IκBε is a key regulator of B cell expansion by providing negative feedback on cRel and RelA in a stimulus-specific manner.

The transcription factor NF-κB is a regulator of inflammatory and adaptive immune responses, yet only IκBα was shown to limit NF-κB activation and inflammatory responses. We investigated another negative feedback regulator, IκBε, in the regulation of B cell proliferation and survival. Loss of IκBε resulted in increased B cell proliferation and survival in response to both antigenic and innate stimulation. NF-κB activity was elevated during late-phase activation, but the dimer composition was stimulus specific. In response to IgM, cRel dimers were elevated in IκBε-deficient cells, yet in response to LPS, RelA dimers also were elevated. The corresponding dimer-specific sequences were found in the promoters of hyperactivated genes. Using a mathematical model of the NF-κB-signaling system in B cells, we demonstrated that kinetic considerations of IκB kinase-signaling input and IκBε's interactions with RelA- and cRel-specific dimers could account for this stimulus specificity. cRel is known to be the key regulator of B cell expansion. We found that the RelA-specific phenotype in LPS-stimulated cells was physiologically relevant: unbiased transcriptome profiling revealed that the inflammatory cytokine IL-6 was hyperactivated in IκBε(-/-) B cells. When IL-6R was blocked, LPS-responsive IκBε(-/-) B cell proliferation was reduced to near wild-type levels. Our results provide novel evidence for a critical role for immune-response functions of IκBε in B cells; it regulates proliferative capacity via at least two mechanisms involving cRel- and RelA-containing NF-κB dimers. This study illustrates the importance of kinetic considerations in understanding the functional specificity of negative-feedback regulators.

Intertwined pathways of programmed cell death in immunity.

Programmed cell death (PCD) occurs widely in species from every kingdom of life. It has been shown to be an integral aspect of development in multicellular organisms, and it is an essential component of the immune response to infectious agents. An analysis of the phylogenetic origin of PCD now shows that it evolved independently several times, and it is fundamental to basic cellular physiology. Undoubtedly, PCD pervades all life at every scale of analysis. These considerations provide a backdrop for understanding the complexity of intertwined, but independent, cell death programs that operate within the immune system. In particular, the contributions of apoptosis, autophagy, and necrosis in the resolution of an immune response are considered.

Lipid-dependent cytotoxicity by the lipase PLRP2 and by PLRP2-positive cytotoxic T lymphocytes (CTLs).

IL-4 induces a lipase, pancreatic lipase related protein 2 (PLRP2), in cytotoxic T lymphocytes (CTLs). Because PLRP2 in semen can mediate lipid-dependent toxicity to sperm, we questioned whether CTL-derived PLRP2 could support similar cytotoxicity toward tumor cells. Recombinant PLRP2 was toxic to P815 tumor cells in 48 h when lipid and another protein, colipase, were present. However, PLRP2-positive CTLs (induced with many lots of IL-4) were unable to mediate lipid-dependent cytotoxicity. Notably, CTLs induced with only one lot of IL-4 had lipid-dependent cytotoxicity. The exceptional lot of IL-4 was effective in multiple experiments at inducing lipid-dependent cytotoxicity. The lipid-dependent cytotoxicity it induced was determined to be perforin-independent. CTLs induced with IL-4 that was unable to induce lipid-dependent cytotoxicity had mRNA for PLRP2 but not mRNA for colipase. Therefore, we added exogenous colipase to the CTL assays but still cytotoxicity was unchanged. We conclude (1) that lipid-dependent cytotoxicity, promoted by the lipase PLRP2 and colipase, will kill tumor cells and (2) that more than PLRP2 alone is required for lipid-dependent cytotoxicity mediated by CTLs.

Pancreatic lipase-related protein 2 (PLRP2) induction by IL-4 in cytotoxic T lymphocytes (CTLs) and reevaluation of the negative effects of its gene ablation on cytotoxicity.

Pancreatic lipase-related protein 2 (PLRP2) is induced by IL-4 in vitro in cytotoxic T lymphocyte (CTL) clones and CTLs from immunized wild-type (WT) PLRP2(+/+) are more cytotoxic than PLRP2(-/-) CTLs, suggesting to previous investigators that the lipase PLRP2 might support CTL functions. Here, we further evaluate PLRP2 in CTLs. We found that PLRP2 was optimally induced in splenocytes by 3.5 x 10(-8) M IL-4 by day 6 after activation and was restricted to CD8(+) T cells. PLRP2 mRNA was detected inconsistently (and at low levels) after activation in the presence of IL-2. Cytotoxicity in 4 h (51)Cr assays of WT CTLs was approximately 3-fold the activity of PLRP2(-/-) CTLs cultured with IL-4 and, with IL-2, was unexpectedly approximately 2 fold the activity of PLRP2(-/-) CTLs. Thus, PLRP2 gene ablation affected short-term (perforin-dependent) cytotoxicity, even under the IL-2 conditions. Other variables failed to account for the reduced cytotoxicity. Granzyme B levels, activation markers, and CD8(+) T cell frequencies were similar for WT vs. PLRP2(-/-) CTLs (with either cytokine). Addition of rPLRP2 to IL-4 induced PLRP2(-/-) CTLs (or to cytotoxic granule extracts) failed to increase lysis, suggesting that the missing mediator is more than released PLRP2. Cytotoxicity of WT and PLRP2(-/-) CTLs was similar in 2-day tumor survival assays with IL-4, which can be mediated by perforin-independent mechanisms. We conclude that extracellular PLRP2 lipase is unable to directly augment the cytotoxicity that was lost by PLRP2 ablation and that after reevaluation, the question of what is PLRP2's role in CD8 T cells is still unanswered.

Hydrolysis of tumor cell lipids after CTL-mediated death.

Contributions of lipases to CTL function have been debated, including if T cell lipases damage target cells. Expression of the lipase pancreatic lipase-related protein 2 (PLRP2) was previously found in IL-4 cultured lymphocyte cell lines but absent from IL-2 cultured lymphocytes. Here, we evaluated IL-2 and IL-4 induced CTLs for hydrolysis of target cell lipids and killing. Using anti-CD3 redirected lysis of [(3)H]-oleic acid-labeled P815 tumor cells, we detected the release of the radioactive fatty acid (FA). When PLRP2(+/+) and PLRP2(-/-) CTLs were compared, there was more killing by the PLRP2(+/+) CTLs. However, [(3)H]-oleic acid release was similar per dead P815, suggesting that lipid hydrolysis was produced by the dead P815s rather than by PLRP2. The FA release and death were completely dependent on perforin and also occurred when P815s were killed by perforin-containing T cell granule extracts that lacked lipase activity. Death by the cytotoxic granules extracts was unaffected by the addition of lipases. A lipase inhibitor, tetrahydrolipstatin, blocked FA release without affecting CTL-mediated cytotoxicity. Also, CTL-mediated death caused as much FA release as death by disruption of cells by freeze-thawing. The released oleic acid may be sufficient to promote secondary apoptotic responses after CTL-induced trauma.

Regulation of perforin lysis: implications for protein disulfide isomerase proteins.

Perforin, a membrane-permeabilizing protein, is important to T cell cytotoxic action. Perforin has potential to damage the T cell in the endoplasmic reticulum (ER), is sequestered in granules, and later is exocytosed to kill cells. In the ER and after exocytosis, calcium and pH favor perforin activity. We found a novel perforin inhibitor associated with cytotoxic T cell granules and termed it Cytotoxic Regulatory Protein 2 (CxRP2). CxRP2 blocked lysis by granule extracts, recombinant perforin and T cells. Its effects lasted for hours. CxRP2 was calcium stable and refractory to inhibitors of granzyme and cathepsin proteases. Through mass spectrometric analysis of active 50-100 kDa proteins, we identified CxRP2 candidates. Protein disulfide isomerase A3 was the strongest candidate but was unavailable for testing; however, protein disulfide isomerase A1 had CxRP2 activity. Our results indicate that protein disulfide isomerases, in the ER or elsewhere, may protect T cells from their own perforin.

Low dose IL-15 induces snap arming of CD44(low) T lymphocytes in the absence of antigen.

It is widely accepted that naïve T cells require two signals, antigen recognition and co-simulation, to become cytotoxic over the course of 3-5days. However, we observed that freshly isolated murine splenocytes without exposure to antigen become cytotoxic within 24h after culture with IL-15. IL-15 is a cytokine that promotes homeostatic proliferation, maintenance and activation of memory T cells. The induced cytotoxicity, measured by anti-CD3 redirected (51)Cr release, represented the combined activity of T cells regardless of their antigen specificity, and proceeded even when CD44(hi) (memory-associated phenotype) CD8(+) T cells were depleted. Cytotoxic capacity was perforin-dependent and occurred without detectable up-regulation of granzyme B or cell division. After induction, the phenotypic markers for the memory subset and for activation remained unchanged from the expression of resting T cells. Our work suggests that T cells may gain cytotoxic potential earlier than currently thought and even without TCR stimulation.

Induction of granzyme B and T cell cytotoxic capacity by IL-2 or IL-15 without antigens: multiclonal responses that are extremely lytic if triggered and short-lived after cytokine withdrawal.

The purpose of these studies was to determine the minimal requirements to induce granzyme B, cytotoxic granules and perforin-dependent lytic capacity. To our surprise, both IL-2 and IL-15 induced not only proliferation, but also profound granzyme B and lytic capacity from CD8+ T cells in the absence of antigen or TCR-stimulation. Mouse splenocytes were incubated with mouse r-IL-2 or r-IL-15 for three days, tested by anti-CD3 redirected lysis and examined for intracellular granzyme B and for T cell activation markers. With 10(-8) M IL-2 or IL-15, there was excellent lytic activity at 1:1 effector to target ratios mediated by T cells from wild-type but not from perforin-gene-ablated mice, consistent with multiclonal activation. Lower interleukin concentrations induced less lytic activity. Granzyme B was undetectable on day 0, and greatly elevated on day 3 in CD44hi CD8+ T cells as detected by flow cytometry. Cytokines alone elevated the granzyme B as much as concanavalin A combined with the cytokines. Some ex vivo CD8+ T cells were CD122+, as were the cultured granzyme B+ cells, thus both populations had low-affinity receptors for the interleukins. Only some of the activated cells were proliferating as detected by CFSE labeling. When the cytokines were withdrawn, the cells lost lytic activity within 24 h and then within the next 24 h, died. Our results suggest that high concentrations of either IL-2 or IL-15 will activate the lytic capacity and granzyme B expression of many T cells and that antigen recognition is not required.

IFN-gamma primes macrophages for enhanced TNF-alpha expression in response to stimulatory and non-stimulatory amounts of microparticulate beta-glucan.

beta-(1-->3)-D-Glucan is an integral cell wall component of a variety of fungi, plants, and bacteria. Like the prototypic inflammatory mediator lipopolysaccharide (LPS), some beta-(1--> 3)-D-glucan-containing preparations have been shown to induce the production of proinflammatory cytokines by macrophages. In the present study, we have tested a new microparticulate form of beta-(1--> 3)-D-glucan (MG) from Saccharomyces cerevisiae for its ability to induce proinflammatory cytokine secretion in mouse peritoneal macrophages in vitro, and we have examined the effect of IFN-gamma. MG was rapidly phagocytized by peritoneal macrophages, and these MG-treated macrophages upregulated TNF-alpha, IL-6, and IL-1beta mRNAs and secreted these proinflammatory cytokines. IFN-gamma treatment alone did not induce unstimulated macrophages to produce TNF-alpha. However, a 4 h IFN-gamma pretreatment augmented TNF-alpha secretion by peritoneal macrophages subsequently treated with an optimally stimulatory dose of MG. IFN-gamma pretreatment for 2 h followed by thorough washing and a further 2 h incubation without IFN-gamma still resulted in enhanced TNF-alpha production in response to MG, suggesting that IFN-gamma can prime macrophages for a subsequent proinflammatory response. Most interestingly, we found that IFN-gamma pretreatment of peritoneal macrophages enhanced the TNF-alpha response to amounts of MG that were poorly stimulatory or non-stimulatory in the absence of IFN-gamma priming. These data suggest that a synergy between IFN-gamma and beta-glucan may have evolved to lower the threshold of sensitivity of the innate immune response to fungal pathogens.