Our lab discovered, within inflammatory cytokine genes, a novel class of intragenic elements that by evoking a cellular stress response, regulate expression of the gene carrying that element at either mRNA splicing or mRNA translation. Once activated by double-helical RNA, the interferon-inducible RNA-dependent stress protein kinase PKR phosphorylates translation initiation factor eIF2α, to inhibit translation, critical for coping with stress. We revealed short, 100-200 nucleotide elements within cellular genes that, once transcribed, fold into RNA structures that potently activate PKR in the vicinity of the RNA and thereby tightly regulate expression in cis. Intragenic RNA activators of PKR can (a) attenuate translation of the encoded mRNA by over an order of magnitude by activating PKR and inducing eIF2α phosphorylation, exemplified by the immune interferon gene (IFN-γ) that through this negative control, avoids hyperinflammation; or (b) potently enhance pre-mRNA splicing efficiency by activating PKR and inducing nuclear eIF2α phosphorylation, thus enabling efficient early-stage spliceosome assembly, exemplified by adult and fetal globin genes and tumor necrosis factor (TNF-α) that activates PKR through an RNA pseudoknot conserved from teleost fish to humans. Our work revealed a novel function for eIF2α phosphorylation in control of mRNA splicing that clarifies the need for PKR activation, extending its role from negative control of translation to positive control of splicing. These opposite outcomes considerably expand the scope of gene regulation enabled by these elements. Our findings explain why genes vital to survival that must be expressed highly efficiently, exemplified by IFN-γ and TNF-α, key mediators of protective immunity and the antitumor response, and the globin genes, indispensable for life, acquired intragenic RNA activators of PKR. Current research focuses on the characterization and role of intragenic RNA activators of PKR in regulating expression of the inflammatory response modulator, suppressor of cytokine signaling (SOCS3), and human immunodeficiency virus (HIV).

Kaempfer Lab studied the molecular biology of cytokine gene expression from its outset. Early focus on regulation of inflammatory cytokine gene expression also allowed us to investigate how it is disturbed in diseases. This laid the foundation for our later work on superantigen toxins and cytokine storm. The Pentagon recruited Ray into biodefense research based on this expertise which provided essential tools. Currently, our biodefense research focuses on wound infections by multidrug-resistant organisms, a major challenge today, at the end of the antibiotics era. Based on detailed insight into molecular mechanisms of human inflammatory signaling, we are designing novel, broad-spectrum therapeutics that protect from lethal infections.

During severe bacterial or viral infections, disease and death are often caused by an overly strong immune response of the human host (‘cytokine storm’). Acute toxic shock is induced by superantigen toxins, a diverse set of proteins secreted by Gram-positive bacterial strains that overstimulate inflammatory gene expression by orders of magnitude. The need to protect from superantigen toxins led to our discoveries that the principal costimulatory receptor, CD28, and its coligand, B7-2 (CD86), previously thought to have only costimulatory function, are actually critical superantigen receptors. Binding of the superantigen into the homodimer interfaces of these costimulatory receptors triggers B7-2/CD28 costimulatory receptor engagement, leading to excessive pro-inflammatory signaling. This finding led to the design of receptor dimer interface mimetic peptides that block binding of superantigen and thus protect from death. It then turned out that such peptides will protect also from Gram-negative bacterial infections and polymicrobial sepsis. One such CD28 mimetic peptide is now advancing in a US Phase 3 clinical trial to protect from lethal necrotizing wound infections by flesh-eating bacteria. Based on fundamental insight into signaling by costimulatory receptors and their structure, we are designing and developing novel therapeutics that show even greater efficacy. These molecules are host-oriented therapeutics that target the human immune system itself, protecting thereby against a broad range of pathogens while avoiding emergence of resistance.

A new project in the cancer field with promising pilot results. Tumor cells evade immune surveillance by silencing expression of human inflammatory genes that provide the natural response against cancer. Antibodies currently used to offset this immune suppression cause severe side effects in patients. We are developing a novel molecular approach that allows for relief of inflammatory silencing while maintaining a normal inflammatory response.