Heterogeneous loss of HIV transcription and proviral DNA from 8E5/LAV lymphoblastic leukemia cells revealed by RNA FISH:FLOW analyses

8E5/LAV cells harbor a single HIV provirus, and are used frequently to generate standards for HIV genome quantification. Using flow cytometry-based in situ mRNA hybridization validated by qPCR, we find that different batches of 8E5 cells contain varying numbers of cells lacking viral mRNA and/or viral genomes. These findings raise concerns for studies employing 8E5 cells for quantitation, and highlight the value of mRNA FISH and flow cytometry in the detection and enumeration of HIV-positive cells

T CELL RECEPTOR SIGNAL STRENGTH AND ANTIGEN AFFINITY DIFFERENTIALLY REGULATE CD8+ MEMORY T CELL DEVELOPMENT

169 pagesCD8+ T cells play a critical role in adaptive immunity by maintaining the ability to differentiate into CD8+ memory T cells, which provide the basis of protective immunity. During an intracellular infection, CD8+ T cells pass through several characteristic phases before becoming mature memory cells. Initial antigen stimulation causes naïve CD8+ T cells to clonally expand and differentiate into short-lived effector cells (SLECs). Subsequently, SLECs undergo a contraction phase, whereby the majority of surviving cells are known as memory precursor effector cells (MPECs); 5-10% of the MPECs survive the initial contraction phase from SLECs to further develop into CD8+ memory T cells. In my dissertation, we show that two distinct parameters: T cell Receptor (TcR) signal strength (regulated by the tyrosine kinase Itk) and antigen affinity, play important but separate roles in modulating the development of memory CD8+ T cells. We found that reducing both TcR signal strength and antigen affinity for the TcR leads to enhanced and accelerated development of CD8+ memory T cells. Additionally, TcR signal strength is able to regulate CD8+ T cell effector cytokine production independent of TcR antigen affinity. Analysis of RNA-sequencing data reveals that genes for inflammatory cytokines/cytokine receptors are significantly altered upon changes in antigen affinity and TcR signal strength. Furthermore, our findings show that the inflammatory milieu is critical in regulating this TcR signal strength mediated increase in memory development as both CpG treatment or co-transfer of WT and Itk-/- T cells eliminates the observed increase in memory cell formation. These findings suggest that TcR signal strength and antigen affinity independently contribute to CD8+ memory T cell development, which is modulated by inflammation, and suggest that manipulating TcR signal strength, along with antigen affinity, may be used to tune the development of CD8+ memory T cells during vaccine development

Non-receptor tyrosine kinase signaling in autoimmunity and therapeutic implications

Autoimmune diseases are characterized by impaired immune tolerance towards self-antigens, leading to enhanced immunity to self by dysfunctional B cells and/or T cells. The activation of these cells is controlled by non-receptor tyrosine kinases (NRTKs), which are critical mediators of antigen receptor and cytokine receptor signaling pathways. NRTKs transduce, amplify and sustain activating signals that contribute to autoimmunity, and are counter-regulated by protein tyrosine phosphatases (PTPs). The function of and interaction between NRTKs and PTPs during the development of autoimmunity could be key points of therapeutic interference against autoimmune diseases. In this review, we summarize the current state of knowledge of the functions of NRTKs and PTPs involved in B cell receptor (BCR), T cell receptor (TCR), and cytokine receptor signaling pathways that contribute to autoimmunity, and discuss their targeting for therapeutic approaches against autoimmune diseases

TCR Signal Strength and Antigen Affinity Regulate CD8+ Memory T Cells

CD8(+) T cells play a critical role in adaptive immunity, differentiating into CD8(+) memory T cells, which form the basis of protective cellular immunity. Vaccine efficacy is attributed to long-term protective immunity, and understanding the parameters that regulate development of CD8(+) T cells is critical to the design of T-cell mediated vaccines. We show here using mouse models that two distinct parameters: T cell Receptor (TcR) signal strength (regulated by tyrosine kinase ITK) and antigen affinity, play important but separate roles in modulating the development of memory CD8(+) T cells. Unexpectedly, our data reveals that reducing TcR signal strength along with reducing antigen affinity for the TcR leads to enhanced and accelerated development of CD8(+) memory T cells. Additionally, TcR signal strength is able to regulate CD8(+) T cell effector cytokine production independent of TcR antigen affinity. Analysis of RNA-sequencing data reveals that genes for inflammatory cytokines/cytokine receptors are significantly altered upon changes in antigen affinity and TcR signal strength. Furthermore, our findings show that the inflammatory milieu is critical in regulating this TcR signal strength mediated increase in memory development as both CpG treatment or co-transfer of WT and Itk(−/−) T cells eliminates the observed increase in memory cell formation. These findings suggest that TcR signal strength and antigen affinity independently contribute to CD8(+) memory T cell development, which is modulated by inflammation, and suggest that manipulating TcR signal strength, along with antigen affinity, may be used to tune the development of CD8(+) memory T cells during vaccine development