronic activation of oncogenes or signal transduction pathways). By maintaining a large dynamic library of different TCRs, a small population of CTLs exist that can induce cell death in cancer cells by recognizing cancer specific pMHC complexes. The ability to harness and augment this specialized killer CTL to launch a persistent, effective, and specific anti-tumor response remains a promising goal in cancer immunology.
Data from animal models and clinical trials have identified three areas to determine whether a cancer immunotherapy strategy is successful or not:
- Generation of a large number of CTLs in peripheral blood
- Traffic and accumulation of these cells at the tumor site
- Acquiring and maintaining effector functions at the tumor site.
Our current understanding of the processes that occur in T cell mediated cancer immunotherapy is fundamentally limited by the rarity of CTLs. Typically, 1mL of peripheral blood contains 109 blood cells and only 30-600 cancer specific CTLs (see figure below). Imagine trying to run an experiment on CTLs but first having to sift through a billion cells to find them! Current technology used to separate cell populations relies heavily on florescence activated cell sorting (FACS). This single cell flow cytometry method is robust but requires a minimum of 105 cells for accurate analysis. Given the rarity of these CTLs in circulation this amounts to large volumes of blood. Clonal expansion and selection of CTLs in vitro with heterogeneous populations of cells derived from blood or cancer sites is a viable alternative, but repeated passaging is prone to genetic drift and primary cells are difficult to keep alive outside the body for extended periods of time.
Further, we have evidence that suggests activated CTLs exit peripheral blood and accumulate at tumor sites (see figure left). These tumor infiltrated lymphocytes (TILs) are integral in promoting melanoma regression in patients treated with CTLA4-antibody that blocks a negative regulatory pathway. Thus an equally important (if not more) area to harvest CTLs is within the tumor. While the limitation of FACS can be overcome for peripheral CTLs by drawing large amounts of blood, investigating CTL activity at tumor sites requires an assay that is sensitive to much smaller populations, even down to a single cell. Further, not all activated CTLs that traffic to the tumor site remain effective; some can be induced into an anergic state by the cancer cells. No single marker can determine whether or not the cell has changed into an effective cell. Therefore, it becomes important to measure the evolution of multiple markers present on cell surface markers, secreted cytokines, mRNAs and intracellular proteins to determine the effector state of the CTLs. Current assays to measure biological signatures include ELISpot, Intracellular Cytokine Staining (ICS), and Cytometric Bead Array (CBA). Some of these assays are variations of FACS and suffer from the same drawbacks. Others are variations of sandwich assays, and require increasing amounts of starting material for multiparameter analysis. Again, the rarity of CTLs in circulation and TILs becomes a major problem.
The goal of NSBCC project 2 is to develop an integrated microfluidic/nanotechnology platform for high-throughput detection and analysis of anti-tumor CTLs and TILs from human cancer immunotherapy trials and murine models of cancer (see discussion of NSBCC technologies). This platform will need to address the following three areas:
- Tumor specific CTL and TIL separation, capture, enumeration and culture from peripheral blood and tumor sites.
- Multiparameter proteomic analysis of cellular surface markers, intracellular and extracellular proteins from small numbers of cells, down to single cells.
- Genomic analysis of mRNA transcripts at the single cell level.
The development of a platform that can handle rare cell separation, capture, and culture at the single cell level will allow detailed characterization of various strategies to promote T cell mediated cancer immunotherapy. For example, the simple ability to count the number of TILs will begin to quantitate the effectiveness of a particular therapy in generating a specific anti tumor response. Further, the ability to manipulate single cells on chip will bypass the need to draw large amounts of blood and will allow analysis of cancerous tissue biopsies. The development of nanoelectronic/nanomechanical protein sensors within these microfluidic platforms would allow simultaneous reading of numerous activation-induced secreted proteins characterizing T cell responses to different antigens. Finally, integration of high-sensitivity transcriptional analysis at single cell level could greatly expand the analytic power of the assay through multiplexing.
Successful completion of this work will require the collaboration and interaction of differently oriented scientific environments represented by the Roukes and Heath groups (microfluidic/nanosensing technologies) and the Witte and Ribas groups (cancer immunotherapy). Findings from the proposed studies will aid in developing enabling nanotech/microfluidic diagnostic and discovery devices applicable not only to the clinical setting of cancer immunotherapy but also to other areas of research, including infectious diseases (acquired immune deficiency syndrome-AIDS, influenza, Epstein-Barr virus and cytomegalovirus infections), allergic diseases, autoimmune diseases and tissue transplantation.