CD4 CTLs Research Articles

IM-11 * GBM-ASSOCIATED EXOSOME-PULSED AUTOLOGOUS DC VACCINE (DC-VEX(R)) INDUCES D-2 APOPTOTIC-STAGE AND BYSTANDER KILLING-EFFECT IN GBM OF THE MESENCHYMAL TYPE

INTRODUCTION: GBM is one of the most lethal cancers due to its resistance to apoptosis or type I PCD after chemo/radiotherapy. Due to its heterogenicity, we must treat this tumour with personalised cancer medicine. METHODS: We obtain relapsed GBM characterised by NF1 deletions and PTEN mutations of the mesenchymal type resistant to chemo/radiation Tx, and then we develop a GBM-model from which we isolate autologous dendritic cells and exosomes expressing CD9/CD91, both of which incubate ex-vivo in culture with granulocyte-macrophage colony stimulating factor,interleukin-4 and tumour necrosis factor-a. RESULTS: Post-treatment with the active specific immunotherapy via the GBM-associated antigen presenting dendritic cells, we observe humoral antibody responses and cellular immunity responses expanding circulating CD4 and CD8 CTLs which express TCRs for the specific GBM antigens which bind to the complex of class I MHC molecule and release IFN-γ,IL-2 and IL-6.RNA sequencing for genomic/epigenomic analysis has exhibited that exosomes carry coding and non-coding genes whose expression was different from the ones detected in the FFPE samples obtained from the initial biopsies exhibiting the most recent gene alterations which take place in the relapsed GBM. The active specific immunotherapy leads to D2 irreversible stage of apoptosis or type-I programmed cell death leading to a bystander killing effect after phagocytosis of the apoptotic bodies by the adjacent relapsed GBM causing their eradication. CONCLUSION: The DC-Vex® autologous dendritic cell vaccine due to pulsed exosomes, it can eradicate with humoral and cellular immunity pathways the relapsed chemo/radioresistant GBM of mesenchymal type.

In Vivo Identification and Characterization of CD4+ Cytotoxic T Cells Induced by Virulent Brucella abortus Infection

CD4+ T cells display a variety of helper functions necessary for an efficient adaptive immune response against bacterial invaders. This work reports the in vivo identification and characterization of murine cytotoxic CD4+ T cells (CD4+ CTL) during Brucella abortus infection. These CD4+ CTLs express granzyme B and exhibit immunophenotypic features consistent with fully differentiated T cells. They express CD25, CD44, CD62L ,CD43 molecules at their surface and produce IFN-γ. Moreover, these cells express neither the co-stimulatory molecule CD27 nor the memory T cell marker CD127. We show here that CD4+ CTLs are capable of cytolytic action against Brucella-infected antigen presenting cells (APC) but not against Mycobacterium-infected APC. Cytotoxic CD4+ T cell population appears at early stages of the infection concomitantly with high levels of IFN-γ and granzyme B expression. CD4+ CTLs represent a so far uncharacterized immune cell sub-type triggered by early immune responses upon Brucella abortus infection.

Patients with B cell chronic lymphocytic leukaemia have an expanded population of CD4+ perforin expressing T cells enriched for human cytomegalovirus specificity and an effector‐memory phenotype

We have previously shown an expansion of cytotoxic antigen-experienced CD4(+)T cells (CTLs) that express perforin (PF) in the peripheral blood of patients with B cell chronic lymphocytic leukaemia (B-CLL). Increased frequencies of CD4(+)CTLs have since been attributed to chronic viral infections, particularly, human cytomegalovirus (HCMV). The present study examined the involvement of CD4(+)CTLs in responses to HCMV in B-CLL, and characterized their differentiation. We studied 36 HCMV seropositive (SP) and seronegative B-CLL patients and 20 healthy age-matched individuals. The HCMV reactivity of CD4(+)PF(+) and CD4(+)PF(-) cells was determined by interferon-gamma expression, and expression of CD45RA and CCR7 was assessed by flow cytometry. Fluorescence in-situ hybridization was used to measure relative telomere lengths. CD4(+)PF(+)T cell expansion in B-CLL patients and controls was strongly associated with HCMV seropositivity. CD4(+)PF(+) compared to CD4(+)PF(-) cells from SP B-CLL patients elicited major histocompatibility complex (MHC) class II-restricted responses to HCMV. CD4(+)PF(+)T cells from patients and controls were enriched with highly differentiated T-effector/memory (CCR7(-)) and revertant (CCR7(-)CD45RA(+)) phenotype. CD4(+)PF(+)T cells from B-CLL patients had shorter telomeres than CD4(+)PF(-)T cells, indicating an extensive replicative history. We conclude that persistent exposure to HCMV antigens in SP B-CLL patients leads to an expansion of the circulating MHC class II-restricted CD4(+)PF(+)T cell population with effector/memory phenotype.

Characterization of CD4(+) CTLs ex vivo.

The cytotoxic potential of CD8(+) T cells and NK cells plays a crucial role in the immune response to pathogens. Although in vitro studies have reported that CD4(+) T cells are also able to mediate perforin-mediated killing, the in vivo existence and relevance of cytotoxic CD4(+) T cells have been the subject of debate. Here we show that a population of CD4(+) perforin(+) T cells is present in the circulation at low numbers in healthy donors and is markedly expanded in donors with chronic viral infections, in particular HIV infection, at all stages of the disease, including early primary infection. Ex vivo analysis shows that these cells have cytotoxic potential mediated through the release of perforin. In comparison with more classical CD4(+) T cells, this subset displays a distinct surface phenotype and functional profile most consistent with end-stage differentiated T cells and include Ag experienced CD4(+) T cells. The existence of CD4(+) cytotoxic T cells in vivo at relatively high levels in chronic viral infection suggests a role in the immune response.

The ectodomain of measles virus envelope glycoprotein does not gain access to the cytosol and MHC class I presentation pathway following virus-cell fusion.

To unravel the intracellular fate of measles virus (MV) haemagglutinin (H) following fusion of the virus envelope with the cell membrane, its presentation by MHC molecules to T cells was explored. After MV infection, murine cells expressing CD46 were lysed by MHC class I-restricted CD8 CTLs specific for the ectodomain of H. In contrast, when sensitized with UV-inactivated MV, they were not lysed by these effectors, but were recognized by H-specific and class II-restricted CD4 CTLs. Thus, after MV binding and fusion, H becomes associated with plasma membrane and its ectodomain can reach the endosomal MHC-II but not the cytosolic MHC-I antigen presentation pathway. From these data and a reappraisal of previous reports, it appears that the ectodomains of both MV haemagglutinin fusion proteins, having undergone the fusion step, are not translocated into the cytosol and end up in the endosomes.