Discussion:
The pathogeneses of ICI toxicities are poorly understood, with few known
risk factors, biomarkers or tailored therapies. As a result, clinicians
cannot identify those at greatest risk or intervene to alter therapy
before morbidity occurs. For ICI-related colitis diagnostic and
treatment algorithms are being derived from classical forms of IBD, and
have not been completely validated in this new clinical setting.
Here we applied multiparameter flow cytometry to the study of
gut-infiltrating and circulating lymphocytes from melanoma patients
treated with combination ipilimumab and nivolumab therapy in the hope of
finding novel insights into disease pathogenesis.
We began by directly investigating the profile of gut-infiltrating
lymphocytes in affected patients (Cohort 1). Here we found IN-COL was
characterised by a predominantly CD8+T cell
lymphocytosis, with the proportion of activated memory
CD8+ T cells greater than observed in active UC, and
supports earlier reports suggesting T cell checkpoint-inhibitor-related
colitis is dominated by CD8+ T cells . The high
proportion of Treg evident in the gastrointestinal tissue of patients
with UC was not evident in IN-COL and we propose this is due to Treg
regulatory mechanisms involving high expression of CTLA-4 . IN-COL was
associated with a higher proportion of activated and granzyme
B+ MAIT cells compared with IN-NAE, changes that were
not found in active UC, again highlighting the distinct pathology of
IN-COL. We confirmed higher rates of MAIT cell activation in IN-COL
using MAIT TCR-specific tetramer staining and paired peripheral blood
and gut-isolated mononuclear cell samples from the same patients.
The comparison of IN-COL and UC patients in Cohort 1 (Table Ic) is
important. The IN-COL patients were older than UC patients and the time
since first diagnosis of colitis was much shorter (1 versus 48 months).
Despite this, the IN-C and UC cohorts scored comparably on both the
UCEIS endoscopic and Nancy histopathological grading system, two scales
commonly used to score severity of UC. The higher levels of
CD4+and CD8+ T cell and MAIT cell
activation measurable by flow cytometry in IN-COL did not correspond
with higher UCEIS or Nancy scores. Therefore, the significant cellular
changes found in IN-COL cannot be simply seen as a function of colitis
severity.
We sought to understand if changes concordant with those seen in local
tissue could be detected in the peripheral blood. We found therapy
results in a widespread “on-treatment” activation of memory T cells,
however these were not predictive of colitis. We additionally determined
that patients with IN-COL could also have pre-existing lower circulating
MAIT cells than those in the IN-NAE group, raising the possibility that
a pre-existing reduction of peripheral MAIT cells might be a risk factor
for IN-COL, as similarly described in other autoimmune diseases of the
gastrointestinal tract . However, this proved an unreliable predictor in
Cohort 3 where the IN-NAE group also had low levels of circulating MAIT
cells. The differences between the IN-NAE groups in Cohort 2 and Cohort
3 could not be explained by significant differences in age, sex or serum
LDH. There is a potential confounder in that the IN-NAE patients in
Cohort 2 were of higher melanoma stage compared with Cohort 3.
IN-COL was not associated with high levels of circulating plasmablasts,
a point of distinction from UC, possibly suggesting there is less of a
role of pathogenic B-cells in IN-COL. Analysis of gut-derived
mononuclear cells demonstrated that IN-COL was associated with high
rates of predominantly activated memory CD8+ T-cells
and granzyme B+ MAIT cells, with a low proportion of
Treg in a cellular pathology that was distinct from both IN-NAE and UC
groups.
Our work provides further evidence that CD8+ T cells
are the predominant cell type in IN-COL. Given that cytotoxic
CD8+ T cells are thought to be the major target of
ICI, it appears that their activation in IN-COL leads to off-target
collateral damage. Important questions remain, including why IN-COL
occurs in some patients and not others, if there is any mechanism to
predict this toxicity prior to treatment, and why steroid and
infliximab-refractory cases occur. Our finding that both
CD8+ T cells and MAIT cells are activated in IN-COL
appears to provide important information as to the initiating antigen
and suggests an interaction with the intestinal microbiome.
MAIT cells provide an important link between microbes and cellular
immunity, being directly activated by vitamin B2 metabolites generated
from bacteria, or indirectly from cytokines (e.g. IFN-α and TNF- α).
Whilst we are unable to confirm that low circulating MAIT cell counts
definitively correlate with subsequent risk of IN-COL, activated MAIT
cells were only seen in the gut-isolated mononuclear cells of IN-COL and
not IN-NAE patients. This raises important questions that centre around
if specific microbes are involved in MAIT cell activation in the setting
of IN-COL, and how MAIT cell trafficking is regulated, particularly
between the blood and gut. There is literature suggesting a host’s
microbiome can impact on the efficacy of ICI therapyand the development
of associated colitis that is relevant to this field and that we are
exploring in a prospective trial. Additionally, there is data supporting
the efficacy of faecal microbiota transplant for ICI-colitis, suggesting
that replacement of microbiome species can ameliorate disease, and one
mechanism of this might be through reducing the provision of
species-specific bacterial-derived MAIT cell ligands.
The presence of the riboflavin operon in bacteria identifies species
that can activate MAIT cells through their TCR. The activation of MAIT
cells in this setting raises a compelling link between host microbiome,
MAIT cells and IN-COL that warrants further investigation, especially in
regard to whether these cells contribute to tissue damage. A report
investigating colitis in the setting of ipilimumab monotherapy found
higher levels of riboflavin (vitamin B2) in the microbiome was
associated with protection from colitis , providing a possible link to
MAIT cells (that are activated by riboflavin metabolites) as protective
in ICI-associated colitis.
MAIT cells can be activated by their TCR or cytokines (IL-12, IL-15,
IL-18, IFNα/β, TNFα) or synergistically by both. We require further work
to determine which pathways are leading to the activation of MAIT cells
in IN-COL and whether this activity contributes to tissue damage .
This work provides an important first report on changes in gut-derived
and peripheral-blood mononuclear cells that occur in ICI-associated
colitis, but our approach has several limitations. This work constitutes
retrospective and cross-sectional analysis conducted in relatively small
numbers of patients that requires confirmation in larger studies. We
also cannot comment at this stage on whether these changes occur in
anti-PD-1 monotherapy-associated colitis.
We are currently recruiting to a prospective single centre study of
oncology patients who have PBMC and GMNC collection, flexible
sigmoidoscopy with gastrointestinal biopsy, and microbiome analysis
prior to commencement of ICI therapy, and at the time of subsequent
development of related colitis. We aim to determine the mechanisms by
which MAIT cells become activated in the gastrointestinal tract. Here we
demonstrate that the levels of activated MAIT cells in the circulation
are not a good indicator of the levels in the colon, and further
investigation into how circulating MAIT cells relate to tissue-based
populations would be of value. It is also unclear at this stage whether
MAIT cell activation contributes to tissue damage or is part of a
homeostatic repair mechanism. It is hoped that further understanding the
mechanisms that activate memory CD8+ T cells and MAIT
cells in the gastrointestinal tract of patients with ICI-related colitis
will lead to the development of improved and novel targeted therapies.