Messenger RNA encoding constitutively active Toll-like receptor 4 enhances effector functions of human T cells.

Adoptive T cell therapy of cancer employs a large number of ex-vivo-propagated T cells which recognize their targets either by virtue of their endogenous T cell receptor (TCR) or via genetic reprogramming. However, both cell-extrinsic and intrinsic mechanisms often diminish the in-vivo potency of these therapeutic T cells, limiting their clinical efficacy and broader use. Direct activation of human T cells by Toll-like receptor (TLR) ligands induces T cell survival and proliferation, boosts the production of proinflammatory cytokines and augments resistance to regulatory T cell (Treg) suppression. Removal of the TLR ligand-binding region results in constitutive signalling triggered by the remaining cytosolic Toll/interleukin-1 receptor (TIR) domain. The use of such TIR domains therefore offers an ideal means for equipping anti-tumour T cells with the arsenal of functional attributes required for improving current clinical protocols. Here we show that constitutively active (ca)TLR-4 can be expressed efficiently in human T cells using mRNA electroporation. The mere expression of caTLR-4 mRNA in polyclonal CD8 and CD4 T cells induced the production of interferon (IFN)-γ, triggered the surface expression of CD25, CD69 and 4-1BB and up-regulated a panel of cytokines and chemokines. In tumour-infiltrating lymphocytes prepared from melanoma patients, caTLR-4 induced robust IFN-γ secretion in all samples tested. Furthermore, caTLR-4 enhanced the anti-melanoma cytolytic activity of tumour-infiltrating lymphocytes and augmented the secretion of IFN-γ, tumour necrosis factor (TNF)-α and granulocyte-macrophage colony-stimulating factor (GM-CSF) for at least 4 days post-transfection. Our results demonstrate that caTLR-4 is capable of exerting multiple T cell-enhancing effects and can potentially be used as a genetic adjuvant in adoptive cell therapy.

HLA-B35 correlates with a favorable outcome following adjuvant administration of an HLA-matched allogeneic melanoma vaccine.

This work presents survival data of 42 melanoma patients at high risk for disease recurrence who received an allogeneic melanoma vaccine composed of three cell lines, each matching at least one allele of the recipient's human leukocyte antigen (HLA)-A and -B loci. The 5-year overall survival (OS) rate and disease-free survival (DFS) compared favorably with the standard interferon-α regimen. Interestingly, patients bearing HLA-B35 had significantly better OS and DFS (OS of 100% and DFS of 90% for HLA-B35 vs 56% and 23%, for the non-B35 patients). In contrast, patients expressing HLA-B07 did not fare well with the vaccine. Although the data include a relatively small cohort of patients, it strongly hints toward a correlation between HLA types and potential benefit from anticancer immunotherapy.

A simplified microtechnique for measuring human lymphocyte proliferation after stimulation with mitogen and specific antigen.

Cutaneous leishmaniasis is a parasitic disease characterized by a marked cell-mediated response. In vitro measurements of this response in humans have so far been used to a limited extent probably because of the relatively large amounts of blood demanded for conventional cell proliferation studies. The microtechnique here described enables lymphocyte proliferation to be performed with small amounts of blood (100 microliter) which can be obtained by finger prick, and do not require Ficoll separation prior to cultivation of the mononuclear cells. The blood was aspirated into sterile capillaries, introduced into tubes containing heparin, and directly distributed into wells. The response to Leishmania major, to tuberculin purified protein derivative (PPD) and to mitogen was compared to the response of mononuclear cells after Ficoll separation, and no marked difference was found between the two methods. Using the described method, individuals cured from a Leishmania major infection showed a high response to the specific antigen, as compared to normal controls. The potential use of this microtechnique for epidemiological studies is discussed.

Development of in vitro parameters of cell-mediated immunity in the course of human cutaneous leishmaniasis infection.

The goal of the present study was to determine whether a correlation between the clinical stage of cutaneous leishmaniasis lesions and in vitro parameters of cell-mediated immunity could be established. For this purpose, we measured lymphocyte proliferation, using a total lymphocyte proliferation (TLP) blood assay, and leishmanicidal effector activity using peripheral blood mononuclear cells (PBMC) in a three-day assay. The parameters of leishmanicidal activity measured included percent infected monocytes and number of amastigotes per 100 infected monocytes 24 and 72 hr after infection. Three groups of people were studied: a group of patients in the course of the disease, a group of immune individuals, and unexposed controls. The results of the study suggested that the ability of PBMC to kill parasites increased in patients as the lesions cured, and was highest in immune individuals. In contrast, the TLP response once positive, did not increase after cure. In approximately 30% of the patients who were retested on several occasions during the course of the disease, a positive response reversed to negative both in the TLP and the effector assays while the lesions were still active. In approximately 50% of these cases, the response eventually became positive again. The data presented show that effector activity and proliferation correlate with immunity, and suggest that marked heterogeneity characterizes the immune response in the course of active disease.

The development of a solid phase radioimmunoassay for the detection of leishmanial parasites in the sand fly.

The development of a solid phase radioimmunoassay inhibition test using anti-leishmanial monoclonal antibodies is described for the detection of leishmanial promastigote infections in sand flies, as an aid to epidemiology. As few as 6-12 Leishmania major promastigotes could be detected in laboratory-bred Phlebotomus papatasi. The test was sensitive and species-specific.

Appraisal of the total blood lymphocyte proliferation assay as a diagnostic tool in screening for tuberculosis.

The total blood lymphocyte proliferation assay (TLP) was evaluated as a screening test for infection with Mycobacterium tuberculosis and was compared with the tuberculin (Mantoux) skin test. The results of TLP assays performed on 33 patients with tuberculosis and 37 non-tuberculous subjects were compared with results of skin tests performed in the previous year. There was a high correlation between skin test responses and TLP responses to PPD which was statistically significant. The sensitivity, specificity and the predictive value of a positive test were also similar for the skin test and TLP test. These findings suggest that the TLP test is as effective in screening for M. tuberculosis infection as tuberculin skin testing. Future research leading to further simplification of the TLP method may lead to it replacing intradermal skin testing.

Cutaneous leishmaniasis acquired during military service in the Middle East.

BACKGROUND: Cutaneous leishmaniasis is endemic in much of the Middle East. Personnel from more than 55 nations are currently participating in Middle East peacekeeping and military activities. OBSERVATIONS: Twenty-three Fijian members of a military observational force in Sinai, Egypt, acquired cutaneous leishmaniasis. They were treated successfully with 1-month courses of ketoconazole. CONCLUSIONS: Soldiers who acquire cutaneous leishmaniasis may return home to nations where cutaneous leishmaniasis is unknown or rarely diagnosed. Cutaneous leishmaniasis, caused by Leishmania major, may be treated with ketoconazole rather than antimonials.

Evaluation of a total lymphocyte proliferation assay as a diagnostic tool for cutaneous leishmaniasis.

A total blood lymphocyte proliferation (TLP) assay, recently developed for cutaneous leishmaniasis (CL), was evaluated as a tool for diagnosis of CL among patients with active lesions and apparently healthy people in an endemic area of the Jordan Valley. It was found that, in patients with lesions less than 3 months old, the TLP assay appeared insensitive, failing to detect about half of the patients in whom a diagnosis of CL had been made clinically. Blood specimens from patients with lesions of at least 3 months duration showed a positive correlation in 8 out of 10 cases. With healthy people resident in an endemic area the TLP assay was positive for 15 of 20 subjects with a past history of CL, and for only 3 of 16 without such a history. With another group of patients, not residents of an endemic area but who had spent 2 weeks together in an endemic area, and with a group of drug-treated patients, the results of the TLP assay showed a similar pattern: in patients with lesions less than 3 months old the test appeared rather insensitive, but in patients with lesions of more than 3 months duration, there was a high correlation between the TLP and clinical diagnosis.

Changing patterns of cutaneous leishmaniasis in Israel and neighbouring territories.

The most frequent form of cutaneous leishmaniasis (CL) in Israel and the neighbouring territories is due to Leishmania major, which is endemic mainly in the Jordan Valley and in the Rift Valley. CL due to L. tropica is much less common, and in the past only sporadic cases have been reported. In this study we present data obtained during the years 1988-1992 regarding CL in the area. Our clinic has diagnosed a total of 371 leishmaniasis cases, most of whom acquired the infection in the Jordan Valley, mainly during June and July. About one-third of the patients had single lesions, and one-third more than 5 lesions. We also describe an outbreak of leishmaniasis in Kfar Adumim, a village 15 km east of Jerusalem, where leishmaniasis was previously unknown. Parasites were characterized by the polymerase chain reaction and by immunostaining, and found to be both L. tropica and L. major. The localization of the homes of the affected people on a slope where hyraxes were abundant suggests that these animals might have been involved in the transmission of L. tropica in this area.

Delayed-type hypersensitivity and lymphocyte proliferation in response to Leishmania major infection in a group of children in Jericho.

The cellular response to Leishmania major was evaluated in vitro with a lymphocyte proliferation microtest, performed on 100 microliters of whole blood obtained by finger prick. The maximum time and optimum conditions for storage of fresh blood before testing were determined, and the ability of the assay to evaluate cellular immunity to Leishmania was compared to that of the classical Montenegro skin test. A positive correlation between the diameter of the skin induration and the stimulation index was demonstrated. Defining a positive skin test by induration greater than or equal to 5 mm, and a positive proliferation assay by a stimulation index greater than 2.6 and a response greater than or equal to 3000 ct/min, we found a significant correlation between the 2 tests. The proliferation assay was less sensitive than the skin test, but somewhat more specific. Diagnostic specificities and sensitivities did not differ for the 2 tests.

Human cutaneous leishmaniasis: in-vitro parasite--mononuclear cell interactions in immune and naive individuals.

The aim of this study was to compare the ability of Leishmania parasites to survive in mononuclear cells from immune individuals with their ability to survive in cells from naive individuals. For this purpose we established an in vitro system based on the co-culture in suspension of human peripheral blood leukocytes derived from immune and naive subjects and L. major promastigotes. The proportion of monocytes containing intracellular parasites and the number of amastigotes per 100 infected monocytes (parasite burden) were determined 24 and 72 h after in-vitro infection. The proportion of infected cells from naive individuals did not change, and the number of amastigotes either did not change or increased by 1.2 to 1.7-fold between 24 and 72 h incubation. In contrast, in the immune subjects, the proportion of infected monocytes 24 h after infection was lower than in the naive individuals, and a 30-90% decrease in both the proportion of infected monocytes and the parasite burden was observed after 72 h incubation. Based on these results, three characteristics of leishmanicidal activity of mononuclear cells from immune individuals were determined: (a) the proportion of infected monocytes 24 h after infection was lower than 22%; (b) there was a decrease of more than 30% in the proportion of infected monocytes between 24 and 72 h after infection; and (c) there was a significant decrease in the number of amastigotes between 24 and 72 h after infection. The results of this study demonstrate an enhanced leishmanicidal activity of mononuclear cells from immune individuals.

Gorging response of culicine mosquitoes (Diptera: Culicidae) to blood fractions.

Although anopheline mosquitoes will ingest plasma without blood cells, Culex spp. and Aedes spp. require the phagostimulatory effect of blood cells; this effect can be duplicated by the addition of adenine-nucleotides to plasma. Because activation of platelets released ADP and ATP into the plasma, they were suspected as the major source of the phagostimulant. This paper describes quantitatively the role of platelets in ingestion by Aedes aegypti (L.) and Culex univittatus Theobald. We found that about 10(6)/mm3 inactivated platelets are required to induce engorgement by 80-90% of the mosquitoes of both species. Thrombin activation of the platelets reduced the effective dose to < 2 x 10(4)/mm3. Other blood fractions also were tested as possible sources of stimulation. A series of washed red blood cells (RBC) dilutions was tested; 5 x 10(5) RBC/mm3 were required to induce 90% engorgement. Several types of leukocytes derived from blood by standard methods also induced engorgement at their physiological concentrations. Macrophages and cultured lymphocytes that do not contain any platelets induced gorging in Cx. univittatus, but not in Ae. aegypti. Because RBC and leukocytes do not release nucleotides unless broken, we suggest that their phagostimulatory effect is due to platelet contamination, which invariably occurs during standard methods of blood fractionation.

Induction of macrophage motility by a T-cell line from Balb/c mice specific for Plasmodium berghei malaria.

A long-term antimalaria T-cell line (AMTL) expressing a helper phenotype (Thy 1.2+, Lyt 2.2-) was established from Plasmodium berghei-recovered Balb/c mice. The ability of this T-line to induce macrophage motility was measured in vivo and in vitro. Adoptive transfer of AMTL cells to normal Balb/c mice showed an increased delayed hypersensitivity response to the homologous antigen, i.e., parasitized erythrocytes (PE). In vitro, AMTL culture supernatant (AMTL-SUP) augmented chemotactic locomotion of macrophages derived from both normal and infected mice. However, the effect on normal macrophages was significantly higher. AMTL cells adoptively transferred to normal mice had no effect on parasitemia levels or mortality rate after subsequent infection with P. berghei. Partial characterization of the AMTL-SUP indicated the involvement of a protein of about 12,600 Daltons in the enhancement of chemotaxis. These findings suggest that the AMTL cells and chemoattractants produced by them can induce macrophage motility, and that the macrophage malfunction in Balb/c with P. berghei infection is not due to defects at the T-lymphocyte level.

Production of interferon gamma in cultures of whole blood obtained in the course of and after healing of cutaneous leishmaniasis.

Whole-blood cultures from individuals with cutaneous leishmaniasis due to Leishmania major, tested under field conditions, showed a good correlation between the production of interferon-gamma and total blood lymphocyte proliferation (TLP), assayed by response to specific leishmania antigen. A group of patients, re-tested on several occasions, converted to positive in both parameters of cell mediated immunity (CMI) at the same time. It is suggested that the TLP assay and the production of interferon-gamma detect similar CMI activity in human cutaneous leishmaniasis. The measurement of interferon-gamma production has the advantage over the TLP assay that the supernatants of the cultures can be frozen and stored for prolonged periods before the assay is performed. For this reason, this could present a suitable method for field studies.

Plasmodium berghei: immunosuppression of the cell-mediated immune response induced by nonviable antigenic preparations.

In this work, plasmodial antigens were examined for their ability to suppress the cellular immune response during lethal Plasmodium berghei infection. Splenic enlargement and the number and function of white spleen cells were assessed after injection of normal mice with irradiated parasitized erythrocytes (IPE) or with parasitized erythrocytes (PE) membranes. Both IPE and PE membranes caused splenomegaly and an increase in the number of splenic white cells with concurrent alteration of the relative proportions of T cells and macrophages. The percentage of T lymphocytes was fractionally diminished, but there was a marked increase in Lyt 2.2 positive (suppressor and cytotoxic) T subsets and in the number of splenic macrophage precursors. The pathological enlargement of the spleen was induced by various plasma membrane-derived antigens containing both proteins and carbohydrates. Splenocytes of mice injected with liposomes containing deoxycholate-treated PE or PE fractions showed both diminished interleukin 2 production and a decreased response to mitogen. It appears that some of the changes in the cellular immune response during P. berghei infection are a consequence of the massive provision of a wide spectrum of antigens, capable of suppressing the immune response. Thus, it may be appropriate to evaluate the possible negative effect of parasite epitopes that are candidates for vaccine.

Plasmodium berghei: lymphocyte and macrophage dynamics in the spleen of Balb/c mice in the course of infection and after rechallenge of cured mice.

The purpose of this study was to determine a possible correlation between the cell populations of the immune system in the spleen, and the failure of normal Balb/c mice to overcome a Plasmodium berghei infection. After primary infection, all splenic white cell populations increased in number. However, the ratios between the different cell populations changed markedly. Macrophages, which comprise 5-10% of normal white spleen cells, rose to 70-80?% during the lethal infection. Most of the macrophages lacked surface Ia antigens. A rise in macrophage precursors in the spleen was also observed. The lymphocyte population was characterized by a preferential increase of T-suppressor-cytotoxic (Tsc) cells resulting in an inversion of the Tsc/Thelper ratio. During chloroquine treatment, administered in order to cure infected mice, all splenic populations remained expanded but their quantitative proportions returned to normal. Secondary infection of cured mice did no cause an additional increase in the number of splenic white cells, nor did it affect the ratios between the various populations. It appears that normal quantitative relations between spleen cell populations is a hallmark of the host's capability to overcome infection with P. berghei. The failure of the immune system, as seen during a lethal infection, may be due to the preferential increase in immature, functionally defective macrophages and possibly T-suppressor lymphocytes. Alternatively, the changes in macrophages and T lymphocytes described could be a result of rather than the cause of the high parasitemia observed during P. berghei infection.