BACKGROUND Immune reconstitution following myeloablative allogeneic hematopoietic stem cell transplant (MA-alloHSCT) is significantly delayed for T cell depleted allografts when compared to T-cell replete allografts, a feature that has been implicated in higher rates and grades of infection, less GVT, and worse overall survival. Orca-T is a high precision cell therapy currently being investigated for the treatment of certain hematological malignancies otherwise treated with MA-alloHSCT. The cellular drug products of Orca-T (HSPCs, Tregs, and Tcons) are administered at high purity, in controlled doses, and on an established schedule with the intent to reconstitute the blood and immune system while controlling GVHD. Here, we present data on the immune reconstitution in 100 adult patients who received Orca-T. METHODS In the context of an ongoing multicenter Phase Ib clinical trial of Orca-T in recipients with hematologic malignancies (NCT04013685), we performed longitudinal measurements (days -28, 28, 56, 100, 180, and 365 post-transplant) of immune reconstitution in the first 100 consecutive patients. With fresh whole blood, clinical 5-part leukocyte differentials were performed at clinical sites, and lymphocyte subset frequencies were measured by flow cytometry in a central lab. Principal component analysis (PCA) was performed to investigate potential associations with recipient sex (male vs. female) and donor relation (related vs. unrelated). RESULTS Longitudinal peripheral blood counts of platelets, WBCs, neutrophils, lymphocytes, monocytes, T cells, B cells, and NK cells are presented in Table 1. T cell and B cell counts were readily observed at days 28 and 56 respectively, and increased with each subsequent time point. Median NK cell levels were observed to be in the normal range at all post-transplant time points. CD4+ T cell and Treg cell counts exhibited similar post-transplant patterns with both being appreciably present at d28 and increasing with each subsequent time point. Strikingly, relative to the level measured in 75 corresponding PBSC donors, the Treg frequency among CD4+ T cells was significantly elevated at all time points post-transplant (Figure 1). Median CD8+ T cell counts increased for the first 6 months post-transplant and then plateaued in the normal range. Upon PCA, very few significant differences were observed in 2-group comparisons of recipient sex and donor relation. CONCLUSIONS Orca-T patients exhibit early immune reconstitution of each of the major leukocyte and lymphocyte subsets hypothesized to control relapse and infection. Concomitantly, elevated Treg frequencies were also observed. This feature of immune reconstitution profiles of Orca-T recipients may be correlated to the reduced occurrence and severity of acute and chronic GVHD in these patients (Meyer et al., EHA 2022, #S237). Similar immune reconstitution profiles were observed in patients of disparate sex and regardless of donor relation. Prospective comparisons of immune reconstitution between Orca-T and standard-of-care patients will be performed in our ongoing phase 3 clinical trial (NCT05316701). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The adaptive immune response to viral infections features the antigen-driven expansion of CD8+ T cells. These cells are widely recognized for their cytolytic activity that is mediated through the secretion of cytokines such as perforin and granzymes. Less appreciated is their ability to secrete soluble factors that restrict virus replication without killing the infected cells. In this study we measured the ability of primary anti-CD3/28-stimulated CD8+ T cells from healthy blood donors to secrete interferon-alpha. Supernatants collected from CD8+ T cell cultures were screened for their ability to suppress HIV-1 replication in vitro and their interferon-alpha concentrations were measured by ELISA. Interferon-alpha concentrations in the CD8+ T cell culture supernatants ranged from undetectable to 28.6 pg/mL. The anti-HIV-1 activity of the cell culture supernatants was observed to be dependent on the presence of interferon-alpha. Appreciable increases in the expression levels of type 1 interferon transcripts were observed following T cell receptor stimulation, suggesting that the secretion of interferon-alpha by CD8+ T cells is an antigen-driven response. In 42-plex cytokine assays, the cultures containing interferon-alpha were also found to contain elevated levels of GM-CSF, IL-10, IL-13, and TNF-alpha. Together, these results demonstrate that the secretion of anti-viral levels of interferon-alpha is a common function of CD8+ T cells. Furthermore, this CD8+ T cell function likely plays broader roles in health and disease.

Significance Our study demonstrates that CD34 + cells generated from human induced pluripotent stem cells (iPSCs) genetically engineered to confer resistance to HIV have engraftment capabilities if they are derived in vivo but not in vitro. We used iPSCs from HIV-infected patients under antiretroviral therapy to form teratomas in immunodeficient mice and isolated CD34 + cells from these teratomas. We showed not only that these CD34 + cells could engraft in recipient mice but also that their cell progeny found in the mouse peripheral blood was resistant to HIV infection. These findings confirm the potential for evaluating in an animal model the clinical development of cure therapies using genetically modified autologous iPSCs from patients with HIV.

OBJECTIVES:The discovery of induced pluripotent stem cells (iPSC) has brought promise to regenerative medicine as it breaks the ethical barrier of using embryonic stem cells. Such cell culture-derived patient-specific autologous stem cells are needed for transplantation. Here we report deriving HIV-1-infected patients' iPSC lines under transgene-free methods and under feeder-free and xeno-free culture conditions to meet the requirement for clinical application.METHODS AND RESULTS:We have reprogrammed patients' peripheral blood mononuclear cells with EBNA1/OriP episomal vectors, or a defective and persistent Sendai virus vector (SeVdp) to ensure a nonintegrating iPSC generation. Both single picked and pooled iPSC lines demonstrated high pluripotency and were able to differentiate into various lineage cells in vivo. The established cell lines could be modified by genetic editing using the TALENs or CRISPR/Cas 9 technology to have a bi-allelic CCR5Δ32 mutations seamlessly. All generated iPSC lines and modified cell lines had no evidence of HIV integration and maintained normal karyotype after expansion.CONCLUSIONS:This study provides a reproducible simple procedure for generating therapeutic grade iPSCs from HIV-infected patients and for engineering these cells to possess a naturally occurring genotype for resistance to HIV-1 infection when differentiated into immune cells.

Objective: To assess the in-vitro CCR5---tropic and CXCR4---tropic HIV---1 infectivity of immune cells, particularly macrophages, derived fromCCR5 gene---edited induced pluripotent stem cells (iPSCs) obtained from the peripheral blood mononuclear cells (PBMC) of HIV---infected patients on antiretroviral therapy (ART). Design: PBMC were obtained from six patients who had been HIV---infected for over 20 years and were on ART for 1---12 years prior to this study. Methods: The PBMC were derived into iPSCs and genetically edited with TALENs or CRISPR---cas9 endonucleases combined with PiggyBac technology to introduce the naturally occurring 32---bp deletion to theCCR5 gene. These iPSCs were differentiated into macrophages, and subsequently challenged with CCR5---tropic or CCR5/CXCR4 dual--- tropic HIV---1 strains. iPSC derivation, gene editing and immune cell differentiation were done in feeder---free, xeno---free in-vitro conditions. Results: Multiple unedited (wild---type) andCCR5 gene---edited (mutant) iPSCs were derived from patients' PBMC. When differentiated into immune cells and HIV---1 challenged, mutant iPSC lines were resistant to CCR5---tropic and to some extent to CCR5/CXCR4 dual---tropic HIV---1 infection when compared to wild---type iPSC lines. Conclusion: Our study demonstrates that iPSC---derived, gene---edited immune cells are resistant to distinct HIV---1 strains. These findings have important implications for both in-vitro stem cell development and therapeutic approaches to cure HIV infection.

CD8+ T lymphocytes can reduce the production of human immunodeficiency virus 1 (HIV-1) by CD4+ T cells by cytotoxic and non-cytotoxic mechanisms. To investigate the involvement of human leukocyte antigen (HLA) class I compatibility in anti-HIV responses, we co-cultured primary CD8+ T cells, isolated from the peripheral blood of HIV-1-infected individuals, with panels of autologous and heterologous acutely HIV-1-infected primary CD4+ T cells. Altogether, CD8+ T cell anti-HIV activity was evaluated in more than 200 co-cultures. Marked heterogeneity in HIV-1 replication levels was observed among the co-cultures sharing a common CD8+ T cell source. The co-cultures that exhibited greater than 50% reduction in HIV production were found to have significantly increased numbers of matching HLA class I alleles (Yates chi-square = 54.21; p < 0.001). With CD8+ T cells from HIV controllers and asymptomatic viremic individuals, matching HLA-B and/or HLA-C alleles were more predictive of strong anti-HIV activity than matching HLA-A alleles. Overall, HLA class I genotype matches were more closely associated with CD8+ T cell anti-HIV activity than supertype pairings. Antibodies against HLA class I and CD3 reduced the CD8+ T cell anti-HIV activity. Stimulated CD8+ T cells exhibited increased anti-HIV activity and reduced dependency on HLA compatibility. These findings provide evidence that the maximal suppression of HIV replication by CD8+ T cells requires the recognition of multiple epitopes. These studies provide insight for HIV vaccine development, and the analytic approach can be useful for the functional characterization of HLA class I alleles and tentative HLA class I supertypes.

A CD8+ cell non-cytotoxic antiviral response (CNAR), mediated by a CD8+ cell antiviral factor (CAF), is associated with a long-term healthy state in human immunodeficiency virus (HIV) infection. CNAR/CAF reduces viral transcription without a known effect on specific viral sequences in the HIV genome. In studies to define the mechanism involved in the block in viral transcription, we now report that transcription from the HIV-LTR reporter is reduced in infected CD4+ cells upon treatment with CAF. In agreement with this observation, the amount of RNA polymerase II (RNAPII) on the HIV promoter and other viral regions was strongly diminished in HIV-infected CD4+ cells co-cultivated with CNAR-expressing CD8+ cells. These results demonstrate further that CNAR/CAF has a specific role in regulating HIV transcription and a step during the preinitiation complex assembly appears to be sensitive to CNAR/CAF.

Individuals homozygous for the C-C chemokine receptor type 5 gene with 32-bp deletions (CCR5Δ32) are resistant to HIV-1 infection. In this study, we generated induced pluripotent stem cells (iPSCs) homozygous for the naturally occurring CCR5Δ32 mutation through genome editing of wild-type iPSCs using a combination of transcription activator-like effector nucleases (TALENs) or RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 together with the piggyBac technology. Remarkably, TALENs or CRISPR-Cas9-mediated double-strand DNA breaks resulted in up to 100% targeting of the colonies on one allele of which biallelic targeting occurred at an average of 14% with TALENs and 33% with CRISPR. Excision of the piggyBac using transposase seamlessly reproduced exactly the naturally occurring CCR5Δ32 mutation without detectable exogenous sequences. We differentiated these modified iPSCs into monocytes/macrophages and demonstrated their resistance to HIV-1 challenge. We propose that this strategy may provide an approach toward a functional cure of HIV-1 infection.

ABSTRACT Broadly neutralizing monoclonal antibodies (bNAbs) 2F5 and 4E10 bind to the membrane proximal external region (MPER) of gp41 and also cross-react with phospholipids. In this study, we investigated if chemical modifications on the MPER adjacent to 2F5 and 4E10 epitopes using mimetics of inflammation-associated posttranslational modifications to induce 2F5- and 4E10-like bNAbs can break tolerance. We synthesized a series of chemically modified peptides spanning the MPER. The serine, threonine, and tyrosine residues in the peptides were modified with sulfate, phosphate, or nitrate moieties and presented in liposomes for rabbit immunizations. All immunizations resulted in high antisera titers directed toward both the modified and unmodified immunogens. Tyrosine modification was observed to significantly suppress antiepitope responses. Sera with strong anti-gp140 titers were purified by affinity chromatography toward the MPER peptide and found to possess a higher affinity toward the MPER than did the bNAbs 2F5 and 4E10. Modest neutralization was observed in the H9 neutralization assay, but neutralization was not observed in the TZM-bl cell or peripheral blood mononuclear cell (PBMC) neutralization assay platforms. Although neutralizing antibodies were not induced by this approach, we conclude that chemical modifications can increase the immune responses to poorly immunogenic antigens, suggesting that chemical modification in an appropriate immunization protocol should be explored further as an HIV-1 vaccine strategy.

Abstract:Antiretroviral therapy (ART) significantly reduced the CD8+ cell noncytotoxic anti-HIV response in 12 HIV-1–infected subjects (P < 0.0001). In separate experiments, CD8+ cells from long-term survivors were cocultured with HIV-infected CD4+ cells using varying concentrations of anti-HIV drugs. The antiviral function of CD8+ cells from 4 of the 14 LTSs was reduced with exposure to 10 &mgr;M of nevirapine (P < 0.05). The antiviral activity of CD8+ cells from 2 LTSs was inhibited by 5 &mgr;M of zidovudine. These studies indicate that nevirapine and probably zidovudine can inhibit the anti-HIV activity of CD8+ cells and thus could influence the effectiveness of antiretroviral therapy.

In HIV-1 infection, plasmacytoid dendritic cell (PDC) numbers and function are decreased. No detailed comparisons of PDC responses to various stimuli in HIV-1-infected patients are available. Using for the first time purified PDCs, we compared PDC responses [interferon (IFN)-α production/cell] to various stimuli in a large number (n=48) of HIV-1-infected patients and healthy volunteers (n=19). Toll-like receptor (TLR)7- and TLR9-induced expression of PDC surface activation and maturation markers was also compared in the two populations. We have confirmed that PDC number coincides with CD4(+) T cell counts and clinical state. Notably, we have shown that a direct association of PDC function in terms of IFN-α production/cell exists with PDC numbers and CD4(+) cell counts when PDCs are exposed to a TLR9 ligand and HIV-infected cells, but not with a TLR7 ligand. Moreover, in the HIV-infected subjects but not the healthy controls, the magnitude of IFN-α release per PDC in response to the TLR7 ligand is significantly (p<0.01) lower than that to the TLR9 ligand. However, in both study populations, the TLR7 stimulation in comparison to TLR9 stimulation induced higher expression of PDC surface activation and maturation markers and significantly (p<0.05) decreased the expression of BDCA-2, a negative regulator of interferon. Furthermore, the cross-ligation of BDCA-2 significantly (p<0.05) inhibited TLR9- but not TLR7-induced IFN-α production by PDCs from both clinical groups. These findings suggest that differences exist in TLR7- and TLR9-induced IFN-α production by PDCs in HIV-infected individuals that are not directly related to BDCA-2 down-modulation.

CD8+ cells can suppress human immunodeficiency virus 1 (HIV-1) replication by releasing soluble factors. In 26 years of intensive research efforts, the identity of the major CD8+ cell antiviral factor has remained elusive. To investigate the mechanism for this antiviral immune response, we performed gene expression analyses on primary CD4+ cells that were exposed to HIV-suppressing CD8+ cells or CD8+ cell-conditioned medium having HIV-suppressing activity. These experiments revealed increased levels of multiple genes stimulated by type I interferons (IFN; eg, IFN-α and IFN-β). Further evaluation revealed that primary CD8+ cells, particularly those from elite controllers and other asymptomatic HIV-1-infected individuals, secrete IFN, and this response directly contributes to the in vitro suppression of HIV replication in CD4+ cells. This novel immune response, likely mediated by memory CD8+ T cells, may play an important role in a wide variety of viral infections, cancers, and autoimmune diseases.

ABSTRACTHIV replication is suppressedin vitroby a CD8+cell noncytotoxic antiviral response (CNAR). This activity directly correlates with an asymptomatic clinical state. The objective of this study was to identify the phenotype of CD8+cell subsets having strong CNAR activity. CD8+cell subset frequencies and CNAR levels were measured for human immunodeficiency virus (HIV)-uninfected individuals and three groups of HIV type 1 (HIV-1)-infected individuals: asymptomatic individuals with low-level viremia (vHIV), antiretroviral-drug-treated subjects with undetectable virus levels (TxHIV), and therapy-naïve aviremic elite controllers (EC). CD8+cells from the vHIV individuals exhibited the highest HIV-suppressing activity and had elevated frequencies of CD45RA−CD27+and PD-1+(CD279+) cells. Functional assessments of CD8+cells sorted into distinct subsets established that maximal CNAR activity was mediated by CD45RA−CCR7−CD27+and PD-1+CD8+cells. T cell receptor (TCR) repertoire profiles of CD8+cell subsets having strong CNAR activity exhibited increased perturbations in comparison to those of inactive subsets. Together, these studies suggest that CNAR is driven by HIV replication and that this antiviral activity is associated with oligoclonally expanded activated CD8+cells expressing PD-1 and having a transitional memory cell phenotype. The findings better describe the identity of CD8+cells showing CNAR and should facilitate the evaluation of this important immune response in studies of HIV pathogenesis, resistance to infection, and vaccine development.

Members of the gammaretroviruses-such as murine leukemia viruses (MLVs), most notably XMRV [xenotropic murine leukemia virus (X-MLV)-related virus-have been reported to be present in the blood of patients with chronic fatigue syndrome (CFS). We evaluated blood samples from 61 patients with CFS from a single clinical practice, 43 of whom had previously been identified as XMRV-positive. Our analysis included polymerase chain reaction and reverse transcription polymerase chain reaction procedures for detection of viral nucleic acids and assays for detection of infectious virus and virus-specific antibodies. We found no evidence of XMRV or other MLVs in these blood samples. In addition, we found that these gammaretroviruses were strongly (X-MLV) or partially (XMRV) susceptible to inactivation by sera from CFS patients and healthy controls, which suggested that establishment of a successful MLV infection in humans would be unlikely. Consistent with previous reports, we detected MLV sequences in commercial laboratory reagents. Our results indicate that previous evidence linking XMRV and MLVs to CFS is likely attributable to laboratory contamination.

Electroporation, a non-virus-mediated gene transfection method, has traditionally had poor outcomes with low gene transfection efficiency and poor cellular viability, particularly in primary human lymphocytes. Herein we have optimized the electroporation conditions for primary CD8+ cells resulting in a maximum rate of 81.3%, and a mean transfection efficiency of 59.6%. After removal of dead cells, the viability of transfected primary CD8+ cells was greater than 90%, similar to untransfected controls. Using this procedure, primary human CD8+ cells transfected with an interferon α8 plasmid produced fluids that inhibited HIV-1 replication by >95%. This transfection protocol is useful for transfection of other primary blood cells, such as CD4+ T cells, and for studying the function of genes in primary human blood cells instead of cell lines. The transfection procedure also has potential application in gene therapy clinical trials to treat diseases utilizing transfected primary human cells.

HIV replication is suppressed in vitro by a CD8 (cid:1) cell noncytotoxic antiviral response (CNAR). This activity directly correlates with an asymptomatic clinical state. The objective of this study was to identify the phenotype of CD8 (cid:1) cell subsets having strong CNAR activity. CD8 (cid:1) cell subset frequencies and CNAR levels were measured for human immunodeficiency virus (HIV)-uninfected individuals and three groups of HIV type 1 (HIV-1)-infected individuals: asymptomatic individuals with low-level viremia (vHIV), antiretroviral-drug-treated subjects with undetectable virus levels (TxHIV), and therapy-naïve aviremic elite controllers (EC). CD8 (cid:1) cells from the vHIV individuals exhibited the highest HIV-suppressing activity and had elevated frequencies of CD45RA (cid:2) CD27 (cid:1) and PD-1 (cid:1) (CD279 (cid:1) ) cells. Functional assessments of CD8 (cid:1) cells sorted into distinct subsets established that maximal CNAR activity was mediated by CD45RA (cid:2) CCR7 (cid:2) CD27 (cid:1) and PD-1 (cid:1) CD8 (cid:1) cells. T cell receptor (TCR) repertoire profiles of CD8 (cid:1) cell subsets having strong CNAR activity exhibited increased perturbations in comparison to those of inactive subsets. Together, these studies suggest that CNAR is driven by HIV replication and that this antiviral activity is associated with oligoclonally expanded activated CD8 (cid:1) cells expressing PD-1 and having a transitional memory cell phenotype. The findings better describe the identity of CD8 (cid:1) cells showing CNAR and should facilitate the evaluation of this important immune response in studies of HIV pathogenesis, resistance to infection, and vaccine development. CNAR assays. To determine the respective levels of CNAR activity, incremen- tal numbers of CD8 (cid:1) cells (without in vitro stimulation) were cocultured with acutely HIV-infected autologous or heterologous CD4 (cid:1) cells and the ensuing level of HIV replication were measured. Briefly, CD4 (cid:1) cells were resuspended (3 (cid:7) 10 6 cells/ml) in growth medium (RPMI 1640 medium supplemented with fetal calf serum [heat inactivated at 56°C for 30 min; 10%, vol/vol], penicillin [100 U/ml], streptomycin [100 (cid:6) g/ml], L -glutamine [2 mM], and recombinant inter- leukin 2 [IL-2; 100 U/ml; Invitrogen]) and stimulated for 3 days in the presence of phytohemagglutinin-leucoagglutinin (PHA-L; 3 (cid:6) g/ml; Sigma) in a 37°C hu- midified incubator. Subsequently, 10 (cid:7) 10 6 cells were treated with Polybrene (2 (cid:6) g/ml; Sigma) for 20 min at 37°C and pelleted. The pelleted cells were resus- pended in 1 ml of HIV-1 SF33 (10,000 50% tissue culture infective doses [TCID 50 ]/ml in PBMC) for 1 h at 37°C with periodic mixing. HIV-1 SF33 , a syncytium-inducing (SI), CXCR4-tropic (X4) strain, has been maintained in primary cells since its isolation, exhibits rapid replication kinetics with a high degree of cytopathicity in cell culture, and is not sensitive to (cid:8) -chemokine-mediated antiviral effects (31). The acutely infected CD4 (cid:1) cells were then washed and resuspended at 10 6 cells/ml of growth medium, and 200- (cid:6) l aliquots were placed into a flat-bottom 96-well tissue culture plate (Falcon 3072; BD) cation CD45RA CD45RA CD28 Comparisons of bulk CD8 (cid:1) cells, which were unstained or stained with antibodies, revealed no substantial differences in CNAR activity (data not shown). Thus, activation of the cells and/or blocking of the surface antigens due to antibody binding was not involved. These results show that is associated with memory CD8 (cid:1) cells, particularly those with a transitional memory phenotype.