Immune elimination of Leishmania major in mice: implications for immune memory, vaccination, and reactivation disease.

Infection of susceptible BALB/c mice with a large, moderate, or low number of Leishmania major parasites respectively results in progressive disease, the formation of substantial but stable lesions, denoted as borderline disease, and the absence of a visible lesion. Infection with a low number of parasites results over the long term in either subclinical infections or an asymptomatic state. Subclinical mice produce a predominant Th1 response and are resistant to challenge, in contrast to their asymptomatic counterparts. Statistical and other evidence suggest that the asymptomatic state can arise from a subclinical state following parasite clearance, with consequent loss of resistance. Cell transfer studies demonstrate unequivocally that immune cells from subclinical mice can protect naive mice against a pathogenic challenge and can clear the parasite, leaving the mice susceptible to a rechallenge infection. This susceptibility is associated with the disappearance of both parasite-specific effector and memory T cells from secondary lymphoid organs. These findings have implications for vaccination, maintenance of memory, and prevention of reactivation disease.

A valid ELISPOT assay for enumeration of ex vivo, antigen-specific, IFNgamma-producing T cells.

We describe an ELISPOT technique for the detection of antigen specific IFNgamma-producing T cells. The technique is performed on spleen cells plated directly ex vivo into ELISPOT trays without an in vitro pre-culture step. Thus, the assay is likely to reflect the in vivo activity of the cells. We have found that very high cell densities (at least 10(6) cells/well) are required for optimal detection of spot forming cells, and only at a high density of cells is the number of spots detected linearly related to the number of primed cells plated. If lower numbers of antigen primed cells are used, then unprimed spleen cells from syngeneic mice can be added to the well to increase the cell density. Under these conditions, we find that the number of spots is linearly proportional to the number of primed cells plated, even if these are well below a million cells/well. Experiments with MHC congenic mice indicate that the high density of spleen cells required to obtain optimal spot formation reflects a requirement for an MHC restricted function, probably efficient antigen presentation to T cells. The formation of IFNgamma spots is antigen dependent and abrogated by depleting the antigen primed cells of T cells. We conclude that this linear assay can be used to efficiently detect ex vivo antigen-specific IFNgamma-producing T cells.