Yakup Tanriver

A T-bet gradient controls the fate and function of CCR6 − RORγt + innate lymphoid cells

At mucosal surfaces, the immune system should not initiate inflammatory immune responses to the plethora of antigens constantly present in the environment, but should remain poised to unleash a potent assault on intestinal pathogens. The transcriptional programs and regulatory factors required for immune cells to switch from homeostatic (often tissue-protective) function to potent antimicrobial immunity are poorly defined. Mucosal retinoic-acid-receptor-related orphan receptor-γt-positive (RORγt+) innate lymphoid cells (ILCs) are emerging as an important innate lymphocyte population required for immunity to intestinal infections. Various subsets of RORγt+ ILCs have been described but the transcriptional programs controlling their specification and fate remain largely unknown. Here we provide evidence that the transcription factor T-bet determines the fate of a distinct lineage of CCR6−RORγt+ ILCs. Postnatally emerging CCR6−RORγt+ ILCs upregulated T-bet and this was controlled by cues from the commensal microbiota and interleukin-23 (IL-23). In contrast, CCR6+RORγt+ ILCs, which arise earlier during ontogeny, did not express T-bet. T-bet instructed the expression of T-bet target genes such as interferon-γ (IFN-γ) and of the natural cytotoxicity receptor NKp46. Mice genetically lacking T-bet showed normal development of CCR6−RORγt+ ILCs, but they could not differentiate into NKp46-expressing RORγt+ ILCs (that is, IL-22-producing natural killer (NK-22) cells) and failed to produce IFN-γ. The production of IFN-γ by T-bet-expressing CCR6−RORγt+ ILCs was essential for the release of mucus-forming glycoproteins required to protect the epithelial barrier during Salmonella enterica infection. Salmonella infection also causes severe enterocolitis that is at least partly driven by IFN-γ. Mice deficient for T-bet or depleted of ILCs developed only mild enterocolitis. Thus, graded expression of T-bet in CCR6−RORγt+ ILCs facilitates the differentiation of IFN-γ-producing CCR6−RORγt+ ILCs required to protect the epithelial barrier against Salmonella infections. Co-expression of T-bet and RORγt, which is also found in subsets of IL-17-producing T-helper (TH17) cells, may be an evolutionarily conserved transcriptional program that originally developed as part of the innate defence against infections but that also confers an increased risk of immune-mediated pathology.

Conferring indirect allospecificity on CD4+CD25+ Tregs by TCR gene transfer favors transplantation tolerance in mice

T cell responses to MHC-mismatched transplants can be mediated via direct recognition of allogeneic MHC molecules on the cells of the transplant or via recognition of allogeneic peptides presented on the surface of recipient APCs in recipient MHC molecules - a process known as indirect recognition. As CD4(+)CD25(+) Tregs play an important role in regulating alloresponses, we investigated whether mouse Tregs specific for allogeneic MHC molecules could be generated in vitro and could promote transplantation tolerance in immunocompetent recipient mice. Tregs able to directly recognize allogeneic MHC class II molecules (dTregs) were obtained by stimulating CD4(+)CD25(+) cells from C57BL/6 mice (H-2(b)) with allogeneic DCs from BALB/c mice (H-2(d)). To generate Tregs that indirectly recognized allogeneic MHC class II molecules, dTregs were retrovirally transduced with TCR genes conferring specificity for H-2K(d) presented by H-2A(b) MHC class II molecules. The dual direct and indirect allospecificity of the TCR-transduced Tregs was confirmed in vitro. In mice, TCR-transduced Tregs, but not dTregs, induced long-term survival of partially MHC-mismatched heart grafts when combined with short-term adjunctive immunosuppression. Further, although dTregs were only slightly less effective than TCR-transduced Tregs at inducing long-term survival of fully MHC-mismatched heart grafts, histologic analysis of long-surviving hearts demonstrated marked superiority of the TCR-transduced Tregs. Thus, Tregs specific for allogeneic MHC class II molecules are effective in promoting transplantation tolerance in mice, which suggests that such cells have clinical potential.

Indefinite mouse heart allograft survival in recipient treated with CD4(+)CD25(+) regulatory T cells with indirect allospecificity and short term immunosuppression.

CD4(+)CD25(+) regulatory T cells (Tregs) play a crucial role in controlling immune responses. It is an appealing strategy to harness Tregs for adoptive cell therapy to induce tolerance to allografts. Several approaches have been developed to expand antigen-specific Tregs. Despite the large body of experimental data from murine studies demonstrating the great potential of these cells for clinical application, Treg adoptive transfer therapy was used in immunodeficient animals or in strain combinations with limited histiocompatibility. The aim of this study was to investigate whether Treg lines can protect from allograft rejection in a fully MHC-mismatched strain combination and whether the presence of Tregs with indirect allospecificity offered an advantage compared to self-reactive Tregs. Treg lines with self-specificity or with indirect allospecificity were generated by stimulating BL/6 CD4(+)CD25(+) T cells with autologous immature DCs either unpulsed or pulsed with K(d) peptide. The Treg lines were injected into recipient mice in combination with temporary depletion of CD8(+) T cells and a short course of Rapamycin. The data demonstrate that Treg lines with indirect allospecificity can be generated and most importantly they can induce indefinite survival of BALB/c hearts transplanted into BL/6 recipients when combined with short term immunosuppression. However, the Treg lines with self-specificity were only slightly less effective. The data presented in this study demonstrate the potential of ex vivo expanded Treg lines for adoptive cell therapy to promote transplantation tolerance.

An endogenous nanomineral chaperones luminal antigen and peptidoglycan to intestinal immune cells

In humans and other mammals, it is known that calcium and phosphate ions are secreted from the distal small intestine into the lumen. However, why this secretion occurs is unclear. Here, we show that the process leads to the formation of amorphous magnesium-substituted calcium phosphate nanoparticles that trap soluble macromolecules, such as bacterial peptidoglycan and orally-fed protein antigens, in the lumen and transport them to immune cells of the intestinal tissue. The macromolecule-containing nanoparticles utilize epithelial M cells to enter Peyer’s patches - small areas of the intestine concentrated with particle-scavenging immune cells. In wild type mice, intestinal immune cells containing these naturally-formed nanoparticles expressed the immune tolerance-associated molecule ‘programmed death-ligand 1 (PD-L1)’, whereas in NOD1/2 double knock-out mice, which cannot recognize peptidoglycan, PD-L1 was undetected. Our results explain a role for constitutively formed calcium phosphate nanoparticles in the gut lumen and how this helps to shape intestinal immune homeostasis.

Targeting MHC Class I Monomers to Dendritic Cells Inhibits the Indirect Pathway of Allorecognition and the Production of IgG Alloantibodies Leading to Long-Term Allograft Survival

T cell depletion strategies are an efficient therapy for the treatment of acute rejections and are an essential part of tolerance induction protocols in various animal models; however, they are usually nonselective and cause wholesale T cell depletion leaving the individual in a severely immunocompromised state. So far it has been difficult to selectively delete alloreactive T cells because the majority of protocols either delete all T cells, subsets of T cells, or subpopulations of T cells expressing certain activation markers, ignoring the Ag specificity of the TCR. We have developed a model in which we were able to selectively deplete alloreactive T cells with an indirect specificity by targeting intact MHC molecules to quiescent dendritic cells using 33D1 as the targeting Ab. This strategy enabled us to inhibit the indirect alloresponse against MHC-mismatched skin grafts and hence the generation of IgG alloantibodies, which depends on indirectly activated T cells. In combination with the temporary abrogation of the direct alloresponse, we were able to induce indefinite skin graft survival. Importantly, the targeting strategy had no detrimental effect on CD4+CD25+FoxP3+ T cells, which could potentially be used as an adjunctive cellular therapy. Transplantation tolerance depends on the right balance between depletion and regulation. For the former this approach may be a useful tool in the development of future tolerance induction protocols in non-sensitized patients.

Tolerogenic Donor-Derived Dendritic Cells Risk Sensitization In Vivo owing to Processing and Presentation by Recipient APCs

Modification of allogeneic dendritic cells (DCs) through drug treatment results in DCs with in vitro hallmarks of tolerogenicity. Despite these observations, using murine MHC-mismatched skin and heart transplant models, donor-derived drug-modified DCs not only failed to induce tolerance but also accelerated graft rejection. The latter was inhibited by injecting the recipient with anti-CD8 Ab, which removed both CD8+ T cells and CD8+ DCs. The discrepancy between in vitro and in vivo data could be explained, partly, by the presentation of drug-modified donor DC MHC alloantigens by recipient APCs and activation of recipient T cells with indirect allospecificity, leading to the induction of alloantibodies. Furthermore, allogeneic MHC molecules expressed by drug-treated DCs were rapidly processed and presented in peptide form by recipient APCs in vivo within hours of DC injection. Using TCR-transgenic T cells, Ag presentation of injected OVA-pulsed DCs was detectable for ≤ 3 d, whereas indirect presentation of MHC alloantigen by recipient APCs led to activation of T cells within 14 h and was partially inhibited by reducing the numbers of CD8+ DCs in vivo. In support of this observation when mice lacking CD8+ DCs were pretreated with drug-modified DCs prior to transplantation, skin graft rejection kinetics were similar to those in non–DC-treated controls. Of interest, when the same mice were treated with anti-CD40L blockade plus drug-modified DCs, skin graft survival was prolonged, suggesting endogenous DCs were responsible for T cell priming. Altogether, these findings highlight the risks and limitations of negative vaccination using alloantigen-bearing “tolerogenic” DCs.

Monitoring of In Vivo Function of Superparamagnetic Iron Oxide Labelled Murine Dendritic Cells during Anti- Tumour Vaccination

Dendritic cells (DCs) generated in vitro to present tumour antigens have been injected in cancer patients to boost in vivo anti-tumour immune responses. This approach to cancer immunotherapy has had limited success. For anti-tumour therapy, delivery and subsequent migration of DCs to lymph nodes leading to effective stimulation of effector T cells is thought to be essential. The ability to non-invasively monitor the fate of adoptively transferred DCs in vivo using magnetic resonance imaging (MRI) is an important clinical tool to correlate their in vivo behavior with response to treatment. Previous reports of superparamagnetic iron oxides (SPIOs) labelling of different cell types, including DCs, have indicated varying detrimental effects on cell viability, migration, differentiation and immune function. Here we describe an optimised labelling procedure using a short incubation time and low concentration of clinically used SPIO Endorem to successfully track murine DC migration in vivo using MRI in a mouse tumour model. First, intracellular labelling of bone marrow derived DCs was monitored in vitro using electron microscopy and MRI relaxometry. Second, the in vitro characterisation of SPIO labelled DCs demonstrated that viability, phenotype and functions were comparable to unlabelled DCs. Third, ex vivo SPIO labelled DCs, when injected subcutaneously, allowed for the longitudinal monitoring by MR imaging of their migration in vivo. Fourth, the SPIO DCs induced the proliferation of adoptively transferred CD4+ T cells but, most importantly, they primed cytotoxic CD8+ T cell responses to protect against a B16-Ova tumour challenge. Finally, using anatomical information from the MR images, the immigration of DCs was confirmed by the increase in lymph node size post-DC injection. These results demonstrate that the SPIO labelling protocol developed in this study is not detrimental for DC function in vitro and in vivo has potential clinical application in monitoring therapeutic DCs in patients with cancer.

Transcription factors controlling development and function of innate lymphoid cells

Innate lymphoid cells (ILCs) are a heterogeneous group of lymphocytes, which play an important role in tissue homeostasis at epithelial surfaces. They are scarce in spleen and lymph nodes, but substantial numbers can be found in the intestinal mucosa even at steady state. There, they represent the first line of defence against invading pathogens and contribute to lymphorganogenesis, tissue repair and, when inappropriately activated, immune pathology. Lineage-specific development, function and maintenance of these cells depend on a restricted set of transcription factors that partially emerged as a result of diversification and selection during vertebrate evolution. The differential expression of transcription factors regulates unique developmental programs, which endow the different ILC subsets with specific effector functions. Despite this division of labour, ILCs are considered to share a common origin, as they all are progeny of the common lymphoid progenitor, rely on the common γ-chain (γc) used by various cytokine receptors and show a developmental requirement for the transcriptional regulator Id2 (inhibitor of DNA binding 2). Here, we review the transcriptional programs required for the development and function of ILCs and give an overview of the evolution of transcription factors and cytokines expressed by ILCs.

Transcriptional control of innate lymphocyte fate decisions.

It has recently emerged that innate lymphocytes are more diverse than previously appreciated. In addition to natural killer cells, various subsets of innate lymphoid cells are now being characterized. It has become apparent that the transcriptional programs underlying lineage specification and cell fate decisions of innate lymphocytes strikingly resemble those of T cell subsets, suggesting that such transcriptional circuitry was already pre-formed in the evolutionary older innate immune system. Here, we will review recent advances in our understanding of the core transcriptional programs driving development and cell fate decisions of innate lymphocytes. We will also discuss whether these transcriptional programs are stable or flexible, thereby allowing for plastic adaptation of immune responses.

Generation and characterization of a tamoxifen‐inducible EomesCreER mouse line

Transgenic mouse lines expressing inducible forms of Cre‐recombinase in a tissue‐specific manner are powerful genetic tools for studying aspects of development and various processes in the adult. The T‐box transcription factor eomesodermin (Eomes) plays critical roles for maintenance and differentiation of different pools of stem and progenitor cells from early embryonic stages to adulthood. These include trophoblast stem cells, epiblast cells during the generation of the primary germ layers, neurogenic intermediate progenitor cells in embryonic and adult cortical neurogenesis, and maturing natural killer and T cells. Here, we report on the generation and analysis of an EomesCreER‐targeted allele by placing the tamoxifen‐activatable Cre‐recombinase (CreER) under the control of the Eomes genomic locus. We demonstrate that CreER expression recapitulates endogenous Eomes transcription within different progenitor cell populations. Tamoxifen administration specifically labels Eomes‐expressing cells and their progeny as demonstrated by crossing EomesCreER animals to different Cre‐inducible reporter strains. In summary, this novel EomesCreER allele can be used as elegant genetic tool that allows to follow the fate of Eomes‐positive cells and to genetically manipulate them in a temporal specific manner. genesis 51:725–733.