Introduction
Human T cell receptor (TCR) immunodeficiencies (TCRID) are rare autosomal recessive
disorders caused by mutations affecting TCR, CD3, or CD247 chains, which share developmental,
functional, and TCR expression defects (1). Their rapid diagnosis is fundamental for
patient survival and early hematopoietic stem cell transplantation. Here, we propose
that studying γδ T cells, which are often neglected, can be helpful for a timely diagnosis.
We thus offer a diagnostic flowchart and some lab tricks based on published cases.
γδ T Cell and TCR Physiopathology
γδ T lymphocytes are a minor subset (1–10%) of human peripheral blood T cells. Most
(>70%) are CD4−CD8− [double negative (DN)], some (30%) are CD8+CD4− and very few (<1%)
are CD4+CD8− [CD8+ or CD4+ single positive (SP), respectively]. Most γδ T cells in
adults express Vδ2/Vγ9 TCR variable regions (65–90%), the rest being mostly Vδ1+,
some Vδ3+ or Vδ5+, all with different Vγ chains (2). As peripheral blood γδ T cells
are scarce, their over-representation is more conspicuous than their under-representation,
which is very rarely reported and normally associated to a single subset, such as
Vδ2+ in granulomatosis (3) or aging (4). Indeed, no selective γδ T cell immunodeficiency
(ID) has been reported to date, although absence of γδ T cells has been described
together with other lymphocyte derangements in rare primary ID (5). The clinical significance
of increased γδ T cells, defined as >10% of peripheral blood T lymphocytes (6), requires
clarification in several diseases including infection, autoimmunity, cancer, and primary
ID.
The human γδTCR (Figure 1A inset) is an octameric protein complex composed of three
heterodimers (TCRγ/TCRδ, CD3γ/CD3ε, and CD3δ/CD3ε) and a single CD247 homodimer (also
termed ζ/ζ). The complex can be abbreviated as γδTCR/γεδεζζ. The TCRγ/TCRδ heterodimer
contains variable regions, which allow for antigen recognition, while the other three
dimers are invariant and are required for surface TCR expression and for intracellular
propagation of the recognition signal (7). Therefore, defects in any chain would expectedly
impact γδTCR expression and γδ T cell selection and function.
Figure 1
γδ T cells in TCRID. (A) Proportion of γδ T cells within the T cell compartment. The
percentage of γδ T cells (mean ± SEM) was defined as γδTCR+ using 11F2, IMMU510, or
anti-TCRδ-1 monoclonal antibody (mAb) within the T lymphocyte gate (defined as CD3+)
and ordered from left to right in decreasing values. The gray band indicates the normal
range for infants (8). Inset: human TCR isotypes. NA: not analyzable (no T cells);
*: leaky mutations (partial defects); n: number of patients for which data was available.
(B) % TCR surface expression (mean ± SEM) in γδ or αβ T cells relative to healthy
donors. TCR surface expression was determined by flow cytometry using different anti-CD3
mAb, γδ T cells were identified as in (A) and αβ T cells as γδTCR−CD3+ or CD4+ cells.
ND: not determined. (C) Our suggested TCRID diagnostic flowchart using absolute lymphocyte
counts for T−B+NK+ or T±B+NK+ phenotype and basic flow cytometry data (top) to point
to the most likely culprit TCR chain (bottom). TCR chains are represented by black
boxes arranged according to the proportion of γδ T cells from (A) and their surface
TCR expression relative to normal controls from (B). The white box indicates normal
value. *: as in (A). Brackets represent expected defects, as γδ T cells values were
not available in these TCRID.
γδ T Cells in TCRID
αβ T cells have been extensively studied in TCRID. In contrast, γδ T cells have been
frequently ignored, in part due to their scarcity but also to the lack of markers
other than the TCR to identify them when TCR expression is reduced, as is the case
in TCRID. Although their functions are still debated, we believe that their accurate
study (relative numbers, Figure 1A, TCR expression, Figure 1B, and main subsets) can
help to diagnose TCRID, as reviewed below and summarized in a practical diagnostic
flowchart in Figure 1C.
TCRα deficient patients showed combined ID and autoimmune features due to a selective
block in αβ T cell development, as TCRα is part of the TCRα/TCRβ (αβTCR, Figure 1A
inset) antigen-binding heterodimer (9). In contrast, the γδTCR was unaffected, as
demonstrated by normal surface expression (Figure 1B), which allowed for normal absolute
but increased relative numbers of γδ T cells (Figure 1A). This is unique among TCRID
and thus a useful feature in the diagnostic flowchart (Figure 1C). Such γδ T cells
were proposed to be in part protective from infections in the two reported patients.
Indeed, γδ T cells are involved in immune responses against a variety of pathogens
including virus, bacteria, and parasites, whereas still other act as antigen-presenting
cells (10) or B cell helper cells (11). Their beneficial effects in vivo have found
recent unexpected recognition in haploidentical allogeneic hematopoietic cell transplantation
after depletion of αβ T and B cells (12), which showed that γδ T cells did not cause
graft vs. host disease and may have helped with host immune maintenance and recovery.
The fact is that, compared to other complete TCRID, symptoms in both TCRα deficient
patients appeared rather late (6 and 15 months of age) and transplantation took place
very late (6–7 years of age).
Similar to TCRα deficient patients, patients with partial CD3δ deficiency (CD3δ* in
Figure 1) due to a leaky splicing mutation showed strongly reduced αβ T cell numbers
and normal absolute but high relative numbers of γδ T cells (Figure 1A), although
with low surface TCR expression [(13) and Figure 1B]. In contrast to TCRα deficiency,
partial CD3δ deficiency showed early severe combined ID (SCID) features and required
very early transplantation (before 2 years of age), thus their γδ T cells were not
protective, perhaps as a consequence of their impaired TCR expression and function
(13). Unexpectedly, partial CD3δ deficiency caused a stronger impact in γδ (Figure
1B) than in αβTCR surface expression (25 vs. 55% relative to controls (13). A detailed
study of their γδ T cells showed an enrichment in a subset of otherwise rare CD4+
γδ T cells, which exhibited an activated phenotype and were refractory to further
TCR stimulation (14). This CD4-expressing γδ T cell subset seems to be pathognomonic
for partial CD3δ deficiency, since: (i) it has been ascertained in three of three
tested patients with this condition and (ii) it was not found in other TCRID (14,
15). Its developmental origin deserves further comment. αβ and γδ T cells differentiate
within the thymus from a late DN common progenitor (16). In humans, development of
most γδ T cells seems to mimic that of αβ T cells: from DN progenitors through a CD4+CD8+
double positive (DP) pathway (17, 18), to DN and either CD4+ or CD8+ SP populations.
DN and CD8+ SP γδ T cells are minor intrathymic subsets but become the major γδ T
cell subsets in the periphery, while CD4+ SP are the main intrathymic subset, followed
by DP (19). Notably, the last two subsets can be found in peripheral blood in pathological
conditions, and most bone marrow and peripheral blood γδ T cells from patients with
γδ T cell acute lymphoblastic leukemia are either CD4+ SP or DP (20). Thus, we believe
that the 10-fold enrichment of CD4+ SP γδ T cells observed in patients with partial
CD3δ deficiency is due to low TCR-dependence for positive selection of CD4− γδ T cells
and disrupted negative selection of CD4+ γδ T cells (14).
CD3γ deficient patients, most of which showed mild ID (21), had normal numbers of
polyclonal peripheral blood γδ T cells [absolute and relative, (22) and Figure 1A]
with low surface TCR [around 30% of control levels (22) and Figure 1B], similarly
to their αβ T cell counterparts (23), likely with an abnormal γδTCR/δεδεζζ stoichiometry.
Despite their high homology, the invariant CD3γ and δ chains show different roles
in human vs. mouse γδ T cell development. Indeed, CD3γ-deficient mice exhibited a
severe γδ T cell developmental block (24).
γδ T cells were studied in only two of three reported CD247 deficient patients (21).
The patients showed SCID features and reduced absolute and relative γδ T cells numbers
(Figure 1A). Surface γδTCR expression was also reduced (4% vs. healthy controls, Figure
1B). The number of αβ T cells was only slightly reduced despite their similarly reduced
surface TCR, with all reported cases showing reduced numbers of CD4+ T cells but normal
or high numbers of CD8+ T cells (21, 25–27).
A single patient with partial CD3ε deficiency (28–30) showed very low surface αβTCR
expression (10% of normal levels, Figure 1B, CD3ε*), mild ID, normal CD8+, and reduced
CD4+ (αβ) T cells, but no γδ T cells as determined with the anti-TCRδ-1 monoclonal
antibody (mAb) (Figure 1A). We have however considered for Figure 1C that surface
γδTCR expression might have been similar to αβTCR expression.
Three studied CD3δ deficient patients [out of 16 reported, all with severe T cell
lymphopenia and SCID (31, 32)], showed a few circulating CD3+ T cells, which were
DN but γδTCR−(33). γδTCR+ cells were indeed undetectable by flow cytometry in peripheral
blood or by immunohistochemistry in the thymus, lymph nodes, spleen, or gut. However,
gene microarray analysis and protein expression of patient thymocytes showed increased
levels of TCRγ and TCRδ transcripts and proteins (33), which could be interpreted
as presence and thus significant selection of γδ T cells unable however to leave the
thymus, perhaps due to insufficient surface TCR compared to partial CD3δ deficiency.
Finally, γδ T cells have not been studied in SCID patients with complete CD3ε deficiency
(31). Nevertheless, given their severe T cell lymphopenia, we can safely presume for
Figure 1C that they were absent.
In summary, the proportion of γδ T cells within total T lymphocytes (Figure 1A) and
the level of surface γδ vs. αβTCR expression (Figure 1B), as well as the severity
of lymphopenia (T−B+NK+ or T±B+NK+ phenotype), can be used to generate a practical
TCRID diagnostic flowchart (Figure 1C). For instance, if an infant has SCID and no
T cells but normal B and NK cell numbers (T−B+NK+ phenotype) and other causes have
been ruled out, CD3δ or CD3ε deficiency should be considered (Figure 1C). In contrast,
if some T cells are present (T±B+NK+ phenotype) and γδ TCR expression is low, TCRα
deficiency can be ruled out. If CD16 expression by NK cells is normal, CD247 deficiency
can be excluded, and the presence or absence of high absolute numbers of CD4+ γδ T
cells will rule out CD3γ or partial CD3δ deficiency, respectively.
Lab Tricks to Identify αβ and γδ T Cells in TCRID
When surface TCR expression is low, αβ T cells can be identified by the expression
of CD4 or CD8αβ (i.e., CD8bright) within the lymphoid subset (23), whereas γδ T cells
are identified only by expression of the γδTCR. We have reported that most CD3+ cells
within normal DN lymphocytes are γδ T cells (34), and this may also help in certain
TCRID.
Despite their reduced numbers and surface TCR expression, an appropriate multicolor
flow cytometry approach can help to identify γδ T cells in TCRID. To avoid underestimation
due to low TCR surface expression, we recommend: (i) the use of bright fluorochromes
such as PE, PE-Cy5.5, PE-Cy7, or APC, rather than FITC, (ii) an appropriate choice
of CD3 mAb such as UCHT-1, F101.01, or S4.1 due to their high signal-to-noise ratio
in TCRID, (iii) two-color stainings with CD3 and γδTCR mAb, which can also help to
single out γδ T cells as a DP subset, and (iv) to avoid mixing αβTCR and CD3 mAb,
as they sometimes compete (UCHT-1, for instance).
CD4 and CD8 expression by γδ T cells should also be tested to rule out partial CD3δ
deficiency (see above). CD4, CD8, γδTCR (IMMU510 or 11F2), and CD3 (UCHT-1 or S4.1)
is a useful combination, to this end. Lastly, intracellular stainings for invariant
TCR chains has been shown to be useful to identify T cells expressing very low surface
TCR, such as those with CD247 (21) or partial CD3δ deficiency (14).
Conclusion and Perspectives
Human γδ T lymphocytes are still puzzling in terms of development, function, and TCR
stoichiometry in ways that mouse models do not wholly recapitulate. Human TCRID share
defects in T cell development and function and in TCR expression. While their αβ T
cells have been studied in detail, γδ T cells have been frequently ignored, in part
due to their scarcity and to the lack of appropriate markers to identify them when
TCR expression is reduced. Here, pooling published studies, we proposed some technical
tricks to identify γδ T cells in TCRID patients and made the point that their careful
analysis can help to inform a rapid differential diagnosis using a flowchart, with
clinical benefit.
Conflict of Interest Statement
The authors declare that the research was conducted in the absence of any commercial
or financial relationships that could be construed as a potential conflict of interest.