Killed but metabolically active microbes: a new vaccine paradigm for eliciting effector T-cell responses and protective immunity.

We developed a new class of vaccines, based on killed but metabolically active (KBMA) bacteria, that simultaneously takes advantage of the potency of live vaccines and the safety of killed vaccines. We removed genes required for nucleotide excision repair (uvrAB), rendering microbial-based vaccines exquisitely sensitive to photochemical inactivation with psoralen and long-wavelength ultraviolet light. Colony formation of the nucleotide excision repair mutants was blocked by infrequent, randomly distributed psoralen crosslinks, but the bacterial population was able to express its genes, synthesize and secrete proteins. Using the intracellular pathogen Listeria monocytogenes as a model platform, recombinant psoralen-inactivated Lm DeltauvrAB vaccines induced potent CD4(+) and CD8(+) T-cell responses and protected mice against virus challenge in an infectious disease model and provided therapeutic benefit in a mouse cancer model. Microbial KBMA vaccines used either as a recombinant vaccine platform or as a modified form of the pathogen itself may have broad use for the treatment of infectious disease and cancer.

No alteration in T lymphocyte expression of CD40 ligand (CD154) in individuals with or at increased risk for insulin-dependent diabetes mellitus.

CD40 ligand (CD40L) regulates multiple phases of the humoral and cellular immune response through binding to CD40. Previous investigations have suggested that insulin-dependent diabetes (IDDM) in both humans and nonobese diabetic mice may be strongly influenced by similar immunoregulatory molecules. As persons with or at increased risk for the disease are characterized by a number of immunological abnormalities, including that of self-reactive autoantibody production (e.g. islet cell cytoplasmic autoantibodies), we analyzed the expression of CD40L on T lymphocytes (CD3+ cells) in a series of individuals with newly diagnosed IDDM (n = 11), nondiabetic relatives of IDDM probands at increased risk for the disease (n = 21; islet cell cytoplasmic autoantibodies positive; Juvenile Diabetes Foundation titer, > or = 20), and healthy controls (n = 13). Both phorbol myristate acetate (PMA)-stimulated and unstimulated peripheral blood mononuclear cells from study subjects were analyzed by flow cytometry with a series of phenotypic antibody markers (CD3, CD40L, and isotype controls). The kinetics of CD3 and CD40L expression on peripheral blood mononuclear cells under PMA-stimulated and unstimulated conditions were similar in the three study groups (6, 24, and 48 h; all P = NS). Similarly, unstimulated and PMA stimulated CD40L expressions (percentage of positive cells and level) on CD3+ cells from newly diagnosed IDDM patients and persons at increased risk for the disease were similar to those in healthy controls (6, 24, and 48 h; all P = NS). These findings do not support abnormal CD40L expression as the mechanism underlying the functional defect(s) in communication between T lymphocytes and antigen-presenting cells that allows for autoantibody production or the inability of individuals to regulate antiself immunity in IDDM.

Increased expression of the lymphocyte early activation marker CD69 in peripheral blood correlates with histologic evidence of cardiac allograft rejection.

BACKGROUND: The human leukocyte membrane protein CD69 is an early activation marker induced in T lymphocytes, B cells, and natural killer cells in response to inflammatory stimuli. Cardiac catheterization and endomyocardial biopsy remain the "gold standard" for diagnosis of rejection after transplantation, and noninvasive methods of rejection surveillance have long been sought. We studied CD69 membrane protein expression in peripheral blood T lymphocytes obtained from pediatric cardiac transplant recipients at the time of biopsy and correlated the results with histologic rejection scores. METHODS: Heparinized whole blood samples were obtained from pediatric cardiac transplant recipients at the time of cardiac biopsy, as well as from control subjects. Lymphocytes were labeled with antibodies for CD3, CD4, CD8, and CD69 and analysis performed using flow cytometric methods. RESULTS: Resting CD69 expression (measured as a percentage of gated events) was significantly increased in patients with concurrent histologic evidence of rejection (International Society for Heart and Lung Transplantation grade > or =3A) when compared to those with minimal or no rejection and controls. Although statistically significant for both lymphocyte subsets, this relationship was more pronounced for CD8+ T cells (P<0.001) than for CD4+ T cells (P=0.001). When data were analyzed by rejection score, a percentage activation of the CD8+ subset (CD69+/CD8+ cells as a percentage of total gated events) exceeding 15% correlated with significant rejection. CONCLUSIONS: Measurement of the expression of the early activation marker CD69 in peripheral blood lymphocytes by flow cytometry may provide a noninvasive means of assessing immune activation and possible rejection in cardiac transplant recipients.

Autoregulation of human monocyte-derived dendritic cell maturation and IL-12 production by cyclooxygenase-2-mediated prostanoid production.

PG added to cell culture profoundly affect the in vitro maturation and function of monocyte-derived dendritic cells (MDC). Because unstimulated monocytes express cyclooxygenase (COX)-1, and COX-2 when activated, we examined whether MDC express these enzymes and produce prostanoids that autoregulate maturation and IL-12 production. Immature MDC (I-MDC) and mature MDC express COX-1, but, unlike monocytes, both MDC populations constitutively express COX-2. However, COX-2 regulation in both MDC populations differs from monocytes, as IL-4 does not suppress enzyme expression. COX-2 is functional in MDC as a specific inhibitor, NS-398, significantly reduces PGE(2) production. I-MDC undergoing maturation with soluble CD40 ligand (sCD40L) increase PGE(2) synthesis, but prostanoid synthesis is switched to COX-1. However, with IFN-gamma present, sCD40L-stimulated PG metabolism is redirected to COX-2, and PGE(2) synthesis increases severalfold. Endogenous PG production by MDC does not regulate CD40, CD80, CD86, or HLA DR expression; however, it does promote MDC maturation, as NS-398 significantly reduces CD83 expression in I-MDC matured with sCD40L/IFN-gamma. PG produced through COX-2 also autoregulate IL-12, but the effects are dependent on the MDC maturation state. Blocking COX-2 reduces I-MDC secretion of IL-12p40, whereas it increases IL-12p40 and p70 production by maturing MDC. COX-2-mediated PG production impacts MDC function as maturing these cells in the presence of NS-398 yields MDC that stimulate significantly more IFN-gamma in an allogeneic mixed lymphocyte response than MDC matured without this inhibitor. These studies demonstrate that MDC express both COX isoforms constitutively and produce prostanoids, which autoregulate their maturation and function.

Activation of CD1d-restricted T cells protects NOD mice from developing diabetes by regulating dendritic cell subsets.

CD1d-restricted invariant NKT (iNKT) cells are immunoregulatory cells whose loss exacerbates diabetes in nonobese diabetic (NOD) female mice. Here, we show that the relative numbers of iNKT cells from the pancreatic islets of NOD mice decrease at the time of conversion from peri-insulitis to invasive insulitis and diabetes. Conversely, NOD male mice who have a low incidence of diabetes showed an increased frequency of iNKT cells. Moreover, administration of alpha-galactosylceramide, a potent activating ligand presented by CD1d, ameliorated the development of diabetes in NOD female mice and resulted in the accumulation of iNKT cells and myeloid dendritic cells (DC) in pancreatic lymph nodes (PLN), but not in inguinal lymph nodes. Strikingly, injection of NOD female mice with myeloid DC isolated from the PLN, but not those from the inguinal lymph nodes, completely prevented diabetes. Thus, the immunoregulatory role of iNKT cells is manifested by the recruitment of tolerogenic myeloid DC to the PLN and the inhibition of ongoing autoimmune inflammation.