Introduction
Tissue-resident memory lymphocytes at mucosal surfaces have been shown to be critical
in long-term protection following mucosal infection. Tissue-resident memory T cells
(Trm) have been well characterized in animal models and in humans, while knowledge
about tissue-resident memory B cells (Brm) is currently more limited. Due to specific
features of tissue-resident memory lymphocytes such as cross-reactivity and polyfunctionality,
Trm and Brm have been demonstrated to be an ideal target for vaccine strategies aiming
to induce protection at mucosal surfaces. The route of vaccine administration, the
choice of antigenic epitopes and the impact of microenvironment appeared to be crucial
parameters in the development of vaccine-induced mucosal tissue-resident memory responses
in animal models. However, it remains significant gaps in understanding systemic and
local signals needed to establish and maintain protective mucosal Trm subsets without
inducing pathogenic populations. In addition, the development of Brm is currently
not well understood. Discovery of innovative recombinant antigens and identification
of safe mucosal adjuvants will be crucial in the development of vaccine formulations
efficient to induce Trm and Brm at mucosal surfaces. This Opinion article will describe
current knowledge about mucosal Trm and Brm and vaccine approaches already tested
to induce tissue-resident memory lymphocytes at mucosal surfaces. It will pinpoint
gaps in knowledge. It will suggest research avenues and highlight considerations to
design vaccine strategies inducing mucosal tissue-resident memory lymphocytes and
it will provide suggestions to improve methodology to quantify mucosal tissue-resident
memory responses.
Characterization and features of Trm at mucosal surfaces
Three memory T cell subsets have been characterized: circulating effector memory T
cells are abundant in non-lymphoid tissues, circulating central memory T cells are
predominant in secondary lymphoid organs and non-circulating tissue-resident memory
T cells (Trm) are able to persist in non-lymphoid tissues. Trm subset was discovered
in parabiosis and tissue transplantation animal studies more than 15 years ago (1)
and it is the most abundant memory T cell population. Trm have the ability to reside
in mucosal tissues such as lung (2), nasal (3), gut (4), skin (5–8) and reproductive
tract tissues (9), following infection (10). Trm were characterized in mice, non-human
primates (NHP) (11) and humans (12–14). They were mostly defined by the high expression
of the adhesion molecule CD69 which can be associated with an upregulation of the
αE integrin CD103 (1), and by the downregulation of molecules preventing tissue exit
such as CCR7 and CD62L (10). Other markers were associated with Trm such as CXCR3,
CD49a (15) (16) or CD44 (1) in specific tissues. Regarding the functions of Trm, antigen-specific
CD8 and CD4 Trm in the respiratory tract were shown to be associated with a better
control of viral and bacterial infections (e.g. reduction of viral load (12), limitation
of intracellular replication of bacteria (17)). In addition, several animal and human
studies described the polyfunctionality of influenza- (18, 19) or respiratory syncytial
virus (RSV)-specific CD8 Trm responses (20) as a key feature of protective Trm. The
cross-reactivity of human Trm via recognition of conserved regions was also reported
in the context of influenza (21, 22) and Severe Acute Respiratory Syndrome Coronavirus
2 (SARS-CoV-2) (3) infections. Another interesting feature of Trm is their potential
innate-like functions helping in mucosal protection. It was demonstrated that CD8
Trm located in lung parenchyma of mice intranasally infected with influenza virus
engineered to express ovalbumin as an antigen, could reduce the severity of a subsequent
pulmonary bacterial infection through neutrophil recruitment (23). A similar bystander
activation of T cells was observed in a Herpes Simplex Virus (HSV) murine challenge
model. Indeed, it was shown that a subset of vaginal CD8 T cells which were unspecific
to the HSV antigen used for immunization, could play a partial role in genital protection.
In this HSV challenge model, both peripheral CD8 T cells able to migrate to inflamed
vaginal tissue and vaginal CD8 Trm of irrelevant antigen specificity were involved
in this innate-like function (24).
Discovery of mucosal Brm
The development of tissue-resident memory B cells (Brm) following infection has been
discovered in murine parabiosis (25), adoptive transfer (26) and depletion studies
(27). The presence of lung Brm early post-infection was reported in mouse models of
influenza (25) and pneumococcal pneumonia (28). It was shown that the induction of
Brm required local encounter with antigen (25) and that Brm contributed to early plasmablast
responses leading to the secretion of cross-neutralizing antibodies against viruses
and bacteria (27, 28). Compared to Trm, Brm have been less characterized and specific
markers have not been fully defined. However, it was shown that Brm expressed CD69,
the hallmark of tissue-resident lymphocytes, in mice (25), NHP and humans (14). Other
markers were also associated with Brm in mice such as CXCR3 and CD44 (25, 26, 28).
In addition, antigen-specific CD73 positive and negative pulmonary Brm subsets were
described in a mouse influenza model (25). In animal studies, the analysis of Brm
is based on the discrimination of resident and circulatory B cells by intravenous
labelling. In animal models and humans, gating strategies based on characterized memory
B cell markers and similarities with Trm transcriptional profiles are also used to
describe Brm subsets (29). Interestingly, a study found a potential intestinal Brm
subset. Indeed, most CD19+CD27+ B cells in human intestine were CD45RB+CD69+ B cells.
In addition, sets of gene expressed in lung Trm were enriched in this gut B cell subset
suggesting that it could be an intestinal-resident population (30). The presence of
Brm in other mucosal tissues such as skin or reproductive tract is currently unclear.
Vaccine approaches to induce tissue-resident memory responses at mucosal sites
For more than two centuries, vaccination has been a successful global strategy to
reduce the burden of several infectious diseases (31). Historically, the immunogenicity
induced by vaccines was associated with systemic humoral response which can be easily
measured in blood using antibody assays (32). However, for several decades, efforts
have been put into the understanding of vaccine-induced cellular and local immune
responses. Could the protective capacities of mucosal Trm and Brm be harnessed to
improve immunity at mucosal barriers?
A range of vaccine approaches have especially been tested in mouse models to improve
mucosal Trm development (33). The route of vaccine administration has been shown to
play a crucial role. Growing evidences suggest that mucosal vaccines on their own
or combined with systemic vaccines could be a promising strategy to enhance the development
of mucosal Trm. For example, a study found that intranasal administration of live-attenuated
influenza virus induced the development of CD4 and CD8 Trm in lungs, whereas systemic
immunisation with live-attenuated influenza virus did not generate similar Trm response
in mice (34). Similarly, intranasal immunization of mice with a chimpanzee adenoviral-based
SARS-CoV-2 vaccine was shown to induce CD103+CD69+CD8 T cells in lungs, while vaccination
by intramuscular route failed to generate pulmonary Trm cells (35). Another strategy
named ‘prime and pull’ was tested to generate Trm in vaginal tract. Mice were subcutaneously
immunized with an attenuated strain of HSV-2 (prime). Then, pro-inflammatory chemokines
were applied to the vagina of mice in order to recruit HSV-specific CD8 T cells to
this mucosal site (pull). Compared to the other experimental groups which were primed
and boosted by intravaginal or subcutaneous routes only, the prime and pull strategy
was the only one leading to the establishment of CD8 Trm in the genital mucosa. This
study suggested that inflammation on its own could lead to the recruitment of Trm
to genital mucosa and that a persistent antigen stimulation was not needed for the
establishment of Trm (36). Similar findings were reported in nasal, upper respiratory
tract (2) and skin (6). However, it was demonstrated that a local antigen encounter
was needed to establish CD8 Trm in lungs (37–39). For instance, mice immunized by
intraperitoneal route with influenza virus (prime) could exclusively generate pulmonary
Trm following an intranasal immunization (pull) with CpG oligodeoxynucleotides combined
with the antigen. The authors of this study suggested that circulating antigen-specific
CD8 T cells could cause local tissue damage, which could play a role in Trm conversion
(39). Interestingly, the importance of vaccine epitopes was pinpointed in a study
describing the sequence design and immunogenicity of a CD8 T cell peptide Coronavirus
Disease 19 (COVID-19) vaccine. This COVID-19 vaccine candidate was based on a range
of 11 structural and non-structural SARS-CoV-2 proteins including conserved regions.
The sequence was designed using SARS-CoV-2 immuno-dominant epitopes determined by
screening and SARS-CoV-2 neoepitopes selected using a computational multi-neoepitopes
based peptide vaccine approach, which had been shown to be safe and efficient in clinical
trials evaluating a vaccine candidate against lung cancer. Following one subcutaneous
vaccination in a mouse model, the COVID-19 vaccine candidate could induce a significant
number of peripheral viral-specific CD8 T cells expressing Trm markers such as CD103
and CD49a, in spleen and draining lymph nodes. Even though the authors did not analyse
Trm in lungs, this study highlights the importance of epitope selection to induce
lymphocytes with a tissue-residency signature (40). Another study compared the functionality
of T cells and Trm in lung biopsies collected for cancer suspicion in SARS-CoV-2 infected
patients or individuals vaccinated with COVID-19 mRNA vaccines. The current spike-based
COVID-19 mRNA vaccines were shown to induce similar SARS-CoV-2 spike-specific IFNγ
CD4 T cell responses in lungs of vaccinees and convalescents, while antigen-specific
CD8 T cell responses were not induced, neither in convalescents, nor in vaccinated
individuals. Regarding tissue-resident memory responses in lungs, polyfunctional CD4
and CD8 Trm induction was shown to be limited post-vaccination compared to post-infection.
A selection of SARS-CoV-2 epitopes, as previously described, could help to improve
tissue-resident memory responses generated by the current COVID-19 mRNA vaccines (41).
Determining the role of local antigen stimulation, mucosal inflammation and antigenic
epitopes in specific vaccine strategies to induce tissue-resident memory lymphocytes
is fundamental. These parameters may impact on the choice of the antigen, the administration
route and the vector used to deliver the vaccine (10).
Regarding Brm, their induction has especially been evaluated in mouse pulmonary tissues
(25). As it was determined that the establishment of pulmonary Brm post-infection
required a local antigen encounter (25), it could be hypothesized that mucosal vaccination
might also be beneficial to generate Brm in lung tissues at least. However, this hypothesis
remains to be demonstrated. Interestingly, evidences revealed that the establishment
of Brm pool did not correlate with the presence of Trm in the reproductive tract of
female mice immunized with HSV (42). These data suggest that Trm and Brm responses
could be induced in an independent manner at least in the reproductive tract. Trm
and Brm development might have different kinetics and/or might require different local
microenvironments. This correlation needs to be studied in other tissues given a lack
of correlation might significantly impact on the development of vaccines targeting
mucosal tissue-resident memory responses. Do specific vaccine approaches need to be
developed to induce either Trm or Brm? It is currently unknown.
Research avenues and considerations to design vaccines inducing protective tissue-resident
memory lymphocytes at mucosal surfaces
Understanding mucosal tissue-resident memory lymphocyte generation
A better understanding of Trm and Brm establishment and maintenance at mucosal surfaces
is needed to tailor efficient vaccine strategies. Based on animal studies, two general
models have been developed to explain Trm formation. The local divergence model suggests
that Trm differentiate within tissues from pluripotent circulating effector T cells.
The systemic divergence model proposes that there is a subset of circulating Trm precursors
intended for migrating into tissues where they finish their differentiation into mature
Trm (32). Regarding Brm formation, their origin has not been fully characterized and
has been mainly based on lung Brm studies in mouse models. Influenza virus infection
models suggest that Brm could originate from germinal centres in mediastinal lymph
nodes or from germinal centre-like structures in the inducible bronchus-associated
lymphoid tissues (25, 43). Identification of circulating Trm and Brm precursors by
flow cytometry and transcriptomics using known tissue-resident memory markers and
genes could be a way to find novel circulating lymphocyte populations sharing residency-promoting
signature with Trm. This type of study could be performed before and after infection
or vaccination in animal models. It would help to validate these models or hypotheses
even though they might be non-exclusive (32), tissue- and context-dependent. Recently,
some evidences have suggested the presence of precursor CD8+ Trm within circulation
(44). In addition, some studies have demonstrated that Trm were able to egress and
migrate to lymph nodes (45) or to distant mucosal sites (46). Understanding potential
movements of tissue-resident memory lymphocytes can be crucial in order to optimize
the routes of administration and to determine whether mucosal vaccination on its own
or whether mucosal vaccination after a systemic prime is the best strategy. The influence
of the priming route should be studied using parabiosis mouse models.
Some studies suggested that specific cytokines or metabolites could impact on Trm
formation. Indeed the role of TGFβ has been described in the generation of lung (47),
nasal/upper respiratory tract (2), gut (48) and skin (7, 49) CD103+ Trm. Modulation
of TGFβ might be a key parameter to optimize vaccine strategies aiming to induce Trm
including lung CD8 Trm (50, 51). A role of other cytokines such as IL-10 (52), IL-21
(53), IL-15 (54, 55) and IL-1/IL-2 (56) was also described in Trm formation in lungs
or other tissues. Recently, it has been demonstrated that a prime of T cells in the
mesenteric lymph node of mice infected with Listeria monocytogenes by oral route,
licensed T cells to differentiate into CD103+ T cells in intestine and that this licensing
was regulated by retinoic acid (57). Systemic and mucosal signals required to generate
tissue-resident memory lymphocytes need to be further elucidated in order to design
vaccine formulations leading to an appropriate microenvironment. The use of knockout
mice for specific cytokines or their receptors, mouse Cre-LoxP system, as well as
reporter systems may help to understand the role of systemic and mucosal signalling
pathways and microenvironments involved in the expansion and responsiveness of tissue-resident
memory lymphocytes post-vaccination at different mucosal sites. Gene-based systems
such as transcription factor profiling of historical activity in specific tissues
(58) or novel DNA-based memory system (59) could help to define the epigenetic state
of potential precursor populations but also early signals linked to tissue-resident
memory lymphocyte formation.
Complicating the picture of tissue-resident memory responses, sub-populations of CD4
Trm have been described based on specific cytokine profiles. Indeed, lung Trm1, Trm2,
Trh, Trm17 have been characterized following different respiratory infections (53)
(60). However, it is unclear whether their differentiation requires different signalling
pathways, specific microenvironments and whether their persistence is similar in mucosal
tissues. It is also important to consider that some particular Trm subsets have been
associated with persistent immunopathology after viral infection or with chronic diseases
(60). For instance, an expansion and activation of Trm17 was identified in bronchoalveolar
lavage fluid from severe COVID-19 patients following SARS-CoV-2 clearance. The characterization
of this subset showed a pathogenic cytokine profile associated with severe disease
and lung damage (61). It was also observed that an enrichment of CD69+CD103- Trm population
in bronchoalveolar lavage fluid collected from patients with post-COVID-19 acute sequelae
negatively correlated with their lung function (62). A specific CD103+CD161+CCR5+CD4+Trm
sub-population was also reported to be predominant in the intestine of Crohn’s disease
patients (63). These examples of pathogenic Trm profiles pinpoint the importance to
clearly define the parameters leading to the establishment of protective tissue-resident
memory lymphocytes at mucosal surfaces following vaccination. The development of protective
or exuberant tissue-resident memory lymphocytes might be related to the type of stimulus,
the persistence of stimulation, the local environment and the type of activated signalling
pathways. Qualitative and quantitative differences between protective and pathological
tissue-resident memory responses need to be elucidated. During the development of
vaccine candidates aiming to induce mucosal Trm or Brm, it will be crucial to determine
the cytokine profile of tissue-resident memory lymphocytes generated at mucosal surfaces
after vaccination in order to evaluate the maintenance of mucosal homeostasis even
though limited inflammation can transiently be induced by vaccination. Spatial transcriptomics
in situ may be a critical approach to detect inflammation associated with tissue-resident
memory lymphocyte populations (64).
Optimizing vaccine formulations
Given the ability of tissue-resident memory lymphocytes to generate cross-reactive
immune responses specific to conserved epitopes, designing recombinant antigens which
include conserved epitopes might be of great interest. Systems vaccinology and artificial
intelligence could be approaches which should be explored to predict epitopes able
to induce tissue-resident memory responses. In addition, the role of adjuvants may
be essential to enhance and tailor tissue-resident memory responses at mucosal sites.
Some promising adjuvants administered by mucosal route have been already identified
in preclinical models. Marinaik et al. showed that acrylic-acid-based adjuvant associated
with a Toll-like receptor agonist glucopyranosyl lipid adjuvant was the most effective
vaccine formulation to induce influenza-specific CD103+ CD8 Trm in lungs of mice immunized
by intranasal route (65). Using the same administration route, it was also shown that
influenza antigens associated with IL-1β enhanced the number of antigen-specific CD103+CD69+
Trm in lungs of mice (66). Interestingly, some adjuvants administered by systemic
route have shown to be able to enhance the induction of mucosal cellular responses.
Indeed, it was reported that all-trans-retinoic acid administered by intraperitoneal
route could enhance the frequency of antigen-specific memory T cells in murine intestine
(67). Woodworth et al. demonstrated that CAF®10b, a liposomal adjuvant administered
by intramuscular route, could prime T cells in order to recall them in the lungs or
skin using the antigen only administered by intratracheal and intradermal routes in
NHP (68). If all-trans-retinoic acid or CAF®10b adjuvants are beneficial to induce
mucosal tissue-resident memory responses, it remains to be confirmed. The main challenge
to design vaccine formulations able to induce mucosal responses including mucosal
Trm/Brm is the current lack of adjuvants licensed for mucosal administration in humans
(69). Identifying effective and safe mucosal adjuvants is a key factor to pursue the
development of vaccine strategies to generate mucosal tissue-resident memory responses.
Unfortunately, the lack of in vitro predictive assays for adjuvants does not help
and in vivo models remain the gold standard for these analyses (70).
Finally, evidences have shown that sex (71) and age (60) (72) could impact on tissue-resident
memory response profile and functionality. These parameters need to be further evaluated
in the context of vaccine development. Vaccine strategies used to generate Trm/Brm
should be tested in both female and male animals, as well as aged animals at some
stages of development. It will help to tailor vaccine strategies aiming to induce
protective Trm and Brm responses in human populations with different pre-existing
chronic mucosal conditions. In addition, the role of microbiota or microbiota-derived
metabolites in the development, maintenance, metabolism and modulation of tissue-resident
memory lymphocytes also have to be considered (73) especially in the context of mucosal
vaccination.
Evaluating tissue-resident memory responses
The gold standard to analyze mucosal Trm and Brm responses remains animal models and
the type of animal models is a crucial parameter. Inbred mice are commonly used in
the studies. However, they may not be the best models to analyze tissue-resident memory
responses at mucosal surfaces. Even though it remains challenging to mimic multiple
mucosal exposures to a range of pathogens impacting on polyfunctional and polyreactive
Trm and Brm in animal models, the use of outbred mice could better recapitulate observations
found in humans as developing more tissue-resident memory lymphocytes in non-lymphoid
tissues (74).
To evaluate the efficacy of vaccine strategies able to enhance Trm and Brm responses
in humans, robust sampling and quantification methods are required to analyze mucosal
tissue-resident memory responses. Analysis of post-mortem tissues or tissues after
resection surgery is currently the best way to study tissue-resident memory responses
at mucosal surfaces by flow cytometry or histological staining (75). Following SARS-CoV-2
infection, human nasal tissue-resident memory T cells have been recently analyzed
using specific device for nasal sampling (76). However, isolation of human Trm and
Brm from mucosal surfaces remains challenging. Consequently, defining correlations
between peripheral markers and mucosal tissue-resident memory responses is crucial
to include the analysis of mucosal tissue-resident memory responses in human vaccine
trials. Peripheral markers could be based on mucosal homing markers expressed on circulating
lymphocyte populations (77) or the circulation of tissue-resident memory precursors.
Conclusion
Vaccines able to induce long-term mucosal responses are needed to improve protection
against infection at mucosal surfaces. Expanding polyfunctional and cross-reactive
tissue-resident memory responses using mucosal vaccination on its own or combined
to systemic vaccination looks a promising way to reach this goal. An ideal vaccine
would induce controlled and balanced Trm and/or Brm responses at mucosal sites (
Figure 1
). For this purpose, a better knowledge is needed to understand the formation of effective
tissue-resident memory responses at mucosal surfaces and to determine the specific
environment needed in each mucosal tissue to generate protective Trm and Brm subsets.
Identifying effective mucosal adjuvants able to induce mucosal tissue-resident memory
responses is a key parameter to optimize vaccine formulations (
Figure 2
). However, it will be challenging to move vaccine candidates into clinical trials
if there are not any standard procedures to quantify Trm and Brm responses in human
mucosal tissues. Identification of peripheral markers correlating with Trm and/or
Brm responses could be an easy way to evaluate mucosal tissue-resident memory responses
post-vaccination in humans.
Figure 1
Generation of tissue-resident memory lymphocytes in mucosal tissues.1. T and B cells
are activated in secondary lymphoid organs (e.g. lymph nodes, mucosa-associated lymphoid
tissues). 2. Following activation, lymphocytes can migrate to mucosal tissues where
they will convert into tissue-resident memory T cells (Trm) and B cells (Brm). 3 &
4. In mucosal tissue, antigen stimulation, cytokine signals and/or cellular interactions
can drive mucosal tissue-resident memory lymphocyte induction and play a role in their
maintenance. The role of these parameters may differ according to the type of mucosal
tissue and there are still significant gaps in the current knowledge. For example,
the role of local antigen encounter (37–39), TGFβ (50, 51), IL-2 (56), IL-15 (54)
or IL-21 (53) in Trm induction, as well as the formation of sub-populations of CD4
Trm have been described in lungs (53, 60). Evidences showing a role of microbiota
or microbiota-derived metabolites in Trm modulation have been reported, especially
in intestine (73). Vaccination may impact on priming, migration, generation and maintenance
of Trm and Brm in mucosal tissues. Trm1: tissue-resident memory CD4 T cells secreting
Th1 cytokines. Trm2: tissue-resident memory CD4 T cells secreting Th2 cytokines. Trm17:
tissue-resident memory CD4 T cells secreting Th17 cytokines. Trh: tissue-resident
memory CD4 T helper cells. The figure was created with BioRender.com.
Figure 2
Challenges and considerations to develop vaccines targeting mucosal tissue-resident
memory responses. Successful vaccine strategies to induce mucosal tissue-resident
memory responses will be based on a better knowledge of the establishment and maintenance
of protective Trm and Brm in mucosal tissues. In addition, understanding their potential
movements to specific mucosal tissues and/or the movements of their precursors, as
well as microenvironments needed to lead to functional Trm and Brm will be crucial.
Interactions between Trm and Brm responses and host parameters such as sex, age or
microbiota should be assessed. The sum of this knowledge will be essential to optimize
vaccine formulations. The figure was created with BioRender.com.
Author contributions
SP and SL have written the manuscript. All authors contributedto the article and approved
the submitted version.