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neoantigen reactive T cells [122] . To improve the efficacy of the T cell therapy, engineering TILs to express the
neoantigen-specific TCR can be a promising next-generation immunotherapy drug [124] . However, to develop
these engineered T cells, identifying paired sequences of both TCR a and b chains from the vast repertoire
of TCRs is a challenge. One way to overcome this challenge is to perform, single-cell TCR profiling to ob-
tain paired TCR α/β sequence information [125] . Using patient samples, neoantigen specific CD8 T cells were
clonally expanded in vitro and multiple paired TCR sequences were identified by single-cell analysis [124] .
Importantly, the transduced T cells expressing TCRs recognized the neoantigen presented by autologous
antigen-presenting cells [124] . Another study using single-cell TCR repertoire analysis revealed that clonally
expanded CD8 T cells were antigen-specific and showed cytotoxic activity against tumors in mouse mod-
els [126] . Intriguingly, the combination of 10x Genomics’ single cell TCR sequencing platform coupled to gene
expression holds enormous potential for assessing and monitoring patient response to cancer vaccines and
immunotherapy drugs.

Monitoring the functional state of CD8 T cells
In the tumor microenvironment, the ability of CD8 T cells to secrete pro-inflammatory cytokines and exert
cytotoxic function can be compromised during persistent immune activation [127] . Such exhausted CD8 T
cells differ profoundly from memory CD8 T cells and co-express multiple co-inhibitory immune check-
point regulators such as PD-1, LAG-3, and TIM-3 and lack successful anti-tumor immune response [127,128] .
Even though various checkpoint inhibitors show clinical efficacy by unleashing cytotoxic T cells activity, a
large fraction of patients fails to respond to these immunotherapies [129] . Therefore, a detailed understanding
of the mechanisms of CD8 T cell exhaustion is required. Further, since the transcriptional signatures of T
cell exhaustion are closely intertwined with their activated T cell state, single-cell analysis is an optimal ap-
proach to identify biomarkers specific to T cell dysfunction. In a single-cell RNA-seq analysis of T cells from
hepatocellular carcinoma patients, 11 unique T cell subsets were identified based on their molecular and
functional properties [130] . Exhaustion signature gene LAYN was identified and associated with inhibition of
IFN-g production [130] . A single-cell RNA-seq of CD8 tumor-infiltrating lymphocytes from murine tumor
models has also aided identification of novel molecular pathways of T cell exhaustion that is uncoupled from
T cell activation [131] .

Profiling of immune suppressive cell types present in the tumor microenvironment
Single-cell transcriptome profiling enables characterization of the complex tumor microenvironment with
its heterogeneous mixture of tumor cells along with stromal and immune cells [132] . Targeting of immuno-
suppressive cell types in the tumor microenvironment can sometimes be key to the efficacy of checkpoint
inhibitors such as anti-CTLA-4 therapy. A variety of cell types including T regulatory cells (Tregs), tumor-
associated macrophages, type 2 NKT cells, M2 macrophages and MDSCs enforce immune suppression in
the tumor helping tumor cells to survive anti-tumor immune attack [133] . Identifying MDSCs has been chal-
lenging from bulk sequencing data due to the absence of unique MDSC markers. In addition, the presence
of over 10 different myeloid subsets further complicates bioinformatics analysis [134] . Tregs are potent immune
modulators and assessing their frequency, phenotype, and function at tissue sites has been profoundly chal-
lenging due to the fact that majority of the defining markers like CD25, FOXP3 and CTLA4 are also pres-
ent in effector T cells [135] . Single-cell analysis of tumor infiltrated immune cells can help circumvent some
of these hurdles in tumor characterization. In a recent single-cell analysis study tumor cells from 11 breast
cancer patients, cancer cells were separated from immune cells based on their copy number variations [132] .
Analysis of the immune cell fraction revealed the presence of immunosuppressive macrophages of M2 phe-
notype and activated T effector cells. Interestingly, the T cells also expressed markers of T cell exhaustion
such LAG3 and TIGIT suggesting that they could be targeted by immune checkpoint inhibitors [132] .

Understanding mechanisms of disease resistance
Resistance to chemotherapy and molecularly targeted therapies is a major barrier to achieving long-term