Commensal Bacteria Maintain a Qa-1^b-restricted Unconventional CD8⁺ T Population in Gut Epithelium

Reviewer #1 (Public Review):

Guan et al. explored the mechanisms responsible for the development, maintenance, and functional properties of a specific subset of unconventional T cells expressing a Va3.2 T cell receptor that recognizes a peptide, QFL, presented by the class Ib protein Qa-1. Prior studies from this group showed that cells from mice deficient in the ER protease ERAAP elicit responses in wild-type animals enriched for Qa-1-restricted CD8 T cells. They further showed that a significant proportion of these responses were directed against the QFL peptide derived from a conserved protein with incompletely understood functions. Many of these so-called QFL T cells expressed Va3.2-Ja21, were present in the spleen of wild-type mice, and exhibited a memory-like phenotype. Due to their relatively low frequency and weak staining with Qa-1 tetramers, analyzing QFL T cells has been challenging. Therefore, the authors generated dextramers, which permitted them to more rigorously identify these cells. They confirmed some of their previous findings and further showed that Va3.2+ and Va3.2- QFL T cells were present in the intestinal epithelium, where they also express CD8alpha homodimers, a characteristic of most small intestinal intraepithelial lymphocytes (siIELs), and most similar to the so-called natural siIELs that acquire their innate functions in the thymus. The authors show that TAP but not Qa-1 or ERAAP expression are required for the development of these cells, and both Qa-1 and ERAAP are required for the natural siIEL phenotype. Some of these findings were confirmed using a new TCR transgenic mouse expressing the QFL TCR. They further show that retention but not homing of QFL T cells to the intestinal epithelium involves commensal microorganisms, and using in silico approaches, they identify a commensal that contains a peptide similar to QFL that can activate QFL T cells. Finally, they show that this organism, P. pentosaceus, can promote gut retention of QFL T cells when it is introduced into germ-free mice. From these findings, the authors conclude that the microbiota influences the maintenance of Qa-1-restricted T cells.

Comments:
1. Overall, the authors employ a number of new reagents and elegant approaches to explore the development, maintenance, and functional properties of QFL T cells.
2. Generally, conclusions made are well supported by the data presented.
3. One limitation of the work is that the immunological functions of QFL T cells remain unclear.
4. The work covers a lot of ground (intestinal IELs, unconventional T cells, innate/virtual memory T cells, Qa-1/HLA-E, etc) that may not be familiar to many readers.
5. A few questions remain:
a) Regarding the results for TAP knockout animals, since Qa-1 does not appear to be required for QFL T cell development, the absence of these cells in TAP KO mice cannot be easily explained.
b) The Va3.2 T cells display similarities with previously identified innate/virtual memory T cells, some of which require IL-4 production by CD1d-restricted NKT cells for their intrathymic development, which is not fully discussed.
c) Qa-1/peptide complexes may also be recognized by CD94/NKG2 (both inhibitory and activating) receptors on NK cells and subsets of CD8 T cells, which may complicate data interpretation, but is not noted in the text.
d) Are these conclusions relevant to the human homolog of Qa-1, HLA-E?

Reviewer #2 (Public Review):

Summary:
CD8+ QFL T cells recognize a peptide, FYAEATPML (FL9), presented on Erap1-deficient cells. QFL T cells are present at a high frequency in the spleen of naïve mice. They express an antigen-experienced phenotype, and about 80% express an invariant TCRα chain Vα3.2Jα21.

Here, Guan and colleagues report that QFL T cells are present not only in the spleen but also in the intestinal epithelium, where they display several phenotypic and functional peculiarities. The establishment of spleen and gut Vα3.2+ QFL T cells is TAP-dependent, and their phenotype is regulated by the presence/absence of Qa-1b and Erap1. Maintenance of gut Vα3.2+ QFL T cells depends on the gut microbiota and is associated with colonization by Pediococcus pentosaceus.

Strengths:
This article contains in-depth studies of a peculiar and interesting subset of unconventional CD8 T cells, based partly on generating two novel TCR-transgenic models.

The authors discovered a clear relation between the gut microbiome and the maintenance of gut QFL T cells. One notable observation is that monocolonization of the gut with Pediococcus pentosaceus is sufficient to sustain gut QFL T cells.

Weaknesses:
In the absence of immunopeptidomic analyses, the presence or absence of the FL9 peptide on various cell types is inferred based on indirect evidence.

Analyses of the homology between the FL9 and bacterial peptides were limited to two amino acid residues (P4 and P6).

The potential function of QFL T cells remains elusive.

Reviewer #3 (Public Review):

The authors investigate the role of commensal microbes and molecules in the antigen presentation pathway in the development and phenotype of CD8 T cells specific for the Qa-1b-restricted peptide FL9 (QFL). The studies track both endogenous QFL-specific T cells and utilize a recently generated TCR transgenic model. The authors confirm that QFL-specific T cells in the spleen and small intestine intraepithelial lymphocyte (IEL) pool show an antigen-experienced phenotype as well as unique phenotypic and innate-like functional traits, especially among CD8+ T cells expressing Va3.2+ TCRs. They find that deficiency in the TAP transporter leads to almost complete loss of QFL-specific T cells but that loss of either Qa1 or the ERAAP aminopeptidase does not impact QFL+ T cell numbers but does cause them to maintain a more conventional, naïve-like phenotype. In germ-free (GF) mice, the QFL-specific T cells are present at similar numbers and with a similar phenotype to SPF animals, but in older animals (>18w) there is a notable loss of IEL QFL-specific cells. This drop can be avoided by neonatal colonization of GF mice with the commensal microbe Pediococcus pentosaceus but not a different commensal, Lactobacillus johnsonii, and the authors show that P. pentosaceus encodes a peptide that weakly stimulates QFL-specific T cells, while the homologous peptide from L. johnsonii does not stimulate such cells.

This study provides new insights into the way in which the differentiation, phenotype, and function of CD8+ T cells specific for Qa-1b/FL9 is regulated by peptide processing and Qa1 expression, and by interactions with the microbiota. The approaches are well designed, the data compelling, and the interpretation, for the most part, appropriate. There are a few relatively minor concerns.

1. For most of the report, the authors use a set of phenotypic traits to highlight the unique features of QFL-specific CD8+ T cells - specifically, CD44high, CD8aa+ve, CD8ab-ve. In Supp. Fig. 4, however, completely distinct phenotypic characteristics are presented, indicating that IEL QFL-specific T cells are CD5low, Thy-1low. No explanation is provided in the text about whether this is a previously reported phenotype, whether any elements of this phenotype are shared with splenic QFL T cells, what significance the authors ascribe to this phenotype (and to the fact that Qa1-deficiency leads to a more conventional Thy-1+ve, CD5+ve phenotype), and whether this altered phenotype is also seen in ERAAP-deficient mice. At least some explanation for this abrupt shift in focus and integration with prior published work is needed. On a related note, CD5 expression is measured in splenic QFL-specific CD8+ T cells from GF vs SPF mice (Supp. Fig. 9), to indicate that there is no phenotypic impact in the GF mice - but from Supp. Fig. 4, it would seem more appropriate to report CD5 expression in QFL-specific cells from the IEL, not the spleen.

2. The authors suggest the finding that QFL-specific cells from ERAAP-deficient mice have a more "conventional" phenotype indicates some form of negative selection of high-affinity clones (this result being somewhat unexpected since ERAAP loss was previously shown to increase the presentation of Qa-1b loaded with FL9, confirmed in this report). It is not clear how this argument aligns with the data presented, however, since the authors convincingly show no significant reduction in the number of QFL-specific cells in ERAAP-knockout mice (Fig. 3a), and their own data (e.g. Fig. 2a) do not suggest that CD44 expression correlates with QFL-multimer staining (as a surrogate for TCR affinity/avidity). Is there some experimental basis for suggesting that ERAAP-deficient lacks a subset of high-affinity QFL-specific cells?

3. The rationale for designing FL9 mutants, and for using these data to screen the proteomes of various commensal bacteria needs further explanation. The authors propose P4 and P6 of FL9 are likely to be "critical" but do not explain whether they predict these to be TCR or Qa-1b contact sites. Published data (e.g., PMID: 10974028) suggest that multiple residues contribute to Qa-1b binding, so while the authors find that P4A completely lost the ability to stimulate a QFL-specific hybridoma, it is unclear whether this is due to the loss of a TCR- or a Qa-1-contact site (or, possibly, both). This could easily be tested - e.g., by determining whether P4A can act as a competitive inhibitor for FL9-induced stimulation of BEko8Z (and, ideally, other Qa-1b-restricted cells, specific for distinct peptides). Without such information, it is unclear exactly what is being selected in the authors' screening strategy of commensal bacterial proteomes. This, of course, does not lessen the importance of finding the peptide from P. pentosaceus that can (albeit weakly) stimulate QFL-specific cells, and the finding that association with this microbe can sustain IEL QFL cells.