1. bookVolume 9 (2021): Issue 1 (January 2021)
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2332-7774
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Toward the Analysis of Lymphocyte Development in Space: PCR-Based Amplification of T-Cell Receptor Excision Circles (TRECs) Aboard the International Space Station

Published Online: 31 Dec 2021
Page range: 159 - 163
Journal Details
License
Format
Journal
eISSN
2332-7774
First Published
30 Jan 2019
Publication timeframe
2 times per year
Languages
English
Abstract

Spaceflight offers vast possibilities for expanding human exploration, whereas it also bears unique health risks. One of these risks is immune dysfunction, which can result in the reactivation of latent pathogens and increased susceptibility to infections. The ability to monitor the function of the immune system is critical for planning successful long-term space travel. T lymphocytes are immune cells that develop in the thymus and circulate in the blood. They can detect foreign, infected, or cancerous cells through T cell receptors (TCRs). The assembly of TCR gene segments, to produce functional TCR genes, can be monitored by measuring the presence of TCR excision circles (TRECs), circular fragments of DNA that are by-products of this assembly process mediated by the V(D)J recombination machinery. In this study, we used polymerase chain reaction (PCR) on the International Space Station (ISS) to detect TRECs in murine peripheral blood. We were able to detect TRECs in the blood of normal healthy mice of different ages, with an efficiency comparable to that achieved in ground controls. As expected, we were unable to detect TRECs in the blood of immunodeficient mice. These results are the first step in optimizing a specific, rapid, safe, and cost-effective PCR-based assay to measure the integrity of mammalian immune systems during spaceflight.

Keywords

INTRODUCTION

Current evidence suggests that spaceflight may impair the immune system and increase the risk of infection (Crucian et al., 2018; Frippiat et al., 2016; Sonnenfeld, 2002). This is likely due to a combination of factors, including microgravity, reduced exposure to environmental antigens, prolonged isolation, and increased radiation exposure. These factors may become exacerbated during long-term space travel, such as planned human expeditions to Mars or the moon (White and Averner, 2001). Robust approaches to monitor the status of immune cell development in astronauts during long-duration missions are needed for early detection of impaired immune function that could lead to serious illness.

T cells, originating from hematopoietic stem cells and maturing in the thymus, are central players of the immune system and are required for critical responses to pathogens. Lack of T cell development or homeostasis, found in conditions including genetic severe common immunodeficiency (SCID) or acquired immunodeficiency syndrome (AIDS), may result in fatal infections unless detected and treated immediately. During T cell maturation, T cell receptor genes are rearranged, creating a wide range of T cell receptors (TCRs) that can recognize a multitude of antigens. The process of rearrangement generates fragments of excised DNA that are circularized and form TCR excision circles (TRECs). TRECs, stable molecules that persist in developing T cells, can be used to estimate the rate of T cell development. A low TRECs level, a proxy for low T cell development, indicates an immunodeficiency and a high risk of infection. Polymerase chain reaction (PCR)-based detection of TRECs in the blood is commonly used on Earth to screen newborns for SCID and to monitor the efficacy of therapy in AIDS patients (Baker et al., 2009). Thus, the TREC detection PCR assay represents a relatively simple and useful technique to monitor the state of immune system development and to detect immunodeficiencies.

This manuscript describes one of the two experiments of the Genes in Space-5 (GiS-5) investigation. The objective of the second study was to establish the feasibility of an in-flight multiplex PCR-based assay to assess and monitor genomic stability by analyzing microsatellite alterations in space. This study aims to better understand the effect of space travel on the immune system. We performed a proof-of-principle study of PCR-based TREC detection on the International Space Station (ISS). The successful results of this study present an initial step towards developing a complete workflow to monitor T-cell development during spaceflight.

MATERIALS AND METHODS
Mice

Animal samples were obtained by Lee Serpas according to the protocol (PI: Boris Reizis) approved by the Institutional Animal Care and Use Committee of New York University School of Medicine. Wild-type control mice of C57BL/6 background were bred in the lab’s animal facility and B6.129S6-Rag2 N12 mice were purchased from Taconic Biosciences. Blood from C57BL/6 wild-type mice was collected from individual 1.5-, 3-, and 5-month-old mice, all raised on Earth. Samples from two to four mice were collected per time point. Thymus tissue was collected from two-month-old C57BL/6 wild-type mice. Blood was also collected from two-month-old Rag2-deficient mice. Rag2 deficiency arrests T and B cell development, rendering the mouse severely immunodeficient (Shinkai et al., 1992).

DNA Extraction and Purification

Blood was drawn from the submandibular vein by puncture with a sterile disposable lancet into EDTA coated tubes from Kent Scientific (product # MTSC-EDTA). In all cases, the blood was collected from mice on the ground, that was not exposed to conditions of microgravity. One hundred microliters of blood were used for DNA isolation. Blood was centrifuged at 1,000×g for 10 min to isolate cells. The red blood cells were lysed for 8 min in red blood cell (RBC) lysis buffer (55 mM NH4Cl, 12 mM NaHCO3, 0.1 mM EDTA). The remaining leukocytes were then resuspended in 200 μL of phosphate-buffered saline (PBS, Gibco). DNA was purified using the QIAamp DNA Mini Kit (Qiagen), according to the manufacturer’s instructions.

PCR Primers

The primers used to amplify the TRECs excised during Tcra gene rearrangement were 5′-CTCTGAGGAACACGGAGTATC-3′ and 5′-TGTCCTCAGCCTTGATCCATC-3′, with an expected amplicon size of 294 bp (Sempowski et al., 2002).

The genomic control is the constant region of Tcra. The primers used to amplify the control were 5′-CTTTGACTCCCAAATCAATGTG-3′ and 5′-TTGGCAGGTGAAGCTTGTCTG-3′, with an expected amplicon size of 142 bp (Broers et al., 2002).

PCR Conditions

TRECs primers (400 nM) and control primers (150 nM) were used to amplify DNA with the NEB Hot Start 2x Master Mix (M0496). The amplification conditions were as follows: 95 °C for 30 s, [95 °C 30 s, 57.5°C for 30 s, 68 °C for 60 s] × 34 cycles, and 68 °C for 5 min.

Sample Preparation

Complete PCR mixes were prepared in quintuplicates on the ground. One replicate was immediately analyzed to confirm TRECs amplification. Two replicates were kept on the ground, and two replicates – the flight samples – were sent to the ISS. The samples were kept frozen at −80°C before and during shipment to Kennedy Space Center, FL, where they were loaded into a CRS Dragon spacecraft. NASA keeps the cold chain storage at −80°C at all times. The samples were launched to the ISS on NASA’s Commercial Resupply Service (CRS-14) mission. Upon successful berthing at the ISS (after 2 days of orbiting Earth), the tubes were transferred to the Minus Eighty Degree Laboratory Freezer (MELFI) in the US National Laboratory. Aboard the ISS, the astronaut crew removed the tubes from the MELFI and allowed them to thaw for 5 min at room temperature before placing them in a miniPCR® mini8 thermal cycler (miniPCR bio) for amplification (Boguraev et al., 2017). After each run was complete, reaction tubes were given 30 min to cool down before being removed from the thermal cycler and then transferred back to the MELFI. Tubes were kept in the MELFI until they were reloaded into the Dragon vehicle for return to the ground. Upon splashdown, samples were returned to Johnson Space Center in unpowered cold bags at −32°C and shipped frozen to miniPCR bio for final analysis. Analysis was completed using gel electrophoresis on a 2% agarose gel at 110 V for 45 min.

RESULTS

To detect TRECs in the peripheral blood of adult mice, we used a PCR assay to amplify TRECs generated from the rearrangement of the murine Tcra locus. We used two primers that anneal at the Vα and Jα elements so that only circular DNA (TRECs) can be amplified (Sempowski et al., 2002) (Figure 1). Primers for the constant region of Tcra were used as control. DNA from the blood of mice raised on the ground was purified on the ground and transported and tested on the ISS.

Figure 1

TREC formation and primer design. Schematic of TREC formation following VJ recombination at the Tcra locus. Primers are depicted by arrows and are designed to face toward each other following formation of the TRECs (Hazenberg et al., 2001). PCR amplification is expected when primers face each other.

To validate the use of this assay in microgravity conditions, a comparative duplex PCR specific for the Tcra TREC and the constant Tcra genomic control locus was performed in-flight on board the ISS and on the ground as previously validated (Walker and McMichael, 2012). A range of concentrations of mouse thymic DNA (0.1–100 ng) was used to establish the limits of detection of the PCR-based amplification method (Figure 2A). The results confirmed a concentration-dependent detection of TREC amplification products in wild-type thymic DNA samples. The TREC amplification product was marginally detected in 1 ng of total blood DNA and robustly detected in 10 ng and 100 ng of DNA on the ISS. An equivalent duplex PCR performed on the ground revealed a comparable pattern of amplification (Figure 2A).

Figure 2

TRECs detected across a range of ages in mice. (A) Agarose gel electrophoresis of TRECs from the thymus of 2-month-old wildtype mice amplified by PCR in flight and on the ground. Numbers indicate starting concentration of purified DNA in nanograms. (B) Agarose gel electrophoresis of PCR-amplified TRECs from peripheral blood obtained from mice over a range of ages. Rag KO = Rag knockout mice. Thymic DNA = DNA isolated from thymus of 2-month-old wildtype mice. In each reaction, the starting concentration of purified DNA was 10 nanograms.

To validate the assay in independent animals of different ages, TRECs were PCR-amplified both on the ground and in-flight from 10 ng of peripheral blood DNA collected from mice 1.5 months to 5 months old. Our results indicate increased TRECs levels in a 3-month-old mouse compared to a 1.5-month-old mouse and decreased TRECs levels in a 5-month-old mouse compared to the 3-month-old mouse (Figure 2B). These results are expected as T cell production decreases with age (Nikolich-Zugich, 2014; Hale et al., 2006). Mice with genetic deletion (knockout) of the recombination-activating gene 2 (Rag2), an essential component of the Rag complex that mediates T cell receptor DNA recombination, fail to rearrange TCR DNA and do not produce TRECs. They lack mature T and B cells and serve as a model of SCID (Schwarz et al., 1996). We used peripheral blood DNA from Rag2 knockout mice to test the ability of our PCR assay to discern wild type from immunodeficient cells in microgravity. We were unable to detect TREC DNA from the Rag2 knockout mice, while the control genomic DNA was amplified normally. On the other hand, both TREC and control DNA were amplified in DNA collected from mice thymi (Figure 2B), suggesting the assay is specific to TRECs. Collectively, our results show that TRECs can be efficiently and specifically detected over a broad range of concentrations on the ISS, providing a way to detect defects of T cell development.

DISCUSSION

In this study, we used a PCR-based assay aboard the ISS to detect TRECs in the peripheral blood and thymus of mice. The TREC detection assay is commonly used to monitor T cell development and detect immunodeficiencies on Earth. Here, we examined its utility in space, in the hope of establishing a method to monitor the development of immune deficiencies commonly experienced during long-duration spaceflight (Crucian et al., 2018; Frippiat et al., 2016; Sonnenfeld, 2002). We selected T cell development as our focus because of the central role T cells play in regulating immune health. Normal T cell development is a primary indicator of immune system homeostasis and can be severely impaired in immunocompromised individuals (Walker and McMichael, 2012).

We used normal mice of different ages as representative healthy subjects and Rag2-deficient mice as a model of SCID. Our study demonstrates efficient detection of TRECs isolated from the peripheral blood of wild-type animals. Our assay detected TRECs in the range of 1–100 ng of total DNA input, establishing the lower limit of detection for the assay. Differences in the intensity of TREC bands between animals of different ages were observed (Figure 2B). Specifically, we observed an increase in TREC levels from a 1.5-month-old (40 day) mouse to a 3-month-old mouse, indicating an increased level of T cell development at 3 months compared to 1.5 months. We observed a decrease in TREC levels from the 3-month-old mouse to the 5-month-old mouse, indicating a decrease in T cell development at 5 months compared to 3 months. These results are generally consistent with the knowledge that naïve T cell production decreases as animals mature (Nikolich-Zugich, 2014; Hale et al., 2006). Detailed characterization of temporal changes in TREC levels will allow us to discern changes due to microgravity from those due to aging. Additional samples across different ages, including very old mice (>18 months of age), could be examined in future experiments.

Due to technical and logistical limitations, we could not draw blood in flight; thus, the experiment was performed using blood collected on the ground and transported to the ISS. In future experiments, tissue should be collected from live mice onboard the ISS to monitor the nature of T cell development during spaceflight. The shorter lifespan of mice relative to humans allows for faster characterization of the effects of prolonged space travel on physiology.

While our assay is robust for the detection of TRECs, further expansion of the ISS’s molecular biology toolkit will enable higher-resolution tracking of T cell development in the future. The use of real-time PCR (Parra et al., 2017) would permit the quantification of TRECs. Additionally, the use of flow cytometry would permit direct quantification of T lymphocytes in the peripheral blood and offer a more direct readout of immune health, and would confirm the validity of the TRECs PCR assay. T cell receptor sequencing can also be conducted to understand changes in TCR repertoire.

This study focused solely on T cell function, but future studies on other immune cells will contribute to a more complete picture of immune health. A recent study indicated the potential for spaceflight to affect human IgM repertoire (Buchheim et al., 2020). Therefore, a complementary future study may assess the homeostasis of B cells to determine if their function may be impaired. If the number of circulating B cells is negatively affected by spaceflight (Ichiki et al., 1996; Tascher et al., 2019), there will be a decrease in circulating immunoglobulins, which can be quantified by the use of an enzyme-linked immunosorbent assay.

Our study establishes a proof of principle that TREC PCR analysis can be used aboard the ISS. This assay can now be further developed to apply to humans and offer insights into the nature of immune defects that occur in microgravity. By using mice as model organisms in this study, we laid the groundwork for human TREC amplification in space, opening a door to the possibility of real-time monitoring of T cell development in astronauts.

Figure 1

TREC formation and primer design. Schematic of TREC formation following VJ recombination at the Tcra locus. Primers are depicted by arrows and are designed to face toward each other following formation of the TRECs (Hazenberg et al., 2001). PCR amplification is expected when primers face each other.
TREC formation and primer design. Schematic of TREC formation following VJ recombination at the Tcra locus. Primers are depicted by arrows and are designed to face toward each other following formation of the TRECs (Hazenberg et al., 2001). PCR amplification is expected when primers face each other.

Figure 2

TRECs detected across a range of ages in mice. (A) Agarose gel electrophoresis of TRECs from the thymus of 2-month-old wildtype mice amplified by PCR in flight and on the ground. Numbers indicate starting concentration of purified DNA in nanograms. (B) Agarose gel electrophoresis of PCR-amplified TRECs from peripheral blood obtained from mice over a range of ages. Rag KO = Rag knockout mice. Thymic DNA = DNA isolated from thymus of 2-month-old wildtype mice. In each reaction, the starting concentration of purified DNA was 10 nanograms.
TRECs detected across a range of ages in mice. (A) Agarose gel electrophoresis of TRECs from the thymus of 2-month-old wildtype mice amplified by PCR in flight and on the ground. Numbers indicate starting concentration of purified DNA in nanograms. (B) Agarose gel electrophoresis of PCR-amplified TRECs from peripheral blood obtained from mice over a range of ages. Rag KO = Rag knockout mice. Thymic DNA = DNA isolated from thymus of 2-month-old wildtype mice. In each reaction, the starting concentration of purified DNA was 10 nanograms.

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