During 2016, we established a single-cell sequencing technology in our laboratory that can simultaneously determine the TCR sequence and 33 functional genes of a single CD8+ T cell, based on a state-of-the-art technology initially developed for CD4+ T cells (Han et al., 2014). The functional gene panel we developed allows us to comprehensively profile the phenotypes (naive, central memory, effector memory, late effector memory and resident memory) and functional states (activation, anergy, exhaustion and apoptosis) of tumor-specific CD8+ T cells at the single-cell level. The establishment of this single-cell technology allows us to deeply decode single tumor antigen-specific CD8+ T cells of lymphoma patients, as we originally proposed in this project.

Samples were obtained through a protocol approved by the Institutional Review Board of the University of Chicago. The samples were single-cell suspensions derived from diagnostic follicular lymphoma (FL) biopsy specimens, which were cryopreserved in liquid nitrogen until the time of analysis. FL grading was determined using the 2008 WHO criteria. In all cases, the neoplastic follicles exhibited an immunophenotype typical for FL, with positive staining for the B- cell marker, CD20, with co-expression of the germinal center B cell markers, CD10 and BCL-6 as well as BCL-2. The peripheral blood mononuclear cells (PBMC) were isolated from healthy donors’ peripheral blood according to PBMC CPT v.009 protocol.

Our single-cell sequencing strategy is composed of three phases of polymerase chain reactions (PCR). In phase-I PCR, the mRNA of the TCR and functional genes from each cell were converted to cDNA and further amplified using a multiplexed panel of TCR primers and phenotyping primers. To enhance PCR efficiency, we performed nested PCR for TCR genes and functional genes separately, and generated a pool composed of short fragments (~200 bp) from genes of interest in phase-II PCR. In this step, two common nucleotide sequences were engineered into the primers for the next run of barcoding PCR. In phase-III PCR, individual barcodes were added to the phase-II products. After phase-III PCR, the DNA concentration of each cell ranged from 13800 pg/ul to 32500 pg/ul. Finally, we combined and purified the products of both TCR genes and functional genes from phase-III PCR and sequenced them using the Illumina MiSeq platform. Both negative and positive controls were included in each experimental setting for quality control.

To test our single-cell sequencing platform, we analyzed the CD4+ regulatory T cells (Tregs) of two FL patients using the previously well-established method that is suitable for Tregs (Han et al., 2014). Single CD3+CD4+ CD25+ CD127- T cells were single-cell sorted by flow cytometry, deep sequenced by illumina platform, and analyzed by our in-house data analysis pipeline. After successfully accomplishing this set of experiments using CD4+ Tregs, we then further tested our newly developed functional gene panel for analyzing CD8+ T cells. We single-cell sequenced CD8+ T cells isolated from two lung cancer patients. The results have shown that we successfully linked TCR sequence to T cell functional phenotype at the single-cell level. We are currently working on analyzing tumor-specific CD8+ T cells form lymphoma patients.

Invited Talks

I was invited by the President of Nanjing Medical University as an honorary speaker to talk in the 2016 symposium “Jinling Medical Symposium of Nanjing Medical University” at the International Conference on Biomedical Engineering and Clinical Medicine, in Nanjing City of China.

I was also an invited speaker to the 2016 Biophysics Program Advance of The University of Chicago at Turkey Run State Park in Marshall, Indiana.

Publications

1. Huang, J.#, Zeng, X., Sigal, N., Lund, P.J., Su, L.F., Huang, H., Chien, Y.H., and Davis, M.M#. (2016). Detection, phenotyping, and quantification of antigen-specific T cells using a peptide-MHC dodecamer. Proceedings of the National Academy of Sciences of the United States of America 113, E1890- 1897.

2. Sasmal, D.K.#, Pulido, E. L., Kasal, S., and Huang, J.# (2016). Single-Molecule Fluorescence Resonance Energy Transfer in Molecular Biology. Nanosclae, 8 (48), 19928-19944.

#Corresponding author

Funding

This award has been used to pay for PCR primers, PCR reagents, PCR thermal cycles, sequencing services, and regular consumables for the proposed project. The generous support of the Young Investigator Award has helped us build, develop and validate our single-cell sequencing technology, and allowed us to continue our proposed research into 2017.

Ongoing Research

With our established single-cell sequencing technology, we are now working on single-cell sequencing tumor-specific CD8+ T cells from 6 follicular lymphoma patients’ tumor lymph nodes. Our focus of last year was to develop a single-cell sequencing technology platform, and this year we aim to sequence tumor-specific CD8+ T cells and analyze the data. Combined with the CD4+ Tregs’ data previously obtained, we aim to decipher the T-cell repertoires of FL patients.

Future Plan

We plan to accomplish the remaining sequencing work for tumor-specific CD8+ T cells. In the meantime, we will analyze experimental data and summarize our research results. After reaching the goals set for this project, I will work with my students to write manuscripts and submit to professional journals for publication. I will also wrap up this project and submit a final report to the Cancer Research Foundation.

With the support of this Young Investigator Award, we have established a flow cytometry sorting based single-cell sequencing technology in our laboratory, which can simultaneously determine the TCR sequence and functional phenotype of a single T cell. The establishment of this single-cell technology allows us to deeply decode single tumor antigen-specific T cells from mice and humans.

Furthermore, we have recently started using an automatic 10x platform to perform high-throughput single-cell sequencing. The preliminary results are very promising and we are currently optimizing experimental conditions and refining the data analysis pipeline. Based on our previous peptide-major histocompatibility complex (pMHC) dodecamer technology (Huang et al, PNAS, 2016), we have simultaneously developed an antibody dodecamer to isolate antigen-specific T cells for mass cytometry and sequencing analyses.

We will apply this technology to study tumor-specific T cells from different cancers. I have presented our research results domestically and internationally. I was invited by the President of Nanjing Medical University as the keynote speaker in the 2016 symposium, “Jinling Medical Symposium of Nanjing Medical University” at the International Conference on Biomedical Engineering and Clinical Medicine. I also presented our dodecamer work at the American Association of Immunologists (AAI) annual meeting.

In addition, I was invited to speak at the 2016 UChicago Biophysics Program, 2017 University of Minnesota-University of Chicago Joint Immunology Retreat, and 2017 UChicago Biomedical Science Cluster Retreat. Using the preliminary data generated from this supported project, we have written and submitted an NIH R21 grant to the National Cancer Institute. Although we were not funded in the first round, the score—20th percentile–was good and the review was generally positive.

We are currently revising the proposal for a second submission. Finally, with the support of this award, we have published the following two papers: 1. Rosenberg J and Huang J. CD8+ T cells and NK cells: parallel and complementary soldiers of immunotherapy. Current Opinion in Chemical Engineering, 2018; 19: 9-20. 2. Wendel et al. Reduced clonal and functional diversity of follicular helper T cells in human lymph nodes during chronic HIV infection. Science Immunology. (in press).