Highly reproducible TCR profiling using RNA from rhesus macaque PBMC
Non-human primates (NHP) such as the rhesus macaque (Macaca mulatta) have long been key translational models in biomedical research because of their genetic and physiological similarity to humans. Studies using rhesus macaques have contributed significantly to our understanding of T-cell responses to vaccines, cancer, and infectious diseases. More recently, these NHP have emerged at the forefront of COVID-19 vaccine research.
Increasingly complex information can now be gleaned from immune system processes. High-throughput TCR sequencing (TCR-seq) profiles T-cell responses in exquisite detail. A comprehensive understanding of immune responses in such a closely related organism as the rhesus macaque would be a significant advance in science.
Numerous tools exist for performing TCR-seq in human samples, but equivalent tools for rhesus samples have been lacking. Because rhesus macaques often serve as surrogates in the lead-up to human studies, there is an industry need for a complete TCR-seq solution for these NHP samples.
In humans and NHP, T cells are an essential component of adaptive immunity. On the surface, each T cell expresses a unique T-cell receptor (TCR) to recognize a specific antigen. Each TCR is a heterodimer. About 95% of T-cells have TCRs composed of an alpha and beta chain. The remaining 5% have TCRs composed of delta and gamma chains.
TCR are encoded by the TRA and TRB genes in the case of αβ T cells, or TRG and TRD genes in the case of ɣ∂ T cells. All of these genes are organized into clusters of segments representing the variable (V), diversity (D; TRB/TRD only), joining (J), and constant (C) regions of the TCR. Developing T cells undergo V(D)J recombination in which single V, D, J, and C gene segments are recombined into mature TRA and TRB loci (or TRG and TRD loci). From these, TCRα and TCRβ chains (or TCRɣ and TCR∂) are expressed. Each clonal population of T cells shares a clonotype, or distinguishing set of loci.
The TCR repertoire—that is, the diverse collection of clonotypes in an organism—plays a major role in shaping immune function, health, and disease progression. Over the last decade, next generation sequencing (NGS) approaches are increasingly used to profile human TRA and TRB genes. Researchers often choose to maximize information by sequencing full-length genes. For high-throughput NGS projects where speed and cost-efficiency are needed, researchers may choose to focus on the CDR3-encoding region of the TRB gene. This region, which contacts the antigen, is the most variable (Figure 1).
The constant regions of the TRA and TRB genes in human and rhesus macaque share significant homology. Therefore, it is likely that tools used to track human T-cell repertoire dynamics can do the same in the rhesus macaque.
SMARTer Human TCR a/b Profiling Kit v2 chemistry
Since the recombined V(D)J segments are located in a region extending to the 5' ends of the TRA and TRB genes, capture of full RNA transcript information is essential for immune profiling. The TCRv2 kit employs SMART (Switching Mechanism at 5' end of RNA Template) technology combined with a 5'-RACE approach. This method ensures efficient incorporation of an RNA's 5'-end via the template-switching mechanism.
Briefly, the SMARTScribe Reverse Transcriptase adds a sequence of nontemplated nucleotides the 5' end of the cDNA. The TCR SMART UMI Oligo (TSUO) binds to these nontemplated nucleotides and also adds the unique molecular identifiers (UMIs). This template-switching step ensures efficient capture of the 5' end of RNA template. Full-length cDNA can be obtained by a combination of the TSUO, the highly efficient long-distance reverse transcriptase, and the use of high-integrity RNA as input.
Subsequent rounds of TCR-specific, nested PCR cycles, depicted in Figure 2, Panel A, amplify the V(D)J portions of the TRA and TRB sequences. These steps rely on primers binding to conserved sequences in the constant regions of TRA and TRB. During the PCR 2 step, unique dual indexes (UDIs) are added. Figure 2, Panel B shows a zoomed-in schematic view of these locations. Sequence homology allows TCRv2 chemistry to amplify rhesus macaque transcripts in an accurate and unbiased manner during both PCR steps.
SMARTer Human TCR a/b Profiling Kit delivers robust and reproducible TCR capture from rhesus macaque RNA input
To test the compatibility of the TCRv2 kit with rhesus macaque RNA, samples from rhesus macaque and human donors were processed in parallel, with no changes to the TCRv2 kit protocol. The TRA and TRB gene homology between rhesus macaque and human precluded any need to change the kit's components. To cover the high and low ends of the RNA input range, 10 ng and 100 ng were chosen as the amounts of starting material. In this case, the sources were total RNA isolated from peripheral blood mononuclear cells (PBMC) of three rhesus macaques or from the control supplied with the TCRv2 kit, Human Blood, Peripheral Leukocytes Total RNA (hereafter referred to as hCtrl).
Libraries were generated in triplicate from each of the rhesus macaque donors or from hCtrl, using high-quality RNA (RIN ≥8). Hereafter, libraries made from 10 ng RNA from rhesus macaque PBMC are referred to as RM-10 while those made from 100 ng are called RM-100.
For both human and rhesus macaque libraries, Bioanalyzer traces showed an amplicon size distribution of 650–1,150 bp, with an average peak size of roughly 690 bp, as would be expected for this type of cDNA sample. Figure 3 shows electroherograms of the SMARTer TCR libraries generated from hCtrl (Panel A)and PBMC RNA from a rhesus macaque (Panel B).
Clonotype capture from rhesus PBMC RNA using the TCRv2 kit is highly reproducible across input levels
The number of clonotypes detected was dependent on the quantity of starting RNA provided (Figure 4, Panel A). Clonotype counts from the TRA and TRB libraries consistently increase as the amount of RNA input increases. Regardless of input amount, the finished TRA/TRB libraries made from rhesus macaque PBMC were highly reproducible, with strong correlation between technical replicates. Analysis of overlapping clones revealed Pearson correlation scores of at least 0.9 for TRA and 0.96 for TRB (Figure 4, Panels B and C). This matches the high standard set by TRA/TRB libraries generated from human RNA samples using the TCRv2 kit, which had Pearson correlation scores of at least 0.91 for TRA and 0.97 for TRB.
Cogent NGS Immune Profiler is compatible with rhesus macaque TCR sequencing data
It is critical that the analysis software that is included with the TCRv2 kit function with these sequences. The software, Takara Bio’s Cogent NGS Immune Profiler v1 (CogentIP), is available for free to customers using the TCRv2 kit. Currently, this user-friendly solution performs its TCR-seq read alignment using the human genome as a reference. Here, we validate CogentIP as an appropriate choice for analyzing sequences of rhesus macaque origin.
As verification of species compatibility, we compared the performance of CogentIP to that of another software platform, MiXCR v3. Available on a third-party website, MiXCR v3 can align TCR-seq reads using the rhesus macaque genome as a reference. Using FASTQ files generated from sequencing RM-10 and RM-100 libraries, CogentIP and MiXCR v3 identified similar numbers of TRA and TRB clonotypes (Figure 5, Panel A). As for specific clonotypes identified, there was significant overlap in the RM-10 and RM-100 datasets across both platforms. A comparison of clonotype rankings according to CogentIP and MiCXR v3 found strong correlation between datasets, regardless of RNA input amount. The TCR-seq results from the RM-10 libraries are compared in Figure 5, Panel B.
The distribution of TCR clonotypes identified in the sequencing data can also be depicted visually using chord diagrams. Figure 6 shows examples representing the observed distributions of TRA and TRB V-J segment combinations identified one representative rhesus macaque donor and one human control. All chord diagrams were generated using the CogentIP analysis software. Data input from both species produced visualizations capturing the full spectrum of TRA and TRB V-J pairs.
CogentIP, despite using human genome reference for alignment, can be therefore used with confidence to analyze TCR-seq data from the rhesus macaque.
SMARTer Human TCR a/b Profiling Kit generates rhesus macaque TCR sequencing libraries compatible with various Illumina sequencing platforms
A key feature of TCRv2 is compatibility with high-throughput Illumina platforms such as NextSeq® and NovaSeq™. Full-length sequencing on the MiSeq (V3 600 cycle kit) is a great choice for those researchers who want information about the full variable (V) region. Researchers who opt for faster and/or more cost-efficient sequencing may prefer to focus on CDR3 only.
We validated this flexibility using both RM-10 and RM-100 libraries. Mixed TRA/TRB libraries were first run on the MiSeq platform, with full-length sequencing performed at 2 x 300 bp reads. The same libraries were also sequenced using the NextSeq platform for CDR3 sequencing at 2 x 150 bp reads (Figure 7).
The number of clonotypes was strongly correlated between samples, regardless of instrument or sequence length. Therefore, TCR-sequencing libraries from the rhesus macaque produce nearly identical results regardless of the Illumina platform used.
We have shown that the SMARTer Human TCR a/b Profiling Kit v2 (TCRv2) chemistry is compatible with rhesus macaque RNA samples. Resulting libraries are of the same high quality as those created from human samples. The Bioanalyzer traces of TRA/TRB-mixed libraries were similar, regardless of whether the RNA input into TCRv2 came from human or rhesus macaque RNA. Of note, these libraries can be generated without compromise to the many features that make TCRv2 the preferred choice for NGS-based bulk TCR profiling.
The Cogent NGS Immune Profiler v1 software (CogentIP), our bioinformatic solution for TCR data analysis, is validated for use with TCR-sequencing of rhesus macaque samples. When used to process TCR-seq data from rhesus macaques, CogentIP showed reproducibility at low and high inputs. There was an increase in the number of clonotypes identified as RNA input increased.
Further, we compared data processed with CogentIP to data processed with third-party software, MiXCR v3. While MiXCR v3 allows read alignment with a rhesus macaque reference, CogentIP used a human reference. Nevertheless, CogentIP was able to identify a highly overlapping set of clonotypes between replicates. Additionally, a diverse range of TRA and TRB V-J pairs could be visualized using data input from both species.
Depending on budget and the level of detail desired, researchers can use TCRv2-generated libraries for full-length TRA/TRB sequencing on the MiSeq platform or choose to sequence the CDR3 region only on high-throughput platforms such as NextSeq.
Together, the SMARTer Human TCR a/b Profiling Kit v2 and Cogent NGS Immune Profiler v1 software represent an industry first: a complete TCR repertoire analysis solution for use with the rhesus macaque.
Mixed TRA/TRB libraries were generated using the TCRv2 kit as per the user manual. Briefly, reverse transcription was carried out using an oligo dT with the template-switching TCR SMART UMI Oligo. Next, two rounds of PCR with primers specific for TRA/TRB genes enriched the cDNA with TCR chain sequences. Human and rhesus macaque libraries were prepared in triplicate using 10 ng or 100 ng RNA, amounts chosen according to the upper and lower input range for this kit.
Library quantification and analysis
Following purification and size selection, library yield and size distribution were determined using the Invitrogen Qubit Fluorometer and Agilent 2100 Bioanalyzer instruments, respectively. The Library Quantification Kit (Cat. # 638324) was used to quantify pooled libraries. Libraries were sequenced on both the Illumina MiSeq (600-cycle V3 cartridge; Cat. # MS-102-3003) and Illumina NextSeq (300-cycle v2.5 Mid Output; Cat. # 20024908).
As directed by TCRv2 protocol, sequencing data analysis was performed with the Cogent NGS Immune Profiler Software v1 using standard parameters, including the human genome as alignment reference. A parallel analysis was performed with third-party MiXCR v3 software, which allowed for read alignment using a rhesus macaque genome reference obtained from IMGT.
Zhang, Z. et al. A greedy algorithm for aligning DNA sequences. J. Comput. Biol. 7, 203–14 (2000).
Giudicelli, V. et al. IMGT/LIGM-DB, the IMGT comprehensive database of immunoglobulin and T cell receptor nucleotide sequences. Nucleic acids res.34, Database issue D781–4 (2006).
Thiel, C. et al. Structure and diversity of the T-cell receptor alpha chain in rhesus macaque and chimpanzee. Hum. immunol.43, 85–94 (1995).
Levinson, G. et al. Sequence and diversity of rhesus monkey T-cell receptor beta chain genes. Immunogenetics35, 75–88 (1992).
Barennes, P. et al. Benchmarking of T cell receptor repertoire profiling methods reveals large systematic biases. Nat. Biotechnol.39, 236–245 (2021).
Bolotin, D. A. et al. MiXCR: software for comprehensive adaptive immunity profiling. Nat. Methods12, 380–381 (2015).