Frequencies Of IFN-γ Producing Cells Research Articles

Abstract 5332: ST316, a clinical peptide antagonist of beta-catenin, induces anti-tumor immune responses by multiple mechanisms of action

Abstract Wnt/β-catenin plays several important roles in cancer, including driving oncogenesis via cell proliferation, survival and metabolic reprogramming of cancer cells, and enhancing immune-desertification of the tumor immune microenvironment (TIME). ST316 is a clinical stage cell-penetrating peptide antagonist of the interaction of β-catenin with its co-activator BCL9, selectively impairing a subset of Wnt target genes and demonstrating potent anti-tumor activity against Wnt-driven tumors in vivo. ST316 is currently being evaluated in a Phase 1-2 study (NCT05848739) enrolling patients with selected advanced solid tumors likely to harbor abnormalities of the Wnt/β-catenin signaling pathway. Here we explore the potential of ST316 to activate the TIME and provide data to support combination treatment with anti-PD-1 and anti-TIGIT therapies. Macrophages derived from human peripheral blood mononuclear cells (hPBMCs) were activated by LPS and IFNγ (M1) or IL-4 (M2) in the presence of ST316 or control and were immunophenotyped by expression of CD80 and CD163 to assess M1 and M2 macrophages, respectively. ST316 induced marked repolarization of hPBMC-derived M2 macrophages to the M1 identity in vitro, as shown by increased CD80 and decreased CD163 staining. Increasing concentration of ST316 in co-cultures of M2 cells with CD8+ T cells induced up to a three-fold increase in IFN-γ expressing cells relative to controls. Additionally, ST316 exposure increased CD155/PVR expression on 4T1 Triple Negative Breast Cancer (TNBC) cells, but not MV411 TNBC that are not Wnt-dependent. When combined with anti-TIGIT treatment, ST316 led to increased T cell activation in an ex vivo assay where 4T1 cells were co-incubated with syngeneic splenic CD8+ cells and frequency of IFN-γ producing cells was assessed. In in vivo experiments, Balb/C mice bearing syngeneic 4T1 TNBC orthotopic tumors were treated with vehicle, ST316, anti-PD-1 or combination. Sub-pharmacologic ST316 enhanced anti-PD-1 suppression of 4T1 tumor growth and induced a substantial decrease of the M2 marker CD209 (DC-SIGN) in the Tumor Associated Macrophage (TAMs). These data support two novel mechanisms of action for the β-catenin antagonist peptide ST316. First, ST316 exposure results in macrophage repolarization towards an immune-active M1 program, both in vitro and in vivo, and increases T-cell activation in co-culture assays. Second, ST316 induced CD155/PVR upregulation and T-cell activation in the presence of anti-TIGIT antibody. Collectively these results suggest a novel immune-modulatory role for ST316 in the TIME and provide rational for combination therapy with checkpoint inhibitors. Citation Format: Claudio Scuoppo, Karen Mendelson, Ricardo Ramirez, Mark Koester, Julia Diehl, Erin Gallagher, Siok Leong, Jerel Gonzales, Zach Mattes, Lila Ghamsari, Gene Merutka, Barry J. Kappel, Abi Vainstein-Haras, Jim A. Rotolo. ST316, a clinical peptide antagonist of beta-catenin, induces anti-tumor immune responses by multiple mechanisms of action [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5332.

Analysis of Humoral and Cellular Immunity of Lung Transplant Recipients Following SARS-CoV-2 Infection and BNT162b2 mRNA Vaccination

PurposeSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2), in lung transplant recipients (LTxR) under immunosuppression carries higher risk with 14-39% mortality. Immune responses of LTxR under immunosuppression following SARS-CoV-2 infection or vaccination remains unknown. Our goal is to determine the humoral and cellular immunity to SARS-CoV-2 in LTxR with infection and following vaccination.MethodsWe performed a single center analysis to determine immune responses of LTxR with infection and following BNT162b2 mRNA vaccination. The results were compared with controls (non-transplant individuals). ELISA was developed to determine the antibody (Ab) concentration (IgG) to SARS-CoV-2 spike (CSP) and nucleocapsid (CNP) antigens. PBMCs from LTxR were isolated by ficoll-hypaque centrifugation to determining the frequency of cells secreting IFNγ and TNFα to CSP and CNP by ELISpot.ResultsConcentration of Abs developed and T-cell frequencies secreting TNFα and IFNγ against CSP and CNP in LTxR and controls are given in Table 1. Infected LTxR and controls developed Abs to both CSP and CNP. In contrast, vaccinated LTxR induced 10 fold less Abs to CSP in comparison to control. Frequencies of cells secreting TNFα for both CSP and CNP were significantly reduced in LTxR with infection. However, vaccination of both LTxR and control induced similar levels of TNFα secreting cells upon stimulation with both CSP and CNP. It is of interest that frequency of IFNγ producing cells against both CSP and CNP were significantly higher in LTxR in comparison to control.ConclusionInfection with SARS-CoV-2 in LTxR and controls produced comparable levels of Abs both against CSP and CNP. However, vaccinated LTxR didn`t induce significant levels of Abs against CSP. Frequency of T-cells, secreting IFNγ were significantly increased by vaccination in LTxR and in controls suggesting that T cell responses against SARS-CoV-2 has been induced in LTxR by mRNA vaccine.

Trypanosoma cruzi-specific IFN-γ-producing cells in chronic Chagas disease associate with a functional IL-7/IL-7R axis.

BackgroundThe severity of cardiac disease in chronic Chagas disease patients is associated with different features of T-cell exhaustion. Here, we assessed whether the ability of T cells to secrete IFN-γ in response to T. cruzi was linked to disruption in immune homeostasis and inflammation in patients with chronic Chagas disease.Methodology/Principal findingsPBMCs from chronic Chagas disease patients and uninfected controls were examined for frequencies of T. cruzi-responsive IFN-γ-producing cells by ELISPOT and cellular expression and function of IL-7R using flow cytometry. Serum levels of IL-7, IL-21, IL-27, soluble IL-7R, and inflammatory cytokines were also evaluated by ELISA or CBA techniques. Patients possessing T. cruzi-specific IFN-γ-producing cells (i.e. IFN-γ producers) had higher levels of memory T cells capable of modulating the alpha chain of IL-7R and an efficient response to IL-7 compared to that in patients lacking (i.e. IFN-γ nonproducers) parasite-specific T-cell responses. IFN-γ producers also showed low levels of soluble IL-7R, high basal expression of Bcl-2 in T cells and low basal frequencies of activated CD25+ T cells. Modulation of IL-7R was inversely associated with serum IL-6 levels and positively associated with serum IL-8 levels. Circulating IL-21 and IL-27 levels were not associated with the frequency of IFN-γ producing cells but were reduced in less severe clinical forms of the disease. In vitro stimulation of PBMCs with IL-7 or IL-27 enhanced IFN-γ production in IFN-γ producers but not in IFN-γ nonproducers.Conclusions/SignificanceAlterations of the IL-7/IL-7R axis and in the levels of inflammatory cytokines were linked to impaired T. cruzi-specific IFN-γ production. These alterations might be responsible of the process of immune exhaustion observed in chronic Chagas disease.

T-Cell Composition of the Lymph Node Is Associated with the Risk for Early Rejection after Renal Transplantation.

The T-cell composition within the lymph node (LN) of end-stage renal disease (ESRD) patients differs from the composition within the circulation. Activation of the alloreactive T-cell response within secondary lymphoid organs is important after organ transplantation. However, to date no data are present on LN T-cell subsets and the risk for acute rejection after kidney transplantation. T cells from LNs of ESRD patients were analyzed for frequency of recent thymic emigrants, relative telomere length, expression of differentiation markers, and were related to the development of early acute rejection (EAR), occurring within 3 months after renal transplantation (RT). Furthermore, the alloreactive potential of mononuclear cells isolated from the LN and peripheral blood of 10 patients was analyzed. Measures of alloreactive potential included proliferation, cytokine production, frequencies of interferon-gamma-producing cells, and the presence of cytotoxic molecules. Patients with EAR were younger (p = 0.019), cytomegalovirus-seropositive (p = 0.037) and usually received dialysis prior to RT (p = 0.030). Next to this, patients with EAR showed a lower CD4:CD8 ratio (p = 0.027) within the LN. T cells from the LN were similar with regard to alloreactive capacity compared with those within the circulation. Univariate regression analysis showed that the CD4:CD8 ratio (OR: 0.67, p = 0.039), patient age (OR: 0.93, p = 0.024), and preemptive RT (OR: 0.11, p = 0.046) were associated with EAR. After a multivariate analysis, only the CD4:CD8 ratio (OR: 0.58, p = 0.019) and preemptive RT (OR:0.05, p = 0.012) were associated with EAR. A lower CD4:CD8 ratio in the LN is associated with a higher risk for the development of rejection within 3 months after RT.

A dose-response study in mice of a tetravalent vaccine candidate composed of domain III-capsid proteins from dengue viruses.

Tetra DIIIC is a subunit vaccine candidate based on domain III of the envelope protein and the capsid protein of the four serotypes of dengue virus. This vaccine preparation contains the DIIIC proteins aggregated with a specific immunostimulatory oligodeoxynucleotide (ODN 39M). Tetra DIIIC has already been shown to be immunogenic and protective in mice and monkeys. In this study, we evaluated the immunogenicity in mice of several formulations of Tetra DIIIC containing different amounts of the recombinant proteins. The Tetra DIIIC formulation induced a humoral immune response against the four DENV serotypes, even at the lowest dose assayed. In contrast, the highest level of cell-mediated immunity, measured as frequency of IFNγ-producing cells, was detected in animals immunized with the lowest dose. The protective capacity of the tetravalent formulations was assessed using the mouse model of dengue virus encephalitis. Upon challenge, vaccinated mice showed significantly reduced virus replication in all tested groups. This study provides new information about the functionality of Tetra DIIIC as a vaccine candidate and also supports the crucial role of cell-mediated immunity in protection against dengue virus.

Canine peripheral blood CD4+CD8+ double-positive T cell subpopulations exhibit distinct T cell phenotypes and effector functions

Canine peripheral blood CD4+CD8α+ double-positive (dp) Tcells represent a small population of T lymphocytes that are not only derived from single-positive (sp) CD4+ but also from CD8+ Tcells. Dp Tcells can be subdivided in three different subpopulations according to their expression levels of CD4 and CD8α, i.e. the CD4dimCD8αbright, CD4brightCD8αbright, and the CD4brightCD8αdim subsets. Previously we could show that total canine peripheral CD4+CD8α+ dp Tcells have features of activated Tcells. Here we demonstrate that the individual dp Tcell subsets distinctly differ in their activation status and phenotype. Thus, among the dp Tcell subsets the CD4dimCD8αbright subpopulation has the lowest and the CD4brightCD8αbright subset the highest frequency of CD25+ cells pointing to the CD4brightCD8αbright subset with the highest activation status. Consistent with that, analysis of CD44 vs. CD62L expression demonstrates that the CD4brightCD8αbright subset contains the highest frequencies of effector-memory Tcells (TEM). Upon in vitro stimulation, the CD4brightCD8αbright and CD4dimCD8αbright dp Tcell subsets show the highest frequencies of IFN-γ producing cells. Expression of granzyme B was found exclusively in the CD4dimCD8αbright dp Tcell subset indicating cytotoxic potential of this dp Tcell subset. Furthermore, T-bet expression was restricted to the CD4dimCD8αbright subset, while Foxp3+ regulatory Tcells were detected in the CD4brightCD8αdim dp Tcell subset. In conclusion, canine dp Tcells are phenotypically and functionally heterogeneous consistent with the diverse characteristics of their single-positive CD4+ and CD8+ Tcell progenitors. The data provide the basis for future in vivo studies to elucidate the role of individual dp Tcell subsets in host defense and/or immune pathological diseases of dogs.

Frequencies of Circulating MAIT Cells Are Diminished in Chronic HCV, HIV and HCV/HIV Co-Infection and Do Not Recover during Therapy.

ObjectiveMucosal-associated invariant T (MAIT) cells comprise a subpopulation of T cells that can be activated by bacterial products and cytokines to produce IFN-γ. Since little is known on MAIT cells during HCV infection, we compared their phenotype and function in comparison to HIV and HCV/HIV co-infected patients, and determined the effect of IFN-α-based and direct-acting antiviral therapy on MAIT cells of HCV patients.MethodsBlood samples from patients with chronic HCV (CHCV), virologically suppressed HIV, acute HCV/HIV co-infection (AHCV/HIV) and healthy individuals were examined by flowcytometry for phenotype and function of MAIT and NK cells.Results and ConclusionsCompared to healthy individuals, the frequency of CD161+Vα7.2+ MAIT cells was significantly decreased in patients with CHCV, HIV and AHCV/HIV co-infection. CD38 expression on MAIT cells was increased in AHCV/HIV patients. MAIT cells were responsive to IFN-α in vitro as evidenced by enhanced frequencies of IFN-γ producing cells. IFN-α-based therapy for CHCV decreased the frequency of IFN-γ+ MAIT cells, which was still observed 24 weeks after successful therapy. Importantly, even after successful IFN-α-based as well as IFN-α-free therapy for CHCV, decreased frequencies of MAIT cells persisted. We show that the frequencies of MAIT cells are reduced in blood of patients with CHCV, HIV and in AHCV/HIV co-infection compared to healthy individuals. Successful therapy for CHCV did not normalize MAIT cell frequencies at 24 weeks follow up. The impact of HIV and HCV infection on the numbers and function of MAIT cells warrant further studies on the impact of viral infections and the antimicrobial function of MAIT cells.

Frequency of IFNγ-producing T cells correlates with seroreactivity and activated T cells during canine Trypanosoma cruzi infection

Vaccines to prevent Trypanosoma cruzi infection in humans or animals are not available, and in many settings, dogs are an important source of domestic infection for the insect vector. Identification of infected canines is crucial for evaluating peridomestic transmission dynamics and parasite control strategies. As immune control of T. cruzi infection is dependent on humoral and cell-mediated immune responses, we aimed to define a serodiagnostic assay and T cell phenotypic markers for identifying infected dogs and studying the canine T. cruzi-specific immune response. Plasma samples and peripheral blood mononuclear cells (PBMCs) were obtained from forty-two dogs living in a T. cruzi-endemic region. Twenty dogs were known to be seropositive and nine seronegative by conventional serologic tests two years prior to our study. To determine canine seroreactivity, we tested sera or plasma samples in a multiplex bead array against eleven recombinant T. cruzi proteins. Ninety-four percent (17/18) of dogs positive by multiplex serology were initially positive by conventional serology. The frequency of IFNγ-producing cells in PBMCs responding to T. cruzi correlated to serological status, identifying 95% of multiplex seropositive dogs. Intracellular staining identified CD4+ and CD8+ T cell populations as the sources of T. cruzi lysate-induced IFNγ. Low expression of CCR7 and CD62L on CD4+ and CD8+ T cells suggested a predominance of effector/effector memory T cells in seropositive canines. These results are the first, to our knowledge, to correlate T. cruzi-specific antibody responses with T cell responses in naturally infected dogs and validate these methods for identifying dogs exposed to T. cruzi.

BCG-treated M2-macrophages enhance IFN-γ production of T cells (P3348)

Abstract M1- and M2-polarized Macrophage (MΦ) subtypes differentially regulate anti-mycobacterial defense. To examine their roles in human infection, we compared the gene expression profile of M1- vs. M2-MΦ to the expression profile of lesional skin from patients with restricted (tuberculoid, T-lep) vs. disseminated (lepromatous, L-lep) leprosy (a mycobacterial skin infection). Rank-Rank Hypergeometric Overlap (RRHO) is a novel algorithm for comparing full gene expression profiles from two independent microarray experiments that generates the most statistically significant overlapping gene list without requiring strict p-value/fold change cutoffs. RRHO analysis showed significant overlap of genes between M2-MΦ and L-lep lesions (n = 1559, max log hypergeometric p-value = 196), indicating a role for M2-MΦ in immune unresponsiveness. Bacillus Calmette-Guerin (BCG) is given to protect household contacts against leprosy. We hypothesized that BCG treatment may affect the function of M2-MΦ and investigated its effects in vitro. We found that BCG-treated M2-MΦ secreted less IL-10 (p < 0.05), with no effect on IL-12p40, compared to untreated M2-MΦ. In addition, BCG-treated M2-MΦ increased the frequency of IFN-γ producing cells in an antigen non-specific manner (p < 0.05), suggesting an adjuvant effect of BCG. Our data indicates M2-MΦ can be reprogrammed by BCG, resulting in increased IFN-γ production to enhance anti-mycobacterial immunity.

P14 Phenotypic and functional profile of CD4posCD25high regulatory T cells in autoimmune hepatitis/systemic lupus erythematosus overlap syndrome

IntroductionAutoimmune hepatitis (AIH) is a severe inflammatory liver disorder characterised by hypergammaglobulinaemia, seropositivity for autoantibodies and interface hepatitis on histology. Though the mechanisms leading to immune-tolerance breakdown have not been...

Baculovirus-based Vaccination Vectors Allow for Efficient Induction of Immune Responses Against Plasmodium falciparum Circumsporozoite Protein

Baculovirus vectors are able to transduce a large variety of mammalian cell types and express transgenes placed under the control of heterologous promoters. In this study, we evaluated the potential of baculovirus vectors for malaria vaccination. To induce efficient CD4(+) and CD8(+) T-cell responses, we produced a series of vectors that display the Plasmodium falciparum circumsporozoite (CS) protein in the virion envelope and/or allow for CS expression upon transduction of mammalian cells. We found that baculovirus vectors can transduce professional antigen-presenting cells and trigger their maturation, which is a prerequisite for efficient antigen presentation. Upon intramuscular injection into mice, the vector that both displayed and expressed CS induced higher anti-CS antibody titers (of the immunoglobulin (IgG)1 and IgG2a type) and a higher frequency of interferon-gamma-producing T cells specific to CS, than the vectors which either only displayed or only expressed CS. The baculovirus CS display/expression vector was also superior in inducing CS-specific CD4(+) and CD8(+) T-cell responses in vitro using human peripheral blood mononuclear cells from naive donors. This, together with the absence of pre-existing immunity to baculoviruses in humans, the absence of viral gene expression in mammalian cells, and the relative low immunogenicity of baculovirus virions, makes these vectors promising tools for vaccination. Furthermore, the ability to produce large amounts in serum-free medium at a low cost adds a further advantage to this vector system.

FTY720 treatment of kidney transplant patients: A differential effect on B cells, naïve T cells, memory T cells and NK cells

FTY720 alters lymphocyte recirculation and homing by interfering with S1P receptors on lymphocytes, possibly in combination with chemokine receptors, and induces a decrease in PBL counts. In fresh, whole blood samples of 14 kidney transplant patients, we analyzed by flow cytometry the effect of FTY on the number of NK cells, monocytes, naïve (CCR7 +) T cells, memory (CCR5 +) T cells and B cells. Patients treated with 0.5, 2.5 or 5 mg FTY/day showed a strong decrease in T and B cell numbers. NK cells and monocytes were not affected. FTY reduced primarily naïve T cells. From the memory T cells (CCR5 +), predominantly CD8 cells, 40–60% remained in the circulation. The majority of the CCR7 + cells disappeared from the circulation within 3–6 h, while a further reduction was achieved later. The more slowly decrease in naïve CCR7 + T cell numbers was also observed in the group treated with 0.25 mg FTY/day. Elispot assays revealed no IL-4 producing cells and a low frequency of IFN-γ producing cells. We suggest that both CCR7 dependent and independent mechanisms are involved in the depletion of T cells from peripheral blood.

Heat shock protein 70-based vaccine stimulates both innate and adaptive immunity against chronic myeloid leukemia

2515 Background: HSP70 chaperones both proteins and antigenic peptides. Vaccination with tumor-derived HSP70-peptide complexes elicits anti-tumor immunity in murine models. We have earlier reported that HSP70 vaccines can be effectively produced from peripheral blood mononuclear cells (PBMCs) of patients with chronic myeloid leukemia (CML) in chronic phase. Moreover, eight weekly injections of 50 μg autologous HSP70 vaccines were associated with no grade II or higher toxicity in any patients and with clinical efficacies in 11 out of 17 patients. In the present study, the immunological responses to this vaccine were studied. Methods: All patients in the study had either cytogenetic or molecular evidence of diseases, despite Imatinib Mesylate, prior to the study entry. Peripheral blood was obtained, before the 1st and 5th vaccines, and two weeks after the 8th vaccine. IFNγ-producing effector cells in PBMCs were measured by an ELISPOT assay using a variety of targets, including HLA negative but Bcr/Abl+ K562 cell lines, HSP70-peptide complexes or K562 lysates. A pan-HLA class I specific antibody W6/32 was used to determine if IFNγ production was MHC restricted or not. Results: The frequency of IFNγ-producing cells in the PBMCs, in response to HSP70-peptide complexes, was increased in nine out of 12 patients analyzed. This activity was significantly blocked by W6/32, indicating that peptides bound to HSP70 can be cross-presented to HLA class I to activate CD8+ T cells. The incomplete blockage by W6/32 indicates that HSP70 can stimulate IFNγ production by other cells, in a peptide-independent manner. Indeed, natural killer (NK) cell activity post vaccinations, as measured against the standard NK target K562, increased markedly in 12 out of 15 patients. We have also confirmed an increased frequency of CML-specific and HLA class I-restricted T cells using K562 cell lysates as targets. Conclusion: The autologous HSP70-based vaccine induces both NK and CML-specific T cell immunity in a majority of patients. The remarkable immunologic activity of HSP70 suggests that it has adjuvant therapeutic values for CML. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Antigenics Antigenics Antigenics

OVER-IMMUNOSUPPRESSION AFTER HLA-IDENTICAL LIVING-RELATED KIDNEY TRANSPLANTATION

P186 Aims: HLA-identical living-related kidney transplant patients still receive standard doses immunosuppression. We wondered why these patients should be exposed to the adverse side-effects of immunosuppression any longer. In this study, we tapered HLA-identical living-related kidney transplant patients who received azathioprine (AZA) in combination with prednisone to half of their AZA-dose. We questioned whether tapering of in vivo immunosuppression influenced the donor-specific T cell reactivity, defined as the reactivity towards minor histocompatibility antigens (mHags). Methods: Patients (n=19) who were at least two years after transplantation were reduced in their immunosuppression from 100% AZA (median: 1.7 mg/kg; range: 1.0-2.2) in two steps to 50% AZA (median: 0.7 mg/kg; range: 0.7-1.1) in combination with 5-10 mg/day prednisone. Recently, we showed that the donor-specific reactivity against mHags can be measured by using an IFN-γ Elispot-assay. Donor-specific reactivity before and after tapering of immunosuppression was compared with the reactivity at 3 months after HLA-identical living-related kidney transplantation (n=16). Results: Three months after transplantation, we found a frequency of 30 donor-specific IFN-γ producing cells/106 PMBC (range: 5-115). At least two years after transplantation, before reduction of immunosuppression, the frequency of IFN-γ producing cells was lower (median: 0/106 PBMC; range: 0-320; p=0.08). Tapering of immunosuppression did not influence the frequency, and was still lower compared to three months after transplantation (median: 5/106 PBMC; range: 0-540; p=0.02). None of these patients developed acute rejection after tapering immunosuppression. Conclusions: In summary, HLA-identical living-related kidney transplant patients can be safely reduced to 50% of their AZA-dose without affecting the immune response. Therefore, we conclude from our Elispot-results, that these patients remain over-immunosuppressed, and we suggest that the immunosuppression could be reduced further or even stopped.

Use of interleukin 15 to enhance interferon-γ production by antigen-specific stimulated lymphocytes from rhesus macaques

The enzyme-linked immune spot (ELISPOT) assay is receiving increased attention as a means for quantifying antigen-specific CD8 T-cell responses in rhesus macaques. Further improving the sensitivity of this assay could aid in the evaluation of vaccine candidates and/or immune therapeutic candidates. Interleukin (IL)-15 has been demonstrated to stimulate expansion of human immunodeficiency virus (HIV)-specific cytotoxic T lymphocytes (CTL) and to regulate homeostatic proliferation of CD8+ memory cells. We evaluated the in vitro effect of IL-15 to increase the detection of interferon-γ (IFN-γ) production by antigen-specific stimulated lymphocytes from a group of rhesus macaques exposed to simian-human immunodeficiency virus (SHIV) and a second group infected with SIVmac251, before and after antiretroviral treatment (ART). Results from these studies demonstrate that the presence of IL-15 during stimulation in a peptide-based ELISPOT assay greatly enhanced IFN-γ production in both SHIV and simian immunodeficiency virus (SIV)-infected macaques. IFN-γ production was mainly mediated by CD8 lymphocytes. The optimal concentrations of IL-15 that give enhancement of IFN-γ production to specific antigen, without a significant increase in the spontaneous IFN-γ release, ranged from 0.5 to 2.5 ng/ml. The mean number of IFN-γ spots was increased 3.1- to 3.6-fold in response to SIV gag or HIV env peptide pools, respectively, in peripheral blood mononuclear cells (PBMC) from SHIV-infected macaques. Similarly, in SIV-infected macaques, IL-15 increased the mean number of IFN-γ spots 2.7-fold in response to both SIV gag and env peptide pools. In samples obtained after ART in the same macaques, the increase factor was 2.5 for SIV gag and 1.8 for the env peptide pools. Thus, the sensitivity of the ELISPOT assay can be enhanced by addition of IL-15. This modified assay will be useful for detection of low frequencies of IFN-γ producing cells in rhesus macaques.

The direct and indirect allogeneic presentation pathway during acute rejection after human cardiac transplantation

Alloreactive T-cells may be activated via a direct or an indirect antigen presentation pathway. T-cells recognise foreign HLA molecules directly on donor cells in the direct presentation pathway, while in the indirect presentation pathway alloantigens are processed and presented in HLA of self APC. We questioned whether the frequency of IFN-γ producing cells determined by Elispot-assay is a good tool to monitor the contribution of the direct and indirect presentation pathway during acute rejection.

Blockade of CTLA-4 Enhances Host Resistance to the Intracellular Pathogen,Leishmania donovani

AbstractCTLA-4 has recently been shown to act as a negative regulator of T cell activation. Here we provide evidence that blockade of CTLA-4 can result in enhanced host resistance to an intracellular pathogen. The administration of anti-CTLA-4 mAb 4F10 to BALB/c mice, 1 day following infection with Leishmania donovani, enhanced the frequency of IFN-γ and IL-4 producing cells in both spleen and liver, and dramatically accelerated the development of a hepatic granulomatous response. The expression of mRNA for the CXC chemokine γIP-10 was also elevated above that seen in control Ab treated mice, and was directly correlated with the frequency of IFN-γ producing cells. In contrast, macrophage inflammatory protein-1α (MIP-1α) and monocyte chemotactic protein-1 (MCP-1) mRNA levels were unaffected by anti-CTLA-4 treatment, suggesting that CTLA-4 blockade may exert selective effects on chemokine expression. These changes in tissue response and cytokine/chemokine production were accompanied by a 50 to 75% reduction of parasite load in the spleen and liver of anti-CTLA-4-treated animals compared to controls. Furthermore, administration of anti-CTLA-4 mAb 15 days after L. donovani infection, when parasite burden is increasing in both organs, also resulted in enhanced resistance. Thus, these studies indicate a potent immunomodulatory and potentially therapeutic role for interventions targeted at CTLA-4.

ALLOSPECIFIC IFN-??-PRODUCING CELLS IN PERIPHERAL BLOOD AS A FUNCTIONAL MEASURE OF HLA HAPLOTYPE MATCH

458 Standard tissue typing methods identify differences in HLA molecules between transplant donors and recipients, with the number of mismatches correlating inversely with the long term outcome of the allograft. It is not clear whether HLA haplotype mismatches correlate with the strength of the alloresponse, however, as the differences may or may not have functional significance. Our laboratory has been interested in identifying a surrogate marker for the strength of the allospecific immune response that would improve upon current matching procedures and thus aid in predicting graft survival. To this end we used a cytokine ELISA spot assay to evaluate the frequency of allospecific, IFNγ-producing cells in the peripheral blood of putative renal transplant recipients and potential living donors. PBLs were obtained from 30 individuals (10 families) at the time of initial crossmatch, and were tested for allospecific IFNγ production using one way MLR ELISA spot assays. The resultant frequencies were correlated with the HLA haplotypes. HLA A and B alleles were defined using microlymphocytotoxicity assays and HLA DR alleles were identified using PCR with sequence specific primers. The PBLs of donor-recipient pairs with identical haplotypes had a significantly lower mean frequency of allospecific IFNγ-producing cells than those with single (3/6 alleles) or full (6/6) haplotype mismatches (p< 0.05 for each), but the number of mismatches did not directly correlate with the number of IFNγ spots detected:Table Many fully mismatched pairs had low frequencies(< 18 spots/300,000 PBLs) of allospecific IFNγ-producing cells, suggesting that the mismatches may not be of functional significance. Furthermore, we often found that the frequency of IFNγ-producing cells was significantly higher in one direction than the other. In one illustrative example, one sibling expressed HLA A2 and a second expressed HLA A30, with the other 5 loci being identical. A2 anti-A30 yielded 68 spots/300,000 PBLs, while A30 anti-A2 yielded only 12. These findings confirm that expression of certain alleles is associated with stronger alloresponses than others despite identical numbers of mismatches. Our studies provide an alternative functional approach for the evaluation of donor-recipient histocompatibility. Long term follow-up of the graft recipients will be required to determine whether the pretransplant frequency of allospecific IFNγ-producing cells provides predictive information regarding graft outcome beyond standard HLA typing.

Frequencies of IFNγ- and IL-4-producing cells during Mycobacterium bovis BCG infection in two genetically susceptible mouse strains: role of [formula omitted] T cells and NK1.1 cells

Frequencies of IFN γ- and IL-4-producing spleen cells in response to Mycobacterium bovis BCG infection were determined in C57BL 6 and BALB c mice. Both mouse strains express equal innate susceptibility to M. bovis BCG (Bcg s), but differ in their NK1.1 and T-cell activities. M. bovis BCG infection induced higher frequencies ( f ≈ 1 500 ) of antigen-induced IFN γ-secreting spleen cells in C57BL 6 mice as compared to BALB c mice ( f ≈ 1 8000 ). Concanavalin A stimulated almost equal numbers of IFN γ-secreting cells in both mouse strains ( f ≈ 1 50 ). Treatment with anti-NK1.1 mAb of M. bovis BCG-infected C57BL 6 mice did not alter frequencies of IFN γ-secreting cells. Equally low numbers of antigen-induced IL-4-producing cells ( f ≈ 1 3000 ) were determined in both C57BL 6 and BALB c mice during M. bovis BCG infection and treatment of C57BL 6 mice with anti-NK1.1 mAb had no measurable effect on IL-4 producers. Finally, frequencies of IFNγ-producing cells were markedly reduced (10-fold) in M. bovis BCG-infected TCR-β − − gene deletion mutants as compared to their heterozygous controls. Our findings verify that M. bovis BCG infection primarily induces IFNγ-secreting α β T cells of TH1 type and show that the frequencies of these IFNγ producers differ in the two Bcg s mouse strains C57BL 6 and BALB c .

Ictal language shift in a polyglot.

<h3>Introduction</h3> Autoimmune hepatitis (AIH) is a severe inflammatory liver disorder characterised by hypergammaglobulinaemia, seropositivity for autoantibodies and interface hepatitis on histology. Though the mechanisms leading to immune-tolerance breakdown have not been fully elucidated, a numerical and functional defect of circulating CD4^posCD25^high regulatory T-cells (T-regs) plays a key role in permitting effector lymphocytes to attack hepatocytes. We have identified a cohort of children with concomitant AIH and systemic lupus erythematosus (SLE), a multi-system autoimmune disease in the context of which most studies have reported numerical and functional T-reg impairment. <h3>Aim</h3> To define the phenotypic and functional profile of T-regs in children with AIH and SLE (AIH/SLE). <h3>Method</h3> 9 AIH/SLE patients (8 females; median age: 13.9 years), 16 AIH patients (13 females; median age: 13.3 years) and 9 healthy subjects (HS, 6 females; median age: 34 years) were studied. T-reg phenotype was determined by flow cytometry after cell incubation with anti-CD4, CD25 and CD127 monoclonal antibodies. Frequency of FOXP3^pos and IFNγ, IL-4, IL-17 and IL-9-producing cells was assessed by intracellular staining. T-reg suppressor function was evaluated as reduction of cell proliferation, measured by ³H-thymidine uptake, in co-culture experiments where CD4^posCD25^highCD127^negT-regs were added to CD25^neg target cells. <h3>Results</h3> The number of T-regs in AIH/SLE patients (7.06±1.1) tended to be higher than in AIH (4.88±1.1; p=0.1) and was similar to HS (7.03±0.6). While the proportion of FOXP3^pos cells within T-regs did not differ, that of CD4^posCD25^highCD127^pos cells was higher in AIH/SLE (2.4±0.99) than in AIH (0.46±0.2; p=0.019) and HS (0.63±0.3; p=0.05). The frequency of IFNγ-producing cells within T-regs was higher in AIH/SLE (6.75±1.7) than in AIH (4.8±1.9; p=0.019) and HS (3.5±0.82; p=0.05); conversely that of IL-4-producing cells within T-regs was lower in AIH/SLE (0±0) than in AIH (0.86±0.3; p=0.02) and HS (0.9±0.47; p=0.06). The frequency of IL-17 and IL-9-producing cells was negligible and did not differ among the groups. Addition of CD127^negT-regs, while decreasing CD25^neg cell proliferation by 31.6% in HS (p=0.08) and by 12.3% (p=0.056) in AIH, did not affect the proliferation of CD25^neg cells isolated from AIH/SLE patients. <h3>Conclusion</h3> Compared to AIH and HS, T-regs from AIH/SLE patients are more activated, are skewed towards a Th1 cytokine profile and are functionally defective. These data suggest that more severe alteration of T-reg phenotype and function may predispose to multiple autoimmune manifestations.