Search

Introduction: Immunotherapies are gaining more and more importance in the treatment of multiple myeloma (MM). Antibodies directed against MM antigens like CD38, SLAMF7 or BCMA are used either in their natural form, conjugated to drugs, or in the form of bispecific T-cell engagers. Cellular therapies make use of cytotoxic lymphocytes, i.e. T cells or NK cells that can also be modified to express chimeric antigen receptors to target MM cells. Combinations of antibody and cellular therapies could further improve the outcome as, for example, NK cells can mediate antibody dependent cellular cytotoxicity (ADCC). However, NK cells also express CD38 and SLAMF7 and would be targeted by the therapeutic antibodies against these antigens. We have recently reported our clinical study infusing multiple doses of ex vivo activated and expanded autologous NK cells in six patients with MM post autologous stem-cell transplantation (EudraCT 2010-022330-83). Here, we report results of a phenotypic analysis of the ex vivo expanded NK cells and peripheral blood NK cells before and after infusion with implications for possible combination therapies. Methods: Ex vivo activated and expanded NK cells and NK cells in peripheral blood of the patients were analyzed by multiparameter flow cytometry. Peripheral blood cells were taken from the non-NK cell infusion arm before and at three different timepoints after infusion. NK-cell sub-populations within these samples were analyzed using t-SNE clustering. Results: Upon ex vivo activation and expansion, we observed that the NK cells gained a unique activated phenotype including populations of CD56 brightCD16 +Ki67 +HLA-DR + NK cells. Interestingly, these NK cells showed a reduced expression of CD38 compared to peripheral blood NK cells. Clustering analyses of data from peripheral blood samples revealed the gradual appearance of a new NK cell population with a similar phenotype in a dose-dependent fashion over four hours following infusion of the NK cell product. Infused NK cells could be detected in circulation up to four weeks after the last infusion. Like the NK cell infusion product, these cells expressed little to none CD38, high levels of NKG2D, 2B4, TIM-3, and TIGIT and similar levels of SLAMF7 compared to peripheral blood NK cells. Conclusions: The persistent high expression of CD16 and the low expression of CD38 in infused NK cells offers the choice to combine ex vivo activated and expanded NK cells with anti-CD38 antibody therapy without concern for antibody-mediated NK-cell death. Based on these findings, we have started a clinical trial testing this combined therapy (NCT04558931). Disclosures Nahi: XNK Therapeutics AB: Consultancy. Chrobook: XNK Therapeutics AB: Consultancy. Meinke: XNK Therapeutics AB: Consultancy, Current holder of stock options in a privately-held company. Gilljam: XNK Therapeutics AB: Current holder of individual stocks in a privately-held company. Stellan: XNK Therapeutics AB: Current holder of individual stocks in a privately-held company. Walther-Jallow: XNK Therapeutics: Other: Shareholder in the company. Liwing: XNK Therapeutics AB: Current Employment. Gahrton: XNK Therapeutics AB: Current holder of individual stocks in a privately-held company; Fujimoto Pharmaceutical Corporation Japan: Membership on an entity's Board of Directors or advisory committees. Ljungman: Takeda: Consultancy, Other: Endpoint committee, speaker; OctaPharma: Other: DSMB; Enanta: Other: DSMB; Merck: Other: Investigator, speaker; AiCuris: Consultancy; Janssen: Other: Investigator. Ljunggren: XNK Therapeutics AB: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees. Alici: XNK Therapeutics AB: Current holder of individual stocks in a privately-held company.

Dendritic cells are able to present viral antigens to T-cells after uptake of apoptotic bodies derived from virus-infected cells. Immunization with virus-infected apoptotic cells was previously shown to induce HIV-specific immune responses in mice. Here we evaluate the safety and immunogenicity of immunization with activated apoptotic cells in non-human primates using autologous T-cells infected with replication defective VSV pseudotyped SIV mac239 Δ env . Animals were immunized with γ-irradiated activated T-cells carrying the VSV env SIV mac239 Δ env pseudovirus. SIV Gag-specific cellular immune responses were induced as early as two weeks after the first immunization eliciting a biased IFN-γ and IL-2 response. In addition, induction of SIV Gag-specific antibody responses and high titer neutralizing activity against the SIV pseudovirus harboring a VSV- env were detected after two immunizations. The vaccinated group and a control group of Chinese rhesus macaques were intravenously challenged with pathogenic SIV mac251. All animals became infected, but SIV-replication was effectively suppressed (below 100 copies/ml) in several animals in both groups. However the group immunized with apoptotic cells revealed better preservation of the gut CD4 + T-cell compartment. Viral control was inversely correlated with an early (4 weeks) but transient increase in the percentage of Ki67 + CD4 + peripheral blood T-cells (Spearman −0.73). We here show that immunizations with activated apoptotic lymphocytes expressing transduced SIV genes result in induction of both cellular and humoral immune responses. This study provides evidence for an immunological principle demonstrating that certain apoptotic cells can be considered as carriers of antigens directing immune responses in macaques.

Dendritic cell derived IL-12p70 stimulates IFN-γ production in naïve T cells, thereby promoting Th1 responses, which counteracts induction of tolerance. Uptake of apoptotic cells by dendritic cells is generally considered to induce tolerance rather than immune activation and has been shown to specifically inhibit IL-12 production. However, we previously demonstrated that the activation state of apoptotic PBMC influence their immunogenic potential. Here we investigated whether dendritic cells that have engulfed apoptotic PBMC are able to produce IL-12p70 after a secondary signal. We show that dendritic cell ability to produce IL-12p70 after uptake of allogeneic apoptotic cells is dependent on the activation state of the apoptotic cells and subsequent CD40 ligation. CD40 ligation by a CD40L-transfected cell-line induced IL-12p70 in DC regardless of previous apoptotic cell uptake. Moreover, dendritic cells that were exposed to allogeneic activated apoptotic PBMC, but not to resting apoptotic PBMC, were able to produce IL-12p70 after co-culture with autologous T cells. These findings show that dendritic cells are able to produce IL-12p70 upon engulfment of apoptotic cells provided that a secondary activating signal such as CD40-ligand is delivered. In addition, resting apoptotic cell but not activated apoptotic cells reduced ongoing IL-12p70 production suggesting that the balance of activated and resting apoptotic lymphocytes influence the amount of IL-12p70 being produced.

Dendritic cells present microbial antigens to T cells after uptake of apoptotic vesicles from infected cells. We previously reported that immunizations with apoptotic HIV-1/murine leukemia virus (MuLV) infected cells lead to induction of both cellular and humoral immune responses as well as resistance to mucosal challenge with live HIV-1/MuLV infected cells. Here we extended those studies and investigated whether apoptotic cells from HIV-1/MuLV infected cells stimulate the production of HIV-1 neutralizing activity. We compared different routes of administration and were able to induce p24- and Nef-specific cellular proliferation after intraperitoneal (i.p.), intranasal (i.n.), subcutaneous (s.c.) and intramuscular (i.m.) immunizations. Serum IgG and IgA antibodies directed against gp160, p24, or Nef were also produced regardless of immunization route used. However, the induction of mucosa-associated IgAs from faeces or vaginal secretions were detected only after either i.p. or i.n. immunizations. We were able to measure neutralizing activity in sera of mice after i.p. and i.n. immunization. Neutralizing reactivity was also detected after s.c. and i.m. immunizations in the presence of the cytokine adjuvant granulocyte macrophage-colony stimulating factor (GM-CSF). Conclusively we show induction of cellular and humoral immune responses including neutralizing activity after immunization with apoptotic HIV-1/MuLV infected cells in mice. The results from this study support further evaluations using apoptotic cells as antigen delivery system for vaccination against HIV-1 in other animal models. Original Publication:Jorma Hinkula, Lilian Walther-Jallow, Anna Lauren, Barbro Makitalo, Monica Oberg, Britta Wahren, Eva-Maria Fenyo and Anna-Lena Spetz, Neutralizing activity and cellular immune responses induced in mice after immunization with apoptotic HIV-1/murine leukemia virus infected cells, 2009, VACCINE, (27), 46, 6424-6431.http://dx.doi.org/10.1016/j.vaccine.2009.06.016Copyright: Elsevier Science B.V., Amsterdam.http://www.elsevier.com/

Human cytomegalovirus (HCMV) encodes the MHC class I-like molecule UL18, which binds with high affinity to the leukocyte Ig-like receptor-1 (LIR-1), an inhibitory receptor commonly expressed on myeloid cells and subsets of NK and T cells. The exact role of UL18 is not known, in particular in relation to its proposed role in HCMV immune escape. Given the ubiquitous expression of LIR-1 on dendritic cells (DCs), we hypothesized that UL18 may affect DC function. To study the effects of UL18 on DC, we made use of UL18 fusion proteins. We demonstrate that UL18 fusion proteins inhibit the chemotaxis of DCs. Furthermore, UL18 interfered with CD40 ligand-induced maturation of DCs, resulting in reduced allogeneic T cell proliferation. Finally, we demonstrate that UL18 proteins up-regulate the expression of the maturation marker CD83 on immature monocyte-derived DCs and induce cytokine production. The capacity of UL18 to affect the function and the phenotype of DCs suggests a novel role for this HCMV-derived protein.

Dendritic cells (DCs) are antigen-presenting cells with the capacity to prime naive T cells for efficient cellular responses against pathogens such as HIV-1. DCs are also susceptible to HIV-1 infection, which may impair their ability to induce immunity. Here, we examined the ability of HIV-1-infected, in vitro-derived DCs to respond to CD40 ligand (CD40L) stimulation with the aim to study events during early HIV-1 infection. HIV-1BaL-infected p24+ DCs were detected after only 3 days of exposure to highly concentrated virus. We show that HIV-1-infected DCs up-regulated costimulatory molecules, but were skewed in their production of effector cytokines in response to CD40L stimulation. CD40L stimulation induced significant secretion of tumor necrosis factor α (TNFα) and interleukin 12 (IL-12) p70 from both HIV-1-exposed and unexposed DCs. Intracellular stainings of HIV-1-exposed DCs revealed that TNFα could be detected in both the p24- and p24+ DCs, but IL-12 p70 could be found only in the p24- DCs. Thus, although p24+ DCs showed a mature phenotype similar to p24- DCs after CD40L stimulation, they appeared to have an impaired cytokine profile. These observations suggest that HIV-1 infection disables DC function, a phenomenon that may be relevant for optimal induction of HIV-1-specific immune responses. (Blood. 2004;104:2810-2817)

Ag-presenting dendritic cells present viral Ags to T cells after uptake of apoptotic bodies derived from virus-infected cells in vitro. However, it is unclear whether apoptotic virus-infected cells are capable of generating immunity in vivo. In this study, we show that inoculation of mice with apoptotic HIV-1/murine leukemia virus (MuLV)-infected cells induces HIV-1-specific immunity. Immunization with apoptotic HIV-1/MuLV-infected syngeneic splenocytes resulted in strong Nef-specific CD8+ T cell proliferation and p24-induced CD4+ and CD8+ T cell proliferation as well as IFN-γ production. In addition, systemic IgG and IgA as well as mucosa-associated IgA responses were generated. Moreover, mice vaccinated with apoptotic HIV-1/MuLV cells were protected against challenge with live HIV-1/MuLV-infected cells, whereas mice vaccinated with apoptotic noninfected or MuLV-infected splenocytes remained susceptible to HIV-1/MuLV. These data show that i.p. immunization with apoptotic HIV-1-infected cells induces high levels of HIV-1-specific systemic immunity, primes for mucosal immunity, and induces protection against challenge with live HIV-1-infected cells in mice. These findings may have implications for the development of therapeutic and prophylactic HIV-1 vaccines.

In this study we compared the efficacy of live attenuated human immunodeficiency virus type 2 (HIV-2) vaccine alone versus boosting with live non-pathogenic HIV-2 following priming with ALVAC HIV-2 (recombinant canarypox virus expressing HIV-2 env, gag and pol). Six monkeys were first inoculated intravenously with live HIV-2SBL-6669 and 7 to 10 months later were challenged intrarectally with 10 MID50 of cell-free simian immunodeficiency virus (SIV) strain SIVsm. One monkey was completely protected against SIV infection and all five monkeys that became SIV-infected showed a lower virus replication and an initial lower virus load as compared with a parallel group of six control animals. In another experiment five monkeys were immunized either three times with ALVAC HIV-2 alone or twice with ALVAC HIV-2 and once with purified native HIV-2 gp125. The monkeys were then challenged with HIV-2 given intravenously and finally with pathogenic SIVsm given intrarectally. After challenge with SIVsm, three of five monkeys were completely protected against SIVsm infection whereas the remaining two macaques became SIV-infected but with limited virus replication. In conclusion, vaccination with an ALVAC HIV-2 vaccine followed by exposure to live HIV-2 could induce cross-protection against mucosal infection with SIVsm and seemed to be more efficient than immunization with a live HIV-2 vaccine only.

The underlying pathophysiological cause of schizophrenia is essentially unknown. Increasing evidence suggests that immunological processes may contribute to the etiology of the disease. Owing to the lack of direct evidence of an infectious cause of schizophrenia much attention has been directed towards cytokines; molecules that initiate immunological responses. However, previous studies measuring the level of cytokines in the serum of patients with schizophrenia have provided an inconsistent picture in this regard.1 The relatively few studies focusing on the level of cytokines in the cerebrospinal fluid (CSF) have been limited to a restricted number of cytokines analysed, to the lack of a control group of healthy volunteers or to the sensitivity of the cytokine assays used. Here, a sensitive assay was used to analyse the CSF concentration of well-characterized cytokines in first-episode patients with schizophrenia and age-matched healthy volunteers.

Dendritic cells (DCs) are activated by signaling via pathogen-specific receptors or exposure to inflammatory mediators. Here we show that co-culturing DCs with apoptotic HIV-infected activated CD4(+) T cells (ApoInf) or apoptotic uninfected activated CD4(+) T cells (ApoAct) induced expression of co-stimulatory molecules and cytokine release. In addition, we measured a reduced HIV infection rate in DCs after co-culture with ApoAct. A prerequisite for reduced HIV infection in DCs was activation of CD4(+) T cells before apoptosis induction. DCs exposed to ApoAct or ApoInf secreted MIP-1α, MIP-1β, MCP-1, and TNF-α; this effect was retained in the presence of exogenous HIV. The ApoAct-mediated induction of co-stimulatory CD86 molecules and reduction of HIV infection in DCs were partially abrogated after blocking TNF-α using monoclonal antibodies. APOBEC3G expression in DCs was increased in co-cultures of DCs and ApoAct but not by apoptotic resting CD4(+) T cells (ApoRest). Silencing of APOBEC3G in DC abrogated the HIV inhibitory effect mediated by ApoAct. Sequence analyses of an env region revealed significant induction of G-to-A hypermutations in the context of GG or GA dinucleotides in DNA isolated from DCs exposed to HIV and ApoAct. Thus, ApoAct-mediated DC maturation resulted in induction of APOBEC3G that was important for inhibition of HIV-infection in DCs. These findings underscore the complexity of differential DC responses evoked upon interaction with resting as compared with activated dying cells during HIV infection.

Neutralizing antibodies (NAb) able to react to heterologous viruses are generated during natural HIV-1 infection in some individuals. Further knowledge is required in order to understand the factors contributing to induction of cross-reactive NAb responses. Here a well-established model of experimental pathogenic infection in cynomolgus macaques, which reproduces long-lasting HIV-1 infection, was used to study the NAb response as well as the viral evolution of the highly neutralization-resistant SIVmac239. Twelve animals were infected intravenously with SIVmac239. Antiretroviral therapy (ART) was initiated ten days post-inoculation and administered daily for four months. Viral load, CD4+ T-cell counts, total IgG levels, and breadth as well as strength of NAb in plasma were compared simultaneously over 14 months. In addition, envs from plasma samples were sequenced at three time points in all animals in order to assess viral evolution. We report here that seven of the 12 animals controlled viremia to below 104 copies/ml of plasma after discontinuation of ART and that this control was associated with a low level of evolutionary divergence. Macaques that controlled viral load developed broader NAb responses early on. Furthermore, escape mutations, such as V67M and R751G, were identified in virus sequenced from all animals with uncontrolled viremia. Bayesian estimation of ancestral population genetic diversity (PGD) showed an increase in this value in non-controlling or transient-controlling animals during the first 5.5 months of infection, in contrast to virus-controlling animals. Similarly, non- or transient controllers displayed more positively-selected amino-acid substitutions. An early increase in PGD, resulting in the generation of positively-selected amino-acid substitutions, greater divergence and relative high viral load after ART withdrawal, may have contributed to the generation of potent NAb in several animals after SIVmac239 infection. However, early broad NAb responses correlated with relatively preserved CD4+ T-cell numbers, low viral load and limited viral divergence., Author Summary In a longitudinal study of clinical and evolutionary responses to transient treatment in 12 experimentally-infected macaques, subjects show clear stratification into two groups based on viral load, immunological response, and evolutionary factors. Subjects that controlled viremia following withdrawal of treatment developed broadly neutralizing antibody responses earlier than subjects with no or transient control of viremia. Moreover, this latter group of macaques with higher viral loads showed greater divergence of SIV sequences, greater numbers of positively-selected amino-acid substitutions and a stronger neutralizing antibody response. The increase in viral genetic diversity started at an early stage of infection. The authors propose that this early phase of evolution is principally responsible for the later failure to control viremia and resulted in the development of potent neutralizing capacity.

Background The kinetics of cellular and humoral immune responses was evaluated in a macaque model after early introduction of antiretroviral treatment. Twelve cynomolgus macaques were infected intravenously with the patogenic SIVmac239 strain. Treatment with the antiretroviral drug Tenofovir was initiated 10 days after inoculation with SIVmac239 and was administered subcutaneously as daily injections for 4 months.

Dendritic cells (DCs) can be activated by signaling via pathogen receptors, by interaction with activated T cells or by exposure to inflammatory mediators. Clearance of apoptotic cells by DCs is generally considered a silent event that is not associated with an inflammatory response. Necrotic cell death, in contrast, leads to induction of inflammation. However, emerging data challenge the view of apoptotic cells as inherently nonimmunogenic. In this study, we report that the activation state of the apoptotic cell may determine whether the exposed DC becomes activated and rendered proficient in Ag presentation. We show that coculture with activated, but not resting, apoptotic PBMCs leads to up-regulation of surface expression of the costimulatory molecules CD80, CD83, and CD86 in human DCs as well as release of proinflammatory cytokines. Furthermore, we show that DCs exposed to allogeneic, activated apoptotic PBMCs induce proliferation and IFN-γ production in autologous T cells. Together, these findings show that activated apoptotic PBMCs per se provide an activation/maturation signal to DCs, suggesting that activated apoptotic PBMCs possess endogenous adjuvant properties.

Leukemia inhibitor factor (LIF) has been shown to potently inhibit HIV-1 replication in vitro and in human organ explant cultures. Furthermore, LIF activates the Jak/Stat signaling pathway with which many viruses, including HIV-1, interfere. We used CXCR4 and the LIF signaling receptor (gp130)-expressing cMAGI cells transfected with CD4, CCR5, and HIV-LTR-beta-galactosidase as a model system to investigate the potential involvement of Stat proteins in the anti-HIV-1 effect of LIF. Pretreatment with recombinant human (rh)LIF resulted in a significantly reduced uptake of HIV-1(BaL) , HIV-1(LAI), and SIVmac251 viral particles without affecting uptake of murine leukemia retroviral particles. HIV-1(BaL), HIV-1(LAI), as well as rhLIF selectively induced phosphorylation of Stat 3 but not Stat 1 or Stat 5. However, treatment of cMAGI cells with rhLIF prior to HIV-1 infection downregulated the HIV-1-mediated Stat 3 phosphorylation. In addition, peripheral blood mononuclear cells (PBMCs) transfected with Stat 3 siRNA prior to HIV-1(LAI) or HIV-1(BaL) infection produced significantly less HIV-1 p24 antigen as compared to nontransfected HIV-1(LAI) and HIV-1(BaL)-infected PBMCs. Thus, the Jak/Stat signaling pathway is important for the HIV-1 replication life cycle and rhLIF excerts its anti-HIV-1 activity by disrupting this signaling cascade.

Suppression of immune activation and increased inflammation are prevalent during viral infection. To investigate the role of inflammation in HIV transmission, we studied the infectious and inflammatory milieu in cervical mucosa from HIV-1- and human papillomavirus (HPV)-coinfected and HPV-monoinfected women. The numbers of cytokine-, chemokine-, and p24-expressing cells were determined using in situ imaging analysis and intracellular staining of p24 antigen. Significantly higher expression of the proinflammatory cytokines, interleukin (IL)-1alpha/beta, was seen in cervical tissue from HIV/HPV-coinfected as compared with HPV-monoinfected tissues, whereas IL-2- and interferon (IFN)-gamma-expressing cells were higher in HPV-monoinfected tissues. IL-10 was low in both groups, whereas IL-4 was significantly higher in HPV-monoinfected and HIV/HPV-coinfected tissues than in HIV/HPV-negative controls. RANTES and macrophage inflammatory protein (MIP)-1beta but not MIP-1alpha were significantly higher in the genital tract of HIV/HPV-coinfected as compared with HPV-monoinfected individuals and controls. HIV/HPV-coinfected tissues had a higher level of human leukocyte antigen D-related (HLA-DR)-expressing dendritic cells (DCs). There was a positive correlation between the number of CD4(+) and CD8(+) T cells as well as CD1a, IL-1alpha, and RANTES expression and p24 antigen-expressing cells in the HIV/HPV-coinfected tissues. These findings suggest the persistence of immune activation and inflammation in the genital tract of women with HPV monoinfection and in HIV-infected women coinfected with HPV.

In the present study, the immunogenicity and protective efficacy of a recombinant vaccinia virus-based simian immunodeficiency virus (SIV) vaccine, given alone or in combination with a protein boost, were investigated. Cynomolgus macaques were immunized intramuscularly with modified vaccinia virus Ankara (MVA) expressing the SIVsmenvandgag–polgenes (MVA–SIVsm) at 0 and 3 months (n=4), at 0, 3 and 8 months (n=4) or at 0 and 3 months followed by purified native SIVsm gp148 and recombinant SIVmac p27 in immunostimulatory complexes at 8 months (n=4). One month after the last immunization, the vaccinees, together with four naive control monkeys and four monkeys immunized with wild-type MVA, were challenged intrarectally with 10 MID50SIVsm. At the time of challenge, antibody titres to SIV Env and lymphocyte proliferation responses to whole viral antigen were highest in vaccinees receiving MVA–SIVsm in combination with protein immunizations. Following rectal challenge, one of these vaccinees was completely protected. A prolonged survival time was observed in two of four monkeys in each of the groups immunized with MVA–SIVsm, in two monkeys given MVA–SIVsm followed by protein and in three of four monkeys given wild-type MVA, compared with naive controls. In conclusion, one monkey given the combined vaccine was protected completely against SIVsm infection. Furthermore, immunization with MVA–SIVsm, as well as wild-type MVA alone, seemed to delay disease progression after mucosal SIV infection in a proportion of the monkeys.

In this study we have evaluated the concordance between serology, using five commercially available antibody assays designed to discriminate between HIV-1 and HIV-2, and the polymerase chain reaction (PCR) for the detection of HIV-1 and HIV-2 dual infection. Thirty-seven HIV-1 and HIV-2 dually reactive serum samples from individuals in Guinea-Bissau with total CD4+ T lymphocyte counts ranging from 9 to 948 x 10(6)/liter were included in the study. All samples were tested by Multispot, Pepti-LAV, and Immunocomb HIV-1 and HIV-2 discriminatory antibody assays. Thirty-two of the 37 samples were also tested by a combination of two HIV type-specific antibody enzyme-linked immunosorbent assays (ELISA; Wellcozyme HIV-1 and Murex HIV-2). Each sample showed dual reactivity in all or any of these assays. A nested PCR based on primer systems in the vif and pol regions of HIV-1 and in the gag and LTR regions of HIV-2 was used to evaluate the serological results. Thirty samples from HIV-1 antibody-positive individuals and 30 samples from HIV-2 antibody-positive individuals were all PCR positive with their corresponding primer systems. The type specificity was 100% for all of the primer systems. The concordance between dual HIV-1 and HIV-2 reactivity on the serological assays and PCR was 77.7% for Multispot, 80% for Pepti-LAV, 81.8% for Immunocomb, and 85.7% for the two ELISAs used in combination. Thus the majority of individuals included in this study appeared to be truly dually infected. The study shows that it is possible, through a careful selection of assays, to reach a high concordance between serological assays and PCR in studying HIV-1 and HIV-2 dual infections.