Immune perturbations in human pancreas lymphatic tissues prior to and after type 1 diabetes onset.

Autoimmune destruction of pancreatic ß cells results in type 1 diabetes (T1D), with pancreatic immune infiltrate representing a key feature in this process. Studies of human T1D immunobiology have predominantly focused on circulating immune cells in the blood, while mouse models suggest diabetogenic lymphocytes primarily reside in pancreas-draining lymph nodes (pLN). A comprehensive study of immune cells in human T1D was conducted using pancreas draining lymphatic tissues, including pLN and mesenteric lymph nodes, and the spleen from non-diabetic control, ß cell autoantibody positive non-diabetic (AAb+), and T1D organ donors using complementary approaches of high parameter flow cytometry and CITEseq. Immune perturbations suggestive of a proinflammatory environment were specific for T1D pLN and AAb+ pLN. In addition, certain immune populations correlated with high T1D genetic risk independent of disease state. These datasets form an extensive resource for profiling human lymphatic tissue immune cells in the context of autoimmunity and T1D.

Phenotypic Characterization of SLex+ and CLA+ CD4+ T Cells.

Recent advances in high-resolution multiparametric flow cytometry enable ever deeper analysis of human lymphocyte subsets that require rigorous methodology development and optimization. Here, we detail methods to characterize glycosylated Sialyl-LewisX (SLeX)- or cutaneous lymphocyte-associated antigen (CLA)-expressing CD4+ T cells using two separate multiparametric flow cytometry panels enabling the identification of memory subsets, Th subsets, and expression of diverse activation markers and chemokine receptors. The proposed protocol allows optimal resolution of the measured parameters while minimizing background in a 25-parameter experiment. For complete details on the use and execution of this protocol, please refer to Colomb et al. (2020).

Deep Immune Profiling of MIS-C demonstrates marked but transient immune activation compared to adult and pediatric COVID-19.

Pediatric COVID-19 following SARS-CoV-2 infection is associated with fewer hospitalizations and often milder disease than in adults. A subset of children, however, present with Multisystem Inflammatory Syndrome in Children (MIS-C) that can lead to vascular complications and shock, but rarely death. The immune features of MIS-C compared to pediatric COVID-19 or adult disease remain poorly understood. We analyzed peripheral blood immune responses in hospitalized SARS-CoV-2 infected pediatric patients (pediatric COVID-19) and patients with MIS-C. MIS-C patients had patterns of T cell-biased lymphopenia and T cell activation similar to severely ill adults, and all patients with MIS-C had SARS-CoV-2 spike-specific antibodies at admission. A distinct feature of MIS-C patients was robust activation of vascular patrolling CX3CR1+ CD8 T cells that correlated with use of vasoactive medication. Finally, whereas pediatric COVID-19 patients with acute respiratory distress syndrome (ARDS) had sustained immune activation, MIS-C patients displayed clinical improvement over time, concomitant with decreasing immune activation. Thus, non-MIS-C versus MIS-C SARS-CoV-2 associated illnesses are characterized by divergent immune signatures that are temporally distinct and implicate CD8 T cells in clinical presentation and trajectory of MIS-C.

Avelumab, an IgG1 anti-PD-L1 Immune Checkpoint Inhibitor, Triggers NK Cell-Mediated Cytotoxicity and Cytokine Production Against Triple Negative Breast Cancer Cells.

The standard treatment for Triple Negative Breast Cancer (TNBC) patients is cytotoxic chemotherapy, but it is restricted since the duration of response is usually short. Blocking the PD-1/PD-L1 pathway through monoclonal antibodies (mAbs) appears to be a promising therapeutic strategy for TNBC patients. Avelumab is a human IgG1 anti-PD-L1 mAb being tested in clinical trials that may also trigger antibody-dependent cell-mediated cytotoxicity (ADCC) against cancer cells as an additional antitumor activity. In the present work, we studied in vitro Avelumab-mediated ADCC against a panel of TNBC cells with different PD-L1 expression using peripheral blood mononuclear cells (PBMC) or purified NK cells from healthy donors. We determined that Avelumab significantly enhanced NK-cell mediated cytotoxicity against TNBC cells and that tumor cells expressing higher levels of PD-L1 were more sensitive to Avelumab-mediated ADCC. IFN-γ treatment upregulated PD-L1 expression in tumor cells but had a variable impact on Avelumab-mediated ADCC, which could be related to the simultaneous effect of IFN-γ on the expression of NK cell ligands. Moreover, IL-2 and IL-15 stimulation of NK cells enhanced Avelumab-triggered cytokine production and degranulation along with increased lytic activity against tumor cells. Improving the treatment of TNBC remains still a considerable challenge. This in vitro study suggests that Avelumab-mediated ADCC, independently of the blockade of the PD-1/PD-L1 pathway, could be a valuable mechanism for tumor cell elimination in TNBC. Avelumab combination with immunomodulators such as IL-15 or IL-2 could be taken into consideration to increase the therapeutic efficacy of Avelumab in TNBC.

Changes in the TCRß Repertoire and Tumor Immune Signature From a Cutaneous Melanoma Patient Immunized With the CSF-470 Vaccine: A Case Report.

The allogeneic therapeutic vaccine CSF-470 has demonstrated a significant benefit over medium-dose IFNα2b in the distant metastasis-free survival for stages IIB-IIC-III cutaneous melanoma patients in a randomized phase II/III clinical trial (CASVAC-0401, NCT01729663). At the end of the 2-year CSF-470 immunization protocol, patient #006 developed several lung and one subcutaneous melanoma metastases; this later was excised. In this report, we analyzed the changes throughout vaccination of immune populations in blood and in the tumor tissue, with special focus on the T-cell repertoire. Immunohistochemistry revealed a marked increase in CD8+, CD4+, and CD20+ lymphocytes infiltrating the metastasis relative to the primary tumor. Lymphocytes were firmly attached to dying-tumor cells containing Granzyme-B granules. Whole-exon sequencing assessment indicated a moderate-to-high tumor mutational burden, with BRAFV600E as the main oncogenic driver. Mutational signature presented large numbers of mutations at dipyrimidines, typical of melanoma. Relevant tumor and immune-related genes from the subcutaneous metastasis were addressed by RNA-Seq analysis, revealing expression of typical melanoma antigens and proliferative tumor-related genes. Stimulatory and inhibitory immune transcripts were detected as well as evidence of active T-cell effector function. Peripheral blood monitoring revealed an increase in CD4+ and CD8+ cells by the end of the immunization protocol. By CDR3-T-cell receptor ß (TCRß) sequencing, generation of new clones and an increase in oligoclonality was observed in the peripheral T-cells immune repertoire throughout immunization. A shift, with the expansion of selected preexisting and newly arising clones with reduction of others, was detected in blood. In tumor-infiltrating lymphocytes, prevalent clones (50%) were both new and preexisting that were expanded in blood following CSF-470 immunization. These clones persisted in time, since 2 years after completing the immunization, 51% of the clones present in the metastasis were still detected in blood. This is the first report of the modulation of the TCRß repertoire from a melanoma patient immunized with the CSF-470 vaccine. After immunization, the changes observed in peripheral immune populations as well as in the tumor compartment suggest that the vaccine can induce an antitumor adaptive immune repertoire that can reach tumor lesions and persists in blood for at least 2 years.

High neutrophil to lymphocyte ratio and decreased CD69+NK cells represent a phenotype of high risk in early-stage breast cancer patients.

PURPOSE: Breast cancer (BC) is a highly heterogeneous disease presenting a broad range of clinical and molecular characteristics. In the past years, a growing body of evidence demonstrated that immune response plays a significant role in cancer outcome. However, immune prognostic markers are not completely validated in clinical practice in BC patients. MATERIALS AND METHODS: With the aim to characterize immune features, several parameters were analyzed in peripheral blood at diagnosis of 85 nonmetastatic BC patients between April 2011 and July 2014. RESULTS: With a median follow-up of 38.6 months, peripheral blood analysis of BC patients (stages I, II, and III) showed that total lymphocyte and T lymphocyte counts were augmented in nonrelapsed patients. Also, a higher neutrophil-to-lymphocytes ratio was associated with prolonged disease-free survival. Natural killer cell receptor analysis revealed that early activation receptor CD69 was associated with a better outcome. CONCLUSION: This preliminary evidence is in accordance with the concept of immune surveillance. We suggest an "immune phenotype" that provides relevant prognostic information in early-stage BC patients and which could be useful in the decision-making process.

Phase II Study of Adjuvant Immunotherapy with the CSF-470 Vaccine Plus Bacillus Calmette-Guerin Plus Recombinant Human Granulocyte Macrophage-Colony Stimulating Factor vs Medium-Dose Interferon Alpha 2B in Stages IIB, IIC, and III Cutaneous Melanoma Patients: A Single Institution, Randomized Study.

The irradiated, allogeneic, cellular CSF-470 vaccine plus Bacillus Calmette-Guerin (BCG) and recombinant human granulocyte macrophage-colony stimulating factor (rhGM-CSF) is being tested against medium-dose IFN-α2b in stages IIB-III cutaneous melanoma (CM) patients (pts) after surgery in an open, randomized, Phase II/III study. We present the results of the Phase II part of the ongoing CASVAC-0401 study (ClinicalTrials.gov: NCT01729663). Thirty-one pts were randomized to the CSF-470 vaccine (n = 20) or to the IFN-α2b arm (n = 11). During the 2-year treatment, immunized pts should receive 13 vaccinations. On day 1 of each visit, 1.6 × 107 irradiated CSF-470 cells plus 106 colony-forming units BCG plus 100 µg rhGM-CSF were administered intradermally, followed on days 2-4 by 100 µg rhGM-CSF. IFN-α2b pts should receive 10 million units (MU)/day/5 days a week for 4 weeks; then 5 MU thrice weekly for 23 months. Toxicity and quality of life (QOL) were evaluated at each visit. With a mean and a maximum follow-up of 39.4 and 83 months, respectively, a significant benefit in the distant metastasis-free survival (DMFS) for CSF-470 was observed (p = 0.022). Immune monitoring showed an increase in antitumoral cellular and humoral response in vaccinated pts. CSF-470 was well tolerated; 20/20 pts presented grades 1-2 dermic reactions at the vaccination site; 3/20 pts presented grade 3 allergic reactions. Other adverse events (AEs) were grade 1. Pts in the IFN-α2b arm presented grades 2-3 hematological (7/11), hepatic (2/11), and cardiac (1/11) toxicity; AEs in 9/11 pts forced treatment interruptions. QOL was significantly superior in the vaccine arm (p < 0.0001). Our results suggest that CSF-470 vaccine plus BCG plus GM-CSF can significantly prolong, with lower toxicity, the DMFS of high-risk CM pts with respect to medium-dose IFN-α2b. The continuation of a Phase III part of the CASVAC-0401 study is encouraged.

Phenotypic and Functional Dysregulated Blood NK Cells in Colorectal Cancer Patients Can Be Activated by Cetuximab Plus IL-2 or IL-15.

The clinical outcome of colorectal cancer (CRC) is associated with the immune response; thus, these tumors could be responsive to different immune therapy approaches. Natural killer (NK) cells are key antitumor primary effectors that can eliminate CRC cells without prior immunization. We previously determined that NK cells from the local tumor environment of CRC tumors display a profoundly altered phenotype compared with circulating NK cells from healthy donors (HD). In this study, we evaluated peripheral blood NK cells from untreated patients and their possible role in metastasis progression. We observed profound deregulation in receptor expression even in early stages of disease compared with HD. CRC-NK cells displayed underexpression of CD16, NKG2D, DNAM-1, CD161, NKp46, and NKp30 activating receptors, while inhibitory receptors CD85j and NKG2A were overexpressed. This inhibited phenotype affected cytotoxic functionality against CRC cells and interferon-γ production. We also determined that NKp30 and NKp46 are the key receptors involved in detriment of CRC-NK cells' antitumor activity. Moreover, NKp46 expression correlated with relapse-free survival of CRC patients with a maximum follow-up of 71 months. CRC-NK cells also exhibited altered antibody-dependent cellular cytotoxicity function responding poorly to cetuximab. IL-2 and IL-15 in combination with cetuximab stimulated NK cell, improving cytotoxicity. These results show potential strategies to enhance CRC-NK cell activity.

Natural killer cells as helper cells in dendritic cell cancer vaccines.

Vaccine-based cancer immunotherapy has generated highly variable clinical results due to differing methods of vaccine preparation and variation in patient populations among other lesser factors. Moreover, these clinical responses do not necessarily correspond with the induction of tumor-specific cytotoxic lymphocytes. Here, we review the participation of natural killer (NK) cells as alternative immune components that could cooperate in successful vaccination treatment. NK cells have been described as helper cells in dendritic cell-based cancer vaccines, but the role in other kinds of vaccination strategies (whole cells, peptide, or DNA-based vaccines) is poorly understood. In this article, we address the following issues regarding the role of NK cells in cancer vaccines: NK cell anti-tumor action sites, and the loci of NK cell interaction with other immune cells; descriptions of new data on the memory characteristics of NK cells described in infectious diseases; and finally phenotypical and functional changes after vaccination measured by immunomonitoring in preclinical and clinical settings.

Cytogenetic characterization of the murine bladder cancer model MB49 and the derived invasive line MB49-I.

Bladder cancer is frequently associated with chromosomal abnormalities, and the complexity of karyotypes increases with tumor progression. The murine model MB49 is one of the most widely studied models of bladder cancer. We developed the invasive cell line MB49-I by successive in vivo passages of MB49 primary tumors. Because little is known about the chromosomal alterations of this model, our goal was to perform cytogenetic analyses of the MB49 and MB49-I lines. The karyotypes of both lines were analyzed by G-banding and fluorescence in situ hybridization techniques. Both lines were composed of two cell subpopulations, a diploid population, which was found mainly in the MB49 line, and the tetraploid population, which was found mainly in the MB49-I line. A translocation between chromosomes 5 and 9 and an isochromosome of chromosome 19 were observed in the subpopulations of both lines. New structural abnormalities and additional chromosomal imbalances were detected in the MB49-I line. Tumor progression in the MB49/MB49-I model was associated with a selection of polyploid cells with accompanying chromosomal abnormalities. This model may be advantageous for the study of the genetic changes associated with the progression of bladder cancer.