Strong Selection of a Few Dominant CD8 Clones in a TLR7-Dependent Autoimmune Mouse Model
←
→
Page content transcription
If your browser does not render page correctly, please read the page content below
Strong Selection of a Few Dominant CD8 Clones in a
TLR7-Dependent Autoimmune Mouse Model
Peter A. Morawski and Silvia Bolland
Downloaded from http://www.immunohorizons.org/ by guest on February 9, 2022
ImmunoHorizons 2019, 3 (2) 61-70
doi: https://doi.org/10.4049/immunohorizons.1800082
http://www.immunohorizons.org/content/3/2/61
This information is current as of February 9, 2022.
References This article cites 42 articles, 17 of which you can access for free at:
http://www.immunohorizons.org/content/3/2/61.full#ref-list-1
Email Alerts Receive free email-alerts when new articles cite this article. Sign up at:
http://www.immunohorizons.org/alerts
ImmunoHorizons is an open access journal published by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
All rights reserved.
ISSN 2573-7732.RESEARCH ARTICLE
Adaptive Immunity
Strong Selection of a Few Dominant CD8 Clones in a
TLR7-Dependent Autoimmune Mouse Model
Peter A. Morawski and Silvia Bolland
Laboratory of Immunogenetics, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health,
Rockville, MD 20852
Downloaded from http://www.immunohorizons.org/ by guest on February 9, 2022
ABSTRACT
Systemic lupus is characterized by the expansion of a self-reactive repertoire of B cells and CD4 cells that together promote
IgG Ab production against common nuclear Ags. Although several studies have suggested roles for CD8+ T cells in lupus, the full
contribution of these lymphocytes to disease remains undefined. In particular, few studies have examined TCR clonotypes of the CD8
pool in lupus. We previously described activated but nonpathogenic CD8+ T cells in a mouse model of systemic autoimmune disease
triggered by increased copy number of the tlr7 gene (TLR7tg mice), in which some of these T cells accumulate in the brain. In this
article, we report, through the analysis of TCRb sequences, that CD8 cells from TLR7tg animals are strongly selected for a small
number of clones, some of them reaching 30% of the repertoire, compared with less than 0.4% for the top clone in any wild type
mice. High frequency clones are variable in sequence among individual TLR7tg mice and are distinct from top clones in the control
animals, whereas CDR3 sequences of spleen and brain-resident T cells from the same TLR7tg animals have perfect concordance.
These results suggest that top CD8 clones are selected in stochastic fashion in each animal but limit further diversification, and that
brain-infiltrating CD8 cells in TLR7tg mice are not selected by a common tissue Ag. This kind of extreme clonal dominance and
narrowing of the CD8+ repertoire might impair anti-viral responses and should be considered as an additional detrimental feature of
chronic autoimmune disease. ImmunoHorizons, 2019, 3: 61–70.
INTRODUCTION We have studied lupus using several disease models including
TLR7tg mice. These animals have a severe phenotype that includes
A substantial component of systemic autoimmune disease such as glomerulonephritis, anemia, and a lymphoproliferative disorder
lupus involves the breakdown of lymphocyte tolerance, primarily that mainly impacts the CD4+ T and B cell pools, whereas the effect
through the expansion of autoreactive CD4+ T cells that provide help on the CD8 compartment is comparatively minimal (9, 10). We
to B cells, which can in turn produce autoantibodies with various recently showed that the CD8+ T cells in these mice are nonpath-
specificities (1). Although comparatively less is understood about ogenic and have a strong effector phenotype compared with wild
how CD8+ T cells contribute to disease progression, perturbations type controls, with the most activated cells accumulating in the
in CTL function are evident in lupus patients (2, 3). Several groups, brain (11). Because of the vast number of potential self-antigens
including ours, have now shown that a functionally competent available in both the periphery and brain, an understanding of the
CD8+ T cell compartment is required to restrict disease severity Ag specificity of these lymphocytes could help to better under-
in lupus-prone mice (4–7), and subsets of CD8+ T cells have been stand their relevance and function.
identified that can suppress excessive CD4+ T follicular helper cell Membrane-bound Ag receptors are required by all lympho-
help to B cells, which is essential for maintenance of self tolerance (8). cytes for the recognition of peptide fragments bound to and presented
Received for publication November 26, 2018. Accepted for publication January 24, 2019.
Address correspondence and reprint requests to: Dr. Silvia Bolland, National Institute of Allergy and Infectious Diseases/National Institutes of Health, 5625 Fishers
Lane, Room 4N03A, Rockville, MD 20852. E-mail address: sbolland@niaid.nih.gov
ORCID: 0000-0001-9159-4032 (P.A.M.).
This work was supported by the Intramural Research Program of the National Institutes of Health, National Institute of Allergy and Infectious Diseases.
Abbreviation used in this article: IMGT, ImMunoGeneTics information system.
This article is distributed under the terms of the CC BY 4.0 Unported license.
https://doi.org/10.4049/immunohorizons.1800082 61
ImmunoHorizons is published by The American Association of Immunologists, Inc.62 CD8+ OLIGOCLONALITY IN A MOUSE MODEL OF SYSTEMIC LUPUS ImmunoHorizons
by MHC proteins. The TCR serves as the specific Ag receptor on lymphocytes with allophycocyanin/Cy7-CD45.2 (1D4; BioLegend)
T lymphocytes and is composed of an a and a b-chain (12). The is done as described below and allows discrimination between
rearrangement of germline encoded variable (V), diversity (D), vessel (dual CD45.2 labeled) and parenchymal (ex vivo fluo-
and joining (J) genes provides the immunological basis for the rophore only) cells.
vast number of possible unique Ag receptors on lymphocytes.
In naive mice (13, 14) and in healthy humans (15, 16), certain Lymphocyte extraction from tissue
common patterns of Vb gene usage can be found in the formation Cell extraction from tissues was modified according to the described
of the TCR repertoire. Previous studies using TCR spectratyping protocol (23). For lymphocyte extraction, organs were digested
and sequencing of T cells isolated from the blood and kidney of for 30 min at 37°C and shaken at 240 rpm in HBSS containing 1
lupus patients revealed deviations from common TCR b-chain mg/ml Collagenase Type I (Life Technologies). Digested mix
usage and alterations in expected CDR3 profiles (17, 18) and was pelleted, washed, and passed through a 70-mM mesh filter,
identified clonally expanded CD8+ T cells associated with renal then resuspended in Percoll (GE Healthcare) diluted to 90% using
disease (19). Studies using next-generation TCR sequencing have HBSS. Layers of 60 and 37% Percoll were then sequentially
now identified the presence of public sequences among lupus overlayed on the 90% cell–Percoll mix. Cell separation was
patients, which could serve as potential biomarkers for disease accomplished by centrifugation at 8°C, 500 3 g for 18 min without
Downloaded from http://www.immunohorizons.org/ by guest on February 9, 2022
(20). Although these data collectively demonstrate that the TCR brakes. Cells were washed and resuspended in staining buffer
repertoire, and thus, Ag specificity, in lupus is important, we still containing 5% FBS in HBBS in preparation for flow cytometry.
understand little about the selective induction of autoreactive
responses in systemic autoimmunity. Lymphocyte enrichment and flow cytometric sorting
In the current study, we address whether the nonpathogenic Single-cell lymphocyte solutions were prepared from organs as
peripheral and brain-resident CD8+ T cell populations in the indicated and resuspended in staining buffer containing 5% FBS in
TLR7tg model of lupus disease are selected by their Ag specificity. HBBS. Pre-enrichment of splenic lymphocytes was performed
We were interested to understand whether these lupus-prone CD8+ using PE-labeled CD8+ T cell magnetic bead positive selection
T cells respond to environmental factors in a polyclonal fashion according to the manufacturer instructions (RoboSep, STEMCELL
or if some strong selective pressures might exist that induce a Technologies). Splenic and brain-resident lymphocytes were
more Ag-specific response. To accomplish this, we sequenced identified using mAbs against the following Ags, all from BioLegend
the TCR b-chains of this population of CD8+ T cells. Our results unless otherwise noted: PE-CD8a (MAR1), FITC-CD4 (RM4-5),
demonstrate strong clonal dominance of CD8+ T cells can arise BV711-CD11b (M1/70), BV421-CD45.2 (1D4), allophycocyanin/
in the context of systemic autoimmune disease, suggesting that Cy7-CD45.2 (1D4), and allophycocyanin-TCRb, (H57-597; eBio
the function of these cells might be compromised in some severe science). Samples were sorted on an FACSAria III SORP (BD
cases of disease. Biosciences) equipped with violet (403 nm, 100 mW), blue (488 nm,
100 mW), yellow-green (561 nm, 50 mW), and red (638 nm, 150 mW)
lasers, then analyzed with FlowJo (Tree Star). Pre- and postsort
MATERIALS AND METHODS purity of CD8+ T cells are shown in Fig. 1A.
Mice and experimental protocols DNA extraction, TCRb gene sequencing, and analysis
The generation of TLR7tg mice has been described previously Frozen cell pellets of sorted wild type B6 and TLR7tg spleen
(9). All experiments in this study used the transgenic line 7.1, (.2 3 105) and TLR7tg brain (.1 3 104) CD8+ T cells were sent on
which harbors 8- to 16-fold TLR7 gene expression above wild dry ice to Adaptive Biotechnologies (Seattle, WA) for genomic
type controls. All mice were maintained on a C57BL/6J back- DNA extraction and subsequent TCRb gene sequencing. Ampli-
ground. Male mice between 12 and 13 wk of age were used for these fication and sequencing of TCRb CDR3 was performed using the
experiments. All animals were housed and studied in accordance Adaptive Biotechnologies ImmunoSEQ Platform, which combines
with the approved National Institutes of Health Animal Study multiplex PCR with high throughput sequencing and a sophisti-
Protocol, and all experimental protocols were approved by and cated bioinformatics pipeline for TCRb CDR3 analysis (24, 25).
performed according to National Institutes of Health Animal Care Data analysis was performed using ImmunoSeq Analyzer 3.0
and Use Committee guidelines. All efforts were made to minimize software, also provided by Adaptive Biotechnologies.
animal suffering and to reduce the number of animals used.
Statistical analysis
Vascular labeling of leukocytes Statistical significance of data were calculated with Prism 6.0
Discrimination of cells in the vasculature versus those in the brain software (GraphPad). For comparisons between two normally
parenchyma was performed as described (21, 22). Briefly, 5 mg of distributed groups, a two-tailed unpaired t test with Welch
BV421-CD45.2 (1D4; BioLegend) diluted in sterile 13 PBS was correction was used. For comparison between more than two
injected i.v. once per mouse. Abs were allowed to circulate for a groups, statistical analysis was performed using a one-way
maximum of 3 min, and then mice were euthanized and organs ANOVA with the Tukey method. Gaussian distribution analy-
were extracted. Subsequent ex vivo colabeling of tissue-extracted sis of CDR3 sequences was used to characterize population
https://doi.org/10.4049/immunohorizons.1800082ImmunoHorizons CD8+ OLIGOCLONALITY IN A MOUSE MODEL OF SYSTEMIC LUPUS 63
Downloaded from http://www.immunohorizons.org/ by guest on February 9, 2022
FIGURE 1. Isolating CD8+ T cells from lupus-prone mice.
(A) Gating strategy (presort) and results (postsort) for flow cytometric isolation of CD8+ T cells from wild type B6 and TLR7tg brain and spleen.
Discrimination of cells in the parenchyma (tissue) from those in the vasculature (blood) was performed only for brain lymphocytes. Splenic
lymphocytes were first positively pre-enriched for CD8a. (B) Absolute count of brain CD8+ T cells of animals from (A) with mean and SD listed. (C)
CD8+/CD4+ T cell ratio in the brain of animals from (A). Error bars indicate mean + SD. Student t test, unpaired (B) or paired (C). *p # 0.05, ***p # 0.001.
clonality as previously described (26). Error bars indicate mean + indicated an average of 13,025 CD8+ T cells per brain of each
SD; *p # 0.05, **p # 0.01, ***p # 0.001, and ****p # 0.0001 TLR7tg mouse compared with an average of only 401 in each
(Student t test). wild type control brain (Fig. 1B). Because we required a minimum
of 1000 cells for effective TCRb sequencing analysis, we did not
include the wild type brain CD8+ T cells in our study. Consistent
RESULTS
with our previous findings (7), CD8+ T cells outnumbered CD4+
T cells in the brain parenchyma by 1.5- to 3-fold, a unique skewing
The CD8+ T cell repertoire in TLR7tg mice is oligoclonal
not present in the vasculature or in other organs of lupus-prone
We previously described the presence of activated nonpathogenic
CD8+ T cells in the spleen and brain of TLR7tg mice, which we hypo- mice (Fig. 1C). Genomic DNA from isolated CD8+ cells was used
thesized could regulate aspects of the systemic disease developed for TCRb sequencing with the Adaptive Biotechnologies Immu-
in this model (7). To gain insight into the clonality of these cells, noSEQ Platform. The number of unique in-frame TCR rearrange-
we performed TCRb sequence analysis of CD8+ lymphocytes ex- ments per sample in TLR7tg spleens was decreased compared with
tracted from the spleens and brains of TLR7tg mice or from spleens wild type spleens (Fig. 2A) despite the total cell input (;2 3 105)
of wild type controls. CD8+ bead enrichment followed by flow cyto- and the sum of all productive templates being the same in both
metric sorting was used to select for CD8+TCRb+CD42CD11b2 groups (Fig. 2B). These data can be collectively summarized
T cells with a final purity of at least 99% (Fig. 1A). using a metric to measure total sample diversity, indicating that
Brain parenchymal CD8+ T cells were distinguished and sepa- the peripheral TLR7tg CD8+ T cell repertoire has an increased
rated from those in the vasculature using a combination of in vivo degree of oligoclonality with respect to wild type controls (Fig.
intravascular labeling and ex vivo staining as previously described 2C). Similar oligoclonality was found in the brain-resident CD8+
(21, 22). A precise cell count provided during FACS isolation T cell pool (Fig. 2A–C), indicating that as a population, these cells
https://doi.org/10.4049/immunohorizons.180008264 CD8+ OLIGOCLONALITY IN A MOUSE MODEL OF SYSTEMIC LUPUS ImmunoHorizons
are largely derived from the same parents clones as those in the lengths within the population. Normal Gaussian distribution of
periphery. Additionally, an equivalent percentage of cells se- the CDR3 lengths is indicative of polyclonal responses (26).
quenced from each of the three groups produced in-frame pro- Consistent with this, we found that CDR3 sequence lengths of
ductive rearrangements without a stop codon (Fig. 2D). CD8+ T cells from healthy control mice demonstrated a very
An analysis of the most abundant 10 clones in wild type strong fit to a Gaussian curve centered on a length of 14 aa (Fig.
controls reveals an average summative frequency per mouse of ,1%, 3A, 3B, top), which is consistent with a recent comprehensive
indicative of a polyclonal response (Fig. 2E, 2F). By comparison, the analysis including B6 control animals (27). By comparison, the
strong oligoclonality of both splenic and brain CD8+ T cells from CDR3 segment lengths of TLR7tg splenic CD8+ T cells had a
TLR7tg mice can also be seen in the summative frequency of the top substantial divergence from expected Gaussian distribution
10 clones in each animal, which can amount to 60.5% of the (Fig. 3A, 3B, middle). Additional variance was found in brain-
repertoire (Fig. 2E, 2F). In this case, the sum of the top clones in isolated cells (Fig. 3A, 3B, bottom). Importantly, despite a narrowing
the TLR7tg brain with respect to the entire repertoire was sta- repertoire, the CDR3 sequence distribution across all TLR7tg
tistically higher relative to the sum of the top clones in the TLR7tg samples tested is not uniformly directed toward a specific set
spleen (Fig. 2F). Remarkably, the top unique rearrangement in of rearrangement processes; each of the eight lupus-prone
the total TLR7tg CD8+ T cell pool was 30.1% of all productive animals exhibit a uniquely skewed abundance of CDR3 lengths.
Downloaded from http://www.immunohorizons.org/ by guest on February 9, 2022
templates, whereas this number never exceeded 0.4% for the There is, however, a similar pattern of CDR3 length usage be-
wild type group (Fig. 2G). tween shared spleen and brain samples within each of the same
TLR7tg animals (Fig. 3A, 3B, middle and bottom, sequentially
Skewed CDR3 length and VDJb gene usage in TLR7tg CD8+ matched bars left to right).
T cells compared with wild type We next analyzed VDJb gene usage in CD8+ T cells from wild
An established method of assessing the relative clonality in a type and TLR7tg mice. The results in Fig. 4 include total Vb gene
given lymphocyte repertoire is through an analysis of the CDR3 segment usage and are presented with two forms of nomenclature.
FIGURE 2. CD8+ T cells from lupus-prone mice demonstrate substantial oligoclonality.
(A–D) Overview of total TCRb rearrangements from wild type B6 and TLR7tg CD8+ T cells from Fig. 1. (A) The count of unique rearrangements in
each sample that are in-frame and do not contain a stop codon. (B) The sum of templates for all productive rearrangements in the sample. (C)
Clonality measure for each sample calculated over all productive rearrangements. (D) The fraction of productive in-frame templates among all
templates. (E–G) The frequency (E) and sum (F) of the top 10 clones and the top clone (G) for each sample relative to all rearrangements. Error bars
indicate mean + SD. ^p = 0.08, *p # 0.05, **p # 0.01, ***p # 0.001, ***p # 0.0001, by Student t test.
https://doi.org/10.4049/immunohorizons.1800082ImmunoHorizons CD8+ OLIGOCLONALITY IN A MOUSE MODEL OF SYSTEMIC LUPUS 65
Downloaded from http://www.immunohorizons.org/ by guest on February 9, 2022
FIGURE 3. Variable CDR3 lengths support the notion of oligoclonality in CD8+ T cells of lupus-prone mice.
(A) Relative abundance and distribution of CDR3 nucleotide sequence lengths (bp) from purified CD8+ T cells of wild type B6 spleen (shades of gray),
TLR7tg spleen (shades of red), and TLR7tg brain (shades of blue). Each shaded bar represents a separate sample of the indicated genotype and
tissue. For each possible CDR3 length, the order of the eight bars from left to right is matched for spleen and brain samples from TLR7tg mice.
(B) Average distribution analysis of compiled samples (n = 8) from (A). Bars indicate minimum and maximum values centered on the mean.
R2 values indicate the fit of each curve to normal Gaussian distribution.
The international ImMunoGeneTics information system (IMGT) in wild type B6 mice was consistent with these published results,
is the current standard used since 2000 and includes all with the majority of cells using Vb12 and Vb13 (Fig. 4A), Db1
pseudogenes in the Vb gene region (28), although many (Fig. 4B), and Jb2.7 (Fig. 4C) gene segments. In contrast, cells
publications and commercial Ab suppliers still adhere to the from the spleen of TLR7tg mice showed highly variable VDJb
original distinctions (29). Naive animals rely on a relatively small usage (Fig. 4A–C) and significant divergence from common gene
pool of TCRb genes in the makeup of their peripheral repertoire segments such as Vb12 and Jb 2.5 (Fig. 4D). The brain CD8+
(30). Our analysis of the VDJb gene makeup of the CD8+ T cells T cells from TLR7tg mice also used statistically less Vb12 and Jb
https://doi.org/10.4049/immunohorizons.180008266 CD8+ OLIGOCLONALITY IN A MOUSE MODEL OF SYSTEMIC LUPUS ImmunoHorizons
Downloaded from http://www.immunohorizons.org/ by guest on February 9, 2022
FIGURE 4. Highly variable VDJb gene usage in lupus-prone CD8+ T cells.
(A–C) Vb gene (A) usage for CD8+ T cells from wild type B6 spleen and TLR7tg spleen and brain. Equivalent IMGT and original (orig.) nomenclature
for Vb genes included. ps, pseudogene. (B) Db gene and (C) Jb gene usage shown as in (A). Bars indicate minimum and maximum values centered
on the mean. (D) Statistical analysis of discovered significant differences between indicated groups for (A–C). Multiple t test grouped analysis used to
generate p values and false discovery rate–adjusted q values. These results are presented with two forms of nomenclature: the comprehensive
international IMGT, and the original, still commonly used nomenclature.
2.5. In place of these common Vb and Jb genes, both the spleen small number of shared identical CDR3 sequences between
and brain cells from lupus-prone mice used other Vb and Jb various wild type B6 splenic samples (Fig. 5A, 5E, 5F). Unexpect-
genes, although no consistent pattern of usage emerged across edly, a significant reduction in sharing across splenic TLR7tg mice
those samples. In fact, substantial mouse-to-mouse variability of was evident (Fig. 5B, 5E, 5F). We found additional reduction in
VDJb genes identified was evident in both TLR7tg splenic and such sharing across all brain-isolated populations (Fig. 5C, 5E, 5F),
brain CD8+ T cell populations (Fig. 4D). in some cases down to only a single shared sequence between two
genetically identical animals. Within the same animal, we did find
The peripheral and brain-infiltrating CD8+ T cell pools considerable instances of complete nucleotide homology in the
within the same lupus-prone mouse show substantial CDR3 CD8+ T cells between spleen and brain tissues (Fig. 5D, 5E),
nucleotide sequence sharing including sharing of the most abundant clone in the two tissues
To better understand the substantial mouse-to-mouse variations (Fig. 6A). These results indicate that lymphocyte expansion in a
in TLR7tg samples despite similar levels of oligoclonality, we chronic-inflammatory autoimmune setting can give rise to an
analyzed absolute CDR3 nucleotide homology across all in-frame oligoclonal population of CD8+ T cells in which the same clones
rearrangements from every animal tested between spleen and are likely to be found in both lymphoid and nonlymphoid tissues,
brain tissues of both genotypes (Fig. 5A–F). We identified a but only within the same animal.
https://doi.org/10.4049/immunohorizons.1800082ImmunoHorizons CD8+ OLIGOCLONALITY IN A MOUSE MODEL OF SYSTEMIC LUPUS 67
Downloaded from http://www.immunohorizons.org/ by guest on February 9, 2022
FIGURE 5. The CD8+ T cell repertoire is largely similar between the spleen and brain of the same lupus-prone animal.
(A–D) Venn-diagram analysis of CDR3 nucleotide sequence homology from three wild type B6 spleens (A), three TLR7tg spleens (B), three TLR7tg
brains (C), and across all three groups (D) comparing in-frame reads. (E and F) CDR3 nucleotide sequence overlap across all in-frame reads of
samples (n = 8) from indicated groups calculated by Morisita Index (E) and summarized for each group (F). Error bars indicate mean + SD. *p # 0.05,
****p # 0.0001, by Student t test.
Dominant clones across lupus-prone mice share regions of homology in these clones, their VDJ gene usage was largely
sequence homology but are distinct from the most common distinct. These results collectively indicate that clonal expansion
clones in healthy control animals in TLR7tg lupus-prone mice selects for particular dominant re-
Remarkably, whereas the most abundant single clone in each wild arrangements that share regions of sequence homology despite
type control sample was only between 0.1 and 0.4% of the entire not deriving from the same parent lymphocyte.
population, in TLR7tg mice, this number reached between 10 and
30% for splenic samples (Fig. 6A). Similar percentages were found DISCUSSION
for brain-isolated CD8+ T cells from TLR7tg mice. The CDR3
amino acid sequences of the top rearrangements from wild type The specificities and clonality of the lymphocytes thought to be
and TLR7tg mice were largely distinct (Fig. 6B), indicating that the involved in systemic lupus are not a clearly understood aspect of
CD8+ T cells selected by the environment in lupus-prone mice are the disease, particularly as they concern CD8+ T cells. Previous
different from the top clones in wild type controls. studies found clonal expansion within the CD8+ T cell pool isolated
Despite the strong oligoclonality we witnessed in the lupus- from the kidneys of lupus patients (19), whereas an analysis of
prone samples, complete nucleotide homology across various T cell Vb gene usage and the Ag-determining CDR3 region
TLR7tg mice was rare (Fig. 5). A possible scenario is that similar revealed significant changes compared with healthy controls,
but not identical CDR3 sequences might be selected by the same indicative of more oligoclonal T cell populations (20, 31). The data
Ag within expanded T cell populations in these animals. We in our current study support the notion of a narrowing TCR
identified several highly similar dominant clones, particularly repertoire in lupus, as we find increased oligoclonality in the TLR7tg
those from TLR7tg mice no. 1, 2, and 6, which contained up to CD8+ T cell pool compared with wild type controls: a reduction
93% amino acid sequence homology while representing nearly of the total number of unique clones (Fig. 2), a diversion of CDR3
one third of the CD8 repertoire in each animal (Fig. 6A, 6C). length away from expected Gaussian distribution (Fig. 3), and skewed
Interestingly, even though there was such a high degree of sequence VDJb gene usage (Fig. 4). Importantly, our data suggest that strong
https://doi.org/10.4049/immunohorizons.180008268 CD8+ OLIGOCLONALITY IN A MOUSE MODEL OF SYSTEMIC LUPUS ImmunoHorizons
Downloaded from http://www.immunohorizons.org/ by guest on February 9, 2022
FIGURE 6. Regions of sequence homology across dominant clones in TLR7tg animals.
(A and B) The CDR3 sequence of the dominant CD8+ T cell clone isolated from the brain of each TLR7tg (n = 8) (A) or spleen of each wild type
B6 (n = 8) (B) mouse is shown. The frequency at which each of these top clones appears across all animals is presented. (C) CDR3 alignment along
with V, D, and J gene usage for each top clone in TLR7tg or wild type B6 mice. An asterisk (*) indicates an instance in which multiple nucleotide
sequences encoding different Vb genes generated identical CDR3 amino acid sequences.
selective pressures exist that shape the T cell repertoire, which The level of clonality we observe in the TLR7tg mice, with a
extend beyond underlying genetics, as there was only partial sequence single clone comprising up to 30% of the entire T cell pool, is
homology of dominant clones across various TLR7tg animals. similar to what has been published in CD8-dependent models of
https://doi.org/10.4049/immunohorizons.1800082ImmunoHorizons CD8+ OLIGOCLONALITY IN A MOUSE MODEL OF SYSTEMIC LUPUS 69
viral infection such as lymphocytic choriomeningitis virus (32) in individuals with chronic disease such as systemic lupus could
and influenza (33). An important distinction between the TLR7tg promote what has been referred to as an “immune risk phenotype”
lupus-prone mouse model and noted viral infection systems is the (40) during senescence. Such a phenotype is predictive of mortality
scope of available Ags to which the T cells can respond. Indeed, in aged individuals who are left more susceptible to constant
only three predominant CD8+ T cell clones have been described immune insult from foreign pathogens. The narrowing of the
against the Armstrong strain of lymphocytic choriomeningitis lymphocyte repertoire, which is thought to impact CD8+ T cells
virus (32). Importantly, although each of these clones contain more than CD4+ T cells, decreases immune defense particularly
unique VDJ genes, CDR3 lengths, and nucleotide sequences, during senescence in both mice and humans (40–42). Because
they are consistently present across all infected animals. In our we see a narrowing lymphocyte repertoire in the context of
lupus model system, despite a similar degree of oligoclonality in autoimmune disease, it will be important in the future to use our
the CD8+ T cell pool there is an unexpectedly broad and variable lupus-prone mice and other such models to study the impact of
usage of VDJ genes, CDR3 lengths, and sequences across animals. chronic systemic disease on the ability of the organism to mount
The importance of this distinction remains unclear, although it is both effective primary and secondary immune responses to
likely indicative of a heterogeneous response against a group pathogens.
of self-antigens. Monozygotic twins concordant for lupus begin The Ag specificity of the TLR7tg CD8+ T cells we identified
Downloaded from http://www.immunohorizons.org/ by guest on February 9, 2022
life with virtually identical TCR repertoires, but their Vb usage remains unknown, making it hard to speculate what might be
specifically in the CD8 pool diverges over time, suggesting that driving the mouse-to-mouse differences despite similar overall
both genetics and a unique history of environmental exposure clonality. Certainly, the frequency and sequence homology among
shape the T cell repertoire over time (34). the most abundant clones suggests a strong selective pressure
In a previous study, we showed that the CD8+ T cells in TLR7tg indicative of some self-antigen–driven expansion of CD8+ T cells.
mice are activated cells that, compared with other lymphocytes in However, how the top clones in the CD8+ T cell pool end up as part
the animal, could traffic to and take up residence in the brain (7). of the response in lupus-prone mice to begin with and why the
Activated lymphocytes gain adhesive properties, allowing them to repertoire is so variable between animals remains unclear and
attach to blood vessel endothelium of tissues into which they will warrants further investigation.
migrate (35). The most highly activated CD8+ T cells in TLR7tg
mice upregulated many adhesion and trafficking markers (7), and
so we posited that these cells would enter the brain and expand in DISCLOSURES
response to some Ag-specific signals. Unexpectedly, our current
data identified a markedly similar TCRb repertoire between the The authors have no financial conflicts of interest.
peripheral and brain-resident CD8+ T cell pools in each respec-
tive animal (Figs. 5D, 5E, 6A). It is clear that of all the possible
ACKNOWLEDGMENTS
comparisons we could make across tissues and genotypes within a
single animal, the strongest degree of sequence homology existed We thank Bethany Scott for technical assistance with animal care and
between cells within different tissues of the same animal. We vascular labeling. We are grateful to Kevin Holmes and the National
conclude from these results that the pool of expanded, oligoclonal, Institute of Allergy and Infectious Diseases Research Technologies Branch
mature lymphocytes we identified in both the spleen and brain of Cytometry section for assistance isolating lymphocytes for sequencing.
TLR7tg mice develop in response to the same selective pressures,
although it remains unclear if the origin of such selection is from
REFERENCES
peripheral or brain Ags.
Nucleotide homology of the Ag-determining CDR3 region can 1. Rahman, A., and D. A. Isenberg. 2008. Systemic lupus erythematosus.
provide some information about the CD8+ T cell repertoire. N. Engl. J. Med. 358: 929–939.
Although absolute homology across TLR7tg mice is rare, particu- 2. Charpentier, B., C. Carnaud, and J. F. Bach. 1979. Selective depression
larly in the brain, it is possible that the high degree of similarity of the xenogeneic cell-mediated lympholysis in systemic lupus
identified across some of the top rearrangements is indicative of erythematosus. J. Clin. Invest. 64: 351–360.
different clones arising with similar specificities, perhaps for some 3. Kis-Toth, K., D. Comte, M. P. Karampetsou, V. C. Kyttaris, L. Kannan,
C. Terhorst, and G. C. Tsokos. 2016. Selective loss of signaling lym-
common self-antigen. This is possible even in cases when the phocytic activation molecule family member 4-positive CD8+ T cells
different VDJ genes are used to make two unique rearrange- contributes to the decreased cytotoxic cell activity in systemic lupus
ments. For example, whereas a close relationship between the erythematosus. Arthritis Rheumatol. 68: 164–173.
CDR3 specificity and the structure of the Ag receptor exists, and 4. Peng, S. L., J. Moslehi, M. E. Robert, and J. Craft. 1998. Perforin protects
most of the T cells with the same MHC:peptide specificity were against autoimmunity in lupus-prone mice. J. Immunol. 160: 652–660.
found to use the same ab-chain combinations (36, 37), rare examples 5. McPhee, C. G., T. J. Sproule, D.-M. Shin, J. A. Bubier, W. H. Schott, M.
P. Steinbuck, L. Avenesyan, H. C. Morse III, and D. C. Roopenian. 2011.
exist of various insulin-specific T cell clones and hybridomas
MHC class I family proteins retard systemic lupus erythematosus au-
using different Va and Vb genes (38, 39). toimmunity and B cell lymphomagenesis. J. Immunol. 187: 4695–4704.
One interesting implication of our data relates to the notion 6. Kim, H.-J., X. Wang, S. Radfar, T. J. Sproule, D. C. Roopenian, and
that shrinking receptor diversity resulting from clonal expansion H. Cantor. 2011. CD8+ T regulatory cells express the Ly49 Class I
https://doi.org/10.4049/immunohorizons.180008270 CD8+ OLIGOCLONALITY IN A MOUSE MODEL OF SYSTEMIC LUPUS ImmunoHorizons
MHC receptor and are defective in autoimmune prone B6-Yaa mice. 2009. Comprehensive assessment of T-cell receptor beta-chain
Proc. Natl. Acad. Sci. USA 108: 2010–2015. diversity in alphabeta T cells. Blood 114: 4099–4107.
7. Morawski, P. A., and S. Bolland. 2017. Expanding the B cell-centric 25. Carlson, C. S., R. O. Emerson, A. M. Sherwood, C. Desmarais,
view of systemic lupus erythematosus. Trends Immunol. 38: 373–382. M.-W. Chung, J. M. Parsons, M. S. Steen, M. A. LaMadrid-Herrmannsfeldt,
8. Kim, H.-J., B. Verbinnen, X. Tang, L. Lu, and H. Cantor. 2010. In- D. W. Williamson, R. J. Livingston, et al. 2013. Using synthetic templates to
hibition of follicular T-helper cells by CD8(+) regulatory T cells is design an unbiased multiplex PCR assay. Nat. Commun. 4: 2680.
essential for self tolerance. Nature 467: 328–332. 26. Pannetier, C., M. Cochet, S. Darche, A. Casrouge, M. Zöller, and
9. Bolland, S., Y.-S. Yim, K. Tus, E. K. Wakeland, and J. V. Ravetch. P. Kourilsky. 1993. The sizes of the CDR3 hypervariable regions of the
2002. Genetic modifiers of systemic lupus erythematosus in Fcgam- murine T-cell receptor beta chains vary as a function of the recom-
maRIIB(-/-) mice. J. Exp. Med. 195: 1167–1174. bined germ-line segments. Proc. Natl. Acad. Sci. USA 90: 4319–4323.
10. Deane, J. A., P. Pisitkun, R. S. Barrett, L. Feigenbaum, T. Town, 27. Cukalac, T., W.-T. Kan, P. Dash, J. Guan, K. M. Quinn, S. Gras,
J. M. Ward, R. A. Flavell, and S. Bolland. 2007. Control of toll-like P. G. Thomas, and N. L. La Gruta. 2015. Paired TCRab analysis of
receptor 7 expression is essential to restrict autoimmunity and den- virus-specific CD8(+) T cells exposes diversity in a previously defined
dritic cell proliferation. Immunity 27: 801–810. ‘narrow’ repertoire. Immunol. Cell Biol. 93: 804–814.
11. Morawski, P. A., C.-F. Qi, and S. Bolland. 2017. Non-pathogenic tissue- 28. Bosc, N., and M. P. Lefranc. 2000. The mouse (Mus musculus) T cell
resident CD8(+) T cells uniquely accumulate in the brains of lupus- receptor beta variable (TRBV), diversity (TRBD) and joining (TRBJ)
prone mice. Sci. Rep. 7: 40838. genes. Exp. Clin. Immunogenet. 17: 216–228.
12. Davis, M. M., and P. J. Bjorkman. 1988. T-cell antigen receptor genes 29. Wilson, R. K., E. Lai, P. Concannon, R. K. Barth, and L. E. Hood. 1988.
Downloaded from http://www.immunohorizons.org/ by guest on February 9, 2022
and T-cell recognition. [Published erratum appears in 1988 Nature Structure, organization and polymorphism of murine and human T-cell
335: 744.] Nature 334: 395–402. receptor alpha and beta chain gene families. Immunol. Rev. 101: 149–172.
13. Barth, R. K., B. S. Kim, N. C. Lan, T. Hunkapiller, N. Sobieck, 30. Behlke, M. A., D. G. Spinella, H. S. Chou, W. Sha, D. L. Hartl, and
A. Winoto, H. Gershenfeld, C. Okada, D. Hansburg, I. L. Weissman, D. Y. Loh. 1985. T-cell receptor beta-chain expression: dependence on
et al. 1985. The murine T-cell receptor uses a limited repertoire of relatively few variable region genes. Science 229: 566–570.
expressed V beta gene segments. Nature 316: 517–523. 31. Thapa, D. R., R. Tonikian, C. Sun, M. Liu, A. Dearth, M. Petri,
F. Pepin, R. O. Emerson, and A. Ranger. 2015. Longitudinal analysis of
14. Ahmed, M., K. G. Lanzer, E. J. Yager, P. S. Adams, L. L. Johnson, and
peripheral blood T cell receptor diversity in patients with systemic
M. A. Blackman. 2009. Clonal expansions and loss of receptor diversity in
lupus erythematosus by next-generation sequencing. Arthritis Res.
the naive CD8 T cell repertoire of aged mice. J. Immunol. 182: 784–792.
Ther. 17: 132.
15. Robins, H. S., S. K. Srivastava, P. V. Campregher, C. J. Turtle,
32. Sourdive, D. J., K. Murali-Krishna, J. D. Altman, A. J. Zajac, J. K. Whitmire,
J. Andriesen, S. R. Riddell, C. S. Carlson, and E. H. Warren. 2010.
C. Pannetier, P. Kourilsky, B. Evavold, A. Sette, and R. Ahmed. 1998. Con-
Overlap and effective size of the human CD8+ T cell receptor rep-
served T cell receptor repertoire in primary and memory CD8 T cell
ertoire. Sci. Transl. Med. 2: 47ra64.
responses to an acute viral infection. J. Exp. Med. 188: 71–82.
16. Hou, X., C. Lu, S. Chen, Q. Xie, G. Cui, J. Chen, Z. Chen, Z. Wu,
33. Deckhut, A. M., W. Allan, A. McMickle, M. Eichelberger,
Y. Ding, P. Ye, et al. 2016. High throughput sequencing of T cell an-
M. A. Blackman, P. C. Doherty, and D. L. Woodland. 1993. Prominent
tigen receptors reveals a conserved TCR repertoire. Medicine (Balti-
usage of V beta 8.3 T cells in the H-2Db-restricted response to an
more) 95: e2839.
influenza A virus nucleoprotein epitope. J. Immunol. 151: 2658–2666.
17. Holbrook, M. R., P. J. Tighe, and R. J. Powell. 1996. Restrictions of T cell 34. Davey, M. P., M. M. Meyer, and A. C. Bakke. 1994. T cell receptor V
receptor beta chain repertoire in the peripheral blood of patients with beta gene expression in monozygotic twins. Discordance in CD8
systemic lupus erythematosus. Ann. Rheum. Dis. 55: 627–631. subset and in disease states. J. Immunol. 152: 315–321.
18. Kolowos, W., U. S. Gaipl, R. E. Voll, C. Frank, J. P. Haas, T. D. Beyer, 35. Nourshargh, S., and R. Alon. 2014. Leukocyte migration into inflamed
J. R. Kalden, and M. Herrmann. 2001. CD4 positive peripheral T cells tissues. Immunity 41: 694–707.
from patients with systemic lupus erythematosus (SLE) are clonally 36. Fink, P. J., L. A. Matis, D. L. McElligott, M. Bookman, and
expanded. Lupus 10: 321–331. S. M. Hedrick. 1986. Correlations between T-cell specificity and the
19. Winchester, R., M. Wiesendanger, H.-Z. Zhang, V. Steshenko, structure of the antigen receptor. Nature 321: 219–226.
K. Peterson, L. Geraldino-Pardilla, E. Ruiz-Vazquez, and V. D’Agati. 37. Hochgeschwender, U., H. G. Simon, H. U. Weltzien, F. Bartels,
2012. Immunologic characteristics of intrarenal T cells: trafficking of A. Becker, and J. T. Epplen. 1987. Dominance of one T-cell receptor in
expanded CD8+ T cell b-chain clonotypes in progressive lupus ne- the H-2Kb/TNP response. Nature 326: 307–309.
phritis. Arthritis Rheum. 64: 1589–1600. 38. Sherman, D. H., P. S. Hochman, R. Dick, R. Tizard, K. L. Ramachandran,
20. Sui, W., X. Hou, G. Zou, W. Che, M. Yang, C. Zheng, F. Liu, P. Chen, R. A. Flavell, and B. T. Huber. 1987. Molecular analysis of antigen rec-
X. Wei, L. Lai, and Y. Dai. 2015. Composition and variation analysis of ognition by insulin-specific T-cell hybridomas from B6 wild-type and
the TCR b-chain CDR3 repertoire in systemic lupus erythematosus bm12 mutant mice. Mol. Cell. Biol. 7: 1865–1872.
using high-throughput sequencing. Mol. Immunol. 67 (2 Pt B): 455–464. 39. Spinella, D. G., T. H. Hansen, W. D. Walsh, M. A. Behlke, J. P. Tillinghast,
21. Galkina, E., J. Thatte, V. Dabak, M. B. Williams, K. Ley, and H. S. Chou, P. J. Whiteley, J. A. Kapp, C. W. Pierce, E. M. Shevach, et al.
T. J. Braciale. 2005. Preferential migration of effector CD8+ T cells into 1987. Receptor diversity of insulin-specific T cell lines from C57BL
the interstitium of the normal lung. J. Clin. Invest. 115: 3473–3483. (H-2b) mice. J. Immunol. 138: 3991–3995.
22. Anderson, K. G., K. Mayer-Barber, H. Sung, L. Beura, B. R. James, 40. Blackman, M. A., and D. L. Woodland. 2011. The narrowing of the
J. J. Taylor, L. Qunaj, T. S. Griffith, V. Vezys, D. L. Barber, and CD8 T cell repertoire in old age. Curr. Opin. Immunol. 23: 537–542.
D. Masopust. 2014. Intravascular staining for discrimination of 41. Messaoudi, I., J. Lemaoult, J. A. Guevara-Patino, B. M. Metzner, and
vascular and tissue leukocytes. Nat. Protoc. 9: 209–222. J. Nikolich-Zugich. 2004. Age-related CD8 T cell clonal expansions
23. Fletcher, A. L., D. Malhotra, S. E. Acton, V. Lukacs-Kornek, A. constrict CD8 T cell repertoire and have the potential to impair im-
Bellemare-Pelletier, M. Curry, M. Armant, and S. J. Turley. 2011. Re- mune defense. J. Exp. Med. 200: 1347–1358.
producible isolation of lymph node stromal cells reveals site-dependent 42. Qi, Q., Y. Liu, Y. Cheng, J. Glanville, D. Zhang, J.-Y. Lee, R. A. Olshen,
differences in fibroblastic reticular cells. Front. Immunol. 2: 35. C. M. Weyand, S. D. Boyd, and J. J. Goronzy. 2014. Diversity and
24. Robins, H. S., P. V. Campregher, S. K. Srivastava, A. Wacher, clonal selection in the human T-cell repertoire. Proc. Natl. Acad. Sci.
C. J. Turtle, O. Kahsai, S. R. Riddell, E. H. Warren, and C. S. Carlson. USA 111: 13139–13144.
https://doi.org/10.4049/immunohorizons.1800082You can also read
