A Hypothetical Model Suggesting Some Possible Ways That the Progesterone Receptor May Be Involved in Cancer Proliferation.

Cancer and the fetal-placental semi-allograft share certain characteristics, e.g., rapid proliferation, the capacity to invade normal tissue, and, related to the presence of antigens foreign to the host, the need to evade immune surveillance. Many present-day methods to treat cancer use drugs that can block a key molecule that is important for one or more of these characteristics and thus reduce side effects. The ideal molecule would be one that is essential for both the survival of the fetus and malignant tumor, but not needed for normal cells. There is a potential suitable candidate, the progesterone induced blocking factor (PIBF). The parent 90 kilodalton (kDa) form seems to be required for cell-cycle regulation, required by both the fetal-placental unit and malignant tumors. The parent form may be converted to splice variants that help both the fetus and tumors escape immune surveillance, especially in the fetal and tumor microenvironment. Evidence suggests that membrane progesterone receptors are involved in PIBF production, and indeed there has been anecdotal evidence that progesterone receptor antagonists, e.g., mifepristone, can significantly improve longevity and quality of life, with few side effects.

MNSFß Regulates TNFα Production by Interacting with RC3H1 in Human Macrophages, and Dysfunction of MNSFß in Decidual Macrophages Is Associated With Recurrent Pregnancy Loss.

Decidual macrophages (dMϕ) are the second largest population of leukocytes at the maternal-fetal interface and play critical roles in maintaining pregnancy. Our previous studies demonstrated the active involvement of monoclonal nonspecific suppressor factor-ß (MNSFß) in embryonic implantation and pregnancy success. MNSFß is a ubiquitously expressed ubiquitin-like protein that also exhibits immune regulatory potential, but its function in human dMϕ remains unknown. Here, we observed that the proportion of CD11chigh (CD11cHI) dMϕ was significantly increased in dMϕ derived from patients with recurrent pregnancy loss (RPL dMϕ) compared to those derived from normal pregnant women (Control dMϕ). The production of MNSFß and TNFα by RPL dMϕ was also significantly increased compared to that by Control dMϕ. Conditioned medium from RPL dMϕ exerted an inhibitory effect on the invasiveness of human trophoblastic HTR8/SVneo cells, and this effect could be partially reversed by a neutralizing antibody against TNFα. Bioinformatics analysis indicated a potential interaction between MNSFß and RC3H1, a suppressor of TNFα transcription. Immunoprecipitation experiments with human Mϕ differentiated from the human monocyte cell line Thp1 (Thp1-derived Mϕ) proved the binding of MNSFß to RC3H1. Specific knockdown of MNSFß in Thp1-derived Mϕ led to a marked decrease in TNFα production, which could be reversed by inhibiting RC3H1 expression. Interestingly, a significant decrease in the protein level of RC3H1 was observed in RPL dMϕ. Together, our findings indicate that aberrantly increased MNSFß expression in dMϕ may promote TNFα production via its interaction with RC3H1, and these phenomena could result in the disruption of the immune balance at the maternal-fetal interface and thus pregnancy loss.

Biologia futura: embryo-maternal communication via progesterone-induced blocking factor (PIBF) positive embryo-derived extracellular vesicles. Their role in maternal immunomodulation.

Paternal antigens expressed by the foetus are recognized as foreign. Therefore,-according to the rules of transplantation immunity-the foetus ought to be "rejected". However, during normal gestation, maternal immune functions are re-adjusted, in order to create a favourable environment for the developing foetus. Some of the mechanisms that contribute to the altered immunological environment, for example, the cytokine balance and NK cell function, with special emphasis on the role of progesterone and the progesterone-induced blocking factor (PIBF) will be reviewed.

Progesterone-induced blocking factor-mediated Th1/Th2 balance correlates with fetal arrest in women who underwent in vitro fertilization and embryo transfer.

The role of progesterone-induced blocking factor (PIBF)-mediated Th1/Th2 balance in delivery outcomes of in vitro fertilization and embryo transfer (IVF-ET) has not been fully elucidated. In this study, 73 infertile women with successful IVF-ET were enrolled (16 fetal arrests and 57 live births). PIBF and IL-4 levels were significantly lower in the fetal arrest group than in the live birth group (p < 0.05). TNF-α level and Th1/Th2 ratios were significantly higher in the fetal arrest group than in the live birth group (p < 0.05). High TNF-α level and Th1/Th2 ratios were risk factors for fetal arrest, whereas increased PIBF and IL-4 levels were protective factors (P < 0.05). Increased TNF-α/IL-4 exhibited relatively strong predictive value for fetal arrest (AUC, 0.855; sensitivity, 93.8%; specificity, 71.9%). In summary, the PIBF-mediated Th1/Th2 balance is closely correlated with delivery outcomes of IVF-ET. TNF-α/IL-4 may be a predictive marker of fetal arrest.

Ex Vivo-Induced Bone Marrow-Derived Myeloid Suppressor Cells Prevent Corneal Allograft Rejection in Mice.

Purpose: To investigate the effects of ex vivo-induced bone marrow myeloid-derived suppressor cells (BM-MDSCs) on allogeneic immune responses in corneal transplantation. Methods: Bone marrow cells from C57BL/6J (B6) mice were cultured with IL-6 and GM-CSF for four days. The ex vivo induction of the BM-MDSCs was assessed using flow cytometry, inducible nitric oxide synthase (iNOS) mRNA expression using reverse transcription-quantitative polymerase chain reaction, and nitric oxide (NO) production in allogeneic stimulation. T-cell proliferation and regulatory T-cell (Treg) expansion were investigated on allogeneic stimulation in the presence of ex vivo-induced BM-MDSCs. IFN-γ, IL-2, IL-10, and TGF-ß1 protein levels were measured using enzyme-linked immunosorbent assays. After subconjunctival injection of ex vivo-induced BM-MDSCs, the migration of the BM-MDSCs into corneal grafts, allogeneic corneal graft survival, neovascularization, and lymphangiogenesis were assessed using flow cytometry, slit-lamp microscopy, and immunohistochemistry. Results: The combination of GM-CSF and IL-6 significantly induced BM-MDSCs with increased iNos mRNA expression. The ex vivo-induced BM-MDSCs promoted NO release in allogeneic stimulation in vitro. The ex vivo-induced BM-MDSCs inhibited T-cell proliferation and promoted Treg expansion. Decreased IFN-γ and increased IL-2, IL-10, and TGF-ß1 production was observed in coculture of ex vivo-induced BM-MDSCs. Injected ex vivo-induced BM-MDSCs were confirmed to migrate into the grafts. The injected BM-MDSCs also prolonged corneal graft survival and prevented angiogenesis and lymphangiogenesis. Conclusions: The ex vivo-induced BM-MDSCs have suppressive effects on allogeneic immune responses and prolong corneal allograft survival via the iNOS pathway, indicating that they may be a potential therapeutic tool for corneal transplantation.

Metabolic support of tumour-infiltrating regulatory T cells by lactic acid.

Regulatory T (Treg) cells, although vital for immune homeostasis, also represent a major barrier to anti-cancer immunity, as the tumour microenvironment (TME) promotes the recruitment, differentiation and activity of these cells1,2. Tumour cells show deregulated metabolism, leading to a metabolite-depleted, hypoxic and acidic TME3, which places infiltrating effector T cells in competition with the tumour for metabolites and impairs their function4-6. At the same time, Treg cells maintain a strong suppression of effector T cells within the TME7,8. As previous studies suggested that Treg cells possess a distinct metabolic profile from effector T cells9-11, we hypothesized that the altered metabolic landscape of the TME and increased activity of intratumoral Treg cells are linked. Here we show that Treg cells display broad heterogeneity in their metabolism of glucose within normal and transformed tissues, and can engage an alternative metabolic pathway to maintain suppressive function and proliferation. Glucose uptake correlates with poorer suppressive function and long-term instability, and high-glucose conditions impair the function and stability of Treg cells in vitro. Treg cells instead upregulate pathways involved in the metabolism of the glycolytic by-product lactic acid. Treg cells withstand high-lactate conditions, and treatment with lactate prevents the destabilizing effects of high-glucose conditions, generating intermediates necessary for proliferation. Deletion of MCT1-a lactate transporter-in Treg cells reveals that lactate uptake is dispensable for the function of peripheral Treg cells but required intratumorally, resulting in slowed tumour growth and an increased response to immunotherapy. Thus, Treg cells are metabolically flexible: they can use 'alternative' metabolites in the TME to maintain their suppressive identity. Further, our results suggest that tumours avoid destruction by not only depriving effector T cells of nutrients, but also metabolically supporting regulatory populations.

Comprehensive Analysis of Immunoinhibitors Identifies LGALS9 and TGFBR1 as Potential Prognostic Biomarkers for Pancreatic Cancer.

Pancreatic cancer (PC) is one of the most deadly cancers worldwide. To uncover the unknown novel biomarker used to indicate early diagnosis and prognosis in the molecular therapeutic field of PC is extremely of importance. Accumulative evidences indicated that aberrant expression or activation of immunoinhibitors is a common phenomenon in malignances, and significant associations have been noted between immunoinhibitors and tumorigenesis or progression in a wide range of cancers. However, the expression patterns and exact roles of immunoinhibitors contributing to tumorigenesis and progression of pancreatic cancer (PC) have not yet been elucidated clearly. In this study, we investigated the distinct expression and prognostic value of immunoinhibitors in patients with PC by analyzing a series of databases, including TISIDB, GEPIA, cBioPortal, and Kaplan-Meier plotter database. The mRNA expression levels of IDO1, CSF1R, VTCN1, KDR, LGALS9, TGFBR1, TGFB1, IL10RB, and PVRL2 were found to be significantly upregulated in patients with PC. Aberrant expression of TGFBR1, VTCN1, and LGALS9 was found to be associated with the worse outcomes of patients with PC. Bioinformatics analysis demonstrated that LGALS9 was involved in regulating the type I interferon signaling pathway, interferon-gamma-mediated signaling pathway, RIG-I-like receptor signaling pathway, NF-kappa B signaling pathway, cytosolic DNA-sensing pathway, and TNF signaling pathway. And TGFB1 was related to mesoderm formation, cell matrix adhesion, TGF-beta signaling pathway, and Hippo signaling pathway. These results suggested that LGALS9 and TGFBR1 might serve as potential prognostic biomarkers and targets for PC.

Inhibitory effects of Vitamin D on inflammation and IL-6 release. A further support for COVID-19 management?

The ongoing worldwide pandemic of Coronavirus disease 2019 (COVID-19), raised the urgency to address knowledge gaps and to establish evidence for improving management and control of this viral infection. Throughout a keen analysis of the World Health Organization (WHO) most updated data, a gender-specific difference in the occurrence of infection was determined, which seems to correlate with patient's vitamin D status. Therefore, our purpose is to provide insights into the nutritional importance of vitamin D for its immunomodulatory effect, in order to help counteracting the COVID-19 pandemic. Novel interesting findings suggest that vitamin D, by inducing progesterone-induced blocking factor (PIBF), might regulate the immune response and also modulate cytokine IL-6, which appears to be increased in COVID-19 infections. Therefore, in addition to the standard recommendations to prevent the infection, supplementation of vitamin D might be considered an approach to help counteracting this global epidemic.

Therapy Aimed to Suppress the Production of the Immunosuppressive Protein Progesterone Induced Blocking Factor (PIBF) May Provide Palliation and/or Increased Longevity for Patients With a Variety of Different Advanced Cancers - A Review.

Progesterone induced blocking factor (PIBF) is a unique protein that is not present in normal cells, but is found predominantly in rapidly growing cells of the fetal placental unit or cancer cells. There is a larger "parent" form that is a nuclear protein involved in cell to cell regulation, allowing tumor cells to proliferate and invade tissues. The parent compound is cleaved into smaller intracytoplasmic isoforms that can suppress cellular immune response, especially, but not limited to natural killer cells. The progesterone receptor antagonist mifepristone can suppress messenger RNA for PIBF, but can also suppress the intracytoplasmic protein. Treating cancer cell lines, intact animals with a variety of spontaneous cancers, and people with various cancers with mifepristone, has been found to inhibit cancer growth, and provide both palliation of symptoms and longevity possibly by suppressing this unique immunomodulatory protein.

The Role of Extracellular Vesicles and PIBF in Embryo-Maternal Immune-Interactions.

Pregnancy represents a unique immunological situation. Though paternal antigens expressed by the conceptus are recognized by the immune system of the mother, the immune response does not harm the fetus. Progesterone and a progesterone induced protein; PIBF are important players in re-adjusting the functioning of the maternal immune system during pregnancy. PIBF expressed by peripheral pregnancy lymphocytes, and other cell types, participates in the feto-maternal communication, partly, by mediating the immunological actions of progesterone. Several splice variants of PIBF were identified with different physiological activity. The full length 90 kD PIBF protein plays a role in cell cycle regulation, while shorter splice variants are secreted and act as cytokines. Aberrant production of PIBF isoforms lead to the loss of immune-regulatory functions, resulting in and pregnancy failure. By up regulating Th2 type cytokine production and by down-regulating NK activity, PIBF contributes to the altered attitude of the maternal immune system. Normal pregnancy is characterized by a Th2-dominant cytokine balance, which is partly due to the action of the smaller PIBF isoforms. These bind to a novel form of the IL-4 receptor, and induce increased production of IL-3, IL-4, and IL-10. The communication between the conceptus and the mother is established via extracellular vesicles (EVs). Pre-implantation embryos produce EVs both in vitro, and in vivo. PIBF transported by the EVs from the embryo to maternal lymphocytes induces increased IL-10 production by the latter, this way contributing to the Th2 dominant immune responses described during pregnancy.

[Modeling shifts and correlation of hematological and immunological parameters in chronic generalized periodontitis]. / Modelirovanie sdvigov i korrelyatsionnykh svyazei gematologicheskikh i immunologicheskikh pokazatelei u bol'nykh khronicheskim generalizovannym parodontitom.

Simulation of shifts and correlations of hematological and immunological parameters of the T-cell immunity in 106 patients with chronic periodontal disease and 65 individuals of the control group allowed us to identify leading diagnostic parameters with the quantitative evaluation and enhanced contingency of intersystem communications in peripheral blood and cellular immunity. Mathematical modelling of systemic immunity revealed the most significant shift in absolute number of T-helpers and T-lymphocytes in peripheral blood. Less significant shifts were seen in relative numbers of T-helpers and T-suppressors as well as neutrophilic activity. The revealed changes may play an important role in both diagnosis and therapy of periodontal disease.

Development of Phage-Based Antibody Fragment Reagents for Affinity Enrichment of Bacterial Immunoglobulin G Binding Proteins.

Disease and death caused by bacterial infections are global health problems. Effective bacterial strategies are required to promote survival and proliferation within a human host, and it is important to explore how this adaption occurs. However, the detection and quantification of bacterial virulence factors in complex biological samples are technically demanding challenges. These can be addressed by combining targeted affinity enrichment of antibodies with the sensitivity of liquid chromatography-selected reaction monitoring mass spectrometry (LC-SRM MS). However, many virulence factors have evolved properties that make specific detection by conventional antibodies difficult. We here present an antibody format that is particularly well suited for detection and analysis of immunoglobulin G (IgG)-binding virulence factors. As proof of concept, we have generated single chain fragment variable (scFv) antibodies that specifically target the IgG-binding surface proteins M1 and H of Streptococcus pyogenes. The binding ability of the developed scFv is demonstrated against both recombinant soluble protein M1 and H as well as the intact surface proteins on a wild-type S. pyogenes strain. Additionally, the capacity of the developed scFv antibodies to enrich their target proteins from both simple and complex backgrounds, thereby allowing for detection and quantification with LC-SRM MS, was demonstrated. We have established a workflow that allows for affinity enrichment of bacterial virulence factors.

IL-37b suppresses T cell priming by modulating dendritic cell maturation and cytokine production via dampening ERK/NF-κB/S6K signalings.

Interleukin 37b (IL-37b) plays a key role in suppressing immune responses, partially by modulating the function of dendritic cells (DCs). However, the precise mechanisms are still largely unknown. Here, we investigated the effects of IL-37b on DC maturation and T cell responses induced by DCs, and explored the involved signaling pathways. It was found that IL-37b down-regulated the expressions of co-stimulatory molecules CD80 and CD86 on DCs in vitro. At the same time, the expressions of pro-inflammatory cytokines, such as TNF-α and IL-6, were suppressed, while the expression of the T cell inhibitory cytokine TGF-ß was increased in IL-37b-treated DCs. In addition, the activation effect of DCs on T cells was impaired by IL-37b. We further revealed that extracellular single-regulated kinase (ERK), nuclear factor-κB (NF-κB), and mTOR-S6K signaling pathways were involved in the inhibition of DCs induced by IL-37b. This was confirmed by the similarly suppressive effect of chemical inhibitors against NF-κB, ERK, and S6K on the expressions of IL-6 and TNF-α in DCs. In conclusion, these results demonstrated that IL-37b suppressed DC maturation and immunostimulatory capacity in T cell priming by involving in ERK, NF-κB, and S6K-based inhibitory signaling pathways.

Impaired gp100-Specific CD8(+) T-Cell Responses in the Presence of Myeloid-Derived Suppressor Cells in a Spontaneous Mouse Melanoma Model.

Murine tumor models that closely reflect human diseases are important tools to investigate carcinogenesis and tumor immunity. The transgenic (tg) mouse strain tg(Grm1)EPv develops spontaneous melanoma due to ectopic overexpression of the metabotropic glutamate receptor 1 (Grm1) in melanocytes. In the present study, we characterized the immune status and functional properties of immune cells in tumor-bearing mice. Melanoma development was accompanied by a reduction in the percentages of CD4(+) T cells including regulatory T cells (Tregs) in CD45(+) leukocytes present in tumor tissue and draining lymph nodes (LNs). In contrast, the percentages of CD8(+) T cells were unchanged, and these cells showed an activated phenotype in tumor mice. Endogenous melanoma-associated antigen glycoprotein 100 (gp100)-specific CD8(+) T cells were not deleted during tumor development, as revealed by pentamer staining in the skin and draining LNs. They, however, were unresponsive to ex vivo gp100-peptide stimulation in late-stage tumor mice. Interestingly, immunosuppressive myeloid-derived suppressor cells (MDSCs) were recruited to tumor tissue with a preferential accumulation of granulocytic MDSC (grMDSCs) over monocytic MDSC (moMDSCs). Both subsets produced Arginase-1, inducible nitric oxide synthase (iNOS), and transforming growth factor-ß and suppressed T-cell proliferation in vitro. In this work, we describe the immune status of a spontaneous melanoma mouse model that provides an interesting tool to develop future immunotherapeutical strategies.

Inhibition of the lymphocyte metabolic switch by the oxidative burst of human neutrophils.

Activation of the phagocytic NADPH oxidase-2 (NOX-2) in neutrophils is a critical process in the innate immune system and is associated with elevated local concentrations of superoxide, hydrogen peroxide (H2O2) and hypochlorous acid. Under pathological conditions, NOX-2 activity has been implicated in the development of autoimmunity, indicating a role in modulating lymphocyte effector function. Notably, T-cell clonal expansion and subsequent cytokine production requires a metabolic switch from mitochondrial respiration to aerobic glycolysis. Previous studies demonstrate that H2O2 generated from activated neutrophils suppresses lymphocyte activation but the mechanism is unknown. We hypothesized that activated neutrophils would prevent the metabolic switch and suppress the effector functions of T-cells through a H2O2-dependent mechanism. To test this, we developed a model co-culture system using freshly isolated neutrophils and lymphocytes from healthy human donors. Extracellular flux analysis was used to assess mitochondrial and glycolytic activity and FACS analysis to assess immune function. The neutrophil oxidative burst significantly inhibited the induction of lymphocyte aerobic glycolysis, caused inhibition of oxidative phosphorylation and suppressed lymphocyte activation through a H2O2-dependent mechanism. Hydrogen peroxide and a redox cycling agent, DMNQ, were used to confirm the impact of H2O2 on lymphocyte bioenergetics. In summary, we have shown that the lymphocyte metabolic switch from mitochondrial respiration to glycolysis is prevented by the oxidative burst of neutrophils. This direct inhibition of the metabolic switch is then a likely mechanism underlying the neutrophil-dependent suppression of T-cell effector function.

From Mysterious Supernatant Entity to miRNA-150 in Antigen-Specific Exosomes: a History of Hapten-Specific T Suppressor Factor.

Soon after the discovery of T suppressor cells by Gershon in 1970, it was demonstrated that one subpopulation of these lymphocytes induced by i.v. hapten injection suppresses contact sensitivity response mediated by effector CD4+ or CD8+ T cells in mice through the release of soluble T suppressor factor (TsF) that acts antigen specifically. Our experiments showed that biologically active TsF is a complex entity consisting of two subfactors, one antigen specific and other non-specific, produced by differently induced populations of cells. In following years, we found that the antigen-specific subfactor is a light chain of IgM antibody that is produced by B1a lymphocytes. However, the exact nature of non-specific part remained a mystery for about 30 years. Our current studies characterized TsF as regulatory miRNA-150 carried by T suppressor cell-derived exosomes that are antigen specific due to a surface coat of IgM antibody light chains produced by B1a cells. The present communication briefly summarizes our studies on TsF that led to discovery of regulating miRNA that acts antigen specifically to suppress immune response.

Myeloid-derived suppressor cells control microbial sepsis.

PURPOSE: To investigate the role of myeloid-derived suppressor cells (MDSCs) during sepsis in mice. MDSCs are a heterogeneous population of cells that expand during cancer, inflammation and infection. These cells, by their ability to suppress T lymphocyte proliferation, regulate immune responses during various diseases. Their role during microbial infections is scarcely known. METHODS: Septic shock was induced by caecal ligation and puncture in adult male BALB/c mice; sham-operated animals served as controls. Animals were killed under anaesthesia to harvest blood and organs. RESULTS: Polymicrobial sepsis induced a progressive accumulation of MDSCs in spleens that were found to be enlarged in surviving mice. MDSCs harvested at day 10 after the onset of infection were highly responsive to LPS in terms of cytokines secretion, NF-kB activation, ROS production and arginase I activity, whereas early-appearing (day 3) MDSCs poorly responded to this stimulus. By contrast, both day 3 and day 10 MDSCs were able to inhibit T cell proliferation. Adoptive transfer of day 10 MDSCs to septic mice attenuated peritoneal cytokine production, increased bacterial clearance and dramatically improved survival rate. CONCLUSION: These results provide new information on the role of MDSCs, suggesting a protective effect during sepsis. Pharmacologic agents known to promote the expansion of MDSCs should thus be further studied for sepsis treatment.

[PIBF - Progesterone-Induced Blocking Factor]. / PIBF - Progesteron induzierter Blockierfaktor.

Pregnancy is a unique immunological situation in which 2 allogeneic organisms live in intimate symbiosis without developing rejection reactions. At different locations, interfaces exist between mother and foetus with direct contact between both individuals: 1) maternal blood surrounds foetal villi, which are covered with syncitiotrophoblast cells; 2) cytotrophoblast cells invade the decidua, in which they touch tissue lymphocytes; 3) trophoblast cells, which substitute endothelium of maternal arterioles filled with maternal blood; and 4) trophoblast particles, which are expressed from syncytiotrophoblast and circulate within the maternal blood until they settle in the lung capillaries, where they become degraded by alveolar macrophages. Several factors are known which support the specific immunotolerance of the mother to her foetus and are focussed by current research in reproductive immunology. One of these factors is progesterone-induced blocking factor (PIBF). Originally, it was discovered as a 34 kDa protein, which is released from lymphocytes of healthy pregnant women under the influence of progesterone. PIBF has immunomodulatory functions in vivo and in vitro, which are important for the establishment of immunotolerance between mother and foetus and, thereby, for the regular course of pregnancy. Finally, during the last years, several tumours have been identified to produce PIBF, which supports their immune escape and which may have the potential to become a novel tumour biomarker and which may lead to the development of new therapeutic strategies.

Immune regulation by peripheral suppressor T cells induced upon homotypic T cell/T cell interactions.

We have shown previously that homotypic interaction of resting memory CD4 T cells with activated T cells induces the production of cytokines with immunoregulatory potential (IL-10, IL-4) from the former. Here, we analyzed the effector functions of these T cells stimulated by homotypic T cell interaction. T cells induced upon homotypic T cell interaction expressed CD25 and reduced levels of CD127 and produced TGF-ß. Functionally, homotypic T cell interaction-induced T cells were anergic and inhibited the proliferation of CD25-negative T cells as potently as naturally occurring CD25-positive Tregs in vitro. They also prevented clonotypic expansion of OVA TCR tg T cells in BALB/c mice upon antigenic challenge in vivo. The generation of suppressor T cells by homotypic T cell contact is anchored and tuned through interactions of LFA-1 and its ligands ICAM-1, ICAM-2, and ICAM-3. Together, the data suggest a negative-feedback mechanism of specific immunity involving bystander-activated memory T cells.