Scaffolding and peer review methods were combined on an online platform to develop scientific writing skills in an undergraduate organic chemistry lab.Collaboration was encouraged throughout the semester.First students anonymously critiqued lab report sections from their peers through the online platform. Later, small groups produced complete reports built up from the sections covered by peer review. Each student served a different role in the group, moving from a collaborative approach towards independent writing. The culmination of the semester was a complete report, produced independent of peer or instructor guidance. Implementation was accomplished through a series of rubrics, worksheets, and instructor feedback. Preliminary assessment of these curriculum changes indicates that student writing skills improved and that student feedback is mixed, but generally positive. The ultimate goal is the development of writing skills in lower-level chemistry labs in preparation for writing-intensive upper-level labs.

The ontogeny of exhausted CD8 T cells as well as the underlying mechanisms that account for the functional inactivation of these cells remains ill-defined. We have utilized cytokine reporter mice, which mark the synthesis of interferon-γ by the expression of Thy1.1, to decipher how activation events during the early stages of a chronic infection dictate the development of exhaustion. We show that, during chronic lymphocytic choriomeningitis virus infection, the precursors of exhausted T cells all upregulate interferon-γ mRNA and become susceptible to depletion with anti-Thy1.1 antibodies. This potent phase of hyperactivation which precedes exhaustion is antigen-dependent and is dictated by viral levels and T cell precursor frequencies. Unlike acute infections, which result in massive expansion of the responding T cells, during chronic infections further expansion of the initial response becomes rapidly attenuated. The exhausted T cells which subsequently emerge in chronically infected hosts attain a distinct CD127lo, KLRG-1lo phenotype but do not silence interferon-γ transcription even though protein expression is abolished. Thus ablation of interferon-γ production by exhausted cells is not due to transcriptional silencing, implicating post-transcription regulatory mechanisms in disabling this effector module.

B cells with potent IL-10-dependent regulatory functions (B10 cells) expand in mice and humans during inflammation and autoimmunity. In this study, the fate of B10 cells following in vivo expansion was examined using two distinct il-10 reporter strains: Tiger mice that express cytoplasmic GFP and 10BiT mice that express cell surface Thy1.1. B10 cells from Tiger mice express cytoplasmic IL-10 and GFP with similar kinetics following stimulation in vitro and in vivo. By contrast, B10 cell Thy1.1 expression in 10BiT mice was delayed relative to IL-10 production and persisted even after B10 cells had lost the capacity to produce IL-10. Thy1.1 also served as a marker for B10 cells that expressed plasma cell-associated transcription factors and had the capacity to differentiate into CD138+B220LO antibody-secreting plasma cells in vivo and in vitro. Antibodies secreted by B10 cells included broadly reactive “natural antibodies” and autoantibodies, and were antigen-specific in immunized mice. At the molecular level, B10 cells predominantly expressed a germline-encoded IgM antibody repertoire with diverse VH and VL gene utilization. These results indicate that B10 cells can differentiate following IL-10 secretion to produce broadly-reactive germline-encoded antibodies that may function to rapidly clear antigens and further reduce inflammation and immunopathology.

Exposure to the opportunistic mold Aspergillus fumigatus may result in a wide range of infectious diseases. Among these infections, invasive aspergillosis is the most common and the most lethal. Our lab has previously shown a protective role for Dectin-dependent IL-22 production. In our current studies we sought to identify potential cell source(s) of IL-22 as well as factors critical for its production during invasive aspergillosis. Employing human CD4 IL-22 reporter mice, we show that iNKT cells and γδ T cells are sources of IL-22 48 h post-A. fumigatus exposure. In subsequent studies, we observed an early impairment in IL-22 production by lung cells from CD1d deficient (iNKT deficient) mice that returned to wild type levels by 48 h. In contrast, mice deficient in γδ T cells had relatively intact IL-22 production early after A. fumigatus exposure that became significantly attenuated by 48 h post-challenge. In order to identify factors critical for IL-22 production we interrogated the role of several pertinent transcription factors including: related orphan receptor-γ (RORγ), the aryl hydrocarbon receptor (Ahr), MyD88, and TRIF. Collectively, our data shows that iNKT cells and γδ T cells support early vs. late IL-22 production, which is fully dependent on RORγ and MyD88, partially dependent on Ahr, and independent of TRIF.

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Insulin-like growth factors (Igfs) are highly conserved proteins similar to pro-insulin in both sequence and structure. They function primarily by binding the Igf1 receptor (Igf1R) and exert pleiotropic effects on many tissues throughout life. These factors were shown to be essential for the normal development and proliferation of B and T lymphocytes, and to positively influence their proliferation and survival following activation. But early attempts to identify potential roles in T cell differentiation and function lead to conflicting results, and the field was largely abandoned. More recent advances in our understanding of T cell biology, however, including the discovery of Th17 and Treg C4s, have permitted a more sophisticated analysis of the role of this system in T cells. Our data indicate that Th17 and Treg CD4s express Igf1R and insulin-like growth factor binding protein 4 (Igfbp4), a critical regulator of Igf activity, at much higher levels than other CD4 T cells, and appear uniquely capable of responding to Igfs. Exogenous Igfs promote Th17 and suppress Treg differentiation in-vitro, yet fail to influence Th1 or Th2 differentiation. Deletion of Igf1R leads to a defect in Stat3 phosphorylation and IL17a production, and enhanced Treg differentiation in-vitro. Mice lacking Igf1R on T cells are substantially protected from Experimental Autoimmune Encephalomyelitis, an effect that can be recapitulated through the in-vivo administration of Igf1R blocking antibodies.

CD4 T cells respond to in vivo activation stimuli, in part, by rapidly producing the homeostatic cytokine IL-2. It is thought that this acute production of IL-2 is necessary for full activation and fate determination of both the conventional CD4 T cells themselves, as well as other components of the adaptive (Tregs/CD8/B cells) and innate immune responses (NK/NKT/DC) to infection. Within a population of responding CD4 T cells, only a percentage of cells will secrete IL-2. Though much is known regarding regulation of IL-2 production by CD4 T cells and the downstream consequences of autocrine and paracrine signaling, little is known about the quantity, quality, or fate of CD4 T cells that produce IL-2. Using L. monocytogenes engineered to express foreign MHCII peptides and peptide-loaded MHCII tetramers, we are able to track and assess the fate of endogenous, antigen-specific CD4 T cells in infected mice. Combining this methodology with novel transgenic IL-2 reporter mouse models, we are able to address questions regarding fate by isolating, depleting, and/or adoptively transferring live, antigen-specific CD4 T cells to secondary hosts based on acute expression of IL-2. Preliminary data suggests IL-2-‘competent’ and -‘incompetent’ cells differ in their ability to traffic to peripheral tissues following an acute, systemic infection, resulting in a potential functional, as well as proliferative and survival advantage for competent cells over those incompetent for IL-2 expression.

Interleukin-21 (IL-21) is critical for host defense against infections with intracellular and extracellular pathogens. Although most infections occur following an initial colonization at mucosal surfaces, no studies have addressed the role of IL-21 in controlling bacterial colonization and dissemination from the colon. Using a novel Il21fl/fl reporter and CD4-Cre conditional knockout mouse, we show that IL-21+CD4+ T cells are the dominant source of IL-21 required for the efficient clearance of the enteric bacterial pathogen Citrobacter rodentium. Consistent with the complete Il21-/- mice, we found that mice with a conditional deletion of Il21 from CD4+ T cells had a greater incidence and severity of colitis, increased bacterial dissemination to the spleen and liver, reductions in IFN-γ+ IL-17A- CD4+ T cells, and a decreased ratio of CXCR5+PD-1+ T follicular helper (Tfh) to FoxP3+ T follicular regulatory cells (Tfr) following infection. In association with impaired T cell differentiation, we found that conditional deletion of Il21 from CD4+ T cells lead to a reduction in the number of plasma cells and a reduction in C. rodentium-specific IgG. Finally, using bone marrow chimeric mice, we found that B cells and T cells require IL-21R expression to mediate the clearance of C. rodentium. These findings establish that IL-21-producing CD4+ T cells can inhibit the dissemination of enteric bacterial pathogens and enhance their clearance from the site of infection via humoral immunity.