T-cell bispecific antibodies in node-positive breast cancer: novel therapeutic avenue for MHC class I loss variants.

BACKGROUND: Tumor-infiltrating lymphocytes (TILs) represent a prognostic factor for survival in primary breast cancer (BC). Nonetheless, neoepitope load and TILs cytolytic activity are modest in BC, compromising the efficacy of immune-activating antibodies, which do not yet compete against immunogenic chemotherapy. PATIENTS AND METHODS: We analyzed by functional flow cytometry the immune dynamics of primary and metastatic axillary nodes [metastatic lymph nodes (mLN)] in early BC (EBC) after exposure to T-cell bispecific antibodies (TCB) bridging CD3ε and human epidermal growth factor receptor 2 (HER2) or Carcinoembryonic Antigen-Related Cell Adhesion Molecule 5 (CEACAM5), before and after chemotherapy. Human leukocyte antigen (HLA) class I loss was assessed by whole exome sequencing and immunohistochemistry. One hundred primary BC, 64 surrounding 'healthy tissue' and 24 mLN-related parameters were analyzed. RESULTS: HLA loss of heterozygosity was observed in EBC, at a clonal and subclonal level and was associated with regulatory T cells and T-cell immunoglobulin and mucin-domain-3 expression restraining the immuno-stimulatory effects of neoadjuvant chemotherapy. TCB bridging CD3ε and HER2 or CEACAM5 could bypass major histocompatibility complex (MHC) class I loss, partially rescuing T-cell functions in mLN. CONCLUSION: TCB should be developed in BC to circumvent low MHC/peptide complexes.

Predictors of responses to immune checkpoint blockade in advanced melanoma.

Immune checkpoint blockers (ICB) have become pivotal therapies in the clinical armamentarium against metastatic melanoma (MMel). Given the frequency of immune related adverse events and increasing use of ICB, predictors of response to CTLA-4 and/or PD-1 blockade represent unmet clinical needs. Using a systems biology-based approach to an assessment of 779 paired blood and tumor markers in 37 stage III MMel patients, we analyzed association between blood immune parameters and the functional immune reactivity of tumor-infiltrating cells after ex vivo exposure to ICB. Based on this assay, we retrospectively observed, in eight cohorts enrolling 190 MMel patients treated with ipilimumab, that PD-L1 expression on peripheral T cells was prognostic on overall and progression-free survival. Moreover, detectable CD137 on circulating CD8+ T cells was associated with the disease-free status of resected stage III MMel patients after adjuvant ipilimumab + nivolumab (but not nivolumab alone). These biomarkers should be validated in prospective trials in MMel.The clinical management of metastatic melanoma requires predictors of the response to checkpoint blockade. Here, the authors use immunological assays to identify potential prognostic/predictive biomarkers in circulating blood cells and in tumor-infiltrating lymphocytes from patients with resected stage III melanoma.

Calreticulin exposure on malignant blasts predicts a cellular anticancer immune response in patients with acute myeloid leukemia.

Experiments performed in mice revealed that anthracyclines stimulate immunogenic cell death that is characterized by the pre-apoptotic exposure of calreticulin (CRT) on the surface of dying tumor cells. Here, we determined whether CRT exposure at the cell surface (ecto-CRT) occurs in human cancer in response to anthracyclines in vivo, focusing on acute myeloid leukemia (AML), which is currently treated with a combination of aracytine and anthracyclines. Most of the patients benefit from the induction chemotherapy but relapse within 1-12 months. In this study, we investigated ecto-CRT expression on malignant blasts before and after induction chemotherapy. We observed that leukemic cells from some patients exhibited ecto-CRT regardless of chemotherapy and that this parameter was not modulated by in vivo chemotherapy. Ecto-CRT correlated with the presence of phosphorylated eIF2α within the blasts, in line with the possibility that CRT exposure results from an endoplasmic reticulum stress response. Importantly, high levels of ecto-CRT on malignant myeloblasts positively correlated with the ability of autologous T cells to secrete interferon-γ on stimulation with blast-derived dendritic cell. We conclude that the presence of ecto-CRT on leukemia cells facilitates cellular anticancer immune responses in AML patients.

Tumor-derived exosomes: a new source of tumor rejection antigens.

Exosomes are small vesicles released by a broad array of hematopoietic cells. Previous studies showed that exosomes released by antigen loaded dendritic cells induce immune-mediated anti-tumor response in mice. Here, we will describe the biochemical properties of tumor-derived exosomes and, their pre-clinical activity as cancer vaccines.

Equilibrium states and ground state of two-dimensional fluid foams.

We study the equilibrium energies of two-dimensional (2D) noncoarsening fluid foams, which consist of bubbles with fixed areas. The equilibrium states correspond to local minima of the total perimeter. We present a theoretical derivation of energy minima; experiments with ferrofluid foams, which can be either highly distorted, locally relaxed, or globally annealed; and Monte Carlo simulations using the extended large-Q Potts model. For a dry foam with small size variance we develop physical insight and an electrostatic analogy, which enables us to (i) find an approximate value of the global minimum perimeter, accounting for (small) area disorder, the topological distribution, and physical boundary conditions; (ii) conjecture the corresponding pattern and topology: small bubbles sort inward and large bubbles sort outward, topological charges of the same signs "repel" while charges of the opposite signs "attract;" (iii) define local and global markers to determine directly from an image how far a foam is from its ground state; (iv) conjecture that, in a local perimeter minimum at prescribed topology, the pressure distribution and thus the edge curvature are unique. Some results also apply to 3D foams.

Tumor-derived exosomes are a source of shared tumor rejection antigens for CTL cross-priming.

The initiation of T-cell-mediated antitumor immune responses requires the uptake and processing of tumor antigens by dendritic cells and their presentation on MHC-I molecules. Here we show in a human in vitro model system that exosomes, a population of small membrane vesicles secreted by living tumor cells, contain and transfer tumor antigens to dendritic cells. After mouse tumor exosome uptake, dendritic cells induce potent CD8+ T-cell-dependent antitumor effects on syngeneic and allogeneic established mouse tumors. Therefore, exosomes represent a novel source of tumor-rejection antigens for T-cell cross priming, relevant for immunointerventions.

Generation of monocyte-derived dendritic cells from patients with renal cell cancer: modulation of their functional properties after therapy with biological response modifiers (IFN-alpha plus IL-2 and IL-12).

The combination of interferon-alpha (IFN-alpha) plus interleukin (IL-2) has been accepted in the treatment of metastatic renal cell carcinoma (MRCC), whereas vaccines based on IL-12 or dendritic cells (DCs) are still being investigated. Here the authors analyzed 1) the feasibility to generate functional monocyte-derived DCs (MDDCs) from patients treated with biological response modifiers (BRMs) who have MRCC, 2) the phenotypic modulations of these MDDCs during BRM treatment. Eight and 13 MRCC patients received IL-2 plus IFN-alpha or IL-12 immunotherapy, respectively. The adherent fraction of mononuclear cells from patients' blood drawn before, during, and after immunotherapy was incubated in clinically approved culture medium supplemented with 5% autologous serum, rhu granulocyte macrophage colony-stimulating factor, and rhuIL-4 for a week. At day 7 or 8 of culture, floating cells were examined in flow cytometric and functional assays (alloreactivity, proliferation assays in the presence of tetanus toxoid or tumor peptides, IL-12 secretion). In all patients except two, MDDCs could be generated but at a lower rate compared with healthy volunteers. Morphologic and phenotypical analyses revealed immature DCs with low levels of CD1a or CD83 expression throughout therapy with BRMs. Capacities in mixed leukocyte reactions were similar to those of healthy volunteers and stable during immunotherapy, whereas presentation of major histocompatibility complex class II tetanus toxoid peptide complexes was slightly enhanced during and after IL-12 therapy. IL-12 expression levels under IFN-gamma and CD40L stimulation were significantly lower in MDDC cultures from patients with MRCC compared with healthy volunteers. Overall, peripheral blood mononuclear cells from a cohort of 21 patients with metastatic disease who were treated with BRMs maintained their ability to differentiate into functional MDDCs with no selective quantitative or qualitative advantage.

Rayleigh-taylor instability with magnetic fluids: experiment and theory

We present experiments showing the Rayleigh-Taylor instability at the interface between a dense magnetic liquid and an immiscible less dense liquid. The liquids are confined in a Hele-Shaw cell and a magnetic field is applied perpendicular to the cell. We measure the wavelength and the growth rate at the onset of the instability as a function of the external magnetic field. The wavelength decreases as the field increases. The amplitude of the interface deformation grows exponentially with time in the early stage, and the growth rate is an increasing function of the field. These results are compared to theoretical predictions given in the framework of linear stability analysis.

Dendritic cells directly trigger NK cell functions: cross-talk relevant in innate anti-tumor immune responses in vivo.

Cytotoxic T lymphocytes and natural killer cells are essential effectors of anti-tumor immune responses in vivo. Dendritic cells (DC) 'prime' tumor antigen-specific cytotoxic T lymphocytes; thus, we investigated whether DC might also trigger the innate, NK cell-mediated anti-tumor immunity. In mice with MHC class I-negative tumors, adoptively transferred- or Flt3 ligand-expanded DC promoted NK cell-dependent anti-tumor effects. In vitro studies demonstrated a cell-to-cell contact between DC and resting NK cells that resulted in a substantial increase in both NK cell cytolytic activity and IFN-gamma production. Thus, DC are involved in the interaction between innate and adaptive immune responses.

Eradication of established murine tumors using a novel cell-free vaccine: dendritic cell-derived exosomes.

Dendritic cells (DCs) are professional antigen presenting cells with the unique capacity to induce primary and secondary immune responses in vivo. Here, we show that DCs secrete antigen presenting vesicles, called exosomes, which express functional Major Histocompatibility Complex class I and class II, and T-cell costimulatory molecules. Tumor peptide-pulsed DC-derived exosomes prime specific cytotoxic T lymphocytes in vivo and eradicate or suppress growth of established murine tumors in a T cell-dependent manner. Exosome-based cell-free vaccines represent an alternative to DC adoptive therapy for suppressing tumor growth.

Diversity of the cytotoxic melanoma-specific immune response: some CTL clones recognize autologous fresh tumor cells and not tumor cell lines.

In the present work, to analyze the heterogeneity of the tumor-specific cytotoxic immune response, a large number of T cell clones were generated from the infiltrate of a tumor-proximal invaded lymph node, and two kinds of melanoma-specific CD8+ CTL clones were derived. The majority of T cell clones (about a hundred) are characterized by a specific lysis of the autologous tumor cell lines. Among 34 of the latter clones, HLA-A2 molecule and MART-1(27-35) peptide have been shown to play a predominant role in tumor recognition. However, no significant amplification at the tumor site was observed for 3 of these CTL. The other kind of tumor-specific CTL (1 oligoclonal and 2 clonal cell lines) did not lyse the autologous melanoma cell lines but lysed the "fresh" autologous tumor cells in a MHC class I-dependent manner. Functional analysis of the two different CTL clones have shown that they did not lyse NK targets, autologous peripheral monocytes, activated T cells, and transformed B cells or any of the few allogeneic cultured and uncultured melanoma cells we tested. TCR repertoire analysis has shown that one of these CTL clones was significantly detectable "in situ" among tumor-infiltrating lymphocytes, while not detectable among PBMC. Such melanoma-specific lymphocytes, which could not have been picked out through conventional screening procedures using tumor cell lines, could potentially play a role in tumor rejection. These results suggest that the immune response analyzed toward melanoma cell lines does not totally reflect the in situ immune status.

TCR alpha/beta and TCR gamma/delta CD4-/CD8- HLA-DR alloreactive CTL clones do not use Fas/Fas ligand pathway to lyse their specific target cells.

The expression of Fas Ligand (FasL) on human TCRalpha/beta and TCRgamma/delta CD4-/CD8- MHC class II-alloreactive clones and Fas/FasL-mediated cytotoxicity were investigated. These clones mediated a HLA-DR2-restricted cytotoxicity toward E418 B cell line (Fas+). Northern blot analysis demonstrated that all the clones expressed FasL mRNA upon stimulation with E418 specific target. FasL surface expression was detected by immunofluorescence analysis using Fas-Fc soluble protein as well as anti-FasL polyclonal antibodies. Cytotoxicity experiments performed in the presence of anti-Fas, anti-FasL and Fas-Fc molecule indicated that these reagents were unable to inhibit T cell clone mediated lysis toward E418. In addition, when emetine, known to inhibit the induction of Fas-mediated killing, was added during the cytolysis effector phase, no inhibition was observed. These data strongly suggest that Fas/FasL pathway is not involved in this particular T-cell clone-mediated lysis. This cytotoxicity is extracellular Ca(2+)-dependent and is abolished in the presence of EGTA suggesting that it is mainly perforin/granzyme-based.

CD48 may serve as an accessory molecule for the activation of a subset of human gamma/delta T cells.

To further assess the role of CD48 in the interaction of human gamma/delta T cells with their specific target, we generated two series of alloreactive clones, L and K. These clones express a V1-D-J1-C delta chain associated to V3-J2-C2 (L) or V2-J2-C2 (K) gamma chain. Functionally they were CTLs able to lyse the sensitizing B-cell line E418. The cytotoxicity of the L and K clones toward E418 was inhibited by anti-CD48 mAb. That of the L clones was also inhibited by anti-HLA class I mAbs. Variation in L and K lysis profile was observed against a panel of CD48+ targets, further strengthening the argument that they display distinct specificities and suggesting that they do not recognize CD48. Heterogeneity in TCR gene segment usage, MHC-dependent recognition of E418 by the L clones, and resistance of some CD48+ targets strongly suggest that CD48 itself does not interact with L and K TCR. Transfection of CHO cells with CD48 induced killing by the K clones. This killing was inhibited by anti-CD48 mAbs. Taking into account the recent reports on CD48 as an accessory molecule, our results suggest that by binding to CD2 (and/or an unknown ligand), CD48 may serve to strengthen E/T interaction and may contribute to the activation of a minor subset of gamma/delta T cells.

Human TCR-gamma/delta alloreactive response to HLA-DR molecules. Comparison with response of TCR-alpha/beta.

We have analyzed the human gamma/delta T cell alloreactive response to class II HLA-DR molecules and attempted to compare this response with that mediated by the TCR-alpha/beta counterparts. Several gamma/delta CTL clones from a healthy individual were generated in mixed lymphocyte reactions against an EBV-transformed B cell line termed E418. Fine specificity and primary TCR structure of 10 representative clones (all CD4- CD8 +/-) were then determined. Functional studies, with the use of B cell lines homozygous for HLA-DR (DR1-10), indicated that all gamma/delta T cell clones specifically reacted with HLA-DR2 molecules. In addition, five clones were able to cross-react with subtypes of HLA-DR8. Extended panel target experiments, including lymphoblastoid cells expressing various HLA-DR2 subtypes, showed that the T cell clones displayed distinct fine specificities. Clones with broad (Dw2, Dw12, Dw21, Dw8.1, and Dw8.2) or in contrast, more restricted (DRB1*1501 or DRB1*1503) specificity were identified. Furthermore, amino acid substitutions at predicted peptide binding site position 30 and TCR-interacting position 67 of the DRB*1 beta-chain seemed to affect alloresponse of some T cell clones. With respect to TCR-gamma/delta structure, diversity in gene segment usage was observed, with the predominance of T cells using a V3-J gamma 2/V1-J delta 1+ receptor. A smaller fraction of the cells expressed TCR comprising V gamma 9 and V delta 1 regions. In contrast, the V delta 3 gene segment was used by a minority of the cells, and the V delta 2 was not expressed by any T cell clone. Together, the present data indicate that similarly to TCR-alpha/beta, human TCR-gamma/delta lymphocytes may recognize in a highly specific fashion a particular HLA-DR heterodimer. T cell clones cross-reacting with other HLA-DR molecules were also identified. Despite some degree of heterogeneity, V gene segment use by alloreactive clones seemed to be nonrandom. No obvious correlation between TCR gene use and HLA-DR alloreactivity could be identified. Moreover, our results suggest that similarly to TCR-alpha/beta cells, foreign MHC-bound peptides may contribute to TCR-gamma/delta alloreactive response.

A physiological amount of zinc supplementation: effects on nutritional, lipid, and thymic status in an elderly population.

Forty-four institutionalized elderly subjects with body mass indexes (BMI) of either > or = 24 or < or = 21 participated in a 16-wk crossover study designed to determine the effects of low-dose zinc supplementation [306 mumol (20 mg)/d] on food intake, anthropometry, and biochemical and immunological indexes. Initial serum zinc concentrations were low in both groups and increased by approximately 20% after zinc supplementation. Zinc supplementation allowed a partial but significant restoration of serum thymulin activity and improved nutritional status (food intake and serum albumin and transthyretin concentrations) but had no effect on anthropometric indexes or serum apolipoproteins, except apolipoprotein CII and apolipoprotein CIII. After zinc supplementation, serum copper concentration decreased but there was no change in the ratio of low-density-lipoprotein cholesterol to high-density-lipoprotein cholesterol. Low-dose zinc supplementation allows restoration, at least partially, of nutritional and thymic status without the known disadvantages of high doses of zinc.