Characterization
of Teicoplanin-Specific T-Cells
from Drug Naïve Donors Expressing HLA-A*32:01
Teicoplanin is a glycopeptide antibiotic deployed to combat Gram-positive bacterial infection and has recently been associated with development of adverse drug reactions, particularly following previous exposure to vancomycin. In this study, we generated teicoplanin-specific monoclonal T-cell populations from healthy volunteers expressing HLA-A*32:01 and defined pathways of T-cell activation and HLA allele restriction. Teicoplanin-responsive T-cells were CD8+, HLA class I-restricted, and cross-reacted with the lipoglycopeptide daptomycin in proliferation and cytokine/cytolytic molecule (granzyme B, Perforin, and FasL) release assays. These data show that teicoplanin activates T-cells, which may play a role in the pathogenesis of teicoplanin-induced adverse events, in HLA-A*32:01 positive donors.
<p>Hypersensitivity
to otherwise
efficacious antibiotics is an area of concern to patients, clinicians,
and researchers in the field of drug development. Prediction of such
reactions is often difficult due to the elicitation of adverse events
arising outside of a drug's known pharmacology. Although rare,
reactions of this nature have been associated with activation of the
adaptive immune system, with T-cells implicated in the pathogenesis
of severe cutaneous adverse reaction, including drug-reaction with
eosinophilia and systemic symptoms (DRESS).1 Glycopeptide antibiotics, such as teicoplanin, have been utilized
for over 30 years with strong efficacy demonstrated against Gram-positive
bacterial infection, including β-lactam resistant strains such
as methicillin-resistant Staphylococcus aureus (MRSA)
and Clostridium difficile.2 Teicoplanin is typically administered as a second line treatment
option and as an alternative to vancomycin. Despite the incidence
of adverse drug reaction (ADR) associated with teicoplanin being substantially
lower (13.9% vs 21.9%3) compared to vancomycin,
the drug still poses a significant risk to patient safety. A recent
GWAS has shown an association between vancomycin-induced DRESS and
HLA-A*32:01 in European populations.4 Case
studies have reported clinical cross-reactivity and subsequent teicoplanin-induced
DRESS following initial vancomycin hypersensitivity.5,6 Preliminary in vitro studies using vancomycin-responsive
T-cells generated from HLA-A*32:01 positive healthy donor PBMCs have
already demonstrated low levels of cross-reactivity with teicoplanin.7 Cross-reactivity has been illustrated further
in patients presenting with suspected vancomycin or teicoplanin-induced
DRESS, with ex vivo data suggesting complex patterns
of immunogenicity within the context of HLA class II presentation.8 The aim of the present study was to investigate
the intrinsic immunogenic potential of teicoplanin in terms of evoking
T-cell responses in healthy donors (HDs), in addition to further exploring
patterns of cross-reactivity to structurally related glycopeptides.</p><p>Teicoplanin-specific T-cell clones (TCCs), generated by serial
dilution,9 were identified in 3 healthy
donors positive for HLA-A*32:01 expression (Figure 1). TCCs generated from CD8+ enriched populations
proliferated to a greater degree (HD-2, 3; SI > 40) and frequency
(HD-1; 118/216 TCC SI > 2) than CD4+ enriched. The presence of
drug-reactive
T-cells that proliferated in a dose-dependent manner to teicoplanin
(data not shown) was restricted to monoclonal populations enriched
for CD8+ T-cells, as upon expansion, CD4+ TCCs did not respond to
teicoplanin following confirmatory dose–response tests. Drug-responsive
clonal populations that exclusively expressed a CD8+ phenotype were
expanded via mitogen driven stimulation for further functional analysis.</p><p>Proliferation
of TCCs generated from HLA-A*32:01 positive donors
following exposure to teicoplanin. T-cell populations were positively
enriched for either CD4+ or CD8+ T-cells via magnetic bead separation
(Miltenyi Biotec, UK). TCCs were rechallenged with 250 Î¼M teicoplanin
or cell culture medium for 48 h in the presence of autologous antigen
presenting cells (Epstein–Barr virus-transformed B-cells; APCs).
[3H]Thymidine was added for the final 16 h of incubation
to measure proliferation, and clones with a stimulation index (SI)
> 2 were deemed to be drug-responsive.</p><p>Following pretreatment of both APCs and T-cells with anti-HLA blocking
antibodies, proliferation of CD8+ TCCs was unaffected after the HLA
class II blockade (HLA-DP, HLA-DQ, and HLA-DR). However, proliferation
was found to be inhibited in the presence of MHC class I blocking
antibodies (Figure 2A) indicating T-cell responses to teicoplanin are driven primarily
by MHC class I complexes. Autologous APCs pulsed with teicoplanin
(30 min, 1 h, 4 h, and 24 h) displayed no proliferative response following
coculture with teicoplanin-reactive TCCs (Figure 2B). After fixation of APCs with glutaraldehyde
and subsequent attenuation of peptide processing pathways, drug-responsive
T-cells exhibited the capacity for proliferation after exposure to
a coculture of fixed APCs and teicoplanin. These data suggest teicoplanin
is able to activate CD8+ TCCs in a processing independent manner in
which direct pharmacological interactions with MHC, concordant with
the p-i concept, evoke T-cell responses to drug.</p><p>HLA restriction and activation
pathway of teicoplanin-responsive
CD8+ TCCs from HD-3. A) Proliferation in response to teicoplanin (250
Î¼M) was measured following blocking of HLA complexes present
on the surface of both APCs and TCCs using anti-HLA antibodies (BD
Pharmingen, San Jose, USA) at a concentration of 10 Î¼g/mL. B)
Autologous APCs were either pulsed with teicoplanin for multiple time-points
and extensively washed to remove unbound drug or fixed with glutaraldehyde
to inhibit APC peptide processing. TCCs were then incubated for 48
h with pulsed APCs or fixed APCs plus teicoplanin (250 Î¼M),
with unmodified autologous APCs used as a positive control. [3H]Thymidine was added for the final 16 h of incubation to
measure proliferative responses. Data is shown for representative
TCCs (n = 3), and statistical significance was determined
using the Mann–Whitney U test (*p < 0.05,
**p < 0.01, ****p < 0.0001).</p><p>Cytokine and cytolytic molecule secretion of teicoplanin-reactive
TCCs was assessed via ELISpot after a drug rechallenge (Figure 3A). Clones were observed to
secrete both Th1 (IFN-γ) and Th2 (IL-5 and IL-13) cytokines.
However, the secretion of Th17 and Th22 associated cytokines such
as IL-17A and IL-22 was not present (data not shown). Interestingly,
secretion of cytolytic molecules was detected in all TCCs profiled.
Most notably, increased secretion of granzyme B, perforin, and FasL
indicated involvement of cytotoxic T-cell responses and potential
for activation of pro-apoptotic pathways. A cross-reactivity study
of clones initially primed and exhibiting proliferative responses
to teicoplanin revealed that memory T-cell responses to teicoplanin
were associated with a greater degree of proliferation. Interestingly,
TCCs exhibited cross-reactivity with the cyclic lipoglycopeptide,
daptomycin, at graded concentrations. However, no cross-reactive T-cells
were identified after exposure to vancomycin (Figure 3B).</p><p>Cytokine/cytolytic molecule secretion profile
and glycopeptide
cross-reactivity of CD8+ teicoplanin-reactive TCCs from HD-3. A) Drug-responsive
clones were incubated with autologous APCs and either teicoplanin
(250 Î¼M) or cell culture medium for 48 h (representative TCCs
shown). T-cell secretion of cytokines (IFN-γ, IL-5, and IL-13)
and cytolytic molecules (granzyme B, perforin, and FasL) was visualized
via the enzyme-linked immunospot (ELISpot) assay using an ELISpot
plate precoated for the cytokines of interest and developed according
to the manufacturer's instructions (Mabtech, Sweden). B) Cross-reactivity
of teicoplanin-responsive T-cells to glycopeptides (vancomycin and
daptomycin) was measured via the proliferation assay as previously
described in Figures 1 and 2. Statistical significance was determined
using a nonparametric t-test (***p < 0.001, ****p < 0.0001).</p><p>In summary, teicoplanin-responsive T-cells displaying a CD8+
phenotype
were generated from 3 drug-naïve healthy donors expressing
the HLA-A*32:01 allele, recently associated with cases of vancomycin-induced
DRESS. Therapeutic concentrations associated with glycopeptide administration
are typically between 10 and 20 Î¼M, substantially lower than
the optimal doses used within this study to elicit maximal T-cell
responses for functional analysis. However, we have observed that
glycopeptide-specific TCCs are capable of eliciting proliferative
responses at lower, more therapeutically relevant doses in line with
concentrations found within the blood plasma of patients. The identification
of TCCs that proliferate and secrete both cytotoxic and DRESS related
cytokines such as IL-5 suggests T-cell involvement within the pathogenesis
of the teicoplanin-induced DRESS syndrome.10</p><p>Mechanistic T-cell assays revealed a processing independent
mechanism
of activation that hinges on drug presentation via direct interaction
with HLA class I molecules. These data are concordant with previous
mechanistic findings relating to T-cell responses to vancomycin for
which it has been hypothesized glycopeptide compounds possess the
capacity to displace and mimic native HLA peptides.7 Proliferative T-cell cross-reactivity of teicoplanin-responsive
TCCs generated from healthy volunteers to daptomycin highlights the
complex patterns of reactivity encountered within clinical settings.
The observed in vitro T-cell cross-reactivity may
be explained by structural similarities between both teicoplanin and
daptomycin, specifically the presence of a hydrophobic lipid chain.
Conversely, vancomycin's structure comprises a heptapeptide
chain that crucially contains a disaccharide, composed of vancosamine
and glucose, instead of the lipid tail found on both teicoplanin and
daptomycin molecules. This potentially explains why some teicoplanin-specific
T-cells are able to proliferate in the presence of daptomycin but
not vancomycin. One intriguing avenue to explore the nature of these
cross-reactive responses involves the study of cellular energetic
parameters, such as glycolysis, which may provide greater sensitivity
for the determination of T-cell activation thresholds upon antigen
presentation. However, to investigate the specificity of teicoplanin
for HLA-A*32:01, additional cloning experiments focusing on individuals
negative for HLA-A*32:01 expression will need to be conducted. Further
genetic studies and functional T-cell analysis following HLA-glycopeptide
binding will be required to determine the full pathway of glycopeptide
cross-reactivity in addition to the extent of interactions with HLA-A*32:01
in order to predict potential susceptibility to severe cross-reactivity
and improve patient safety.</p><p>This work was
conducted with funding from a BBSRC/GSK Industrial Partnership award
(Grant No. BB/R008108/1). The project also received core funding from
the MRC Centre for Drug Safety Science (Grant No. G0700654).</p><p>The authors declare no
competing financial interest.</p>