No description available.

<p>Diacylglycerol (DAG), a lipid messenger generated upon T cell receptor (TCR) engagement, mediates signaling through the IKK/NF-&#954;B and Ras/ERK pathways. Further downstream of the Ras/ERK pathway are mammalian target of rapamycin (mTOR) and MAP kinase signal integrating kinases Mnk1 and Mnk2. While mTOR acts as a critical regulator of T cell metabolism, homeostasis and function, Mnk1 and Mnk2 phosphorylate the initiation factor eIF4E that plays an important role in cap-dependent mRNA translation. Diacylglycerol kinases (DGKs) terminate DAG-mediated signals by phosphorylating DAG into phosphatidic acid. T cells that lack both &#945; and &#950; isoforms of DGK accumulate excess DAG upon activation, resulting in hyper-activation of the IKK/NF-&#954;B, Ras/ERK and mTOR pathways, hypersensitivity to TCR stimulation, and loss of self-tolerance. Here, we have examined the mechanisms by which dysregulated DAG-mediated signaling affects T cell function. To this end, we studied the effects of hyper-activating individual DAG-mediated pathways (IKK/NF-&#954;B and TSC/mTOR) on T cell function. We also examined the role of ERK-activated kinases Mnk1 and Mnk2 in T cell function.</p><p>Using mice with T cell-specific expression of a constitutively active form of IKK&#946; (`IKK' mice), we found that uncontrolled IKK&#946;/NF-&#954;B signaling promotes T cell apoptosis and attenuates responsiveness to TCR stimulation. Defective IL-2 production and increased FasL expression contributed to enhanced IKK T cell apoptosis. Impaired IKK T cell activation and proliferation were associated with defects in TCR signaling, and upregulation of the cell surface inhibitory receptor PD1. In vivo, IKK T cells mounted a compromised antigen-specific CD8 T cell response with curtailed expansion and exaggerated contraction phases. Notably, expression of transcriptional repressor Blimp1 (a regulator of T cell exhaustion) was increased in IKK T cells, and conditionally deleting Blimp1 was able to largely restore responsiveness to TCR stimulation.</p><p>Investigating Mnk1/2 double knockout (DKO) mice, we found that Mnk1 and Mnk2 are dispensable for T cell development and function, but important for the pathogenesis of experimental autoimmune encephalomyelitis (EAE). TCR engagement activated Mnk1/2 in a Ras/ERK-dependent manner in primary T cells, and was inhibited by DGK &#945; and &#950;. Mnk1/2 deficiency did not affect the development of conventional &#945;&#946; T cells, regulatory T cells, or invariant NKT cells. Mature T cells from DKO mice showed normal activation and CD4 TH differentiation ex vivo, but DKO mice developed lower clinical scores than WT counterparts in an EAE model, correlating with a smaller pool of MOG-reactive IL-17-producing and IFN&#947;-producing CD4 cells. These results suggest that Mnk1/2 may play a minimal role in T cell development and function but may control non-T cell lineages to regulate TH1 and TH17 differentiation in vivo. </p><p>To determine the effect of constitutive mTOR complex 1 activity on anti-bacterial CD8 responses, we investigated mice with T cell-specific deletion of TSC1, a suppressor of mTOR complex 1 activity. Using an established model system of transgenic (OT1) CD8 cell adoptive transfer and challenge with Listeria monocytogenes expressing a cognate antigen, we found that TSC1 deficiency impairs antigen-specific CD8 responses. Fewer TSC1-deficient OT1 cells were present in the peripheral blood and spleen at the peak of the response and fewer memory cells were found at later time points, in individual and competitive adoptive transfer experiments with WT counterparts. Weak expansion of TSC1-deficient cells was correlated with defects in survival and proliferation in vivo, while exaggerated contraction was associated with an increased ratio of SLECs to MPECs in the effector cell population. This perturbation in effector-memory differentiation was concomitant with enhanced T-bet expression and decreased Eomes expression among activated TSC1 KO cells. Upon competitive adoptive transfer with WT counterparts and antigen re-challenge, TSC1-deficient memory cells showed moderate defects in expansion but not cytokine production. Taken together, these findings provide direct evidence of a CD8 cell-intrinsic role for TSC1 in regulating antigen-specific primary and memory responses.</p><p>In sum, findings from these studies provide deeper insight into the regulation of T cell function by DAG-mediated pathways, and may have implications for the design of immune-modulation strategies during vaccination, autoimmunity and cancer immunotherapy.</p> / Dissertation

T-cell receptor (TCR) polyclonal mature T cells are surprisingly resistant to oncogenic transformation after retroviral insertion of T-cell oncogenes. In a mouse model, it has been shown that mature T-cell lymphoma/leukemia (MTCLL) is not induced upon transplantation of mature, TCR polyclonal wild-type (WT) T cells, transduced with gammaretroviral vectors encoding potent T-cell oncogenes, into RAG1-deficient recipients. However, further studies demonstrated that quasi-monoclonal T cells treated with the same protocol readily induced MTCLL in the recipient mice. It has been hypothesized that in the TCR polyclonal situation, outgrowth of preleukemic cells and subsequent conversion to overt malignancy is suppressed through regulation of clonal abundances on a per-clone basis due to interactions between TCRs and self-peptide-MHC-complexes (spMHCs), while these mechanisms fail in the quasi-monoclonal situation. To quantitatively study this hypothesis, we applied a mathematical modeling approach. In particular, we developed a novel ordinary differential equation model of T-cell homeostasis, in which T-cell fate depends on spMHC-TCR-interaction-triggered stimulatory signals from antigen-presenting cells (APCs). Based on our mathematical modeling approach, we identified parameter configurations of our model, which consistently explain the observed phenomena. Our results suggest that the preleukemic cells are less competent than healthy competitor cells in acquiring survival stimuli from APCs, but that proliferation of these preleukemic cells is less dependent on survival stimuli from APCs. These predictions now call for experimental validation.

No description available.

HIV Tat protein downregulates surface expression of the interleukin-7 receptor alpha-chain (CD127) on CD8 T cells resulting in impaired T cell proliferation and cytolytic capacity. Once taken up by CD8 T cells, Tat binds directly to the cytoplasmic tail of CD127 inducing receptor internalization and degradation. Given the important roles of CD127 in proper immune function, the Tat/CD127 interactions were characterized and the mechanisms required to induce receptor loss from the surface of CD8 T cells were investigated. Tat deletion mutants were generated each sequentially lacking a region of the protein. CD8 T cells isolated from HIV negative volunteers were exposed to exogenous or intracellular Tat proteins before surface CD127 expression was analyzed by flow cytometry. To characterize Tat/CD127 physical interactions, wild type Tat and Tat mutants were incubated with lysates from a CD127+ Jurkat cell line followed by CD127/Tat co-immunoprecipitation. The effect of Tat on CD127 post-translational modifications was also investigated. Removal of the N-terminus of Tat (aa 1-10 or aa 17-21) prevented Tat from downregulating CD127 and prevented Tat from binding CD127 as assessed by co-immunoprecipitation. Deletion of the basic region (aa 48-59) also prevented Tat from downregulating CD127 but did not prevent Tat from interacting physically as demonstrated by co-immunoprecipitation. Strikingly, endogenously expressed Basic Tat acted as a dominant negative mutant, causing an accumulation of CD127 at the cell surface. These observations suggest that Tat may bind CD127 via its N-terminus to disrupt the normal recycling of the receptor, and then recruit cellular endocytic machinery to the receptor via it’s basic region, to remove the receptor from the cell surface and target it for degradation. Furthermore, Tat encourages the ubiquitination of CD127 by recruiting the cytokine-inducible SH2 containing (CIS) protein to the receptor, possibly leading to accelerated CD127 internalization and proteasomal degradation. I propose a model whereby Tat binds CD127 via its N-terminal region then recruits CIS via its basic region. CIS in turn recruits a cellular E3 ubiquitin ligase to ubiquitin tag the receptor for internalization and proteasome degradation. This research may lead to novel treatments designed to maintain IL-7 signalling and strengthen CD8 T cell function in HIV+ persons.

Early during an immune response, dendritic cells (DC) interact closely with CD4+ T cells, and cross-talk between these cells can come in the form of tumour necrosis factor (TNF) superfamily ligand-receptor interactions. These signals are critical for the maturation, function and survival of DC, and thereby dictate the capacity of DC to prime a robust T cell response. Among these cues, helper T cell-expressed CD40L interaction with DC-expressed CD40 is required to fully mature DC for cross-priming of help-dependent CD8+ T cell responses. The lymphotoxin-beta receptor (LTβR) is another TNF family receptor on DC, and it’s ligands LTα1β2 and LIGHT are expressed on activated T cells. Since abrogated LTβR signaling impairs T cell immunity, we have examined whether LTαβ represents another possible helper T cell-derived cue for full DC maturation. However the LT pathway controls lymphoid tissue organization and DC homeostasis, a second possible mechanism explaining the necessity of LTβR signaling for T cell immunity. Here we dissect the role of helper T cell-expressed LTβR ligands and DC-intrinsic LTβR signaling, independent of DC homeostasis or lymphoid organization, in DC function and T cell immunity. Absence of LTα1β2 and not LIGHT on helper T cells results in compromised T cell priming by DC ex vivo, and LTβ-/- CD4+ T cell responses are impaired in vivo. Ag-specific CD4+ T cell-expressed LTα1β2 and DC-intrinsic LTβR signaling are required for an optimal cytotoxic T lymphocyte (CTL) response in vivo. While CD40 induces IL-12 and is required for CTL function, DC-intrinsic LTβR signaling is necessary for CTL activation and expansion, early up-regulation of CD86 and IFNα/β production. Our results reveal non-redundant roles for distinct TNF family receptors in enabling DC to program different features in Ag-specific CD8+ T cells.

Cancer vaccines can fail despite the induction of large numbers of CD8+ T cells. Two categories of memory CD8+ T cells have been defined; central memory (TCM, IL-7RαhighCD44highCD62Lhigh) and effector memory (TEM, IL-7RαhighCD44highCD62Llow). It is clear that the memory phenotype of CD8+ T cells can affect vaccine potential; however methods to augment a beneficial phenotype are not clear. I have compared three vaccine delivery systems: Listeria monocytogenes, Salmonella enterica serovar Typhimurium and the particulate liposomal adjuvant, archaeosomes, for their efficacy to protect against murine melanoma. My study revealed that the anti-tumour response is strongly influenced by the kinetics, phenotype, and lymph node homing potential of CD8+ T cells. Listeria monocytogenes-ovalbumin (LM-OVA) induced TCM cells were adept at long lasting protection against B16-OVA melanoma due to their increased homeostatic and antigen-induced proliferation, interleukin-2 production, and ability to extravasate into tumour draining lymph nodes. Conversely, although Salmonella Typhimurium-ovalbumin (ST-OVA) induced TEM, produced IFN-γ, and killed target cells, this was insufficient for long-term tumour protection. Selectin-ligand engagements of TCM cells influenced their homing potential and efficacy against murine melanoma. Fucosyltransferase deficient (FtDKO) mice, lacking functional selectin ligands, were vaccinated with LM-OVA; despite the activation of cytotoxic CD8+ T cells, there was a reduced protection against murine melanoma compared to wild-type. FtDKO CD8+ T cells exhibited reduced extravasation into FtDKO lymph nodes compared to wild-type. Additionally, fewer FtDKO CD8+ T cells compared to wild-type migrated into tumour sites. Archaeosome vaccination was used to compare the influence of CD8+ T cell quantity versus phenotype. Single or multiple therapeutic vaccinations with archaeosome-OVA yielded transient melanoma tumour protection, despite an increased frequency of circulating and tumour infiltrating CD8+ T cells. This correlated with increased expression of Program death receptor-1 (PD-1) on CD8+ T cells and induction of regulatory T cells. Prophylactic archaeosome-OVA vaccination resulted in a maximal frequency of antigen-specific CD8+ T cells of ~50-60 % with just three injections, and ~50 % of the mice were of mice were afforded long-term tumour protection (> 90 days). Overall, my study shows that the choice of vaccine adjuvant and/or vector can profoundly influence CD8+ T cell quality and cancer vaccine efficacy.

Glycosylation is one of the most abundant post-translational modifications of proteins. Glycoproteins participate in virtually all aspects of cellular functions. ST6Gal I is a glycosyltransferase highly expressed by B and T cells. Here, we interrogated the role of ST6Gal I in viral specific B and T cell immune responses, as well as examined how loss of this enzyme impacted viral pathogenesis. First, to understand how loss of ST6Gal I expression impacted viral specific humoral responses, we infected ST6Gal I-/- mice with influenza virus. We discovered that loss of ST6Gal I expression results in both reduced influenza specific antibodies levels and decreased viral-specific antibody secreting cells numbers. Following influenza infection, mice that received ST6Gal I-/- B cells showed reduced influenza-specific IgM responses compared to mice that received wild-type B cells. These experiments demonstrated that the expression of ST6Gal I by B cells is required for optimal viral-specific humoral response. We further examined how loss of ST6Gal I expression impacted the anti-influenza IgA response. We observed that immune ST6Gal I-/- mice displayed higher viral specific IgA levels and altered sialylation of IgG and IgA, which have been implicated in a human disease, IgA nephropathy. Moreover, ST6Gal I-/- mice exhibited increased immunoglobulin deposition in kidney glomeruli following influenza infection. These data suggest that ST6Gal I deficiency, together with influenza infection, may result in the initiation of a kidney disease. Finally, we examined how ST6Gal I expression regulated CD8 T cell responses. We discovered that ST6Gal I is differentially expressed during CD8 T cell activation. To understand its relevance, we infected ST6Gal I-/- mice and demonstrated that the early expansion of effector T cells was impaired in a cell intrinsic manner. Moreover, in the absence of ST6Gal I, the differentiation of CD8 T cells skewed towards memory precursor cells, whereas terminal effector cell expansion was impaired. Mechanistically, we identified delayed surface expression of IL-2Ralpha on ST6Gal I-/- CD8 T cells due to impaired IL-2/IL-2R signaling. These studies implicate that ST6Gal I expression enhances early proliferation of terminal effector CD8 T cells by promoting the rapid surface expression of IL2Ralpha during acute viral infection. 

Human cytomegalovirus (HCMV) is a β-herpesvirus that establishes a life-long presence in the infected host. The adaptive immune response is indispensable in controlling HCMV infection. Consequently, healthy individuals show no or mild symptoms following primary infection. In contrast, immunocompromised individuals who develop primary infection or recrudescence of HCMV can experience severe morbidity, and sometimes mortality. HCMV-specific T cell populations undergo changes in the architecture of their T cell receptor (TCR) repertoire following each episode of viral reactivation. A diverse TCR repertoire is thought to be required to provide the most efficient protection against virus infection. Perturbation to this repertoire, as can occur in immunocompromised individuals following transplantation, can lead to an increase risk of developing virus-associated clinical disease. Therefore, the study of factors influencing TCR selection is critically important in both healthy and immunocompromised individuals. To further understand the factors governing TCR selection in a persistent virus infection, the current thesis examined this process in different settings. CD8+ T cell responses to persistent viral infections are characterised by the accumulation of T cells exhibiting an oligoclonal T cell repertoire, with a parallel reduction in the naïve T cell pool. However, the precise mechanism for this phenomenon remains elusive. Here, we showed that HCMV-specific CD8+ T cells recognising distinct epitopes from the pp65 protein and restricted through an identical HLA class I allele (HLA B*3508) exhibited either a highly conserved public T cell repertoire, or a private, diverse T cell response, which was uniquely altered in each donor following in vitro antigen exposure. Selection of a public TCR was co-incident with an atypical peptide-MHC (pMHC) structure, whereby the epitope adopted a helical conformation that bulged from the peptide-binding groove, whilst a diverse TCR profile was observed in response to the epitope that formed a flatter, more ‘featureless’ landscape. Clonotypes with biased TCR usage demonstrated more efficient recognition of virus-infected cells, a greater CD8 dependency, and were more terminally differentiated in their phenotype when compared to the T cells expressing diverse TCR. These findings provide new insights into our understanding of how the biology of antigen presentation, in addition to the structural features of the pMHC, might shape the T cell phenotype and its corresponding repertoire architecture. Next, the role of HCMV in shaping the global and antigen-specific TCR repertoire in healthy donors was examined. First, exposure to HCMV led to an inflation of terminally differentiated CD57-expressing T cells. This effect was not seen in HCMV seronegative individuals who showed evidence of exposure to another persistent herpesvirus, Epstein-Barr virus (EBV). More importantly, these terminally differentiated CD8+ T cells in HCMV-exposed individuals displayed a highly skewed architecture of their peripheral blood T cell repertoire, with large monoclonal/oligoclonal expansions. However, ex vivo analyses of HCMV-specific T cells revealed a heterogeneous pattern of CD57 expression that showed no correlation to the antigenic source of its cognate epitope. Based on these observations, we proposed that exposure to HCMV drives the differentiation of not only the global T cell population, but select HCMV-specific T cell populations as well, and that expression of CD57 by these cells was co-incident with an oligoclonal T cell repertoire. Finally, the TCR repertoire was examined in a cohort of solid organ transplant (SOT) recipients, where primary infection or recrudescence of latent virus infection can be manifested either as asymptomatic or symptomatic disease. We examined 18 SOT recipients, and observed that symptomatic HCMV or EBV infection or recrudescence following solid organ transplantation was co-incident with a dramatic skewing of the TCR repertoire, with expansions of monoclonal/oligoclonal clonotypes. As the clinical symptoms resolved, the peripheral blood repertoire reverted to a more diverse distribution. In contrast, SOT recipients with asymptomatic or no HCMV/EBV infection or recrudescence showed minimal or no skewing of the TCR repertoire, and maintained TCR repertoire diversity. Interestingly, this disparate repertoire showed no correlation with levels of viral load in the peripheral blood. More importantly, we showed that large monoclonal/oligoclonal repertoire expansions was linked to the loss of antigen-specific T cell function observed in SOT patients undergoing symptomatic viral infection or recrudescence, while SOT recipients who maintained peripheral blood TCR repertoire diversity and functional antigen-specific T cell responses could resist clinical symptomatic disease in spite of high levels of viral load. Therefore, the work presented in this thesis provides additional evidence on the factors governing TCR selection in HCMV-exposed healthy individuals, as well as the consequences that perturbation to the TCR repertoire has on the functionality of the T cell compartment in immunocompromised individuals.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 6 uppsatser.