@phdthesis{Fichtner2020,
  author    = {Fichtner, Alina Suzann},
  title     = {Alpaca, armadillo and cotton rat as new animal models for nonconventional T cells: Identification of cell populations and analysis of antigen receptors and ligands},
  doi       = {10.25972/OPUS-16910},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-169108},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {In this thesis, three species were investigated for the conservation of two non-conventional T cell systems, the CD1d/ iNKT cell system and the BTN3/ Vγ9Vδ2 T cell system. Non-conventional T cells are αβ or γδ T cells that do not fit into the classical mode of antigen recognition and adaptive responses. These T cells recognize antigens different from classical peptide antigens and are not restricted to the polymorphic MHC molecules but rather to non-polymorphic antigen-presenting molecules. The iNKT cell subset is restricted by the lipid antigen-presenting molecule CD1d and carries out immunomodulatory functions by rapid cytokine secretion. The molecular basis of this system, the semi-invariant iNKT TCR chains and CD1d were proven to be expressed and compared to homologs in human and rodents. Cotton rats possess multiple members of the AV14 and BV8 family and only one isoform of CD1d which is comparable to findings in the rat. Moreover, the reactivity of primary cells to glycolipid antigens could be shown, and an iNKT cell-like population was detected in primary cells using newly developed cotton rat CD1d oligomers. These were also applied to test the capacity of CD1d to present typical glycolipid antigens to iNKT TCR transductants. In addition, expression of cotton rat iNKT TCR α and β chains in TCR-negative cell lines was used to show successful pairing and detection of glycolipids in the context of CD1d. In summary, the conservation of a functional CD1d/iNKT cell system in the cotton rat could be shown, and tools were developed to study this cell subset in the course of infectious diseases. The Vγ9Vδ2 T cell subset is the major γδ T cell subset in human peripheral blood and has the unique ability to contribute to immune surveillance by detecting pyrophosphorylated metabolites of isoprenoid synthesis that indicate cell stress, transformation or infection. Up to this date, phosphoantigen-reactive γδ T cells have only been shown in primate species. However, evidence for the existence and functional conservation of the genes implied in the BTN3/Vγ9Vδ2 T cell system was found in several placental mammal species, and two candidate species were chosen for further investigation. The nine-banded armadillo, a valuable model for leprosy research, was shown to possess homologous genes to TRGV9, TRDV2 and BTN3. In this study, the expression of productive rearrangements of TRDV2 gene segments could be shown in peripheral blood samples, but no evidence was found for the expression of a functional TRGV9 rearrangement or BTN3 molecules. Moreover, determinants of phosphoantigen-reactive Vγ9Vδ2 T cells and functional BTN3 molecules were found to still be prevalent in armadillo genes. This makes the armadillo an interesting model to study the structural determinants that allow phosphoantigen recognition by a functional Vγ9Vδ2 T cell subset although this species is merely a witness for a functional system in a placental mammal ancestor. In contrast, alpacas were shown to express functional Vγ9Vδ2 T cells which conserved many features of the human counterpart. Expression of Vγ9Vδ2 pairings could be shown by single-cell PCR and functional phosphoantigenreactive pairings were observed. This phosphoantigen reactivity was also shown in PBMC cultures with a newly developed antibody specific for alpaca Vδ2Jδ4 chains. Moreover, a more detailed study of the alpaca TCR repertoire showed similarities to "γδ high" species like camelids and cattle which possess an extended family of TRDV genes. The γ and δ loci of alpaca TCR genes were drafted based on genomic information and cDNA studies and provide an overview for more detailed studies. Conservation of phosphoantigen recognition by the single BTN3 molecule of alpacas was shown in 293T knock out cell lines, and BTN3 detection on PBMCs was investigated with a newly developed alpaca BTN3-specific antibody. These findings prove the existence of a functional BTN3-dependent phosphoantigen-reactive Vγ9Vδ2 T cell subset and provide a basis for the future study of this cell system in a non-primate species. Moreover, as the first non-primate candidate species with the BTN3/Vγ9Vδ2 T cell system the alpaca is an important outgroup for research in this field. The use of a single BTN3 variant in contrast to three human isoforms that work together renders the alpaca a unique and to this date indispensable model for Vγ9Vδ2 T cells. In conclusion, this study provides an overview of the applicability of new animal models in the study of the non-conventional T cell subsets iNKT cells and Vγ9Vδ2 T cells and leads the way for a better understanding of structural and functional relationships.},
  subject      = {T-Lymphozyt},
  language  = {en}
}
@phdthesis{JarickneeOttmueller2020,
  author    = {Jarick [n{\´e}e Ottm{\"u}ller], Katja Julika},
  title     = {Migration of allogenic T cells in intestinal lymphoid structures during acute Graft-versus-Host Disease},
  doi       = {10.25972/OPUS-17875},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-178758},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {T cell infiltration into the intestine occurs after priming and activation in the mesenteric lymph nodes and Peyer's patches and subsequent trafficking via the blood circulation. We hypothesized that additionally to the vascular trafficking route, a fraction of T cells in the Peyer's patches directly migrate into the adjacent lamina propria of the small intestine. To test this hypothesis, we employed a mouse model of acute Graft-versus-Host Disease to study the direct T cell migration from the Peyer's patches to the adjacent lamina propria. First, we analyzed the border of Peyer's patches on histological sections and found that the Peyer's patch is not enclosed by a capsule or basement membrane. Thus, the tissue architecture allows for direct access to the surrounding tissue. With whole-mount light sheet fluorescence microscopy we quantified a three-dimensional gradient of T cells around Peyer's patches on day 2.5 and day 3 after transplantation. This gradient evened out at day 4 and day 6 when high numbers of T cells started to evenly infiltrate the intestine from the blood circulation. We confirmed that gradient-forming T cells around Peyer's patches resided within the tissue parenchyma of the lamina propria and not inside lymphatic vessels. To positively prove that the recently activated donor T cells around Peyer's patches have egressed directly from that patch, we established a protocol for intravital photoconversion of T cells inside Peyer's patches. 12 h after photoconversion inside a single Peyer's patch, photoconverted T cells resided only around this particular Peyer's patch and not elsewhere in the small intestine. This indicated that the T cells did not infiltrate via the blood but migrated to the adjacent lamina propria of the small intestine. Dynamic intravital two-photon microscopy revealed that these T cells next to the Peyer's patch migrated in a random pattern. This suggested that these cells did not follow a positive chemoattractive gradient once they had reached the lamina propria. Laser-capture microdissection combined with RNA sequencing of the mucosa near the Peyer's patch identified a wide range of migration-promoting factors. These included chemokines, co-stimulatory receptors and migration-associated intracellular molecules, which are candidates to promote this direct migration from Peyer's patches. Altogether, we demonstrate for the first time that additionally to the vascular trafficking route, a fraction of T cells migrates directly from the Peyer's patch to the surrounding mucosa. This mechanism implies so far unrecognized regional specification of Peyer's-patch-primed T cells. Our findings may impact treatment strategies to avoid intestinal inflammation or foster immunity after oral vaccination.},
  subject      = {T-Lymphozyt},
  language  = {en}
}
@phdthesis{Koenig2022,
  author    = {K{\"o}nig, Anika},
  title     = {The role of the transcriptional regulators NFATc1 and Blimp-1 in follicular T-cells},
  doi       = {10.25972/OPUS-20972},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-209727},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {The defense against invading pathogens is, amongst other things, mediated via the action of antibodies. Class-switched antibodies and antibodies of high affinity are produced by plasma cells descending from germinal center B (GCB) cells. GCB cells develop in the germinal center (GC), a specialized microstructure found in the B-cell follicle of secondary lymphoid organs. GCB-cell maturation and proliferation are supported by follicular T- helper (Tfh) cells. On the other hand, follicular regulatory T (Tfr) cells control this process in quantity and quality preventing, for instance, the formation of autoantibodies directed against endogenous structures. The development of GCB, Tfh and Tfr cells essentially depends on the migration into the GC, which is mediated via the expression of the chemokine receptor CXCR5. One transcription factor highly expressed in follicular T cells, comprising Tfh and Tfr cells, is NFATc1. Tfr cells additionally express the transcriptional repressor Blimp-1, which is not expressed in Tfh cells. We found that NFATc1 is transactivating Cxcr5 via response elements in the promoter and enhancer in vitro. Blimp-1 binds to the same elements, transactivating Cxcr5 expression in cooperation with NFATc1, whilst mediating Cxcr5- repression on its own. In Tfr cells Blimp-1 suppresses CXCR5 expression in the absence of NFATc1. Blimp-1 itself is necessary to restrict Tfr-cell frequencies and to mediate Tfr- cell function as in mice with Blimp-1-ablated Tregs high frequencies of Tfr cells do not reduce GCB- or Tfh cell frequencies. NFATc1 and Blimp-1 double deficient Tfr cells show additional loss of function, which becomes visible in clearly expanded antibody titers. To evaluate the function of NFATc1 in Tfr cells, we not only deleted it, but also overexpressed a constitutive active form of NFATc1/aA (caNFATc1/aA) in regulatory T cells (Tregs). The latter is leading to an upregulation of CXCR5 per cell, without changing Tfh or Tfr-cell frequencies. However, the high density of surface CXCR5 enhances the migration of Tfr cells deep into the GC, which results in a tighter control of the antigen- specific humoral immune response. Additionally, caNFATc1/aA increases the expression of genes coding for Tfr effector molecules like Il1rn, Il10, Tigit and Ctla4. Interestingly, this part of the transcriptional change is dependent on the presence of Blimp-1. Furthermore, Blimp-1 regulates the expression of multiple chemokine receptor genes on the background of caNFATc1/aA. In contrast, when caNFATc1/aA is overexpressed in all T cells, the frequencies of Tfh- and GCB cells are dominantly reduced. This effect seems to stem from the conventional T- cell (Tcon) side, most probably originating from increased secretion of interleukin-2 (IL- 2) via the caNFATc1/aA overexpressing Tcons. IL-2 is known to hinder the germinal center reaction (GCR) and it might in its abundance not be neutralizable by Tfr cells. Taken together, NFATc1 and Blimp-1 cooperate to control the migration of Tfr cells into the GC. Tfr cells in the GC depend on NFATc1 and Blimp-1 to perform their proper function. Overexpression of caNFATc1 in Tregs strengthens Tfr function in a Blimp-1-dependent manner, whilst overexpression of caNFATc1 in all T cells dominantly diminishes the GCR.},
  subject      = {Signaltransduktion},
  language  = {en}
}
@phdthesis{CruzdeCasas2024,
  author    = {Cruz de Casas, Paulina},
  title     = {Sphingolipids as modulators of T cell function},
  doi       = {10.25972/OPUS-35969},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-359698},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {The immune system is responsible for the preservation of homeostasis whenever a given organism is exposed to distinct kinds of perturbations. Given the complexity of certain organisms like mammals, and the diverse types of challenges that they encounter (e.g. infection or disease), the immune system evolved to harbor a great variety of distinct immune cell populations with specialized functions. For instance, the family of T cells is sub-divided into conventional (Tconv) and unconventional T cells (UTCs). Tconv form part of the adaptive arm of the immune system and are comprised of αβ CD4+ or CD8+ cells that differentiate from na{\"i}ve to effector and memory populations upon activation and are essential during infection and cancer. Furthermore, UTCs, which include γδ T cells, NKT and MAIT, are involved in innate and adaptive immune responses, due to their dual mode of activation, through cytokines (innate-like) or TCR (adaptive), and function. Despite our understanding of the basic functions of T cells in several contexts, a great number of open questions related to their basic biology remain. For instance, the mechanism behind the differentiation of na{\"i}ve CD4+ and CD8+ T cells into effector and memory populations is not fully understood. Moreover, the exact function and relevance of distinct UTC subpopulations in a physiological context have not been fully clarified. Here, we investigated the factors mediating na{\"i}ve CD8+ T cell differentiation into effector and memory cells. By using flow cytometry, mass spectrometry, enzymatic assays, and transgenic mouse models, we found that the membrane bound enzyme sphingomyelin-phosphodiesterase acid-like 3b (Smpdl3b) is crucial for the maintenance of memory CD8+ T cells. Our data show that the absence of Smpdl3b leads to diminished CD8+ T cell memory, and a loss of stem-like memory populations due to an aggravated contraction. Our scRNA-seq data suggest that Smpdl3b could be involved in clathrinmediated endocytosis through modulation of Huntingtin interacting protein 1 (Hip1) levels, likely regulating TCR-independent signaling events. Furthermore, in this study we explored the role of UTCs in lymph node-specific immune responses. By using transgenic mouse models for photolabeling, lymph node transplantation models, infection models and flow cytometry, we demonstrate that S1P regulates the migration of tissue-derived UTC from tissues to draining lymph nodes, resulting in heterogeneous immune responses mounted by lymph nodes draining different tissues. Moreover, our unbiased scRNAseq and single lineage-deficient mouse models analysis revealed that all UTC lineages (γδ T cells, NKT and MAIT) are organized in functional units, based on transcriptional homogeneity, shared microanatomical location and migratory behavior, and numerical and functional redundancy. Taken together, our studies describe additional cell intrinsic (Smpdl3b) and extrinsic (S1Pmediated migration) functions of sphingolipid metabolism modulating T cell biology. We propose the S1P/S1PR1/5 signaling axis as the potential survival pathway for Smpdl3b+ memory CD8+ T cells and UTCs, mainly in lymph nodes. Possibly, Smpdl3b regulates S1P/S1PR signaling by balancing ligandreceptor endocytosis, while UTCs migrate to lymph nodes during homeostasis to be exposed to specific levels of S1P that assure their maintenance. Our results are clinically relevant, since several drugs modulating the S1P/S1PR signaling axis or the levels of Smpdl3b are currently used to treat human diseases, such as multiple sclerosis and B cell-mediated diseases. We hope that our discoveries will inspire future studies focusing on sphingolipid metabolism in immune cell biology.},
  subject      = {T-Lymphozyt},
  language  = {en}
}
@phdthesis{PenaMosca2024,
  author    = {Pe{\~n}a Mosca, Mar{\´i}a Josefina},
  title     = {Local regulation of T-cell immunity in the intestinal mucosa},
  doi       = {10.25972/OPUS-35266},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-352665},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {After priming in Peyer's patches (PPs) and mesenteric lymph nodes (mLN) T- cells infiltrate the intestine through lymphatic draining and homing through the bloodstream. However, we found that in mouse models of acute graft-versus-host disease (GvHD), a subset of alloreactive T-cells directly migrates from PPs to the adjacent intestinal lamina propria (LP), bypassing the normal lymphatic drainage and vascular trafficking routes. Notably, this direct migration occurred in irradiated and unirradiated GvHD models, indicating that irradiation is not a prerequisite for this observed behavior. Next, we established a method termed serial intravascular staining (SIVS) in mouse models to systematically investigate the trafficking and migration of donor T- cells in the early stages of acute GvHD initiation. We found that the direct migration of T-cells from PPs to LP resulted in faster recruitment of cells after allogeneic hematopoietic cell transplantation (allo-HCT). These directly migrating T-cells were found to be in an activated and proliferative state, exhibiting a TH1/TH17-like phenotype and producing cytokines such as IFN-γ and TNF-α. Furthermore, we observed that the directly migrating alloreactive T-cells expressed specific integrins (α4+, αE+) and chemokine receptors (CxCR3+, CCR5+, and CCR9+). Surprisingly, blocking these integrins and chemokine-coupled receptors did not hinder the direct migration of T- cells from PPs to LP, suggesting the involvement of alternative mechanisms. Previous experiments ruled out the involvement of S1PR1 and topographical features of macrophages, leading us to hypothesize that mediators of cytoskeleton reorganization, such as Coro1a, Dock2, or Cdc42, may play a role in this unique migration process. Additionally, we observed that directly migrating T-cells created a local inflammatory microenvironment, which attracts circulating T-cells. Histological analysis confirmed that alloreactive PPs-derived T-cells and bloodborne T-cells colocalized. We employed two experimental approaches, including either photoconversion of T-cells in PPs or direct transfer of activated T-cells into the vasculature, to demonstrate this colocalization. We hypothesize that cytokines released by migrating T-cells, such as IFN-γ and TNF-α, may play a role in recruiting T-cells from the vasculature, as inhibiting chemokine-coupled receptors did not impair recruitment.},
  subject      = {T-Lymphozyt},
  language  = {en}
}