T Cells
20250270506 · 2025-08-28
Assignee
Inventors
Cpc classification
A61K40/11
HUMAN NECESSITIES
International classification
Abstract
The invention relates to T cells, and to methods of producing tissue-resident memory T cells (T.sub.RM). The invention concerns tissue-resident memory T cells (T.sub.RM) per se which have been obtained from the methods of the invention, compositions comprising these T.sub.RM cells, and the use of these T.sub.RM cells and the compositions in therapy, such as in immuno-therapy for treating cancer.
Claims
1. A method for producing a tissue-resident memory T cell (T.sub.RM), the method comprising culturing a lymphocyte in the presence of transforming growth factor beta (TGF) and/or co-culturing the lymphocyte with a regulatory T cell.
2. The method according to claim 1, wherein the lymphocyte is cultured in the presence of TGF, preferably wherein the lymphocyte is not co-cultured with a regulatory T cell.
3. The method according to claim 1, wherein the lymphocyte is a nave, effector or memory CD8+ T lymphocyte.
4. The method according to claim 1, wherein the TGF .sub.is present at a concentration of between 0.01 ng/ml and 50 ng/ml.
5. The method according to claim 1, wherein the lymphocyte has been obtained from tissue of a human or non-human animal, optionally wherein the tissue may be selected from the group consisting of: blood, spleen, lymph node, lung, gastrointestinal tract, skin, prostate mammary gland tissue, liver, bone marrow and pancreas.
6. The method according to claim 1, wherein the T.sub.RM is characterised by expression of cluster of differentiation 8 (CD8), cluster of differentiation 69 (CD69), Hobit, aryl hydrocarbon receptor (AhR) and/or cluster of differentiation 103 (CD103).
7. The method according to claim 1, wherein the T.sub.RM is characterised by the absence of expression of killer cell lectin-like receptor subfamily G member (KLRG1) and/or Eomesodermin (Eomes).
8. The method according to claim 1, wherein the method comprises culturing the lymphocyte in the presence of IL-2, IL-4, IL-7, IL-12, IL-15 and/or IL-21, optionally, wherein the method further comprises comprising culturing the lymphocyte in the presence of interleukin 33 (IL-33) and/or at least one interleukin 1 family member, optionally wherein the at least one interleukin 1 family member is IL-1a, IL-1b and/or IL-18.
9-14. (canceled)
15. The method according to claim 1, wherein the lymphocyte is cultured in a culture media comprising at least one aryl hydrocarbon receptor (AhR) ligand.
16. The method according to claim 9, wherein the AhR ligand is selected from a halogenated aromatic hydrocarbon, a polycyclic aromatic hydrocarbon, a dietary derived aryl hydrocarbon, a heme metabolite, an indigoid, StemRegenin 1 and a tryptophan metabolite.
17. The method according to claim 1, wherein the lymphocyte is cultured in a culture media comprising at least one lipid.
18. The method according to claim 1, wherein the lymphocyte is cultured in a culture media comprising an antigen, optionally wherein the antigen is a tumour antigen.
19. The method according to claim 1, wherein the regulatory T cell is characterised by expression of Foxp3, or is absent.
20. The method according to claim 1, wherein the method further comprises culturing the lymphocyte with a dendritic cell.
21. The method according to claim 1, wherein the method further comprises expanding a population of tissue-resident memory T cells (T.sub.RM).
22. The method according to claim 15, wherein the method further comprises culturing the T.sub.RM cells in the presence of IL-2, IL-4, IL-7, IL-12, IL-15 and/or IL-21.
23. A tissue-resident memory T cell (T.sub.RM) derived from a lymphocyte cultured in the presence of transforming growth factor beta (TGF) and/or co-cultured the with a regulatory T cell.
24-25. (canceled)
26. A method of preventing, treating or ameliorating of cancer or an infection in a subject in need thereof, the method comprising administering a tissue-resident memory T cell derived from a lymphocyte cultured in the presence of transforming growth factor beta (TGF) and/or co-cultured the with a regulatory T cell, to the subject.
27. The tissue-resident memory T cell (T.sub.RM) according to claim 23 or an expanded population thereof, wherein the tissue-resident memory T cell (T.sub.RM) or the expanded population thereof is comprised in a pharmaceutical composition, optionally wherein the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.
28. (canceled)
Description
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EXAMPLES
[0190] The inventors hypothesised that T.sub.REG cells are important in the generation of T cells, T.sub.RM cells, that are able to penetrate deeply into tissues and that are highly effective against solid tumours. The inventors aimed to determine the factors required to generate T.sub.RM cells to enable the generation of T.sub.RM cells in vitro with the ultimate aim to adapt current culture protocols to generate anti-tumour T cells to provide these with tissue penetrating properties to target both primary tumours and to provide critical organ-wide immunosurveillance directed against metastasis that have migrated to tissues away from the primary tumour. The inventors also aimed to assess whether the generation of T.sub.RM cells was possible in the absence of T.sub.REG cells in the medium as detailed in
Materials and Methods
Mice:
[0191] C57Bl/6J and C57Bl/6J CD45.1 mice were purchased from Charles River, France. Tbx21.sup.f/f (Tbx21.sup.tm2Srnr) and Eomes.sup.fl/fl (Eomes.sup.tmtSrnr) were kindly provided by Dr Reiner.sup.14, 82, Foxp3eYFP-Cre (Foxp3.sup.tm4(YFP/icre)Ayr) was kindly provided by Dr Rudensky.sup.83, Rosa26-tdRFP was kindly provided by Dr Fehling.sup.84, Rag2.sup./, IL15R.sup./ (Jackson labs). Mice were bred at the Instituto de Medicina Molecular, Lisbon, Portugal. Male and female mice, aged and sex matched, at 8-18 weeks of age were used. Animals were housed in IVC cages with temperature-controlled conditions under a 12-hours light/dark cycle with free access to drinking water and food. All mice were kept in specific-pathogen-free conditions. All mice in the Foxp3eYFP-Cre Rosa26-tdRFP lines were stringently genotyped by PCR and those in which a knock out allele was detected were discarded (20%), appropriate Tbx21 presence was confirmed by blood typing for CD4 T cells expressing CXCR3. In addition, mice were counter screened for inappropriate expression of RFP in relation of eYFP (10% discarded) (
[0192] Cell isolation: Intestinal cells were isolated as previously described 90. Intestine was flushed with PBS to remove contents and opened longitudinally. After cutting into 1 cm pieces, it was incubated in PBS containing 20 mM Hepes, 100 U/ml penicillin, 100 g/ml streptomycin, 1 mM Pyruvate, 10% FCS, 100 g/ml polymyxin B and 10 mM EDTA for 30 min at 37 C. while shaking to release IELs. IEL single-cell suspensions were further purified using 37.5% isotonic Percoll. To isolate LPLs, intestinal tissue was then digested in IMDM medium containing 0.5 mg/ml of Collagenase D (Roche) and 0.2 mg/ml of DNaseI (Roche) for 25 min at 37 C. while shaking. Liver lymphocytes were isolated by mashing the organ through a 70 m filter, followed by cell purification with 37.5% isotonic Percoll. Lungs were shredded in small pieces with scissors and digested in PBS containing 1 mg/ml Collagenase D, 37 C. during 30 minutes. The cell suspension containing the lymphocytes was obtained after passing through a 50 m cell strainer.
[0193] Adoptive cell transfers: CD8+ T cells and/or CD25+ cells (Treg) were purified from a single cell suspension of spleen and lymph nodes. Briefly, cells were labelled with anti-CD8-APC or anti-CD25-APC antibody and selected with anti-APC MACS microbeads, according to the manufacturer's instructions. After counting, purity was determined by flow cytometry and cell numbers adjusted. To ensure a wide TCR diversity in the population transferred a minimum of 210.sup.6 CD8 T cells were used. Some of the recipient mice received in addition 0.4-110.sup.6 Treg cells. Infection was performed one day after cell transfer (day 0).
[0194] Infection challenges: Animals were infected with Eimeria vermiformis (Ev) as previously described in detail.sup.91. Briefly, oocysts were washed 3 times with deionized water, floated in sodium hypochloride and counted using a Fuchs-Rosenthal chamber. Mice received 500 oocysts of E.vermiformis by oral gavage in 100 l of water and were analysed after the infection was cleared (from week 3 p.i). To determine burden of infection, animals were caged individually and faeces collected daily until oocysts were no longer detected. Animals were infected with Yersinia pseudotuberculosis (Yptb), kindly provided by Dr T. Bergsbaken, as previously described.sup.58. Animals were infected with 10.sup.6 Yptb by oral gavage in 100 l of water. Analysis of tissues was performed on days 15-19.
[0195] Flow cytometry: Single cell suspensions from spleen, lymph nodes, intestine, lung and liver were prepared and stained with antibodies (see list), according to the agreed standards.sup.92 and with indicated gating strategy (
[0196] Quantitative RT-PCR: RNA was isolated using the Qiagen RNeasy Mini kit and cDNA generated using the High Capacity RNA-to-cDNA kit from Applied Biosystems. Amplification was performed using the SYBR Select Master Mix (Applied Biosystems) and the QuantiTect Primer Assays Mm_Cxcl10_1_SG, Mm_Tgfb1_1_SG, Mm_Itgb8_1_SG and Mm_Hprt_1_SG (Qiagen).
[0197] Immunohistochemistry and microscopy: Intestinal tissues were rolled into a Swiss roll, fixed in 10% formalin, rehydrated in 30% glucose and frozen in OCT media. Tissues were cut at 10 m and sections treated with 4% paraformaldehyde. Blocking was performed using 10% BSA and the following antibodies were used for detection: CD45.1 (A20, Biolegend) and FOXP3 (FJK-16s, eBioscience). Slides were mounted in Fluoromount (Invitrogen) and imaged using a Zeiss LSM 880 microscope. Analysis was performed using Fiji software.
[0198] scRNA-Seq analysis Original data was produced and analysed in55. From the initial data-set, T.sub.REG cells were selected based on Foxp3, excluding Tmems, stressed and low-quality cells. In order to analyse this subset, we followed a similar approach as.sup.55, using the R package Seurat.sup.93. Normalization of the data using the LogNormalize method and using a scale factor of 10.sup.5; and scale the data based on Negative Binomial Model and using UMI's. Subtypes of T.sub.REG were defined using the following criteria: Type 1 (Cells with raw counts assigned to the genes Tbx21, Stat1 and Cxcr3); Type 2 (Cells with raw counts assigned to the genes Gata3, Stat6 and Il.sup.1rl1); Type 3 (cells with raw counts assigned to the genes Rorc, Stat3 and Ccr6); other (Cells with no raw counts assigned to the genes Tbx21, Gata3 and Rorc).
In Vitro Cultures:
[0199] Effector CD8 T cells were obtained from C57BL6/J or CD45.1 C57BL6/J mice previously i.p. injected with 25 g anti-CD38. Cells were isolated via AutoMACS bead selection and cultured at 200.000 cells per flat bottom 96-well plates in IMDM medium. 100.000 BMDC, cultured via standard protocol using GM-CSF were added in indicated conditions. Cells were restimulated with 0.25 g/ml anti-CD38, 10 ng/ml IL-15, and 0.5 ng/ml TGF, and where indicated 10-20 ng/ml IL-2 or IL-7. Cells were grown for 3 days before analysis or adoptive transfers into full C57BL6/J mice to test for tissue homing. Cells were assessed for the T.sub.RM markers CD69, CD103, the absence of KLRG-1, and expression of CTLA-4.
[0200] In vitro differentiated cells were transferred into mice through intravenous injection. At the indicated time points, animals were sacrificed and lymphocytes from spleen, lungs and small intestine (both IEL and LPL fractions) were isolated following standard methods. Cell populations were analysed by flow cytometry. Transferred cells were distinguished from endogenous cells by their expression of the congenic marker CD45.1 and cell counts were performed using flow cytometry counting beads.
Example 1
Results
Deletion of Tbx21 in FoxP3.sup.+ Cells Reduces Type 1 T.sub.REG Cells
[0201] T.sub.RM cells express T-bet but not Eomes (
[0202] The inventors confirmed that Foxp3-specific targeting, CD4.sup.+CXCR3.sup.+Foxp3, but not CD4.sup.+CXCR3.sup.+Foxp3.sup.+, T cells were present in Foxp3.sup.Tbx21 mice (
Excision of Tbx21 or Eomes in T.SUB.REG .Cells Alters CD8 T Cell Distribution
[0203] The splenic CD8.sup.+ T cell compartment of Foxp3.sup.Tbx21 mice show an increase in effector (T.sub.eff)/T.sub.EM T cells (
[0204] Despite altered numbers of CD4.sup.+Foxp3-T cells and T.sub.REG cells in the LPL compartment, and irrespective of the Foxp3-dependent excision of Tbx21 or Eomes, the proportion of CD4.sup.+ T cells and T.sub.REG cells remains stable (
Tbx21.sup.+ and Eomes.sup.+ T.sub.REG Cells Influence the CD8 T Cell Memory Compartment
[0205] The reduction of T.sub.RM cells in the intestine and increased proportion of circulating effector/T.sub.EM cells in the absence of T-bet-sufficient T.sub.REG cells suggested a potential role for these cells in the generation or maintenance of T.sub.RM cells. Although there is a reduction in IEL numbers (
[0206] In the LPL compartment of Foxp3WT and Foxp3.sup.Eomes animals, most CD8 T cells express the TRIM markers CD69 and CD103 (
Tbx21.sup.+ T.sub.REG Cells Influence T.sub.RM Cell Development in Multiple Tissues
[0207] Upon skin infections, KLRG1.sup.+CD103.sup.CD8.sup.+ effector T cells have been reported in the dermis early, but not late, nor in the epidermis.sup.19. In agreement with the population of CD103.sup.CD8.sup.+ T cells observed in the small intestine of Foxp3.sup.Tbx21 animals under steady state conditions, a marked population of KLRG1.sup.+CD8.sup.+ T cells, around 20% of the total CD8 T cell population, in all sections of the small intestine was observed (
[0208] Co-staining with CD103 confirmed the KLRG1 protein to be expressed in a mutually exclusive form with CD103.sup.19, 26, 43, and primarily present in Foxp3.sup.Tbx21 mice (
[0209] The presence of T.sub.RM cells is described in many tissues.sup.4. Consistent with results in the intestine, the liver and lungs of Foxp3.sup.Tbx21 mice contained an increased proportion of effector CD8 T cells, expressing high levels of KLRG1 and Eomes, compared with Foxp3.sup.Eomes and Foxp3.sup.WT animals (
Compromised T.SUB.RM .Cell Compartment Reduces Protection Against Pathogen Invasion
[0210] The inventors hypothesised that reduced T.sub.RM cell numbers in Foxp3.sup.Tbx21 mice could reduce protection against new infections, since bystander-mediated activation of T.sub.RM cells is an important defence mechanism limiting pathogen invasion.sup.47, 48,49. We tested the acute response of intestinal CD8+ T cells by administrating anti-CD3 antibody upon which the LPL T cell response was assessed two days later. Eomes.sup.+CD8 T cells displayed a reduced activity profile compared with Eomes CD8 T cells with increased expression of PD-1 and reduced granzyme B (
[0211] Next, the inventors challenged mice with the intracellular protozoan parasite Eimeria vermiformis (Ev), which infects murine small intestinal epithelial cells. In this infection model lymphocytes reduce parasite burden (
Type 1 T.sub.REG Cells Enhance T.sub.RM Development
[0212] T cells from Foxp3.sup.Tbx21 or Foxp3.sup.Eomes mice where indistinguishable from Foxp3WT controls with respect to the expression of Tbet or Eomes, at steady state, upon activation or upon T.sub.RM cell establishment (
[0213] To understand if T-bet-expressing T.sub.REG cells have specific functional attributes that may explain their role in TRY development, the inventors made use of a recent publically available set of single T.sub.REG cell sequencing data.sup.55. In line with previous reports.sup.42, 56, although small trends may exists, the inventors did not uncover significant differences in T.sub.REG cell effector molecules such as IL-10, IL-35, TGF, CD25, LAG3 or CTLA-4 across T.sub.REG cell subsets defined by the presence of the characteristic lineage transcription factors Tbx21, Gata3 or Rorc (
T.sub.RM Development Relies on T.sub.REG Recruitment to Make TGF Bio-Available Locally
[0214] The inventor's observations relied on the microbial presence under specific pathogen free conditions and the intracellular small intestinal parasite Ev, which provokes a very local response. The inventors made use of our CD8.sup.CD45.1 T cell transfer system (
[0215] The transfer model of CD8.sup.CD45.1 T cells into Foxp3.sup.Tbx21 mice enabled the inventors to investigate the contribution T.sub.REG cells make to promote T.sub.RM cell development. In the absence of T-bet, T.sub.REG cells express similar levels of CD103, CCR6 and P-selectin.sup.35, but are unable to express CXCR3, important for localisation of T cells to areas of infection in non-lymphoid tissues.sup.7,35,39,60. The local inflammatory environment controls recruitment of T.sub.RM precursor cells.sup.58, 61, and T.sub.REG cells (
[0216] Aggregates of CD8.sup.+ and CD4.sup.+ T cells together with other immune cells such as macrophages and dendritic cells but without B cells, in areas of microbial invasion are commonly observed.sup.58, 62, 63, 64, 65. Interactions between CD4.sup.+ and CD8.sup.+ T cells, although not required for T cell maintenance.sup.59, likely constitute distinct microenvironments that may support T.sub.RM differentiation. In line, the inventors frequently observed transferred CD8.sup.CD45.1 T cells in close proximity with Foxp3-expressing T.sub.REG cells in Foxp3.sup.WT mice, which were not readily observed in Foxp3.sup.Tbx21 animals despite similar CD8 T cell infiltration (
[0217] The requirement of CXCR3 to promote T.sub.RM development suggests T.sub.REG provide a short range acting or cell-bound effector molecule. Type 1 cytokines, such as IL-12, can maintain high levels of T-bet and Eomes, thereby preventing the differentiation of T.sub.RM cells and the expression of CD103. IL12RB2-deficient CD8+ T cells have increased proportions of cells expressing CD103 and T cell clusters have higher TGF transcripts.sup.59. IL-10 could reduce IL-12 expression and dendritic cell maturation.sup.66. However, IL-10-deficient T.sub.REG cells are able to assist in the efficient development of T.sub.RM cells (
[0218] Many cells, especially at the mucosal barrier, are able to make TGF.sup.67. Yet, TGF is produced as an inactive precursor, which requires cleavage from its latency-associated peptide. TGF- has potent cell modulation activity, acting on numerous immune and non-immune cell types, hence its availability is strictly regulated in the local microenvironment. It was recently shown that T.sub.REG cells can activate TGF via the integrin v8 and that this protein is upregulated in activated/effector T.sub.REG cells, thereby reducing local bioactive TGF68, and the inventors hypothesised that specific recruitment of CXCR3-expressing T.sub.REG cells, which do not show differential expression for TGF1 or Itgb8 under steady state (
Discussion
[0219] The induction of long-lived cellular immunity in non-lymphoid tissues is important to protect against reinfection, as well as a major aim in vaccine design. The inventor's data supports a model in which CD8+ T cells home to tissues as effector cells or memory cell precursors, which subsequently differentiate into T.sub.RM cells upon receiving local cues.sup.69. T cell activation in SLOs induces the expression of a large variety of tissue homing receptors that guide activated T cells to non-lymphoid tissues ensuring that effector T cells inspect most peripheral tissues.sup.69, 70. The unique profile of T.sub.RM cells suggests that factors in the tissue microenvironment instruct the differentiation of effector cells into T.sub.RM cells.
[0220] The inventor's data is based on localised infection models and a polyclonal TCR repertoire. Using localised infection models, it has been shown that optimal T.sub.RM cell development, but not maintenance, requires inflammation-mediated trafficking and cognate antigen in the local microenvironment.sup.61, 71, 72. Although this is in contrast with observations using systemic viral infections where IEL numbers remain stable.sup.69, the inventor's observations are in line with previously reported small intestinal infection.sup.58, and suggest this is a characteristic of local inflammation. Local cues, such as cytokines and secondary antigen encounter may be required for T.sub.RM cell differentiation from recruited effector or memory precursor T cells. The inventor's data supports this model and extends it with the need to recruit T.sub.REG cells to the site of inflammation and their ability to raise bioactive TGF levels that facilitate effector-to-memory development. Upon total T.sub.REG cell depletion, numbers of T.sub.RM cells in the central nervous system were reduced upon viral infection.sup.73, suggesting a role of T.sub.REG cells in the development or maintenance of T.sub.RM cells. The inventors extend this observation by showing that local recruitment of type 1 T.sub.REG cells is critical, whereupon expression of Itg8 promotes T.sub.RM development, which critically relies on locally supplied TGF and its bioavailability.sup.23, 26, 59. Local antigen can retain CD8+ T cells in tissues, which form stable contacts with infected cells and whereupon CD69 is re-expressed, a process that requires CXCR3 expression.sup.74. Although in non-inflamed tissues the inventors report a reduction in CD4 and T.sub.REG cells, CD4 T cells are recruited upon inflammation. Although the supplementation of T.sub.REG cells is sufficient to enhance T.sub.RM cell development, this does not exclude a supportive role of CD4 T cells. The inventors show that CXCR3 expression, also identifying type 1 T.sub.REG cells in humans.sup.75, is required to recruit T.sub.REG cells to the site of inflammation providing a rational for the requirement of the upstream transcription factor T-bet. In the absence of CXCR3, T.sub.REG cell tissue recruitment is limited, resulting in enhanced type 1 immunity and immunopathology.sup.42, 76. Although the absence of T.sub.REG cells is able to enhance immune responses, even resulting in sterile immunity, loss of subsequent immunity was reported.sup.53. The inventors show here that type 1 T.sub.REG cells provide TGF and make it available locally with the expression of v8 integrin, thereby facilitating the development of T.sub.RM cells, supporting life-long immune surveillance and increasing tissue protection against invading microorganisms.
[0221] The inventors observed reduced T.sub.RM differentiation in several tissues assessed in the absence of T-bet-expressing T.sub.REG cells. This highlights that specific tissue microenvironments do not play a critical role in the development of T.sub.RM cells or the recruitment of type 1 T.sub.REG cells. Nevertheless, tissue specific differences may alter the amplitude of T.sub.RM development or their phenotype. T.sub.RM cells in the intestine are known to predominantly produce IFN, while those in the epidermis have been shown to be able to produce IL-17 after microbial challenge. Furthermore, additional tissue insults can alter epidermal T.sub.RM cell function, contributing to wound repair.sup.77,78. The colon was the notable exception in which the inventors did not find alterations in T.sub.RM or effector cells, nor were ratios in T cell subsets altered, in the absence of type 1 T.sub.REG cells. Although T.sub.REG cells require T-bet to express CXCR3 and to be recruited to inflammatory sites, T-bet does not seem to control CXCR3 expression in the colon.sup.42. The disjunction between T-bet and CXCR3 in the colon suggests alternative immune regulation in the organ harbouring the largest content of microbes.
[0222] TGF is a potent driver of CD103 expression on CD8+ T cells in vitro and in vivo.sup.22, and has been shown to reduce KLRG1 expression.sup.43. Furthermore, the importance of reducing inflammation for T.sub.RM cell development is suggested by diminished CD103-expression during chronic infection.sup.23,79. In addition, TGFRII-deficient CD8 T cells fail to become or remain T.sub.RM cells.sup.19, 58. In addition to CD103.sup.+ T.sub.RM cells, a CD103.sup. T.sub.RM cell population has been reported.sup.22, 58, 62. The stability of this population may depend on tissue type and antigen persistence. In the inventor's models in control mice, looking at steady state under specific pathogen-free conditions, as well as after Ev challenge, CD103-T cells were a minor population. Instead, CD103-T cells observed in Foxp3.sup.Tbx21 mice expressed KLRG1 and high levels of Eomes, characteristics of T cells in a transition phase to express CD103 and switch off Eomes.sup.80. Without wishing to be bound to any particular theory, the inventor's data does reveal an important role for type 1 T.sub.REG cells in T.sub.RM cell development, but a smaller T.sub.RM cell population could still be generated, which suggests other cells may make an additional contribution in releasing TGF. Alternative sources of generation of bioactive TGF have been reported, including stromal epithelial cells, important for the maintenance of T.sub.RM cells.sup.81.
[0223] T.sub.REG cells are critical in dampening excessive immune responses, thereby preventing autoimmunity and immunopathology, and may reduce the amplitude of responses upon infection and vaccination as measured in blood. However, the inventor's data highlights their important role in efficiently generating tissue resident memory T cells from effector or memory precursors, which would otherwise become exhausted. T.sub.REG cells thereby ensure that critical numbers of T cells are available for immunosurveillance in tissues to prevent or reduce re-infection as well as reducing pathogen load of new infections.
Example 2
Results
[0224] The inventors assessed whether the generation of T.sub.RM cells was possible in the absence of T.sub.REG cells in the medium, as detailed in
[0225] Referring to
[0226] Referring to
[0227] Referring to
[0228] Furthermore, the inventors assessed if the cells produced in vitro maintained their therapeutic properties, in particular, their ability to migrate and survive in vivo inside the tissues, the experimental setup detailed in
[0229] The inventors have recreated the in vivo conditions in an in vitro setup consisting of effector CD8+ T cells and bone marrow derived dendritic cells (BMDC). The T cells are stimulated in vitro and expanded in a similar manner to the produce a large amounts of cells for T cell therapies. The inventors show that the addition of bioactive TGF can replace the role of T.sub.REG cells in the development of T cells resembling T.sub.RM cells, with continued expression of the markers and tissue retention factors CD69 and CD103 (
[0230] The data resulting from transferring the in vitro generated and expanded T cells in vivo show that effector cells, those generated in the absence of TGF are not found in substantial numbers in the tissues (
SUMMARY
[0231] In summary, the inventors have developed a highly novel and innovative protocol to generate T cells (known as T.sub.RM cells) that are able to deeply penetrate tumours (and especially solid tumours) to contribute to a step-change in T cell therapies against tumours or infections. The inventor's protocol results in vitro generated and expanded cells with the phenotype of migratory and tissue penetrating cells based on the expression of CTLA-4, CD69 and CD103. Conform their phenotype and in contrast to effector T cells, the generated cells are readily found in a variety of lymphoid and non-lymphoid tissues at least 40 days after adoptive transfer into a full mouse host. The inventors' work will make important inroads for efficacious treatment of organ infections and for those cancer patients suffering from solid tumours, which are much harder to treat. However, the inventors believe that the tissue-penetrating ability of the T.sub.RM cells will go beyond the targeting of infections or primary tumours and may provide critical organ-wide immunosurveillance directed against metastasis that have migrated to, often less accessible, tissues away from the primary tumour.
REFERENCES
[0232] 1. Stary, G. et al. VACCINES. A mucosal vaccine against Chlamydia trachomatis generates two waves of protective memory T cells. Science 348, aaa8205 (2015). [0233] 2. Masopust, D., Vezys, V., Marzo, A. L. & Lefrancois, L. Preferential localization of effector memory cells in nonlymphoid tissue. Science 291, 2413-2417. (2001). [0234] 3. Reinhardt, R. L., Khoruts, A., Merica, R., Zell, T. & Jenkins, M. K. Visualizing the generation of memory CD4 T cells in the whole body. Nature 410, 101-105. (2001). [0235] 4. Mueller, S. N. & Mackay, L. K. Tissue-resident memory T cells: local specialists in immune defence. Nature reviews. Immunology 16, 79-89 (2016). [0236] 5. Konjar, S., Ferreira, C., Blankenhaus, B. & Veldhoen, M. Intestinal Barrier Interactions with Specialized CD8 T Cells. Front Immunol 8, 1281 (2017). [0237] 6. Ariotti, S. et al. Tissue-resident memory CD8+ T cells continuously patrol skin epithelia to quickly recognize local antigen. Proceedings of the National Academy of Sciences of the United States of America 109, 19739-19744 (2012). [0238] 7. Schenkel, J. M., Fraser, K. A., Vezys, V. & Masopust, D. Sensing and alarm function of resident memory CD8(+) T cells. Nature immunology 14, 509-513 (2013). [0239] 8. Slutter, B., Pewe, L. L., Kaech, S. M. & Harty, J. T. Lung airway-surveilling CXCR3 (hi) memory CD8(+) T cells are critical for protection against influenza A virus. Immunity 39, 939-948 (2013). [0240] 9. Wu, T. et al. Lung-resident memory CD8 T cells (TRM) are indispensable for optimal cross-protection against pulmonary virus infection. J Leukoc Biol 95, 215-224 (2014). [0241] 10. Konjar, S. et al. Mitochondria maintain controlled activation state of epithelial-resident T lymphocytes. Sci Immunol 3, eaan2543 (2018). [0242] 11. Intlekofer, A. M. et al. Effector and memory CD8+ T cell fate coupled by T-bet and eomesodermin. Nature immunology 6, 1236-1244 (2005). [0243] 12. Pearce, E. L. et al. Control of effector CD8+ T cell function by the transcription factor Eomesodermin. Science 302, 1041-1043 (2003). [0244] 13. Banerjee, A. et al. Cutting edge: The transcription factor eomesodermin enables CD8+ T cells to compete for the memory cell niche. Journal of immunology 185, 4988-4992 (2010). [0245] 14. Intlekofer, A. M. et al. Requirement for T-bet in the aberrant differentiation of unhelped memory CD8+ T cells. The Journal of experimental medicine 204, 2015-2021 (2007). [0246] 15. Joshi, N. S. et al. Inflammation directs memory precursor and short-lived effector CD8(+) T cell fates via the graded expression of T-bet transcription factor. Immunity 27, 281-295 (2007). [0247] 16. Pipkin, M. E. et al. Interleukin-2 and inflammation induce distinct transcriptional programs that promote the differentiation of effector cytolytic T cells. Immunity 32, 79-90 (2010). [0248] 17. Joshi, N. S. et al. Increased numbers of preexisting memory CD8 T cells and decreased T-bet expression can restrain terminal differentiation of secondary effector and memory CD8 T cells. Journal of immunology 187, 4068-4076 (2011). [0249] 18. Kaech, S. M. & Ahmed, R. Memory CD8+ T cell differentiation: initial antigen encounter triggers a developmental program in naive cells. Nature immunology 2, 415-422. (2001). [0250] 19. Mackay, L. K. et al. The developmental pathway for CD103 (+) CD8+tissue-resident memory T cells of skin. Nature immunology 14, 1294-1301 (2013). [0251] 20. Wakim, L. M. et al. The molecular signature of tissue resident memory CD8 T cells isolated from the brain. Journal of immunology 189, 3462-3471 (2012). [0252] 21. Nakayamada, S. et al. Early Th1 cell differentiation is marked by a Tfh cell-like transition. Immunity 35, 919-931 (2011). [0253] 22. Casey, K. A. et al. Antigen-independent differentiation and maintenance of effector-like resident memory T cells in tissues. Journal of immunology 188, 4866-4875 (2012). [0254] 23. Zhang, N. & Bevan, M. J. Transforming growth factor-beta signaling controls the formation and maintenance of gut-resident memory T cells by regulating migration and retention. Immunity 39, 687-696 (2013). [0255] 24. Sheridan, B. S. et al. Oral infection drives a distinct population of intestinal resident memory CD8(+) T cells with enhanced protective function. Immunity 40, 747-757 (2014). [0256] 25. Laidlaw, B. J. et al. CD4+ T cell help guides formation of CD103+ lung-resident memory CD8+ T cells during influenza viral infection. Immunity 41, 633-645 (2014). [0257] 26. Mackay, L. K. et al. T-box Transcription Factors Combine with the Cytokines TGF-beta and IL-15 to Control Tissue-Resident Memory T Cell Fate. Immunity 43, 1101-1111 (2015). [0258] 27. Laidlaw, B. J., Craft, J. E. & Kaech, S. M. The multifaceted role of CD4(+) T cells in CD8(+) T cell memory. Nature reviews. Immunology 16, 102-111 (2016). [0259] 28. Feau, S., Arens, R., Togher, S. & Schoenberger, S. P. Autocrine IL-2 is required for secondary population expansion of CD8(+) memory T cells. Nature immunology 12, 908-913 (2011). [0260] 29. Yu, F., Sharma, S., Edwards, J., Feigenbaum, L. & Zhu, J. Dynamic expression of transcription factors T-bet and GATA-3 by regulatory T cells maintains immunotolerance. Nature immunology 16, 197-206 (2015). [0261] 30. McNally, A., Hill, G. R., Sparwasser, T., Thomas, R. & Steptoe, R. J. CD4+CD25+ regulatory T cells control CD8+ T-cell effector differentiation by modulating IL-2 homeostasis. Proceedings of the National Academy of Sciences of the United States of America 108, 7529-7534 (2011). [0262] 31. de Goer de Herve, M. G., Jaafoura, S., Vallee, M. & Taoufik, Y. FoxP3(+) regulatory CD4 T cells control the generation of functional CD8 memory. Nature communications 3, 986 (2012). [0263] 32. Pace, L. et al. Regulatory T cells increase the avidity of primary CD8+ T cell responses and promote memory. Science 338, 532-536 (2012). [0264] 33. Laidlaw, B. J. et al. Production of IL-10 by CD4(+) regulatory T cells during the resolution of infection promotes the maturation of memory CD8(+) T cells. Nature immunology 16, 871-879 (2015). [0265] 34. Kalia, V., Penny, L. A., Yuzefpolskiy, Y., Baumann, F. M. & Sarkar, S. Quiescence of Memory CD8(+) T Cells Is Mediated by Regulatory T Cells through Inhibitory Receptor CTLA-4. Immunity 42, 1116-1129 (2015). [0266] 35. Koch, M. A. et al. The transcription factor T-bet controls regulatory T cell homeostasis and function during type 1 inflammation. Nature immunology 10, 595-602 (2009). [0267] 36. Hall, A. O. et al. The cytokines interleukin 27 and interferon-gamma promote distinct Treg cell populations required to limit infection-induced pathology. Immunity 37, 511-523 (2012). [0268] 37. Reis, B. S., Hoytema van Konijnenburg, D. P., Grivennikov, S. I. & Mucida, D. Transcription factor T-bet regulates intraepithelial lymphocyte functional maturation. Immunity 41, 244-256 (2014). [0269] 38. Lord, G. M. et al. T-bet is required for optimal proinflammatory CD4+ T-cell trafficking. Blood 106, 3432-3439 (2005). [0270] 39. Levine, A. G. et al. Stability and function of regulatory T cells expressing the transcription factor T-bet. Nature 546, 421-425 (2017). [0271] 40. Obar, J. J. & Lefrancois, L. Memory CD8+ T cell differentiation. Ann N Y Acad Sci 1183, 251-266 (2010). [0272] 41. Omilusik, K. D. et al. Sustained Id2 regulation of E proteins is required for terminal differentiation of effector CD8(+) T cells. The Journal of experimental medicine 215, 773-783 (2018). [0273] 42. Tan, T. G., Mathis, D. & Benoist, C. Singular role for T-BET+CXCR3+ regulatory T cells in protection from autoimmune diabetes. Proceedings of the National Academy of Sciences of the United States of America 113, 14103-14108 (2016). [0274] 43. Schwartzkopff, S. et al. TGF-beta downregulates KLRG1 expression in mouse and human CD8(+) T cells. European journal of immunology 45, 2212-2217 (2015). [0275] 44. Janssen, E. M. et al. CD4+ T-cell help controls CD8+ T-cell memory via TRAIL-mediated activation-induced cell death. Nature 434, 88-93 (2005). [0276] 45. Oh, S. et al. IL-15 as a mediator of CD4+help for CD8+ T cell longevity and avoidance of TRAIL-mediated apoptosis. Proceedings of the National Academy of Sciences of the United States of America 105, 5201-5206 (2008). [0277] 46. Madakamutil, L. T. et al. CD8alphaalpha-mediated survival and differentiation of CD8 memory T cell precursors. Science 304, 590-593 (2004). [0278] 47. Lertmemongkolchai, G., Cai, G., Hunter, C. A. & Bancroft, G. J. Bystander activation of CD8+ T cells contributes to the rapid production of IFN-gamma in response to bacterial pathogens. Journal of immunology 166, 1097-1105 (2001). [0279] 48. Chu, T. et al. Bystander-activated memory CD8 T cells control early pathogen load in an innate-like, NKG2D-dependent manner. Cell reports 3, 701-708 (2013). [0280] 49. Fernandez-Ruiz, D. et al. Liver-Resident Memory CD8(+) T Cells Form a Front-Line Defense against Malaria Liver-Stage Infection. Immunity 45, 889-902 (2016). [0281] 50. Herndler-Brandstetter, D. et al. KLRG1(+) Effector CD8(+) T Cells Lose KLRG1, Differentiate into All Memory T Cell Lineages, and Convey Enhanced Protective Immunity. Immunity 48, 716-729 e718 (2018). [0282] 51. Rose, M. E., Joysey, H. S., Hesketh, P., Grencis, R. K. & Wakelin, D. Mediation of immunity to Eimeria vermiformis in mice by L3T4+ T cells. Infection and immunity 56, 1760-1765 (1988). [0283] 52. Rose, M. E., Wakelin, D. & Hesketh, P. Gamma interferon controls Eimeria vermiformis primary infection in BALB/c mice. Infection and immunity 57, 1599-1603 (1989). [0284] 53. Belkaid, Y., Piccirillo, C. A., Mendez, S., Shevach, E. M. & Sacks, D. L. CD4+CD25+ regulatory T cells control Leishmania major persistence and immunity. Nature 420, 502-507 (2002). [0285] 54. Ramsburg, E., Tigelaar, R., Craft, J. & Hayday, A. Age-dependent requirement for gammadelta T cells in the primary but not secondary protective immune response against an intestinal parasite. The Journal of experimental medicine 198, 1403-1414 (2003). [0286] 55. Miragaia, R. J. et al. Single-Cell Transcriptomics of Regulatory T Cells Reveals Trajectories of
[0287] Tissue Adaptation. Immunity 50, 493-504 e497 (2019). [0288] 56. Zemmour, D. et al. Single-cell gene expression reveals a landscape of regulatory T cell phenotypes shaped by the TCR. Nature immunology 19, 291-301 (2018). [0289] 57. Xiong, Y., Ahmad, S., Iwami, D., Brinkman, C. C. & Bromberg, J. S. T-bet Regulates Natural Regulatory T Cell Afferent Lymphatic Migration and Suppressive Function. Journal of immunology 196, 2526-2540 (2016). [0290] 58. Bergsbaken, T. & Bevan, M. J. Proinflammatory microenvironments within the intestine regulate the differentiation of tissue-resident CD8(+) T cells responding to infection. Nature immunology 16, 406-414 (2015). [0291] 59. Bergsbaken, T., Bevan, M. J. & Fink, P. J. Local Inflammatory Cues Regulate Differentiation and Persistence of CD8(+) Tissue-Resident Memory T Cells. Cell reports 19, 114-124 (2017). [0292] 60. Harris, T. H. et al. Generalized Levy walks and the role of chemokines in migration of effector CD8+ T cells. Nature 486, 545-548 (2012). [0293] 61. Khan, T. N., Mooster, J. L., Kilgore, A. M., Osborn, J. F. & Nolz, J. C. Local antigen in nonlymphoid tissue promotes resident memory CD8+ T cell formation during viral infection. The Journal of experimental medicine 213, 951-966 (2016). [0294] 62. Wakim, L. M., Woodward-Davis, A. & Bevan, M. J. Memory T cells persisting within the brain after local infection show functional adaptations to their tissue of residence. Proceedings of the National Academy of Sciences of the United States of America 107, 17872-17879 (2010). [0295] 63. Iijima, N. & Iwasaki, A. T cell memory. A local macrophage chemokine network sustains protective tissue-resident memory CD4 T cells. Science 346, 93-98 (2014). [0296] 64. Natsuaki, Y. et al. Perivascular leukocyte clusters are essential for efficient activation of effector T cells in the skin. Nature immunology 15, 1064-1069 (2014). [0297] 65. Collins, N. et al. Skin CD4(+) memory T cells exhibit combined cluster-mediated retention and equilibration with the circulation. Nature communications 7, 11514 (2016). [0298] 66. Corinti, S., Albanesi, C., la Sala, A., Pastore, S. & Girolomoni, G. Regulatory activity of autocrine IL-10 on dendritic cell functions. Journal of immunology 166, 4312-4318 (2001). [0299] 67. Li, M. O., Wan, Y. Y., Sanjabi, S., Robertson, A. K. & Flavell, R. A. Transforming growth factor-beta regulation of immune responses. Annual review of immunology 24, 99-146 (2006). [0300] 68. Worthington, J. J. et al. Integrin alphavbeta8-Mediated TGF-beta Activation by Effector Regulatory T Cells Is Essential for Suppression of T-Cell-Mediated Inflammation. Immunity 42, 903-915 (2015). [0301] 69. Masopust, D. et al. Dynamic T cell migration program provides resident memory within intestinal epithelium. The Journal of experimental medicine 207, 553-564 (2010). [0302] 70. Butcher, E. C. & Picker, L. J. Lymphocyte homing and homeostasis. Science 272, 60-66. (1996). [0303] 71. Gebhardt, T. et al. Memory T cells in nonlymphoid tissue that provide enhanced local immunity during infection with herpes simplex virus. Nature immunology 10, 524-530 (2009). [0304] 72. Mackay, L. K. et al. Long-lived epithelial immunity by tissue-resident memory T (T.sub.RM) cells in the absence of persisting local antigen presentation. Proceedings of the National Academy of Sciences of the United States of America 109, 7037-7042 (2012). [0305] 73. Graham, J. B., Da Costa, A. & Lund, J. M. Regulatory T cells shape the resident memory T cell response to virus infection in the tissues. Journal of immunology 192, 683-690 (2014). [0306] 74. Hickman, H. D. et al. CXCR3 chemokine receptor enables local CD8(+) T cell migration for the destruction of virus-infected cells. Immunity 42, 524-537 (2015). [0307] 75. Duhen, T., Duhen, R., Lanzavecchia, A., Sallusto, F. & Campbell, D. J. Functionally distinct subsets of human FOXP3+ Treg cells that phenotypically mirror effector Th cells. Blood 119, 4430-4440 (2012). [0308] 76. Paust, H. J. et al. CXCR3+ Regulatory T Cells Control TH1 Responses in Crescentic GN. J Am Soc Nephrol 27, 1933-1942 (2016). [0309] 77. Harrison, O. J. et al. Commensal-specific T cell plasticity promotes rapid tissue adaptation to injury. Science 363 (2019). [0310] 78. Ariotti, S. & Veldhoen, M. Immunology: Skin T Cells Switch Identity to Protect and Heal. Curr Biol 29, R220-R223 (2019). [0311] 79. Beura, L. K. et al. Lymphocytic choriomeningitis virus persistence promotes effector-like memory differentiation and enhances mucosal T cell distribution. J Leukoc Biol 97, 217-225 (2015). [0312] 80. Mueller, S. N., Gebhardt, T., Carbone, F. R. & Heath, W. R. Memory T cell subsets, migration patterns, and tissue residence. Annual review of immunology 31, 137-161 (2013). [0313] 81. Mohammed, J. et al. Stromal cells control the epithelial residence of DCs and memory T cells by regulated activation of TGF-beta. Nature immunology 17, 414-421 (2016). [0314] 82. Intlekofer, A. M. et al. Anomalous type 17 response to viral infection by CD8+ T cells lacking T-bet and eomesodermin. Science 321, 408-411 (2008). [0315] 83. Rubtsov, Y. P. et al. Regulatory T cell-derived interleukin-10 limits inflammation at environmental interfaces. Immunity 28, 546-558 (2008). [0316] 84. Luche, H., Weber, O., Nageswara Rao, T., Blum, C. & Fehling, H. J. Faithful activation of an extra-bright red fluorescent protein in knock-in Cre-reporter mice ideally suited for lineage tracing studies. European journal of immunology 37, 43-53 (2007). [0317] 85. Hancock, W. W. et al. Requirement of the chemokine receptor CXCR3 for acute allograft rejection.
[0318] The Journal of experimental medicine 192, 1515-1520 (2000). [0319] 86. Kuhn, R., Lohler, J., Rennick, D., Rajewsky, K. & Muller, W. Interleukin-10-deficient mice develop chronic enterocolitis. Cell 75, 263-274 (1993). [0320] 87. Nieuwenhuis, E. E. et al. Disruption of T helper 2-immune responses in Epstein-Barr virus-induced gene 3-deficient mice. Proceedings of the National Academy of Sciences of the United States of America 99, 16951-16956 (2002). [0321] 88. Travis, M. A. et al. Loss of integrin alpha(v)beta8 on dendritic cells causes autoimmunity and colitis in mice. Nature 449, 361-365 (2007). [0322] 89. Azhar, M. et al. Generation of mice with a conditional allele for transforming growth factor beta 1 gene. Genesis 47, 423-431 (2009). [0323] 90. Li, Y. et al. Exogenous stimuli maintain intraepithelial lymphocytes via aryl hydrocarbon receptor activation. Cell 147, 629-640 (2011). [0324] 91. Figueiredo-Campos, P., Ferreira, C., Blankenhaus, B. & Veldhoen, M. Eimeria vermiformis Infection Model of Murine Small Intestine. Bio-Protocol 8 (2018). [0325] 92. Cossarizza, A. et al. Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition). Eur J Immunol 49, 1457-1973 (2019). [0326] 93. Butler, A., Hoffman, P., Smibert, P., Papalexi, E. & Satija, R. Integrating single-cell transcriptomic data across different conditions, technologies, and species. Nat Biotechnol 36, 411-420 (2018). [0327] 94. Savas, P., Virassamy, B., Ye, C. et al. Single-cell profiling of breast cancer T cells reveals a tissue-resident memory subset associated with improved prognosis. Nat Med 24, 986-993 (2018). [0328] 95. Zhi-Qiang Wang, Katy Milne, Heather Derocher, John R. Webb, Brad H. Nelson and Peter
[0329] H. Watson. CD103 and Intratumoral Immune Response in Breast Cancer. Clin Cancer Res (22) (24) 6290-6297 (2018). [0330] 96. John R. Webb, Katy Milne, Peter Watson, Ronald J. deLeeuw and Brad H. Nelson. Tumor-Infiltrating Lymphocytes Expressing the Tissue Resident Memory Marker CD103 Are Associated with Increased Survival in High-Grade Serous Ovarian Cancer. Clin Cancer Res (20) (2) 434-444 (2014). [0331] 97. Jarem Edwards et al. CD103.sup.+ Tumor-Resident CD8.sup.+ T Cells Are Associated with Improved Survival in Immunotherapy-Nave Melanoma Patients and Expand Significantly During Anti-PD-1 Treatment. Clin Cancer Res (24) (13) 3036-3045 (2018). [0332] 98. Mami-Chonaib P. Blane C, Corgnac S. et al. Resident memory T cells, critical components in tumor immunology. Journal for ImmunoTherapy of Cancer 6:87 2018. [0333] 99. Biliana Lozanoska-Ochser, Nigel J. Klein, Guo C. Huang, Raymond A. Alvarez and Mark Peakman. Expression of CD86 on Human Islet Endothelial Cells Facilitates T Cell Adhesion and Migration. J Immunol. 181 (9) 6109-6116 (2008), [0334] 100. Knieke K, Hoff H, Maszyna F, Kolar P. Schrage A, Hamann A, et al. (2009) CD152 (CTLA-4) Determines CD4 T Cell Migration In Vitro and In Vivo. PLOS ONE 4 (5): 5702. [0335] 101. Brunner-Weinzierl Monika C., Rudd Christopher E. CTLA-4 and PD-1 Control of T-Cell Motility and Migration: Implications for Tumor Immunotherapy. JOURNAL=Frontiers in Immunology