Kawasaki disease (KD) is a multisystem vasculitis with predilection for the coronary arteries. Although the cause of KD remains elusive, there is evidence to suggest a superantigenic trigger. When T-cells are activated by a superantigen (SAg) they undergo massive proliferation but eventually apoptose; however, in KD, we hypothesize that these T-cells persist and infiltrate the coronary arteries. Previous studies have shown that enhanced costimulation through CD28 or 4-1BB rescues T-cells from apoptosis and exacerbates disease in a mouse model of KD. Our results suggest that this signal needs to be initiated close in timing to that of the SAg. In addition, the two molecules can act independently of one another, but are not additive. Also, stimulation of the 4-1BB pathway in the presence of a SAg elicits a Th1 phenotype. Lastly, TRAF1 regulates this enhanced survival downstream of 4-1BB. Thus, these results provide new insights into the effects of costimulation in SAg-mediated disease, and suggest that these pathways need to be targeted early to abrogate the enhanced survival of SAg-activated T-cells.

Kawasaki disease (KD) is a multisystem vasculitis with predilection for the coronary arteries. Although the cause of KD remains elusive, there is evidence to suggest a superantigenic trigger. When T-cells are activated by a superantigen (SAg) they undergo massive proliferation but eventually apoptose; however, in KD, we hypothesize that these T-cells persist and infiltrate the coronary arteries. Previous studies have shown that enhanced costimulation through CD28 or 4-1BB rescues T-cells from apoptosis and exacerbates disease in a mouse model of KD. Our results suggest that this signal needs to be initiated close in timing to that of the SAg. In addition, the two molecules can act independently of one another, but are not additive. Also, stimulation of the 4-1BB pathway in the presence of a SAg elicits a Th1 phenotype. Lastly, TRAF1 regulates this enhanced survival downstream of 4-1BB. Thus, these results provide new insights into the effects of costimulation in SAg-mediated disease, and suggest that these pathways need to be targeted early to abrogate the enhanced survival of SAg-activated T-cells.

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A common bipotent thymocyte precursor gives rise to both lineages of T cells, αβ and γδ. This thesis addresses how the interplay between intrinsic T cell receptor (TCR) signals and cell extrinsic signals provided by Notch and TCR ligands help to assign and support a final lineage fate decision. Emerging data supports a model in which differential TCR signaling capacity plays an instructional role in specifying lineage fate, particularly through induction of the ERK - early growth response gene (Egr) - inhibitor of DNA binding 3 (Id3) pathway. In particular, Id3 expression serves to regulate adoption of the γδ fate. Moreover, Id3 is both necessary and sufficient to enable γδ-lineage cells to differentiate independently of Notch signaling and become competent interferon (IFN)-γ-producing effectors. These findings identify Id3 as a central player that controls both adoption of the γδ fate and their maturation in the thymus. While loss of Notch signaling in γδTCR-expressing CD4-CD8- (DN)3 cells does not affect development, Notch signals are critical for pre-TCR-bearing cells to transition to the CD4+CD8+ (DP) stage of αβ T cell development. Notch signals affect the activation of the PI3K/Akt pathway, which is required for pTα/TCRβ (pre-TCR)-induced survival, differentiation and proliferation of developing αβ-lineage thymocytes. Here, I identify the key molecular players responsible for the interaction between the Notch and PI3K pathways at this critical developmental stage. Notch induction of Hes1 expression is necessary to repress the expression of the PI3K/Akt pathway inhibitor, PTEN, which in turn facilitates pre-TCR-induced differentiation. c-Myc, another critical target of Notch, is required for proliferation during β-selection. Lastly, I find that the majority of DN3 cells expressing both pre-TCR and γδTCR follow the signal strength model for lineage development, and commit and mature along the γδ-lineage. However, manipulation of signal strength, through γδTCR ligand availability or Id3 expression, can skew this development outcome. Taken together, the results from this thesis provide a detailed examination of the molecular mechanisms that are instrumental in determining lineage fate, survival, and proliferation of differentiating thymocytes. Central to these outcomes is the interplay between the Notch, TCR, PI3K, and MAPK signaling pathways.

MUC1, a protein of epithelial and carcinoma cells, has recently been shown on activated T cells where it inhibits CD3-stimulated proliferation. Two immunoregulatory domains similar to ITAM and ITIMs are present on its cytoplasmic tail, suggesting that MUC1 can act as both a costimulatory and coinhibitory molecule of T cells. In my work, I have examined immunoregulatory function of MUC1 on human T cells. We first showed that MUC1, when ligated in a population of unpurified T cells with an anti-CD3 and a crosslinking antibody, enhances proliferative and cytokine responses in a NF-AT-dependent manner by recruiting the AP-1 transcription factor and translocating it into the nucleus. With purified CD3+ T cells, we instead observed inhibition after MUC1/CD3 coligation and crosslinking. Reconstituting with irradiated CD3- cells, we discovered that MUC1 costimulation is dependent on the amount of accessory cells. These data imply a novel role for MUC1 in T cell immunoregulation. / Experimental Surgery

The oncogene Ahi-1 was recently identified through provirus insertional mutagenesis in murine leukemias and lymphomas. Its involvement in human leukemogenesis is demonstrated by gross perturbations in its expression in several leukemic cells lines, particularly in cutaneous T-cell lymphoma (CTCL) cell lines (Hut 78 and Hut 102). Hut 78 is derived from a patient with Sezary syndrome, a common leukemic variant of the human CTCL mycosis fungoides. Aberrant expression of AHI-1 mRNA and protein has been found in CD4⁺CD7⁻ leukemic Sezary cells from patients with Sezary syndrome. Moreover, stable suppression of AHI-1 using retroviral-mediated RNA interference in Hut 78 cells inhibits their transforming activity in vitro and in vivo. In an effort to identify genes involved in AHI-1-mediated leukemic transformation in CTCL, microarray analysis was performed to compare six RNA samples from AHI-1 suppressed Hut 78/sh4 cells to five samples from Hut 78 control cells. Limma and dChip analyses identified 218 and 95 differentially expressed genes, respectively, using a fold change criteria of > or < 2 and a p-value threshold of ≤ 0.01. After evaluation of both analyses, 21 genes were selected based upon interesting structural and functional information, specificity to hematopoietic cells or T-cells, and previous connections to cancer. Expression patterns of these 21 genes were validated by qRT-PCR with p-values < 0.05 ranging from 1.97 x 10⁻¹⁰ to 6.55 x 10⁻³, with the exception of BRDG1 at 5.88 x 10⁻². The observed up-regulation of both BIN1 and HCK in AHI-1 suppressed Hut 78/sh4 cells as compared to control cells further confirmed at the protein level. The tumor suppressor BIN1 is known to physically interact with c-MYC, which also exhibits differential protein expression in these cells. Characterization of BIN1 identified 4 isoforms all of which contain exon 10 and demonstrate alternative splicing of exons 12A and 13. Additionally, qRT-PCR results from primary Sezary samples indicate there is clinical significance in the expression changes detected for BIN1, HCK, REPS2, BRDG1, NKG7 and SPIB. These findings identify several new differentially expressed genes that may play critical roles in AHI-1-mediated leukemic transformation of human CTCL cells.

Cytotoxic T-lymphocytes are important for the control of viral replication during HIV infection, however the magnitude and breadth of HIV-specific CD8+ T-cell response does not correlate well. The purpose for this study was the examination of the HLA-B*2705-specific CD8+ T-cell response to the KRWIILGLNK (KK10) epitope as a definitive model of immune control over HIV replication. The breadth of the T-cell receptor (TCR) repertoire was determined for an association between the qualitative nature of this response and immune escape and therefore, disease progression. Methodology was developed and validated for TCR repertoire analysis in formaldehyde fixed antigen-specific CD8+ T-cells. The TCR repertoire for the KK10-specific CD8+ T-cell response was defined in cross-section and longitudinally for 6 HLA-B*2705+ patients. Comparison was made to cognate HLA-A*0201 CMV NV9 and HLA-B*2705 EBV RL9-specific CD8+ T-cell populations using the Simpson??s diversity index and the Morisita-Horn similarity index for standardized repertoire analysis. HLA-B*2705 KK10-specific TCR repertoire was not found to be a determinant of control. Greater clonotype variation was found within CMV-specific CD8+ T-cell populations, suggesting an association with reactivation of CMV and disease state. An association was found between KK10-specific population diversity and the prevalence of cognate KK10 epitope in vivo. Cross-reactivity observed for dominant KK10-specific clonotypes suggested that avidity of CD8+ T-cells was important for in vivo survival. Phenotype and function was tested through multiparameter analysis of HIV and CMV-specific CD8+ T-cells. Increased frequency of CD127 (IL-7R) and Bcl-2 expression within dominant populations was suggestive of selective advantage. Division of dominant and sub-dominant CMV-specific CD8+ T-cell populations into ??early?? and ??late?? differentiation phenotypes indicated virus-specific mechanisms of clonotype turn over. No simple association of TCR expression was found for HIV and CMV-specific CD8+ T-cells with published examples of definitive TCR bias. Over-represented TCR ??-chain families of patients were found in association with public clonotypes. Convergent recombination of TCR genes was demonstrated as a mechanism for the prevalence of shared clonotypes. Standardized assessment of T-cell repertoire successfully identified mechanisms of antigen-specific CD8+ T-cell recruitment. A substantial increase in sample numbers is required before this methodology can be used to accurately demonstrate the importance of TCR repertoire usage in the control of human viral infection.

Diss. (sammanfattning) Umeå : Univ., 2004. / Härtill 5 uppsatser.

Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted.