IL-15 superagonist N-803 improves IFNγ production and killing of leukemia and ovarian cancer cells by CD34+ progenitor-derived NK cells.

Allogeneic natural killer (NK) cell transfer is a potential immunotherapy to eliminate and control cancer. A promising source are CD34 + hematopoietic progenitor cells (HPCs), since large numbers of cytotoxic NK cells can be generated. Effective boosting of NK cell function can be achieved by interleukin (IL)-15. However, its in vivo half-life is short and potent trans-presentation by IL-15 receptor α (IL-15Rα) is absent. Therefore, ImmunityBio developed IL-15 superagonist N-803, which combines IL-15 with an activating mutation, an IL-15Rα sushi domain for trans-presentation, and IgG1-Fc for increased half-life. Here, we investigated whether and how N-803 improves HPC-NK cell functionality in leukemia and ovarian cancer (OC) models in vitro and in vivo in OC-bearing immunodeficient mice. We used flow cytometry-based assays, enzyme-linked immunosorbent assay, microscopy-based serial killing assays, and bioluminescence imaging, for in vitro and in vivo experiments. N-803 increased HPC-NK cell proliferation and interferon (IFN)γ production. On leukemia cells, co-culture with HPC-NK cells and N-803 increased ICAM-1 expression. Furthermore, N-803 improved HPC-NK cell-mediated (serial) leukemia killing. Treating OC spheroids with HPC-NK cells and N-803 increased IFNγ-induced CXCL10 secretion, and target killing after prolonged exposure. In immunodeficient mice bearing human OC, N-803 supported HPC-NK cell persistence in combination with total human immunoglobulins to prevent Fc-mediated HPC-NK cell depletion. Moreover, this combination treatment decreased tumor growth. In conclusion,  N-803 is a promising IL-15-based compound that boosts HPC-NK cell expansion and functionality in vitro and in vivo. Adding N-803 to HPC-NK cell therapy could improve cancer immunotherapy.

Characterization of human immunodeficiency virus type 1-specific cytotoxic T lymphocyte clones isolated during acute seroconversion: recognition of autologous virus sequences within a conserved immunodominant epitope.

Virus-specific cytotoxic T lymphocytes (CTL) are involved in protective immunity to many virus infections. It has recently been shown that CTL are detectable early during primary infection with the primate lentiviruses, human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus. To better characterize the CTL response during acute HIV-1 infection, HIV-1-specific CTL clones were generated from two patients during symptomatic HIV-1 seroconversion. These CTL clones demonstrated specificity for env of HIV-1 and recognized sequences within gp41. Two human histocompatibility leukocyte antigen (HLA) A31-restricted clones from the same individual were found to have differing virus strain specificities. Both clones recognized the 11-amino acid peptide RLRDLLLIVTR from position 770-780 of gp41. A change from T to V at position 779 in this epitope abrogated lysis by one clone but not the other. A CTL clone from the other patient, restricted by a different class I HLA allele, recognized the nine-amino acid peptide HRLRDLLLI from position 769-777 of gp41. Of note, the peptide RLRDLLLIVTR has been shown by others to be presented to CTL by HLA-A3.1. Autologous virus sequences from seroconversion and up to 15 wk after presentation in these two patients were recognized by the CTL clones isolated during acute infection. None of the CTL clones recognized the MN strain of HIV-1, indicating the problems inherent in relying on a single virus strain in the development of a vaccine. These studies have identified an immunodominant and promiscuous area for the generation of CTL responses within gp41. This recognition of autologous virus sequences by the initial CTL response is consistent with the hypothesis that a single virus strain is transmitted to the seroconverter and that the CTL response is involved in the initial control of that virus. These studies indicate the importance of the CTL response to HIV-1 infection and have implications in the design of vaccines.

Dramatic rise in plasma viremia after CD8(+) T cell depletion in simian immunodeficiency virus-infected macaques.

To determine the role of CD8(+) T cells in controlling simian immunodeficiency virus (SIV) replication in vivo, we examined the effect of depleting this cell population using an anti-CD8 monoclonal antibody, OKT8F. There was on average a 99.9% reduction of CD8 cells in peripheral blood in six infected Macaca mulatta treated with OKT8F. The apparent CD8 depletion started 1 h after antibody administration, and low CD8 levels were maintained until day 8. An increase in plasma viremia of one to three orders of magnitude was observed in five of the six macaques. The injection of a control antibody to an infected macaque did not induce a sustained viral load increase, nor did it significantly reduce the number of CD8(+) T cells. These results demonstrate that CD8 cells play a crucial role in suppressing SIV replication in vivo.

Adaptive evolution of human immunodeficiency virus-type 1 during the natural course of infection.

The rate of progression to disease varies considerably among individuals infected with human immunodeficiency virus-type 1 (HIV-1). Analyses of semiannual blood samples obtained from six infected men showed that a rapid rate of CD4 T cell loss was associated with relative evolutionary stasis of the HIV-1 quasispecies virus population. More moderate rates of CD4 T cell loss correlated with genetic evolution within three of four subjects. Consistent with selection by the immune constraints of these subjects, amino acid changes were apparent within the appropriate epitopes of human leukocyte antigen class I-restricted cytotoxic T lymphocytes. Thus, the evolutionary dynamics exhibited by the HIV-1 quasispecies virus populations under natural selection are compatible with adaptive evolution.

The immunology of primary HIV infection: which immune responses control HIV replication?

In the majority of patients, HIV-1 replication is quickly and efficiently controlled after the initial burst of viremia. Recent studies have begun to elucidate the underlying immune responses that may be responsible for this dramatic reduction in viral load. These responses include those of HIV-specific CD8+ cytotoxic T lymphocytes and antibodies capable of binding the virus.

Sensitivity of resistant human tumor cell lines to tumor necrosis factor and adriamycin used in combination: correlation between down-regulation of tumor necrosis factor-messenger RNA induction and overcoming resistance.

A number of human tumor cell lines of various histological origin were examined for their sensitivity and resistance to tumor necrosis factor-alpha (TNF) and Adriamycin (ADR). Six ovarian lines, and one each of a renal, lung, and B-cell line, were tested for putative mechanisms of resistance to these agents. Cytotoxicity resulting from TNF or ADR showed no overall correlation in these lines. The combination of TNF and ADR produced enhanced cytotoxicity against these tumor lines and furthermore resulted in overcoming the resistance of TNF or ADR alone or in combination. A proposed mechanism of TNF resistance in tumor cells is the endogenous production of TNF mRNA and protein. There was a positive correlation between resistance to TNF and the constitutive production of TNF mRNA and protein. The TNF-resistant lines that did not constitutively produce TNF mRNA and protein and the three TNF-sensitive tumor lines exhibited up-regulation of their TNF mRNA in the presence of TNF or phorbol myristate acetate/ionophore, but did not secrete any detectable protein. Due to the enhanced cytotoxicity seen with the combination of TNF and ADR, the effect of this combination on the level of TNF mRNA was examined. ADR alone reduced the constitutive level of TNF mRNA and in combination with TNF reduced the level of induction produced by TNF. This down-regulation of TNF mRNA by ADR may play a role in the enhanced cytotoxicity seen with the combination of these 2 agents.

hu-PBL-SCID mice can be protected from HIV-1 infection by passive transfer of monoclonal antibody to the principal neutralizing determinant of envelope gp120.

OBJECTIVE: To determine whether passive transfer of a monoclonal antibody specific for the principal neutralizing determinant in the V3 region of HIV-1IIIB gp120 can protect mice with severe combined immunodeficiency (SCID) transplanted with normal human peripheral blood leukocytes (hu-PBL), designated hu-PBL-SCID mice, from subsequent challenge with the homologous viral strain. DESIGN AND METHODS: hu-PBL-SCID mice were given intraperitoneal injections of an anti-HIV-1 neutralizing murine monoclonal antibody (BAT123), its mouse-human chimeric form (CGP 47 439), or a control murine antibody (PNTU), at a dose of 40 mg/kg. The mice were then challenged intraperitoneally with 10 mouse infectious doses of HIV-1IIIB. Three weeks later the mice were killed, and spleen cells and peritoneal lavage collected for determination of infection by coculture for viral isolation and by detection of HIV-1 DNA using polymerase chain reaction (PCR). RESULTS: All three antibodies had similar serum half-lives of 9-12 days. No toxicity was observed in the animals. HIV-1 was recovered by coculture from five out of the six mice given PNTU, and by PCR from two out of the six mice given PNTU, but was not recovered by either technique from any of the 12 mice given BAT123 or CGP 47 439. CONCLUSION: BAT123 and CGP 47 439, which are specific for the principal neutralizing determinant of HIV-1IIIB, protect hu-PBL-SCID mice from infection by this viral strain. Our findings support the use of the hu-PBL-SCID mouse as an in vivo model for studying protection against HIV-1 infection by passive immunization with anti-HIV-1 neutralizing antibodies.

CD8+ T-cell-mediated suppression of HIV replication in the first year of life: association with lower viral load and favorable early survival.

OBJECTIVE AND DESIGN: To study the role and development of non-cytotoxic CD8+ T-cell-mediated suppression of HIV replication in early perinatal HIV infection in a prospective study of vertically infected infants. CD8 T-cell-mediated HIV suppression was measured several times during the first year of life and correlated with viral load, cytotoxic T-cell (CTL) activity, in vitro antibody production (IVAP) and clinical outcome. METHODS: CD8+ T-cell-mediated HIV suppression was measured by comparing the amount of p24 antigen produced by endogenously infected lymphocytes with cultures of the same number of autologous CD4+ T cells from which CD8+ cells were removed immunomagnetically. CD8 viral suppressive activity (VSA) was defined as a > or = 50% reduction in p24 antigen in the cultures containing CD8+ cells. RESULTS: CD8+ T-cell-mediated HIV VSA was detected in 11/16 infants in the first year of life, including six/nine infants studied before 6 months and as early as 3 weeks of age. Infants who demonstrated CD8 VSA had a lower early peak and 6-month 'setpoint' plasma HIV RNA concentration than infants who lacked CD8 VSA [1.51 versus 4.94 and 0.094 versus 0.639 x 10(6) copies/ml, respectively, and higher CD4 percentage at 1 year of age. Survival of infants lacking CD8 VSA (four/six were rapid progressors) was shorter than for infants who demonstrated CD8 VSA (none out of 10 were rapid progressors). CD8 VSA was present before CTL and before or at the same time as IVAP in two of two and 11 of 14 infants studied, respectively. CONCLUSIONS: CD8+ T-cell-mediated VSA can be demonstrated in a large proportion of HIV-infected infants early in the course of infection. This non-cytolytic HIV-suppressive immune response appears to play an important protective role in the early control of perinatal HIV infection at a time when other immune responses are either absent or deficient.

Quantitative assessment of human immunodeficiency virus type 1 replication in human xenografts of acutely infected Hu-PBL-SCID mice.

Replication of the IIIB strain of human immunodeficiency virus type 1 (HIV-1IIIB) in CB.17 scid/scid mice reconstituted with human peripheral blood lymphocytes (Hu-PBL-SCID) was investigated. Hu-PBL-SCID mice could be infected, in a dose-dependent manner, when HIV-1IIIB was injected intraperitoneally. Replication-competent HIV-1 could be recovered from spleen, peripheral blood, bone marrow, thymus, lymph node, and peritoneal cavity, indicating the ability of the infection to spread to human tissue throughout the chimeric animals. HIV-1 infection was quantitated using p24 determination, end-point dilution culture, and polymerase chain reaction amplification. The level of HIV-1 replication in Hu-PBL-SCID mice was found to approximate the level reported in human infection. No HIV-1-specific immune response was generated to this infection, but nonspecific immune activation did occur, as also reported in human infection. Similarities therefore exist between HIV-1 infection of humans and Hu-PBL-SCID mice, which makes the latter an in vivo model in which to study HIV-1 replication.

Identification of subdominant cytotoxic T lymphocyte epitopes encoded by autologous HIV type 1 sequences, using dendritic cell stimulation and computer-driven algorithm.

Conventional analysis of the cytotoxic T lymphocyte (CTL) response to HIV-1 may underestimate the true breadth of CTL epitopes recognized. This underestimation could be due to several reasons, including (1) the use of laboratory-adapted stains of HIV or consensus sequences, which would lead to the identification of only highly conserved epitopes, (2) the use of EBV-transformed B cells (B-LCLs) and vaccinia virus constructs in standard assays that may obscure low level CTL responses due to high EBV or vaccinia reactivity, and (3) relatively insensitive assays wherein PBMCs instead of professional APCs are used to stimulate CTL responses. To address these problems, we first identified an immunodominant HLA-B7-restricted CTL epitope, by standard cloning methods, in a long-term nonprogressor (LTNP). To determine whether the patient had CTLs specific for autologous viral sequences other than the dominant epitope, proviral DNA was cloned and sequenced. A matrix-based epitope algorithm (EpiMatrix) was used to identify the top 2% of peptides from the viral sequences with the highest likelihood of binding to HLA-B7. These 55 peptides were synthesized and tested for HLA-B7 binding in a T2/B7 cell line; 10 peptides were able to stabilize HLA-B7 on the cell surface. By using peptide-pulsed autologous dendritic cells as a more sensitive method of CTL stimulation, we found three additional subdominant CTL epitopes.

Induction of humoral immune responses following vaccination with envelope-containing, formaldehyde-treated, thermally inactivated human immunodeficiency virus type 1.

The lack of success of subunit human immunodeficiency virus type 1 (HIV-1) vaccines to date suggests that multiple components or a complex virion structure may be required. We previously demonstrated retention of the major conformational epitopes of HIV-1 envelope following thermal treatment of virions. Moreover, antibody binding to some of these epitopes was significantly enhanced following thermal treatment. These included the neutralizing epitopes identified by monoclonal antibodies 1b12, 2G12, and 17b, some of which have been postulated to be partially occluded or cryptic in native virions. Based upon this finding, we hypothesized that a killed HIV vaccine could be derived to elicit protective humoral immune responses. Shedding of HIV-1 envelope has been described for some strains of HIV-1 and has been cited as one of the major impediments to developing an inactivated HIV-1 vaccine. In the present study, we demonstrate that treatment of virions with low-dose formaldehyde prior to thermal inactivation retains the association of viral envelope with virions. Moreover, mice and nonhuman primates vaccinated with formaldehyde-treated, thermally inactivated virions produce antibodies capable of neutralizing heterologous strains of HIV in peripheral blood mononuclear cell-, MAGI cell-, and U87-based infectivity assays. These data indicate that it is possible to create an immunogen by using formaldehyde-treated, thermally inactivated HIV-1 virions to induce neutralizing antibodies. These findings have broad implications for vaccine development.

Hierarchy of in vitro sensitivity and resistance of tumor cells to cytotoxic effector cells, cytokines, drugs and toxins.

Drug resistance of tumor cells has led to the development of other therapeutic modalities including biological response modifiers, lymphokine-activated killer cells (LAK), and cytokines alone and in combination. The premise of these alternative modalities is that drug resistance can be overcome by other cytotoxic agents or cytotoxic effector cells. However, the relationship between tumor cell sensitivity to these different agents and the cytotoxicity caused by drugs is not known or well understood. Thus, understanding the relationship between these different systems of tumor cell cytotoxicity is essential for optimal therapeutic intervention. To this end, we compared the tumor cell cytotoxicity mediated by recombinant tumor necrosis factor (rTNF), cytotoxic effector cells (natural killer cells, monocytes, LAK cells), chemotherapeutic drugs, and microbial toxins. Human tumor cell lines sensitive and resistant to rTNF or drugs were used to evaluate the effectiveness of the other cytotoxic modalities. Sensitivity was considered as tumor cell cytotoxicity above 15% while resistance refers to that below 10%. Cell lines tested consisted of several histological types such as brain, lung, colon and ovarian tumors. In our experiments, cell lines made resistant to rTNF by coculture were also relatively resistant to unactivated monocytes and their supernatants. These lines were sensitive to all other methods tested including activated monocytes, natural killer and LAK cells, drugs, and toxins. The tumor lines naturally resistant to rTNF were found to have various degrees of sensitivity and resistance to these other systems. Upon the analysis of our data, a pattern emerged that suggested a hierarchy of sensitivity and resistance of the tumor cells to the cytotoxic mechanisms explored. From a majority of cell lines resistant to rTNF to a minority of lines resistant to LAK, we found an interesting gradation of sensitivity and/or resistance to the other cytotoxic modalities employed. The hypothesis of an underlying common mechanism of action within these systems is discussed.

Synergistic effect of tumor necrosis factor-alpha- and diphtheria toxin-mediated cytotoxicity in sensitive and resistant human ovarian tumor cell lines.

Recent studies have demonstrated that diphtheria toxin (DTX) also mediates target cell lysis, and the mechanism of cytotoxicity has many features similar to those of cytotoxicity mediated by TNF-alpha. Thus, we hypothesized that DTX and TNF-alpha, used in combination, may result in either additive or synergistic cytotoxic activity. This was examined on three human ovarian carcinoma cell lines chosen for their differing sensitivities to TNF-alpha and DTX, i.e., 222, which is sensitive to both TNF-alpha and DTX, 222TR, a TNF-alpha-resistant DTX-sensitive variant of 222, and SKOV-3, which is resistant to both DTX and TNF-alpha. The simultaneous use of DTX and TNF-alpha at suboptimal concentrations resulted in synergistic cytotoxic activity against all three lines tested, thus overcoming the TNF-alpha resistance of 222TR and the double resistance of SKOV-3. DNA fragmentation was observed in all three lines treated with DTX and TNF-alpha and occurred as early as 4 h after treatment. Cycloheximide, actinomycin D, or emetine, at concentrations causing greater than 90% protein synthesis inhibition, did not result in cytotoxicity alone or synergy with TNF-alpha, suggesting that synergy by DTX was not due to its ability to inhibit protein synthesis. The use of energy poisons and pH conditions that inhibit DTX-mediated cytotoxicity resulted in the abrogation of synergy. These findings show that the two cytotoxic agents TNF-alpha and DTX, when used at suboptimal concentrations, synergize in their cytotoxic activity against sensitive and resistant cell lines. Because the SKOV-3 cell line used here is also resistant to chemotherapeutic drugs, combination treatment with DTX and TNF-alpha may be beneficial in overcoming drug resistance.

Sensitivity of human renal cell carcinoma lines to TNF, adriamycin, and combination: role of TNF mRNA induction in overcoming resistance.

We have examined 6 human renal cell carcinoma (RCC) cell lines for their sensitivity and resistance to the cytolytic effect of tumor necrosis factor (TNF) and adriamycin (ADR), alone or in combination. The results of cytotoxicity mediated by TNF and ADR showed no direct correlation as TNF resistant lines were sensitive to ADR while the TNF sensitive lines were resistant. The combination of TNF and ADR resulted in enhanced cytotoxicity against the tumor lines. Induction of TNF mRNA and protein has been suggested as a mechanism of resistance to TNF in certain tumors. Resistant and sensitive lines were capable of upregulating TNF mRNA after treatment with TNF or PMA+ionophore for periods as short as 1 hour, but only the resistant lines were able to secrete detectable levels of TNF protein. Therefore, a positive correlation existed between resistance to TNF and production and secretion of TNF by the cell lines. In the presence of the protein synthesis inhibitor cycloheximide (CHX), the TNF mRNA level in the TNF resistant lines was increased while the sensitive lines required an additional signal, such as exogenous TNF, to upregulate the mRNA. Due to the enhanced cytotoxicity seen with the combination of TNF and ADR, we determined the effect of this combination on the levels of TNF mRNA. As examined in a constitutively TNF expressing line, ADR alone reduced the constitutive mRNA level and, in combination with TNF, reduced the level of induction produced by TNF. This downregulation of TNF mRNA by ADR may play a role in the enhanced cytotoxicity seen with combined TNF and ADR treatment. The present study demonstrates that RCC cell lines differ in their sensitivity and/or resistance to TNF. Further, ADR and/or TNF resistant RCC lines can be rendered sensitive by combining TNF and ADR. The constitutive and/or inductive secretion of TNF by certain lines and its relationship to tumor pathogenesis as well as overcoming resistance are discussed.

Sensitivity of drug-resistant human ovarian tumor cell lines to combined effects of tumor necrosis factor (TNF-alpha) and doxorubicin: failure of the combination to modulate the MDR phenotype.

We have examined four human ovarian tumor lines (A2780, AD10, OVC-8, and SKOV-3) selected for their sensitivity and/or resistance to the recombinant human tumor necrosis factor alpha (TNF-alpha) and the chemotherapeutic drug doxorubicin (DOXO). The tumor lines were either sensitive to both agents, resistant to one or the other, or resistant to both. Of the four lines examined only the DOXO-resistant line AD10 exhibited the multidrug-resistance (MDR) phenotype. Enhanced cytotoxicity was seen with the combination of TNF-alpha and DOXO in each line regardless of their sensitivity or resistance patterns and, thus, demonstrates that drug resistance due to the expression of the MDR phenotype or its absence can be overcome by TNF-alpha and DOXO. We then examined whether TNF-alpha or TNF-alpha and DOXO modulated the MDR phenotype in AD10 as a possible mechanism of overcoming drug resistance. TNF-alpha had no effect on either DOXO intake or efflux as measured by flow cytometry. Further, TNF-alpha treatment showed no effect on the level of MDR-1 mRNA. These results suggest that the enhanced cytotoxicity seen with the combination of TNF-alpha and DOXO is not the result of any modulation of drug influx or efflux levels by TNF-alpha. Overall, these findings suggest that combination treatment with TNF-alpha and DOXO can overcome resistance inflicted by different mechanisms.

Pre- and postexposure protection against human immunodeficiency virus type 1 infection mediated by a monoclonal antibody.

Monoclonal antibody BAT123 was passively transferred into SCID mice reconstituted with human peripheral blood lymphocytes (hu-PBL-SCID) to study passive antibody protection against human immunodeficiency virus type 1 (HIV-1) infection. BAT123 is specific for the third variable loop of the gp120 of HIV-1LAI. Animals were protected against subsequent infection with LAI strain, but not other virus strains, when BAT123 (1 mg/kg; 25 micrograms/mouse) was given 1 h before virus inoculation. This resulted in a peak serum concentration of 16 micrograms/mL of the antibody, which should be easily attainable in humans. In addition, postexposure protection was observed when the antibody was given within 4 h of virus inoculation. No therapeutic effect was observed, however, when BAT123 was administered after infection had been established. These results indicate that passive antibody prophylaxis against HIV-1 infection may be possible in certain clinical situations.

Defining antibody protection against HIV-1 transmission in Hu-PBL-SCID mice.

Multiple monoclonal and polyclonal antibody preparations have been shown to neutralize HIV-1 infection in vitro. Upon direct testing in humans, however, many of these have failed to demonstrate clinical efficacy. Hu-PBL-SCID mice offer a model system in which to test the pre-clinical efficacy of antibody preparations. Testing in hu-PBL-SCID mice has shown that some antibodies are able to mediate pre- and post-exposure protection against HIV-1 infection, at concentrations that should be attainable in humans. Despite differences in the route and mode of transmission in humans and in hu-PBL-SCID mice, several aspects of the model make it a favorable model for future testing of antibody protection against HIV-1 infection. These include the architecture of the peritoneal cavity, the mixture of human cells that engraft, the density of human target cells for HIV-1 infection, and the presence of complement and NK cells that can interact with antibody preparations in blocking HIV-1 infection. The use of this model in testing newer antibody preparations for efficacy against primary isolates should enhance our knowledge of the mechanisms of antibody protection against HIV-1 infection in vivo and speed the pre-clinical evaluation of potential immunoprophylactic agents against HIV-1.