Modulation of T cell function via accessory cell surface receptors CD43 and CD71

T-Zellen spielen eine entscheidende Rolle bei der Initiierung und Regulation einer adaptiven Immunantwort auf Fremdantigene. Auch wenn das Signal über den T-Zell-Rezeptor bei der Initiation der T-Zell-Antwort der entscheidende Schritt ist, sind akzessorische Zelloberflächenrezeptoren entscheidend für die Feinabstimmung der T-Zell-Aktivierung und Funktion. CD43 ist eines der häufigsten Glykoproteine, das auf der Zelloberfläche von den verschiedenen T-Zell-Subtypen exprimiert wird. Bisherige Analysen der physiologischen Bedeutung von CD43-Expression auf T-Zellen lieferten kein kohärentes Bild, da sowohl eine positive wie auch eine negative Regulation der T-Zell-Aktivierung über CD43 beobachtet werden konnte. Im ersten Teil meiner Dissertation konnte ich nun zeigen, dass CD43 sowohl eine positive kostimulatorische Rolle als auch eine negative koinhibitorische Rolle bei der Regulation einer Immunantwort spielen kann, je nachdem welches Epitop von CD43 involviert ist. Kostimulation von T-Zellen aus dem peripheren Blut (PB) über zwei verschiedene CD43-Epitope, die von mAb CD43-6E5 (T6E5-act) und CD43-10G7 (T10G7-act) erkannt werden, induziert die Proliferation in verschiedenen T-Zell-Subpopulationen, einschließlich CD4+, CD8+ und naiven T-Zellen. Allerdings führte die Kostimulation über diese beiden CD43-Epitope zu einem Unterschied im Clusterverhalten der T-Zellen, unterschiedlicher Zytokinproduktion und auch nachfolgender Effektor-Funktion. T6E5-act produzieren ein hohes Maß an IL-22 und IFN-[gamma], ähnlich wie über CD28 aktivierte T-Zellen (TCD28-act) und beherrschen weiters eine zytotoxische Funktion. Auf der anderen Seite sezernieren T10G7-act geringe Mengen an proinflammatorischen Zytokinen, aber höhere Mengen an TGF-[beta] und IL-35. Im Vergleich zu T6E5-Act oder TCD28-Act, zeigen T10G7-Act einen hypo-proliferative Phänotyp nach Restimulation. Dabei erlangen T10G7-Act eine T-Zell-unterdrückende Funktion, die über Zell-Zell-Kontakt vermittelt wird. Diese inhibitorische Wirkung exekutieren T10G7-Act nicht direkt über Responder-T-Zellen, sondern über Antigen-präsentierenden Zellen (APC) in einer allogenen Leukozyten-Reaktion (MLR). T10G7-act formen stabile heterotypische Cluster mit dendritischen Zellen (DC) über CD2 und hemmen dadurch die Aktivierung von Responder-T-Zellen. Die Daten dieser Arbeit belegen daher, dass CD43 eine einzigartige Rolle in der Aktivierung von T-Zellen spielt und eine bidirektionale Polarisierung von T-Zellen ausüben kann. Der Transferrin-Rezeptor (TfR1 / CD71) ist von grundlegender Bedeutung für den Eisentransport ins Inneren von Zellen und ist weit verbreitet auf der Oberfläche von proliferierenden Zellen exprimiert, wie z.B. auf aktivierten T-Zellen. Trotz seiner zentralen Rolle in der Zellbiologie ist über die immun-modulatorische Rolle von CD71 auf T-Zellen noch wenig bekannt. Daher untersuchte ich im zweiten Teil meiner Dissertation die Wirkung von CD71 auf Signalwege, Proliferation und die Produktion von Zytokinen. Die Blockade von CD71 auf aktivierten T-Zellen mit mAb VIP-1 führte zu einer Inhibition der Transferrin (Tf)-Aufnahme und in Folge zu einer gehemmten T-Zellproliferation. VIP-1 behandelte T-Zellen (TVIP-1) zeichneten sich durch die einzigartige Fähigkeit aus, dass diese T-Zellen trotz ihres Wachstumsstopps dennoch Zytokine produzierten und sezernierten. Weiters konnte gezeigt werden, dass die Signalwege in Jurkat-T-Zellen verändert waren und es im Vergleich zu unbehandelten Zellen zu einer Überaktivierung von NFAT kommt. Ausserdem zeigten VIP-1-behandelte Jurkat-T-Zellen eine verminderte Expression von Proteinen, die am intrazellulären Transport wie z.B.: Rab-5C, Rab-9A und Rab-11B oder im Zellzyklus wie z.B.: Histondeazetylase 2 (HDAC2) beteiligt sind. Der Wachstumsstopp in TVIP-1-Zellen konnte weder durch eine wiederhergestellte CD71 Zelloberflächenexpression noch durch Zugabe von exogenem IL-2 rückgängig gemacht werden nicht. Zusammenfassend zeigen die Daten, dass die Aufnahme von Eisen über CD71 während der T-Zell-Aktivierung zwar zu einer Hemmung des Wachstums der T-Zellen führt jedoch nicht zur Inhibition wichtiger Effektor-Funktionen von T-Zellen, wie z.B. die Produktion von Zytokinen.T cells are pivotal in the initiation and regulation of adaptive immune responses to foreign antigens. Even though signalling via T cell receptor is the key step in initiation of a T cell response, accessory cell surface receptors play a decisive role in fine-tuning of T cell activation and function. CD43 is one of the abundant cell surface glycoproteins expressed on various T cell subsets. The physiological role of CD43 in T cells is not completely clear, with several studies indicating positive and negative regulation of T cell activation via CD43. In the first part of this thesis I demonstrate that CD43 can play a positive co-stimulatory role and a negative role in down-regulation of an immune response, depending on its targeted epitope. Peripheral blood (PB) T cell co-stimulation via two diverse CD43 epitopes recognized by mAb CD43-6E5 (T6E5-act) and CD43-10G7 (T10G7-act) could potently induce proliferation in various T cell subsets including CD4+, CD8+ as well as naïve T cells. However, T cell co-stimulation via these two CD43 epitopes differentially regulated T cell homotypic clustering, cytokine production and also subsequent effector function. T6E5-act produced high levels of IL-22 and IFN-[gamma] similar to T cells activated via CD28 (TCD28-act) and further displayed proficient cytotoxic function. On the other hand, T10G7-act produced low levels of inflammatory cytokines, but higher levels of regulatory cytokines, TGF-[beta] and IL-35. Compared to T6E5-act or TCD28-act, T10G7-act exhibited a hypo-proliferative phenotype as analyzed during re-stimulation assays and subsequently acquired cell-cell contact dependent T cell suppressive function. T10G7-act did not directly inhibit responder T cells but rather routed their suppressive effect via antigen-presenting cells (APC) when added to allogeneic mixed leukocyte reaction (MLR). T10G7-act formed stable heterotypic clusters with dendritic cells (DC) via CD2 to inhibit activation of responder T cells. Together, the data suggest a unique role of CD43 in bidirectional polarization of T cell function. Transferrin receptor (TfR1/CD71) is fundamental for transport of iron into cells and is widely expressed on all proliferating cells, including activated T cells. Despite its central role in cell biology, the immune-modulatory role of CD71 in T cells is poorly understood. Therefore, in the second part of my thesis, I assessed the effect of targeting CD71 during T cell activation on downstream signalling pathways, T cell proliferation and cytokine production. Targeting of CD71 on activated T cells via VIP-1 mAb inhibited transferrin (Tf)-mediated iron uptake and further inhibited T cell proliferation. VIP-1-treated T cells (TVIP-1) were characterized by the unique ability to secrete T cell cytokines despite their inhibited growth. Analysis of downstream signalling pathways revealed over-activation of NFAT in VIP-1-treated Jurkat T cells, compared to mock-treated cells. Furthermore, VIP-1-treated Jurkat T cells were deficient in expression of proteins involved in intracellular trafficking such as Rab-5C, Rab-9A and Rab-11B as well as those involved in cell cycle progression such as Histone deacetylase 2 (HDAC2). Indeed, despite restored expression of CD71 on cell surface, TVIP-1 cells responded poorly and acquired IL-2 unresponsiveness during re-stimulation assays. In summary, the data show that targeting iron transport function of TfR1 during T cell activation alters the expression of distinct targets that are not exclusively associated with iron metabolism and leads to IL-2 unresponsiveness in T cells that is not related to iron deficiency.submitted by Madhura Meghsham Modak, MResZusammenfassung in deutscher SpracheMedizinische Universität Wien, Dissertation, 2016OeB

Expression and regulation of Schlafen (SLFN) family members in primary human monocytes, monocyte-derived dendritic cells and T cells

Schlafen (SLFN/Slfn) family members have been investigated for their involvement in fundamental cellular processes including growth regulation, differentiation and control of viral replication. However, most research has been focused on the characterization of Slfns within the murine system or in human cell lines. Since little is known about SLFNs in primary human immune cells, we set out to analyze the expression and regulation of the six human SLFN genes in monocytes, monocyte-derived dendritic cells (moDCs) and T cells. Comparison of SLFN gene expression across these three cell types showed high mRNA expression of SLFN11 in monocytes and moDCs and high SLFN5 expression in T cells, indicating functional importance within these cell types. Differentiation of monocytes to moDCs leads to the gradual upregulation of SLFN12L and SLFN13 while SLFN12 levels were decreased by differentiation stimuli. Stimulation of moDCs via human rhinovirus, lipopolysaccharide, or IFN-α lead to strong upregulation of SLFN gene expression, while peptidoglycan poorly stimulated regulation of both SLFNs and the classical interferon-stimulated gene MxA. T cell activation was found to downregulate the expression of SLFN5, SLFN12 and SLFN12L, which was reversible upon addition of exogenous IFN-α. In conclusion, we demonstrate, that SLFN gene upregulation is mainly dependent on autocrine type I interferon signaling in primary human immune cells. Rapid decrease of SLFN expression levels following T cell receptor stimulation indicates a role of SLFNs in the regulation of human T cell quiescence

Immunology / Engagement of distinct epitopes on CD43 induces different co-stimulatory pathways in human T cells

Co-receptors, being either co-stimulatory or co-inhibitory, play a pivotal role in T-cell immunity. Several studies have indicated that CD43, one of the abundant T-cell surface glycoproteins, acts not only as a potent co-receptor but also as a negative regulator for T-cell activation. Here we demonstrate that co-stimulation of human peripheral blood (PB) T cells through two distinct CD43 epitopes recognized by monoclonal antibodies (mAb) CD43-6E5 (T6E5-act) and CD43-10G7 (T10G7-act) potently induced T-cell proliferation. However, T-cell co-stimulation through two CD43 epitopes differentially regulated activation of nuclear factor of activated T cells (NFAT) and nuclear factor-kappa B (NF-kappa B) transcription factors, T-cell cytokine production and effector function. T6E5-act produced high levels of interleukin-22 (IL-22) and interferon-gamma (IFN-gamma) similar to T cells activated via CD28 (TCD28-act), whereas T10G7-act produced low levels of inflammatory cytokines but higher levels of regulatory cytokines transforming growth factor-beta (TGF-beta) and interleukin-35 (IL-35). Compared with T6E5-act or to TCD28-act, T10G7-act performed poorly in response to re-stimulation and further acquired a T-cell suppressive function. T10G7-act did not directly inhibit proliferation of responder T cells, but formed stable heterotypic clusters with dendritic cells (DC) via CD2 to constrain activation of responder T cells. Together, our data demonstrate that CD43 is a unique and polarizing regulator of T-cell function.P 22869-B11(VLID)311063

STAT1 is a sex‐specific tumor suppressor in colitis‐associated colorectal cancer

The interferon‐inducible transcription factor STAT1 is a tumor suppressor in various malignancies. We investigated sex‐specific STAT1 functions in colitis and colitis‐associated colorectal cancer (CRC) using mice with specific STAT1 deletion in intestinal epithelial cells (STAT1∆IEC). Male but not female STAT1∆IEC mice were more resistant to DSS‐induced colitis than sex‐matched STAT1flox/flox controls and displayed reduced intraepithelial infiltration of CD8+ TCRαβ+ granzyme B+ T cells. Moreover, DSS treatment failed to induce expression of T‐cell‐attracting chemokines in intestinal epithelial cells of male but not of female STAT1∆IEC mice. Application of the AOM‐DSS protocol for induction of colitis‐associated CRC resulted in increased intestinal tumor load in male but not in female STAT1∆IEC mice. A sex‐specific stratification of human CRC patients corroborated the data obtained in mice and revealed that reduced tumor cell‐intrinsic nuclear STAT1 protein expression is a poor prognostic factor in men but not in women. These data demonstrate that epithelial STAT1 is a male‐specific tumor suppressor in CRC of mice and humans

Identification of bone morphogenetic protein 7 (BMP7) as an instructive factor for human epidermal Langerhans cell differentiation

Human Langerhans cell (LC) precursors populate the epidermis early during prenatal development and thereafter undergo massive proliferation. The prototypic antiproliferative cytokine TGF-β1 is required for LC differentiation from human CD34(+) hematopoietic progenitor cells and blood monocytes in vitro. Similarly, TGF-β1 deficiency results in LC loss in vivo. However, immunohistology studies revealed that human LC niches in early prenatal epidermis and adult basal (germinal) keratinocyte layers lack detectable TGF-β1. Here we demonstrated that these LC niches express high levels of bone morphogenetic protein 7 (BMP7) and that Bmp7-deficient mice exhibit substantially diminished LC numbers, with the remaining cells appearing less dendritic. BMP7 induces LC differentiation and proliferation by activating the BMP type-I receptor ALK3 in the absence of canonical TGF-β1-ALK5 signaling. Conversely, TGF-β1-induced in vitro LC differentiation is mediated via ALK3; however, co-induction of ALK5 diminished TGF-β1-driven LC generation. Therefore, selective ALK3 signaling by BMP7 promotes high LC yields. Within epidermis, BMP7 shows an inverse expression pattern relative to TGF-β1, the latter induced in suprabasal layers and up-regulated in outer layers. We observed that TGF-β1 inhibits microbial activation of BMP7-generated LCs. Therefore, TGF-β1 in suprabasal/outer epidermal layers might inhibit LC activation, resulting in LC network maintenance