Recent advances in two-photon microscopy have provided a new way of visualizing the behavior of fluorescently tagged cells within their natural microenvironment. This technology has allowed for generating a detailed picture of the cellular interaction dynamics operant in the activation of T cells and B cells during primary immune responses within secondary lymphoid organs. In contrast, relatively little is known about the migratory and interactive behavior of effector T cells within peripheral organs. We have recently developed a two-photon microscopy model that enables tracking of cytotoxic T cells within tumors. We have demonstrated that tumor-infiltrating T lymphocytes (TILs) follow random migratory paths and that their migratory properties depend on signals from the T-cell receptor. We further showed that TILs underwent short- and long-term interactions with tumor cells as well as macrophages. Recently, we succeeded in dynamic imaging of the distribution of fluorescently tagged molecules within TILs at subcellular resolution, which will be instrumental for defining the composition of the lytic synapse as well as the targeted release of cytotoxic granules by these cells. The purpose of this review is to put our findings into the context of the current literature and to point out the molecular cues mediating effector T-cell function as candidates for future investigation.

Severe persistent asthma (SPA) and chronic obstructive pulmonary disease (COPD) are both associated with non-reversible airflow limitation and airway neutrophilia.

To compare inflammatory cell profiles and T lymphocyte subsets between SPA and COPD patients with similar severity of airflow limitation.

Sputum induction and lung function tests were performed in 15 COPD patients aged (mean+/-SD) 68+/-8 years, ex-smokers, mean forced expiratory volume in 1 s (FEV1) 45% of predicted (% pred) and 13 SPA aged 55+/-10 years, non-smokers, mean FEV(1) 49% pred. All patients were on inhaled steroid treatment. Eight asthmatics exhibited irreversible airflow limitation. Differential cell count, metachromatic cell count and double immunocytochemistry for the analysis of T lymphocyte subsets were performed on sputum slides.

COPD patients had increased sputum neutrophils in comparison with SPA (P<0.03), but similar to SPA with fixed obstruction. In COPD sputum neutrophils negatively correlated with the lung transfer factor for carbon monoxide (KCO) (r=-0.462, P=0.04). SPA showed significantly increased eosinophils and metachromatic cells vs. COPD patients (P<0.04, P<0.007, respectively). Increased CD4/CD8 and decreased CD4-IFN-gamma/CD4-IL4+ cell ratio (P<0.001) were found in SPA vs. COPD. In SPA, CD4/CD8+ cell ratio correlated with sputum eosinophils (r=0.567, P=0.04).

In spite of treatment with inhaled steroids, SPA and COPD exhibit distinct sputum inflammatory cell patterns, although SPA with fixed airflow limitation and COPD patients have similar numbers of neutrophils.

Oral administration of antigen induces a state of tolerance that is associated with activation of CD8+ T cells that can transfer unresponsiveness to naïve syngeneic hosts. These T cells are not lytic, but they inhibit development of antibody, CD4+ T helper cell, and CD8+ cytotoxic T lymphocyte (CTL) responses upon adoptive transfer into naïve, syngeneic mice. In addition, we have shown that depletion of gammadelta T cells by injection of the anti-delta chain antibody (GL3) down modulates the expression of gammadelta T-cell receptor (TCR) and inhibits the induction of oral tolerance to ovalbumin. Oral administration of antigen also fails to induce tolerance in TCR delta-chain knockout mice suggesting that gammadelta T cells play a critical, active role in tolerance induced by orally administered antigen. To further study the contribution of gammadelta T cells to tolerance, murine gammadelta T cells were isolated from intraepithelial lymphocytes (IEL) of the small intestine by stimulation with splenic filler cells, concanavalin A and growth factors. gammadelta IEL lines demonstrated lytic activity in a redirected lysis assay. gammadelta T-cell clones express different gammadelta TCR genes and secrete large amounts of interleukin (IL)-10, but little or no IL-2, IL-4, or interferon-gamma. gammadelta IEL clones expressed transforming growth factor-beta1 and macrophage migration inhibitory factor, as well as IL-10, mRNA. Moreover, gammadelta T-cell clones potently inhibited the generation of CTL responses by secreted molecules rather than by direct cell-to-cell contact.

We investigated the relationship between transforming growth factor-beta (TGF-beta)-secreting T-regulatory (Tr) cells and anti-B16 melanoma immunity, and studied the association of early cytokines expressed at tumour sites with the generation of Tr cells. A large number of CD4(+) Tr cells producing interleukin (IL)-4, IL-10 and TGF-beta accumulated with functionally depressed CD8(+) cytotoxic T lymphocytes (CTLs) at tumour sites on day 20 after subcutaneous (s.c.) inoculation of B16 tumour cells. Tr cells consisted of two populations, which were termed T helper 3 (Th3) and Tr1 cells. B16-infiltrating Tr cells strongly inhibited the generation of B16-specific T helper 1 (Th1) cells in a TGF-beta-dependent manner and were assumed to suppress effective generation of CTLs. In addition, B16 cells markedly progressed in mice transferred adoptively by the cultured B16-infiltrating Tr cells compared with untreated mice. The capacity of these Tr cells to produce TGF-beta was hampered by neutralizing anti-IL-10 and partly anti-IL-4 monoclonal antibodies (mAbs) injected intralesionally during the early development of B16 tumours, and this treatment markedly attenuated B16 growth. Furthermore, a lesional injection of recombinant mouse IL-10 at an early tumour site resulted in the vigorous progression of B16 tumours. These results provide evidence that Tr cells, belonging to the T helper 3/T-regulatory 1 (Th3/Tr1) type, are activated in B16-bearing hosts under the influence of T helper 2 (Th2) cytokines, mainly IL-10 (produced at early tumour lesions), and that this regulatory T-cell population functions as a suppressor of anti-B16 immunity.

Allograft rejection remains the single largest impediment to success in corneal transplantation. This article briefly reviews our current understanding of some fundamental aspects of corneal immunology and the pathogenetic mechanisms underlying corneal graft rejection. As knowledge increases, it is hoped that a better understanding of the immunobiology may result in improved preventive and therapeutic measures.

A non-human primate model for acquired immunodeficiency syndrome (AIDS), the simian immunodeficiency virus (SIV)-infected rhesus monkey, was used to explore the role of the AIDS virus-specific cytotoxic T-lymphocyte (CTL) response in disease pathogenesis. This CTL response was measured using the major histocompatibility complex (MHC) class I/peptide tetramer technology. Large numbers of tetramer-binding CD8+ T lymphocytes were demonstrable not only in the peripheral blood, but in lymph nodes and even in semen of chronically SIV-infected monkeys. The central role of these effector T lymphocytes in containing SIV spread during primary infection was demonstrated by showing that early SIV clearance during primary infection correlated with the emergence of the tetramer binding CD8+ T lymphocytes and that in vivo depletion of CD8+ lymphocytes eliminated the ability of the infected monkeys to contain SIV replication. These observations suggest that an effective AIDS vaccine should elicit a potent virus-specific CTL response. In fact, a live, recombinant SIV vaccine constructed using the attenuated pox virus vector modified vaccinia Ankara (MVA) elicited a high-frequency CTL response, comparable in magnitude to that elicited by SIV infection itself. This suggests that vaccine modalities such as MVA may prove useful in creating an effective human immunodeficiency virus (HIV) vaccine. These studies also indicate the power of both the SIV/macaque model and MHC class I/peptide tetramers for assessing AIDS vaccine strategies.

In order to determine the mechanism of tumour destruction by tumour-infiltrating lymphocytes (TIL), we examined the ability of both CD4+ and CD8+ effector TIL, and TIL clones, to manifest granzyme-mediated and Fas-mediated destruction of tumour targets. In many in vitro studies TIL have been shown to manifest anti-tumour reactivity, yet many tumours escape immunological destruction. To investigate the role of Fas expression and the concomitant sensitivity to the inducibility of apoptotic death, we derived TIL from four melanomas and one glioma. The glioma, and all but one of the melanomas, expressed Fas, but Fas-mediated apoptosis could only be detected if the targets were treated with cyclohexamide. The melanomas and the glioma all expressed detectable cytoplasmic Bcl-2 protein, known to exert anti-apoptotic activity. Lysis of tumours by CD8-enriched cultures and CD8+ clones was Ca2+-dependent and could not be modified by an anti-Fas MoAb. In CD4-enriched cultures or CD4+ clones with cytotoxic potential against tumour cells, cytotoxicity was also Ca2+-dependent. As Ca2+-dependent cytotoxicity is usually the result of secretion of perforin/granzyme-B, we investigated the presence of perforin in cytotoxic CD4+ clones and demonstrated the presence of granular deposits of this enzyme in some of the CD4+ clones. Although an anti-Fas MoAb did not block the lysis of melanoma targets by CD4+ clones, the examination of Fas-dependent targets demonstrated that these clones also had the potential to kill by the Fas/Fas ligand system. These data suggest that the predominant mechanism in tumour killing by TIL appears to be perforin-granzyme-dependent, and that the solid tumour cell lines we studied are less susceptible to Fas-mediated apoptosis. As non-apoptotic pathways may enhance tumour immunogenicity, exploitation of the perforin-granzyme-dependent cytotoxic T lymphocyte (CTL) pathways may be important for achieving successful anti-tumour responses.

When T effector cells meet antigen-bearing target cells, there is a specific accumulation of T-cell receptors, co-receptors and structural proteins at the point of cell-cell contact. Ly49 inhibitory receptors bind to murine major histocompatibility complex (MHC) class I molecules and prevent natural killer-(NK) cell cytotoxicity. In this study we have tested whether inhibitory receptors accumulate at the point of cell-cell contact when NK cells encounter target cells bearing MHC class I ligands for those inhibitory receptors. We have used RNK-16 effector cells that express Ly49A receptors and have found that there was a specific accumulation of Ly49A receptors at the point of NK cell-target cell contact when the target cells expressed H-2Dd. We also observed that engagement of Ly49A on NK cells resulted in an altered redistribution of potential triggering receptors CD2 and NKR-P1. These data indicate that inhibitory receptors, like activating receptors, may specifically aggregate at the point of cell-cell contact which may be necessary for them to mediate their full inhibitory effect.

The genes that encode molecules involved in antigen presentation within the class I and class II regions of the mammalian major histocompatibility complex (MHC) include several that are highly polymorphic. There is evidence that this polymorphism is maintained by positive selection, most likely overdominant selection, relating to their role in presenting foreign peptides to T cells. This selection can maintain allelic lineages for much longer periods of time than neutral polymorphisms are expected to last, but sharing of polymorphic amino acid motifs among species of different mammalian orders is due to independent (or convergent) evolution rather than common ancestry. It has been suggested that interallelic recombination (gene conversion) plays a role in enhancing polymorphism, but there is evidence of striking differences among loci with respect to the rate at which such recombination has contributed to current polymorphism. Recent attempts to interpret linkage relationships in the MHC region as evidence of ancient genomic duplications are not supported by phylogenetic analysis. Rather, natural selection may have played a role in the linkage of other genes to those of the MHC.

Large granular lymphocytes (LGL) proliferation is characterized by expansion of cytotoxic lymphocytes and associated with neutropenia. In a case of CD3+ LGL-proliferation the epinephrine stimulation test (EST) induced a striking elevation of CD3+, CD8+, CD57+ LGL in peripheral blood from 2.7 x 10(9)/l to 20 x 10(9)/l and might be an additional diagnostic tool in patients with normal or low absolute numbers of circulating LGL. After treatment with steroids, plasma elastase--a marker of neutrophil destruction--decreased from 162 to 40 micrograms/l (normal < 47 micrograms/l) which correlated well with a simultaneous increase in peripheral neutrophil counts from 0.14 to 1.0 x 10(9)/l. This finding supports the hypothesis that neutropenia in CD3+ LGL proliferation is due to neutrophil destruction, possibly mediated by LGL.