Primary pulmonary cytotoxic T lymphocytes induced by immunization with a vaccinia virus recombinant expressing influenza A virus nucleoprotein peptide do not protect mice against challenge

The nucleoprotein (NP) of influenza A virus is the dominant antigen recognized by influenza virus-specific cytotoxic T lymphocytes (CTLs), and adoptive transfer of NP-specific CTLs protects mice from influenza A virus infection. BALB/c mouse cells (H-2d) recognize a single Kd-restricted CTL epitope of NP consisting of amino acids 147 to 155. In the present study, mice were immunized with various vaccinia virus recombinant viruses to examine the effect of the induction of primary pulmonary CTLs on resistance to challenge with influenza A/Puerto Rico/8/34 virus. The minigene ESNP(147-155)-VAC construct, composed of a signal sequence from the adenovirus E3/19K glycoprotein (designated ES) and expressing the 9-amino-acid NP natural determinant (amino acids 147 to 155) preceded by an alanine residue, a similar minigene NP(Met 147-155)-VAC lacking ES, and a full-length NP-VAC recombinant of influenza virus were analyzed. The two minigene NP-VAC recombinants induced a greater primary pulmonary CTL response than the full-length NP-VAC recombinant. However, NP-specific CTLs induced by immunization with ESNP(147-155)-VAC did not decrease peak virus titer or accelerate clearance of virus in the lungs of mice challenged intranasally with A/PR/8/34. Furthermore, NP-specific CTLs induced by immunization did not protect mice challenged intranasally with a lethal dose of A/PR/8/34. Sequence analysis of the NP CTL epitope of A/PR/8/34 challenge virus obtained from lungs after 8 days of replication in ESNP(147-155)-VAC-immunized mice showed identity with that of the input virus, demonstrating that an escape mutant had not emerged during replication in vivo. Thus, in contrast to adoptively transferred CTLs, pulmonary NP-specific CTLs induced by recombinant vaccinia virus immunization do not have protective in vivo antiviral activity against influenza virus infection

STING agonist promotes CAR T cell trafficking and persistence in breast cancer

CAR T therapy targeting solid tumors is restrained by limited infiltration and persistence of those cells in the tumor microenvironment (TME). Here, we developed approaches to enhance the activity of CAR T cells using an orthotopic model of locally advanced breast cancer. CAR T cells generated from Th/Tc17 cells given with the STING agonists DMXAA or cGAMP greatly enhanced tumor control, which was associated with enhanced CAR T cell persistence in the TME. Using single-cell RNA sequencing, we demonstrate that DMXAA promoted CAR T cell trafficking and persistence, supported by the generation of a chemokine milieu that promoted CAR T cell recruitment and modulation of the immunosuppressive TME through alterations in the balance of immune-stimulatory and suppressive myeloid cells. However, sustained tumor regression was accomplished only with the addition of anti-PD-1 and anti-GR-1 mAb to Th/Tc17 CAR T cell therapy given with STING agonists. This study provides new approaches to enhance adoptive T cell therapy in solid tumors

Classification of current anticancer immunotherapies

During the past decades, anticancer immunotherapy has evolved from a promising therapeutic option to a robust clinical reality. Many immunotherapeutic regimens are now approved by the US Food and Drug Administration and the European Medicines Agency for use in cancer patients, and many others are being investigated as standalone therapeutic interventions or combined with conventional treatments in clinical studies. Immunotherapies may be subdivided into “passive” and “active” based on their ability to engage the host immune system against cancer. Since the anticancer activity of most passive immunotherapeutics (including tumor-targeting monoclonal antibodies) also relies on the host immune system, this classification does not properly reflect the complexity of the drug-host-tumor interaction. Alternatively, anticancer immunotherapeutics can be classified according to their antigen specificity. While some immunotherapies specifically target one (or a few) defined tumor-associated antigen(s), others operate in a relatively non-specific manner and boost natural or therapy-elicited anticancer immune responses of unknown and often broad specificity. Here, we propose a critical, integrated classification of anticancer immunotherapies and discuss the clinical relevance of these approaches

Identification, isolation and in vitro expansion of human and nonhuman primate T stem cell memory cells

The T cell compartment is phenotypically and functionally heterogeneous; subsets of naive and memory cells have different functional properties, and also differ with respect to homeostatic potential and the ability to persist in vivo. Human stem cell memory T (TSCM) cells, which possess superior immune reconstitution and antitumor response capabilities, can be identified by polychromatic flow cytometry on the basis of the simultaneous expression of several naive markers together with the memory marker CD95. We describe here a protocol based on the minimum set of markers required for optimal identification of human and nonhuman primate (NHP) TSCM cells with commonly available flow cytometers. By using flow sorters, TSCM cells can thereby be isolated efficiently at high yield and purity. With the use of the 5.5-h isolation procedure, depending on the number of cells needed, the sorting procedure can last for 2-15 h. We also indicate multiple strategies for their efficient expansion in vitro at consistent numbers for functional characterization or adoptive transfer experiments

Identification of a CD11b(+)/Gr-1(+)/CD31(+) myeloid progenitor capable of activating or suppressing CD8(+) T cells

Apoptotic death of CD8(+) T cells can be induced by a population of inhibitory myeloid cells that are double positive for the CD11b and Gr-1 markers. These cells are responsible for the immunosuppression observed in pathologies as dissimilar as tumor growth and overwhelming infections, or after immunization with viruses. The appearance of a CD11b(+)/Gr-1(+) population of inhibitory macrophages (iMacs) could be attributed to high levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) in vivo. Deletion of iMacs in vitro or in vivo reversed the depression of CD8(+) T-cell function. We isolated iMacs from the spleens of immunocompromised mice and found that these cells were positive for CD31, ER-MP20 (Ly-6C), and ER-MP58, markers characteristic of granulocyte/monocyte precursors. Importantly, although iMacs retained their inhibitory properties when cultured in vitro in standard medium, suppressive functions could be modulated by cytokine exposure. Whereas culture with the cytokine interleukin 4 (IL-4) increased iMac inhibitory activity, these cells could be differentiated into a nonadherent population of fully mature and highly activated dendritic cells when cultured in the presence of IL-4 and GM-CSF. A common CD31(+)/CD11b(+)/Gr-1(+) progenitor can thus give rise to cells capable of either activating or inhibiting the function of CD8(+) T lymphocytes, depending on the cytokine milieu that prevails during antigen-presenting cell maturation. (Blood. 2000;96:3838-3846

MicroRNA-155 Is Required for Effector CD8(+) T Cell Responses to Virus Infection and Cancer.

MicroRNAs (miRNAs) regulate the function of several immune cells, but their role in promoting CD8(+) T cell immunity remains unknown. Here we report that miRNA-155 is required for CD8(+) T cell responses to both virus and cancer. In the absence of miRNA-155, accumulation of effector CD8(+) T cells was severely reduced during acute and chronic viral infections and control of virus replication was impaired. Similarly, Mir155(-/-) CD8(+) T cells were ineffective at controlling tumor growth, whereas miRNA-155 overexpression enhanced the antitumor response. miRNA-155 deficiency resulted in accumulation of suppressor of cytokine signaling-1 (SOCS-1) causing defective cytokine signaling through STAT5. Consistently, enforced expression of SOCS-1 in CD8(+) T cells phenocopied the miRNA-155 deficiency, whereas SOCS-1 silencing augmented tumor destruction. These findings identify miRNA-155 and its target SOCS-1 as key regulators of effector CD8(+) T cells that can be modulated to potentiate immunotherapies for infectious diseases and cancer

Transcriptional profiles reveal a stepwise developmental program of memory CD8(+) T cell differentiation.

The generation of CD8(+) T-cell memory is a major aim of vaccination. While distinct subsets of CD8(+) T-cells are generated following immunization that differ in their ability to confer long-term immunity against infection, the transcriptional profiles of these subsets within endogenous vaccine-induced CD8(+) T cell responses have not been resolved. Here, we measure global transcriptional profiles of endogenous effector (TEFF), effector memory (TEM) and central memory (TCM) CD8(+) T-cells arising from immunization with three distinct prime-boost vaccine regimens. While a proportion of transcripts were uniquely regulated within distinct CD8(+) T cell populations, we observed progressive up- or down-regulation in the expression of a majority of differentially expressed transcripts when subsets were compared in the order TN>TCM>TEM>TEFF. Strikingly, when we compared global differences in gene expression between TN, TCM, TEM and TEFF cells with known transcriptional changes that result when CD8(+) T cells repetitively encounter antigen, our analysis overwhelmingly favored a model whereby cumulative antigen stimulation drives differentiation specifically from TN>TCM>TEM>TEFF and this was common to all vaccines tested. These findings provide insight into the molecular basis of immunological memory and identify potential biomarkers for characterization of vaccine-induced responses and prediction of vaccine efficacy

MiR-155 augments CD8+ T-cell antitumor activity in lymphoreplete hosts by enhancing responsiveness to homeostatic γc cytokines

Lymphodepleting regimens are used before adoptive immunotherapy to augment the antitumor efficacy of transferred T cells by removing endogenous homeostatic “cytokine sinks.” These conditioning modalities, however, are often associated with severe toxicities. We found that microRNA-155 (miR-155) enabled tumorspecific CD8+ T cells to mediate profound antitumor responses in lymphoreplete hosts that were not potentiated by immune-ablation. miR-155 enhanced T-cell responsiveness to limited amounts of homeostatic γc cytokines, resulting in delayed cellular contraction and sustained cytokine production. miR-155 restrained the expression of the inositol 5-phosphatase Ship1, an inhibitor of the serinethreonine protein kinase Akt, and multiple negative regulators of signal transducer and activator of transcription 5 (Stat5), including suppressor of cytokine signaling 1 (Socs1) and the protein tyrosine phosphatase Ptpn2. Expression of constitutively active Stat5a recapitulated the survival advantages conferred by miR-155, whereas constitutive Akt activation promoted sustained effector functions. Our results indicate that overexpression of miR-155 in tumorspecific T cells can be used to increase the effectiveness of adoptive immunotherapies in a cell-intrinsic manner without the need for life-threatening, lymphodepleting maneuvers.126261sciescopu

Defining 'T cell exhaustion'.

'T cell exhaustion' is a broad term that has been used to describe the response of T cells to chronic antigen stimulation, first in the setting of chronic viral infection but more recently in response to tumours. Understanding the features of and pathways to exhaustion has crucial implications for the success of checkpoint blockade and adoptive T cell transfer therapies. In this Viewpoint article, 18 experts in the field tell us what exhaustion means to them, ranging from complete lack of effector function to altered functionality to prevent immunopathology, with potential differences between cancer and chronic infection. Their responses highlight the dichotomy between terminally differentiated exhausted T cells that are TCF1 <sup>-</sup> and the self-renewing TCF1 <sup>+</sup> population from which they derive. These TCF1 <sup>+</sup> cells are considered by some to have stem cell-like properties akin to memory T cell populations, but the developmental relationships are unclear at present. Recent studies have also highlighted an important role for the transcriptional regulator TOX in driving the epigenetic enforcement of exhaustion, but key questions remain about the potential to reverse the epigenetic programme of exhaustion and how this might affect the persistence of T cell populations