Human ß-defensin 2 ameliorates acute GVHD by limiting ileal neutrophil infiltration and restraining T cell receptor signaling.

Acute graft-versus-host disease (aGVHD), which is driven by allogeneic T cells, has a high mortality rate and limited treatment options. Human ß-defensin 2 (hBD-2) is an endogenous epithelial cell-derived host-defense peptide. In addition to its antimicrobial effects, hBD-2 has immunomodulatory functions thought to be mediated by CCR2 and CCR6 in myeloid cells. In this study, we analyzed the effect of recombinant hBD-2 on aGVHD development. We found that intestinal ß-defensin expression was inadequately induced in response to inflammation in two independent cohorts of patients with aGVHD and in a murine aGVHD model. Treatment of mice with hBD-2 reduced GVHD severity and mortality and modulated the intestinal microbiota composition, resulting in reduced neutrophil infiltration in the ileum. Furthermore, hBD-2 treatment decreased proliferation and proinflammatory cytokine production by allogeneic T cells in vivo while preserving the beneficial graft-versus-leukemia effect. Using transcriptome and kinome profiling, we found that hBD-2 directly dampened primary murine and human allogeneic T cell proliferation, activation, and metabolism in a CCR2- and CCR6-independent manner by reducing proximal T cell receptor signaling. Furthermore, hBD-2 treatment diminished alloreactive T cell infiltration and the expression of genes involved in T cell receptor signaling in the ilea of mice with aGVHD. Together, we found that both human and murine aGVHD were characterized by a lack of intestinal ß-defensin induction and that recombinant hBD-2 represents a potential therapeutic strategy to counterbalance endogenous hBD-2 deficiency.

Recombinant human ß-defensin 2 delivery improves smoking-induced lung neutrophilia and bacterial exacerbation.

Treatment of the cigarette smoke-associated lung diseases, such as chronic obstructive pulmonary disease (COPD), has largely focused on broad-spectrum anti-inflammatory therapies. However, these therapies, such as high-dose inhaled corticosteroids, enhance patient susceptibility to lung infection and exacerbation. Our objective was to assess whether the cationic host defense peptide, human ß-defensin 2 (hBD-2), can simultaneously reduce pulmonary inflammation in cigarette smoke-exposed mice while maintaining immune competence during bacterial exacerbation. Mice were exposed to cigarette smoke acutely (4 days) or chronically (5 days/wk for 7 wk) and administered hBD-2 intranasally or by gavage. In a separate model of acute exacerbation, chronically exposed mice treated with hBD-2 were infected with nontypeable Haemophilus influenzae before euthanasia. In the acute exposure model, cigarette smoke-associated pulmonary neutrophilia was significantly blunted by both local and systemic hBD-2 administration. Similarly, chronically exposed mice administered hBD-2 therapeutically exhibited reduced pulmonary neutrophil infiltration and downregulated proinflammatory signaling in the lungs compared with vehicle-treated mice. Finally, in a model of acute bacterial exacerbation, hBD-2 administration effectively limited neutrophil infiltration in the lungs while markedly reducing pulmonary bacterial load. This study shows that hBD-2 treatment can significantly attenuate lung neutrophilia induced by cigarette smoke exposure while preserving immune competence and promoting an appropriate host-defense response to bacterial stimuli.

Fish oil replacement prevents, while docosahexaenoic acid-derived protectin DX mitigates end-stage-renal-disease in atherosclerotic diabetic mice.

Diabetic nephropathy (DN) remains the major cause of end-stage renal disease (ESRD). We used high-fat/high-sucrose (HFHS)-fed LDLr-/- /ApoB100/100 mice with transgenic overexpression of IGFII in pancreatic ß-cells (LRKOB100/IGFII) as a model of ESRD to test whether dietary long chain omega-3 polyunsaturated fatty acids LCω3FA-rich fish oil (FO) could prevent ESRD development. We further evaluated the potential of docosahexaenoic acid (DHA)-derived pro-resolving lipid mediators, 17-hydroxy-DHA (17-HDHA) and Protectin DX (PDX), to reverse established ESRD damage. HFHS-fed vehicle-treated LRKOB100/IGFII mice developed severe kidney dysfunction leading to ESRD, as revealed by advanced glomerular fibrosis and mesangial expansion along with reduced percent survival. The kidney failure outcome was associated with cardiac dysfunction, revealed by reduced heart rate and prolonged diastolic and systolic time. Dietary FO prevented kidney damage, lean mass loss, cardiac dysfunction, and death. 17-HDHA reduced podocyte foot process effacement while PDX treatment alleviated kidney fibrosis and mesangial expansion as compared to vehicle treatment. Only PDX therapy was effective at preserving the heart function and survival rate. These results show that dietary LCω3FA intake can prevent ESRD and cardiac dysfunction in LRKOB100/IGFII diabetic mice. Our data further reveals that PDX can protect against renal failure and cardiac dysfunction, offering a potential new therapeutic strategy against ESRD.

Targeting of non-dominant antigens as a vaccine strategy to broaden T-cell responses during chronic viral infection.

In this study, we compared adenoviral vaccine vectors with the capacity to induce equally potent immune responses against non-dominant and immunodominant epitopes of murine lymphocytic choriomeningitis virus (LCMV). Our results demonstrate that vaccination targeting non-dominant epitopes facilitates potent virus-induced T-cell responses against immunodominant epitopes during subsequent challenge with highly invasive virus. In contrast, when an immunodominant epitope was included in the vaccine, the T-cell response associated with viral challenge remained focussed on that epitope. Early after challenge with live virus, the CD8+ T cells specific for vaccine-encoded epitopes, displayed a phenotype typically associated with prolonged/persistent antigenic stimulation marked by high levels of KLRG-1, as compared to T cells reacting to epitopes not included in the vaccine. Notably, this association was lost over time in T cells specific for the dominant T cell epitopes, and these cells were fully capable of expanding in response to a new viral challenge. Overall, our data suggests a potential for broadening of the antiviral CD8+ T-cell response by selecting non-dominant antigens to be targeted by vaccination. In addition, our findings suggest that prior adenoviral vaccination is not likely to negatively impact the long-term and protective immune response induced and maintained by a vaccine-attenuated chronic viral infection.

Midline 1 directs lytic granule exocytosis and cytotoxicity of mouse killer T cells.

Midline 1 (MID1) is a microtubule-associated ubiquitin ligase that regulates protein phosphatase 2A activity. Loss-of-function mutations in MID1 lead to the X-linked Opitz G/BBB syndrome characterized by defective midline development during embryogenesis. Here, we show that MID1 is strongly upregulated in murine cytotoxic lymphocytes (CTLs), and that it controls TCR signaling, centrosome trafficking, and exocytosis of lytic granules. In accordance, we find that the killing capacity of MID1(-/-) CTLs is impaired. Transfection of MID1 into MID1(-/-) CTLs completely rescued lytic granule exocytosis, and vice versa, knockdown of MID1 inhibited exocytosis of lytic granules in WT CTLs, cementing a central role for MID1 in the regulation of granule exocytosis. Thus, MID1 orchestrates multiple events in CTL responses, adding a novel level of regulation to CTL activation and cytotoxicity.

The availability of a functional tumor targeting T-cell repertoire determines the anti-tumor efficiency of combination therapy with anti-CTLA-4 and anti-4-1BB antibodies.

It has previously been found that combination therapy with anti-CTLA-4 and anti-4-1BB antibodies may enhance tumor immunity. However, this treatment is not efficient against all tumors, and it has been suggested that variations in tumor control may reflect differences in the immunogenicity of different tumors. In the present report, we have formally tested this hypothesis. Comparing the efficiency of combination antibody therapy against two antigenically distinct variants of the B16.F10 melanoma cell line, we observed that antibody therapy delayed the growth of a variant expressing an exogenous antigen (P<0.0001), while this treatment failed to protect against the non-transfected parental line (P = 0.1850) consistent with published observations. As both cell lines are poorly immunogenic in wild type mice, these observations suggested that the magnitude of the tumor targeting T-cell repertoire plays a major role in deciding the efficiency of this antibody treatment. To directly test this assumption, we made use of mice expressing the exogenous antigen as a self-antigen and therefore carrying a severely purged T-cell repertoire directed against the major tumor antigen. Notably, combination therapy completely failed to inhibit tumor growth in the latter mice (P = 0.8584). These results underscore the importance of a functionally intact T-cell population as a precondition for the efficiency of treatment with immunomodulatory antibodies. Clinically, the implication is that this type of antibody therapy should be attempted as an early form of tumor-specific immunotherapy before extensive exhaustion of the tumor-specific T-cell repertoire has occurred.