Natasa Strbo

Perforin Is Required for Innate and Adaptive Immunity Induced by Heat Shock Protein Gp96

Tumor-secreted gp96-Ig is highly immunogenic and triggers CD8 T cell-mediated tumor rejection. In vivo secreted gp96-Ig and gp96-myc cause NK activation and clonal expansion of specific CD8(+) CTL in wild-type and in Fas-ligand-deficient (gld) mice but not in perforin- (PKO) or IFN-gamma-deficient (GKO) mice. Transfer of perforin-competent NK cells restores the ability of PKO mice to clonally expand CD8 CTL in response to gp96-Ig. The data demonstrate an essential role for perforin-mediated functions in the activation of innate and adaptive immunity by heat shock protein gp96-peptide complexes. Crosspresentation of antigens by heat shock proteins seems to require a perforin-dependent positive feedback loop between NK and DC for both sustained NK activation and clonal CTL expansion. The studies also explain how depressed NK activity in patients with tumors or after viral infections could diminish CTL responses.

Identification of a Subpopulation of Macrophages in Mammary Tumor–Bearing Mice That Are Neither M1 nor M2 and Are Less Differentiated

Systemic and local immune deficiency is associated with cancer, and the role of M2 tumor-associated macrophages in this phenomenon is well recognized. However, the immune status of macrophages from peripheral compartments in tumor hosts is unclear. Peritoneal macrophages (PEM) are derived from circulating monocytes and recruited to the peritoneal cavity where they differentiate into macrophages. We have previously shown that PEMs from mice bearing D1-DMBA-3 mammary tumors (T-PEM) are deficient in inflammatory functions and that this impairment is associated with diminished expression of transcription factors nuclear factor kappaB and CAAT/enhancer-binding protein. We now provide evidence that T-PEMs display neither M1 nor M2 phenotypes, yet exhibit deficiencies in the expression of several inflammatory cytokines and various proinflammatory signaling pathways. Moreover, due to nuclear factor kappaB down-regulation, increased apoptosis was observed in T-PEMs. We report for the first time that macrophage depletion is associated with increased macrophage progenitors in bone marrow. Furthermore, T-PEMs have a lower expression of macrophage differentiation markers F4/80, CD68, CD115, and CD11b, whereas Gr-1 is up-regulated. Our results suggest that T-PEMs are less differentiated and represent a newly derived population from blood monocytes. Lastly, we show that transforming growth factor-beta and prostaglandin E(2), two immunosuppressive tumor-derived factors, may be involved in this phenomenon.

Molecular and Cellular Requirements for Enhanced Antigen Cross-Presentation to CD8 Cytotoxic T Lymphocytes

MHC class I-mediated cross-priming of CD8 T cells by APCs is critical for CTL-based immunity to viral infections and tumors. We have shown previously that tumor-secreted heat shock protein gp96-chaperoned peptides cross prime CD8 CTL that are specific for genuine tumor Ags and for the surrogate Ag OVA. We now show that tumor-secreted heat shock protein gp96-chaperoned peptides enhance the efficiency of Ag cross-priming of CD8 CTL by several million-fold over the cross-priming activity of unchaperoned protein alone. Gp96 also acts as adjuvant for cross-priming by unchaperoned proteins, but in this capacity gp96 is 1000-fold less active than as a peptide chaperone. Mechanistically, the in situ secretion of gp96-Ig by transfected tumor cells recruits and activates dendritic cells and NK cells to the site of gp96 release and promotes CD8 CTL expansion locally. Gp96-mediated cross-priming of CD8 T cells requires B7.1/2 costimulation but proceeds unimpeded in lymph node-deficient mice, in the absence of NKT and CD4 cells and without CD40L. Gp96-driven MHC I cross-priming of CD8 CTL in the absence of lymph nodes provides a novel mechanism for local, tissue-based CTL generation at the site of gp96 release. This pathway may constitute a critically important, early detection, and rapid response mechanism that is operative in parenchymal tissues for effective defense against tissue damaging antigenic agents.

Progesterone Induced Blocking Factor (PIBF) Mediates Progesterone Induced Suppression of Decidual Lymphocyte Cytotoxicity

Laškarin G, Tokmadžić VS, Štrbo N, Bogović T, Szekeres‐Bartho J, Randić L, Podack ER, Rukavina D. Progesterone induced blocking factor (PIBF) mediates progesterone induced suppression of decidual lymphocyte cytotoxicity. AJRI 2002; 48:201–209

Progesterone directly and indirectly affects perforin expression in cytolytic cells

Laskarin G, Faust Zs, Štrbo N, SotoŠek V, Szekeres‐Bartho J, Podack ER, Rukavina D. Progesterone directly and indirectly affects perforin expression in cytolytic cells. AJRI 1999; 42:312–320

IL-21 augments natural killer effector functions in chronically HIV-infected individuals

Objective:This study addresses the interleukin (IL)-21 effects on resting peripheral blood natural killer (NK) cells in chronically HIV-infected individuals. Design:The effects of IL-21 on perforin expression, proliferation, degranulation, interferon (IFN)-γ production, cytotoxicity and induction of STAT phosphorylation in NK cells were determined in vitro. Methods:Peripheral blood mononuclear cells from HIV-infected and healthy individuals were incubated in vitro for 6 h, 24 h or 5 days with IL-21 or IL-15. Percentages of perforin, IFN-γ, CD107a, NKG2D and STAT3–5 positive cells were determined within NK cell populations. K562 cells were used as target cells in NK cytotoxicity assay. Results:Frequency of CD56dim cells in chronically HIV-infected individuals was diminished. Perforin expression in CD56dim and CD56bright was comparable in healthy and HIV-infected individuals. IL-15 upregulated perforin expression primarily in CD56bright NK cells, whereas IL-21 upregulated perforin in both NK subsets. IL-21 and IL-15 upregulated CD107a and IFN-γ, as well as NK cytotoxicity. IL-15 predominantly activated STAT5, whereas IL-21 activated STAT5 and STAT3. IL-15, but not IL-21 increased NK cell proliferation in uninfected and HIV-infected individuals. Conclusion:IL-21 augments NK effector functions in chronically HIV-infected individuals and due to its perforin enhancing properties it has potential for immunotherapy or as a vaccine adjuvant.

Surmounting tumor-induced immune suppression by frequent vaccination or immunization in the absence of B cells

Tumor-induced immune suppression is one of the most difficult obstacles to the success of tumor immunotherapy. Here, we show that established tumors suppress CD8 T cell clonal expansion in vivo, which is normally observed in tumor-free mice upon antigen-specific glycoprotein (gp) 96-chaperone vaccination. Suppression of CD8 T-cell expansion by established tumors is independent of tumor-associated expression of the antigen that is recognized by the CD8–T-cell receptor. Vaccination of tumor-bearing mice is associated with increased cellular recruitment to the vaccine site compared with tumor-free mice. However, rejection of established, suppressive tumors required frequent (daily) gp96 vaccination. B cells are known to attenuate T helper cell-1 responses. We found that in B-cell deficient mice, tumor rejection of established tumors can be achieved by a single vaccination. Accordingly, in tumor-free B-cell deficient mice, cognate CD8 cytotoxic T lymphocyte clonal expansion is enhanced in response to gp96-chaperone vaccination. The data have implications for the study of tumor-induced immune suppression and for translation of tumor immunotherapy into the clinical setting. Frequent vaccination with cellular vaccines and concurrent B-cell depletion may greatly enhance the activity of anticancer vaccine therapy in patients.

CD30 - Governor of memory T cells?

Abstract: CD30 is well recognized as a marker expressed by a heterogeneous group of lymphomas and in several immune and autoimmune disorders. However, the function of CD30 in theses diseases or in the normal immune response has remained unclear. Studying gene expression patterns induced by stimulating CD30 signals on a large granular lymphoma cell line, YT, with an agonistic anti‐CD30 antibody, we found that CD30 signals affected proapoptotic and antiapoptotic genes, regulated cytotoxicity, and controlled molecules regulating T cell traffic. Creating CD30‐L deficient mice and studying CD8 CTL activation and memory responses, it was observed that the absence of CD30‐L resulted in diminished primary clonal expansion of CD8 cells. In addition the absence of CD30‐L abolished clonal contraction after primary expansion and interfered with secondary expansion upon boosting. The studies suggest that CD30 regulates CD8 CTL function and survival during memory responses and is important for clonal contraction.

An insight into the dendritic cells at the maternal-fetal interface.

The conditions that permit the genetically distinct fetus to survive and develop within the mother are among the most fascinating immunologic puzzles. The presence of dendritic cells in the maternal decidua pointed to a biologic role of antigen‐presenting cells in maternal–fetal interaction. The method of study included recent findings on the lineage, maturity, phenotype and function of dendritic cells at the maternal–fetal interface. The increment of uterine dendritic cells occurs simultaneously with the decisive phase of gestation, when implantation takes place. Decidual dendritic cells of the first trimester pregnancy, with a phenotype characteristic of the mature myeloid lineage, express MHC class II, co‐stimulatory and adhesion molecules, control Th1/Th2 balance and activate the proliferative response of autologous NK cells. Dentritic cells are specifically equipped to control immunity, to trigger immune response and also to maintain tolerance, avoiding the rejection of the conceptus by the maternal immune system.

Early pregnancy decidual lymphocytes beside perforin use Fas ligand (FasL) mediated cytotoxicity.

Decidual natural killer (NK) cells are the predominant lymphocytes at the maternal-fetal interface. They are involved in defense against virally infected, parasitized and transformed cells and may contribute to the control of trophoblast invasion. The presence of perforin and other possible cytolytic mediators suggests these functions. Cytolytic mechanisms of unstimulated and Th1 cytokine stimulated decidual lymphocytes (DL), as well as purified decidual CD56(+) cells, were analyzed against NK sensitive and resistant targets. DL were isolated from decidual mononuclear cells (DMC) cultured in the medium only or in the presence of Th1 cytokines: IL-2, IL-12, IL-15, IL-18 and their combinations (IL-12/IL-18 or IL-15/IL-18). Fas ligand (FasL), perforin and granzyme B mRNAs expression and cytotoxicity were analyzed by flow cytometry and/or RT-PCR. DL (containing 72.19+/-7.53% of CD56(+) cells), obtained from 18h-cultured DMC in the medium only, expressed perforin, FasL and granzyme B mRNAs and lysed the NK-sensitive K-562 cell line, and also the NK-resistant P815 and P815-Fas transfected cell lines. Concanamycin A, a blocker of granule exocytosis, decreased significantly K-562 lysis, but not P815 lysis. However, the addition of anti-FasL antibody diminished significantly P815 lysis as well. IL-2 and IL-15, known inducers of perforin and FasL mRNAs and protein expression, could not additionally increase P 815 cell lysis by DL cultured within DMC. These results suggest that DL cultured in DMC for 18h, have the characteristics of lymphokine-activated killer (LAK) cells and are able to use efficiently both the perforin and the FasL cytolytic pathways.