Cell Precursor Frequency Research Articles

Glomerular filtration rate predicts pro-vascular progenitor cell depletion: insights from the IPE-PREVENTION and ORIGINS-RCE studies

Abstract Background People living with cardiometabolic disease and impaired renal function are at increased risk of experiencing adverse cardiovascular outcomes(1,2). Recent evidence indicates that individuals living with atherosclerotic cardiovascular disease (ASCVD) and type 2 diabetes (T2D) exhibit a reduction in circulating vascular regenerative (VR) cells that are essential for maintaining blood vessel homeostasis(3). However, there is limited data on whether VR cell content changes in response to cardiometabolic and kidney disorders. Purpose We sought to determine if there is an association between estimated glomerular filtration rate (eGFR) and VR cell content in individuals with ASCVD and/or diabetes. Data were from participants enrolled in the IPE-PREVENTION and ORIGINS-RCE studies. Methods IPE-PREVENTION participants had moderately elevated triglycerides (1.5 - 5.65 mmol/L) with established ASCVD and/or T2D. ORIGINS-RCE participants had established ASCVD or T2D and at least one additional cardiovascular risk factor. Circulating mononuclear cells were isolated from peripheral blood and characterized using side scatter properties (SSC) and high aldehyde dehydrogenase activity to quantify pro-angiogenic early myeloid progenitor cells (ALDHhi), and primitive versus lineage-specific cell surface marker co-expression. Participants were stratified according to those with an eGFR <60 mL/min/1.73m², or ≥60 mL/min/1.73m². Results Median [IQR] eGFR of the pooled cohort (n=169) was 75 [59, 88] mL/min/1.73m²; 45% had an eGFR that was <60 mL/min/1.73m². The stratum with eGFR <60 mL/min/1.73m² had fewer circulating ALDHhiSSClow progenitor cells (0.037 vs. 0.051%; P<0.01) (Figure 1A) and ALDHhiSSClow cells expressing CD34+ and CD133+ (49 vs. 54%; P=0.03) (Figure 1B) than that with an eGFR ≥60 mL/min/1.73m². The predictive value of eGFR on ALDHhiSSClowCD34+CD133+ frequency was conserved even after controlling for age, sex, LDL-C, BMI, and HbA1c (P<0.01) (Table 1). The frequency of ALDHhiSSCmid monocyte precursor cells did not differ based on eGFR status. Conclusions Low eGFR was predictive for the depletion of ALDHhiSSClow primitive progenitor cells that co-express the stem cell markers CD133 and CD34 in individuals with ASCVD and/or T2D suggesting that diminished kidney function is associated with a decrease in vessel reparative pro-angiogenic progenitor cell content. Strategies that improve circulating VR cell content in individuals living with kidney and cardiometabolic disease may provide clinically meaningful benefits.Figure 1Table 1

792-P: GLP-1 RA Therapy Increases Circulating Vascular Regenerative Cell Content and Decreases Inflammatory Burden in People with T2D

CV outcome trials have established that long-term use of GLP-1 receptor agonists (GLP-1RAs) yields important CV benefits in people with T2D. Similar to SGLT2i, GLP-1RAs are recommended for people with T2D who have established CVD and heart failure regardless of their A1C. In a randomized trial of people with T2D and coronary artery disease, use of the SGLT2i empagliflozin for 6 months increased the frequency of vascular regenerative (VR) cells in the circulation. To determine if GLP-1RA therapy has a similar impact on the same VR cell populations, we retrospectively analyzed data from an ongoing research program with adults >40 years of age with T2D. Peripheral blood mononuclear cells were isolated and profiled with an established flow cytometry assay that was founded on the activity of the anti-oxidative enzyme aldehyde dehydrogenase (ALDH) that is highly expressed in progenitor (ALDHhi) versus mature (ALDHlow) cells. At baseline, 17 participants were on a GLP-1RA (9 of whom were also on an SGLT2i), 33 were on an SGLT2i but not a GLP-1RA, and 28 were on neither drug class. Mean age, weight, BMI, A1C, and duration of T2D was balanced across the groups. The frequency of ALDHhiSSClow cells (primitive hematopoietic and endothelial progenitor cells that coordinate angiogenesis) was higher in participants who were on a GLP-1RA (0.064%) or an SGLT2i (0.048%) relative to those not on either drug class (0.037%; P<0.05 for both). The frequencies of VR ALDHhiSSCmid monocytes in the three groups were comparable. The frequency of pro-inflammatory ALDHhiSSChi granulocyte precursor cells in participants on a GLP-1RA (2.9%) was lower than that in individuals on an SGLT2i (4.3%; P=0.05) or not on either drug class (5.2%; P<0.01). GLP-1RA therapy in people with T2D was associated with greater VR cell content and lower inflammatory burden. Augmented vessel repair mediated by higher VR cell content may account in part for the CV benefits observed with GLP-1RA in the setting of T2D. Disclosure A.Krishnaraj: None. B.Park: None. E.Bakbak: None. Y.Pan: None. A.Quan: None. H.Teoh: None. S.Verma: Advisory Panel; Amgen Canada, AstraZeneca, Bayer Inc., Boehringer Ingelheim and Eli Lilly Alliance, HLS Therapeutics Inc., Janssen Pharmaceuticals, Inc., Novartis Canada, Novartis, Novo Nordisk, Novo Nordisk Canada Inc., Consultant; AstraZeneca, Other Relationship; Amarin Corporation, AstraZeneca, Bayer Inc., Boehringer Ingelheim and Eli Lilly Alliance, Boehringer Ingelheim International GmbH, Canadian Medical and Surgical Knowledge Translation Research Group, EOCI Pharmacomm, HLS Therapeutics Inc., Janssen Pharmaceuticals, Inc., Novartis Canada, Novo Nordisk, Novo Nordisk Canada Inc., Pfizer Inc., PhaseBio Pharmaceuticals, Inc., S & L Solutions Event Management Inc, Sanofi, Sun Pharmaceutical Industries Ltd., Toronto Knowledge Translation Working Group, Research Support; Amarin Corporation, Amgen Canada, AstraZeneca, Bayer Inc., Boehringer Ingelheim International GmbH, HLS Therapeutics Inc., Novartis, Novo Nordisk, Pfizer Inc., PhaseBio Pharmaceuticals, Inc. D.A.Hess: None.

Expansion of CD8+ T cell population in Lassa virus survivors with low T cell precursor frequency reveals durable immune response in most survivors.

Lassa virus is a priority pathogen for vaccine research and development, however the duration of cellular immunity and protection in Lassa fever (LF) survivors remains unclear. We investigated Lassa virus specific CD8+ T cell responses in 93 LF survivors. Peripheral blood mononuclear cells from these individuals were infected with recombinant vesicular stomatitis virus encoding Lassa virus antigens and virus specific T cell responses were measured after 18-hour incubation. Participants who had undetectable CD8+ T cell response underwent further analysis using a 10-day T cell proliferation assays to evaluate for low T cell precursor frequency. Forty-five of the 93 LF survivors did not have a Lassa virus specific CD8+ T cell response. Of those with responses and a known date of onset of LF (N = 11), 9 had LF within the last ten years. Most participants without a measurable CD8+ T cell response were more than 10 years removed from a clinical history of LF (N = 14/16). Fourteen of 21 patients (67%) with undetectable CD8+ T cell response had a measurable Lassa virus specific CD8+ T cell response with the 10-day assay. Despite reports of strong CD8+ T cell responses during acute Lassa virus infection, circulating Lassa virus-specific CD8+ T cells declined to undetectable levels in most Lassa fever survivors after ten years when evaluated with an 18-hour T cell stimulation. However, when Lassa virus-specific T cells were expanded prior to restimulation, a Lassa virus-specific CD8+ T cell response could be detected in many if the samples that were negative in the 18-hour stimulation assay, suggesting that prolonged cellular immunity does exist in Lassa fever survivors at low frequencies.

B cells expressing IgM B cell receptors of HIV-1 neutralizing antibodies discriminate antigen affinities by sensing binding association rates.

HIV-1 envelope (Env) proteins designed to induce neutralizing antibody responses allow study of the role of affinities (equilibrium dissociation constant [KD]) and kinetic rates (association/dissociation rates) on B cell antigen recognition. It is unclear whether affinity discrimination during B cell activation is based solely on Env protein binding KD and whether B cells discriminate among proteins of similar affinities that bind with different kinetic rates. Here, we use a panel of Env proteins and Ramos B cell lines expressing immunoglobulin M (IgM) B cell receptors (BCRs) with specificity for CD4-binding-site broadly neutralizing antibodies to study the role of antigen binding kinetic rates on both early (proximal/distal signaling) and late events (BCR/antigen internalization) in B cell activation. Our results support a kinetic model for B cell activation in which Env protein affinity discrimination is based not on overall KD but on sensing of association rate and a threshold antigen-BCR half-life.

B cells promote CD8 Tcell primary and memory responses to subunit vaccines.

SUMMARYThe relationship between B cells and CD4 T cells has been carefully studied, revealing a collaborative effort in which B cells promote the activation, differentiation, and expansion of CD4 T cells while the so-called “helper” cells provide signals to B cells, influencing their class switching and fate. Interactions between B cells and CD8 T cells are not as well studied, although CD8 T cells exhibit an accelerated contraction after certain infections in B-cell-deficient mice. Here, we find that B cells significantly enhance primary CD8 T cell responses after vaccination. Moreover, memory CD8 numbers and function are impaired in B-cell-deficient animals, leading to increased susceptibility to bacterial challenge. We also show that interleukin-27 production by B cells contributes to their impact on primary, but not memory, CD8 responses. Better understanding of the interactions between CD8 T cells and B cells may aid in the design of more effective future vaccine strategies.

Negative Feedback by NUR77/ <i>Nr4a1</i> Restrains B Cell Clonal Dominance During Early T-Dependent Immune Responses

B cell clones compete for entry into and dominance within germinal centers (GC), where the highest affinity BCRs are selected. However, diverse and low affinity B cells can enter and reside in GCs for extended periods. To reconcile these observations, we hypothesized that a negative feedback loop may operate within B cells to preferentially restrain high affinity clones from monopolizing the early GC niche. Here we report a role for the nuclear receptor NUR77/Nr4a1 in this process. We previously showed that NUR77 expression scales with antigen stimulation and restrains B cell expansion when T cell help is limiting. Here we show that, although NUR77 is dispensable for regulating GC size when GC are elicited in a largely clonal manner, it serves to curb immunodominance under conditions where diverse clonal populations must compete for a constrained niche. Moreover, this is independent of B cell precursor frequency and reflects, at least in part, a B cell-intrinsic role for NUR77. We propose that this is important to preserve early B cell clonal diversity in order to limit holes in the post-immune repertoire and to optimize GC selection.

HLA Class II Genotype Does Not Affect the Myelin Responsiveness of Multiple Sclerosis Patients.

Background: When aiming to restore myelin tolerance using antigen-specific treatment approaches in MS, the wide variety of myelin-derived antigens towards which immune responses are targeted in multiple sclerosis (MS) patients needs to be taken into account. Uncertainty remains as to whether the myelin reactivity pattern of a specific MS patient can be predicted based upon the human leukocyte antigen (HLA) class II haplotype of the patient. Methods: In this study, we analyzed the reactivity towards myelin oligodendrocyte glycoprotein (MOG), myelin basic protein (MBP) and proteolipid protein (PLP) peptides using direct interferon (IFN)-γ enzyme-linked immune absorbent spot (ELISPOT). Next, the HLA class II haplotype profile was determined by next-generation sequencing. In doing so, we aimed to evaluate the possible association between the precursor frequency of myelin-reactive T cells and the HLA haplotype. Results: Reactivity towards any of the analyzed peptides could be demonstrated in 65.0% (13/20) of MS patients and in 60.0% (6/10) of healthy controls. At least one of the MS risk alleles HLA-DRB1*15:01, HLA-DQA1*01:02 and HLA-DQB1*06:02 was found in 70.0% (14/20) of patients and in 20.0% (2/10) of healthy controls. No difference in the presence of a myelin-specific response, nor in the frequency of myelin peptide-reactive precursor cells could be detected among carriers and non-carriers of these risk alleles. Conclusion: No association between HLA haplotype and myelin reactivity profile was present in our study population. This complicates the development of antigen-specific treatment approaches and implies the need for multi-epitope targeting in an HLA-unrestricted manner to fully address the wide variation in myelin responses and HLA profiles in a heterogeneous group of MS patients.

B cells expressing authentic naive human VRC01-class BCRs can be recruited to germinal centers and affinity mature in multiple independent mouse models

Animal models of human antigen-specific B cell receptors (BCRs) generally depend on "inferred germline" sequences, and thus their relationship to authentic naive human B cell BCR sequences and affinities is unclear. Here, BCR sequences from authentic naive human VRC01-class B cells from healthy human donors were selected for the generation of three BCR knockin mice. The BCRs span the physiological range of affinities found in humans, and use three different light chains (VK3-20, VK1-5, and VK1-33) found among subclasses of naive human VRC01-class B cells and HIV broadly neutralizing antibodies (bnAbs). The germline-targeting HIV immunogen eOD-GT8 60mer is currently in clinical trial as a candidate bnAb vaccine priming immunogen. To attempt to model human immune responses to the eOD-GT8 60mer, we tested each authentic naive human VRC01-class BCR mouse model under rare human physiological B cell precursor frequency conditions. B cells with high (HuGL18HL) or medium (HuGL17HL) affinity BCRs were primed, recruited to germinal centers, and they affinity matured, and formed memory B cells. Precursor frequency and affinity interdependently influenced responses. Taken together, these experiments utilizing authentic naive human VRC01-class BCRs validate a central tenet of germline-targeting vaccine design and extend the overall concept of the reverse vaccinology approach to vaccine development.

Factors in B cell competition and immunodominance.

The majority of all vaccines work by inducing protective antibody responses. The mechanisms by which the B cells responsible for producing protective antibodies are elicited to respond are not well understood. Interclonal B cell competition to complex antigens, particularly in germinal centers, has emerged as an important hurdle in designing effective vaccines. This review will focus on recent advances in understanding the roles of B cell precursor frequency, B cell receptor affinity for antigen, antigen avidity, and other factors that can substantially alter the outcomes of B cell responses to complex antigens. Understanding the interdependence of these fundamental factors that affect B cell responses can inform current vaccine design efforts for pathogens with complex proteins as candidate immunogens such as HIV, influenza, and coronaviruses.

Steady-state and regenerative hematopoiesis occurs normally in mice in the absence of GDF11

AbstractTightly regulated production of mature blood cells is essential for health and survival in vertebrates and dependent on discrete populations of blood-forming (hematopoietic) stem and progenitor cells. Prior studies suggested that inhibition of growth differentiation factor 11 (GDF11) through soluble activin receptor type II (ActRII) ligand traps or neutralizing antibodies promotes erythroid precursor cell maturation and red blood cell formation in contexts of homeostasis and anemia. As Gdf11 is expressed by mature hematopoietic cells, and erythroid precursor cell expression of Gdf11 has been implicated in regulating erythropoiesis, we hypothesized that genetic disruption of Gdf11 in blood cells might perturb normal hematopoiesis or recovery from hematopoietic insult. Contrary to these predictions, we found that deletion of Gdf11 in the hematopoietic lineage in mice does not alter erythropoiesis or erythroid precursor cell frequency under normal conditions or during hematopoietic recovery after irradiation and transplantation. In addition, although hematopoietic cell-derived Gdf11 may contribute to the pool of circulating GDF11 protein during adult homeostasis, loss of Gdf11 specifically in the blood system does not impair hematopoietic stem cell function or induce overt pathological consequences. Taken together, these results reveal that hematopoietic cell–derived Gdf11 is largely dispensable for native and transplant-induced blood formation.

A novel dendritic cell-based direct ex vivo assay for detection and enumeration of circulating antigen-specific human T cells.

Although a variety of assays have been used to examine T cell responses in vitro, standardized ex vivo detection of antigen-specific CD4+ T cells from human circulatory PBMCs remains constrained by low-dimensional characterization outputs and the need for polyclonal, mitogen-induced expansion methods to generate detectable response signals. To overcome these limitations, we developed a novel methodology utilizing antigen-pulsed autologous human dendritic target cells in a rapid and sensitive assay to accurately enumerate antigen-specific CD4+ T cell precursor frequency by multiparametric flow cytometry. With this approach, we demonstrate the ability to reproducibly quantitate poly-functional T cell responses following both primary and recall antigenic stimulation. Furthermore, this approach enables more comprehensive phenotypic profiling of circulating antigen-specific CD4+ T cells, providing valuable insights into the pre-existing polarization of antigen-specific T cells in humans. Combined, this approach permits sensitive and detailed ex vivo detection of antigen-specific CD4+ T cells delivering an important tool for advancing vaccine, immune-oncology and other therapeutic studies.

Anti–HIV-1 B cell responses are dependent on B cell precursor frequency and antigen-binding affinity

The discovery that humans can produce potent broadly neutralizing antibodies (bNAbs) to several different epitopes on the HIV-1 spike has reinvigorated efforts to develop an antibody-based HIV-1 vaccine. Antibody cloning from single cells revealed that nearly all bNAbs show unusual features that could help explain why it has not been possible to elicit them by traditional vaccination and instead would require a sequence of different immunogens. This idea is supported by experiments with genetically modified immunoglobulin (Ig) knock-in mice. Sequential immunization with a series of specifically designed immunogens was required to shepherd the development of bNAbs. However, knock-in mice contain superphysiologic numbers of bNAb precursor-expressing B cells, and therefore how these results can be translated to a more physiologic setting remains to be determined. Here we make use of adoptive transfer experiments using knock-in B cells that carry a synthetic intermediate in the pathway to anti-HIV-1 bNAb development to examine how the relationship between B cell receptor affinity and precursor frequency affects germinal center (GC) B cell recruitment and clonal expansion. Immunization with soluble HIV-1 antigens can recruit bNAb precursor B cells to the GC when there are as few as 10 such cells per mouse. However, at low precursor frequencies, the extent of clonal expansion is directly proportional to the affinity of the antigen for the B cell receptor, and recruitment to GCs is variable and dependent on recirculation.

Humoral and cell-mediated immune responses to H5N1 plant-made virus-like particle vaccine are differentially impacted by alum and GLA-SE adjuvants in a Phase 2 clinical trial

The hemagglutinination inhibition (HI) response remains the gold standard used for the licensure of influenza vaccines. However, cell-mediated immunity (CMI) deserves more attention, especially when evaluating H5N1 influenza vaccines that tend to induce poor HI response. In this study, we measured the humoral response (HI) and CMI (flow cytometry) during a Phase II dose-ranging clinical trial (NCT01991561). Subjects received two intramuscular doses, 21 days apart, of plant-derived virus-like particles (VLP) presenting the A/Indonesia/05/2005 H5N1 influenza hemagglutinin protein (H5) at the surface of the VLP (H5VLP). The vaccine was co-administrated with Alhydrogel® or with a glucopyranosyl lipid adjuvant-stable emulsion (GLA-SE). We demonstrated that low doses (3.75 or 7.5 μg H5VLP) of GLA-SE-adjuvanted vaccines induced HI responses that met criteria for licensure at both antigen doses tested. Alhydrogel adjuvanted vaccines induced readily detectable HI response that however failed to meet licensure criteria at any of three doses (10, 15 and 20 μg) tested. The H5VLP also induced a sustained (up to 6 months) polyfunctional and cross-reactive HA-specific CD4+ T cell response in all vaccinated groups. Interestingly, the frequency of central memory Th1-primed precursor cells before the boost significantly correlated with HI titers 21 days after the boost. The ability of the low dose GLA-SE-adjuvanted H5VLP to elicit both humoral response and a sustained cross-reactive CMI in healthy adults is very attractive and could result in significant dose-sparing in a pandemic situation.

P028 Immunogenicity of minor histocompatibility antigens may be dependent on the frequencies of naive precursor cells in the donor repertoire

Aim Out of 60+ minor histocompatibility antigens (MiHA) discovered so far some are immunodominant, i.e. almost universally cause allogeneic response when mismatched, while others are much less immunogenic. Before transplantation MiHA specific T cells reside in naive T cell (TN) subpopulation of the donor. One potential explanation for unequal MiHA immunogenicity is the variation in frequencies of TN specific to different antigens in the donor T cell repertoires. To test this hypothesis we compared the frequencies of naive T cells, specific for highly immunogenic minor antigen HA-2 and less immunogenic ACC-1Y in individuals negative for these antigens. Methods We genotyped a panel of healthy volunteers for HA-2 and ACC-1Y polymorphisms and selected donors negative for these minor antigens, but bearing restricting HLA alleles (HLA-A∗02:01 and HLA-A∗24:02 respectively). Donor naive T cells were magnetically purified and stimulated by autologous monocyte derived dendritic cells, loaded with appropriate peptides. Expanding MiHA-specific lymphocytes were detected using MHC-multimers and flow cytometry. Frequencies of HA-2 and ACC-1Y-specific TN cells were calculated by extreme limiting dilution analysis. Results Frequency of specific TN was found to be substantially higher for HA-2 than for ACC-1Y (5 × 10−6 vs. 2 × 10−7, p = 3 × 10−9), which correlates with HA-2 immunodominance and confirms the initial hypothesis. Conclusions Further studies are needed to elucidate the reason for major differences in TN frequencies between various MiHA. It is possible that the variation could be explained by the peptide properties or by the influence of the negative selection on initial T cell repertoire. In contrast with HA-2, ACC-1Y has alternative allelic variant (ACC-1C) presented in the thymus, which leads to elimination of all cross-reactive clones. Creating a model of MiHA immunogenicity would be very useful for predicting allogeneic immune response after transplantation. Funding: Russian Science Foundation grant 17-15-01512.

Oxidized Hemoglobin Is Antigenic and Immunogenic in Lupus.

Hemolysis-associated anemia is characteristic of diseases such as atherosclerosis, lupus, malaria, and leishmaniasis; the toxic effects of free hemoglobin (Hb) have been extensively described. This study was based on the premise that release of this sequestered, inflammatory molecule can result in deleterious immunological consequences, particularly in the context of pre-existing lupus. IgG anti-Hb responses were detected in the sera of lupus patients. Lupus-prone mice exhibited heightened plasma Hb levels, and ferric (Fe3+) Hb triggered preferential release of lupus-associated cytokines from splenocytes derived from aging lupus-prone mice. Anti-Hb B cell precursor frequencies were heightened in such mice, which also expressed increased titers of anti-Hb antibodies in serum and in kidney eluates. Fe3+ Hb preferentially increased the functional maturation of bone marrow-derived dendritic cells (BMDCs) from lupus-prone mice, effects abrogated upon the inhibition of Stat3. Hb interacted with lupus-associated autoantigens extruded during apoptosis and coincubation of Hb and apoptotic blebs had additional maturation-inducing effects on lupus BMDCs. Immunization with Hb in lupus-prone mice induced antigen spreading to lupus-associated moieties; Hb-interacting autoantigens were preferentially targeted and increased complement deposition and glomerulosclerosis were observed. Hb therefore demonstrates both antigenicity and immunogenicity and triggers specific immuno-pathological effects in a lupus milieu.

Dissection of CD8+ T cell immunodominance hierarchy in human influenza virus infection

Abstract Based on magnitude, virus-specific CD8+ cytotoxic T lymphocyte (CTL) responses can be grouped as immunodominant and subdominant. Such immunodominance hierarchies are governed by multiple factors, including naïve T cell precursor frequency, proliferative capacity and clonotypic diversity of Ag-specific CTLs. However, how these factors determine together inter/intra-individual immunodominance remains elusive. In this study, we dissected CTL immunodominance hierarchy for universally conserved influenza (IAV)-specific epitopes. Our data from 59 donors covering the HLAI types presenting universally conserved IAV-specific peptides, HLA-A* 0201, A*0301, B*2705, B*5701, B*1801 and B*0801, revealed that CTLs directed against HLA-A* 0201:M158 were immunodominant, except in A*0201+B*2705+ heterozygous individuals, where the HLA-B* 2705-NP383 recalls higher magnitude CTL responses. Subsequently, we further dissected the basis of B*2705 immundomination over A*0201 by analyses of T cell Receptor (TCRαβ) repertoire, TCR avidity and kinetics of killing and proliferation. Our data show that following in vitro peptide stimulation, B*2705:NP383 CTLs proliferate rapidly acquiring higher levels of polyfunctionality than A*02:M158 specific CTLs. Furthermore, ex vivo single-cell paired CDR3αβ analysis revealed that public A*0201:M158 TCRs characterized by Vβ19/Vα27 profiles in A*0201 individuals are significantly reduced/absent in A*0201+B*2705+ individuals. Clonal analysis of M158 dominant public TCRs suggested B*2705-mediated removal of self-reactive TCRs and is under investigation. Our study provides insights into the importance of human IAV-specific immunodominance at quantitative and qualitative levels.

Natural killer cells support myeloid suppressor cell expansion during persistent viral infection

Abstract Persistent viral infections in mice (e.g. lymphocytic choriomeningitis virus, LCMV) and humans (e.g. HIV, hepatitis C virus) are characterized by chronic inflammation. This sustained inflammatory milieu promotes T cell exhaustion, which limits harmful tissue damage but prevents effective viral control. We found that natural killer (NK) cells aid in the establishment of this tolerogenic state through cytolytic elimination of antiviral T cells. In parallel, others shown that chronic infection encourages expansion of immunoregulatory IL-10-expressing myeloid cells and bona fide myeloid-derived suppressor cells (MDSCs) that contribute to T cell inhibition immune exhaustion and cell injury. We now report that depletion of NK cells prior to persistent infection with the clone 13 strain of LCMV abrogates the expansion of immunosuppressive myeloid cells. In the absence of NK cells, the levels of myelo-stimulatory cytokines, including TNF-a, MCP-1, and IL-6 were also reduced. Furthermore, frequencies of myeloid precursor cells were reduced in the bone marrow during infection in the absence of NK cells. Our results suggest that NK cells support an inflammatory milieu promoting expansion of the myeloid niche and specifically suppressive myeloid cells during chronic infection. This cross-talk between NK cells and myeloid cells during inflammation has the potential to contribute to immune dysfunction during both chronic infection and in cancer.

Most Closely HLA-Matched Third Party Allogeneic CMV Specific T-Cells Generated Using the Cytokine Capture System for the Treatment of CMV Infections after Allogeneic Stem Cell Transplantation

Adoptive immunotherapy with ex vivo expanded virus-specific T-cell lines from transplant donors has emerged as a potentially curative approach for the treatment of drug-refractory viral infections complicating allogeneic hematopoietic cell transplants (HSCT). In these studies, viral-specific T cells are generated in compliance with current good manufacturing practice (cGMP) following repeated rounds of antigen-driven stimulation over a number of weeks. Such protocols are logistically and technically demanding, requiring access to specialized GMP units and are difficult to apply in urgent clinical settings.The cytokine-capture (gamma-catch) procedure is a fast and simple method that takes <30 hours to isolate polyclonal IFN-gamma-secreting CD8+ and CD4+ viral-specific T cells and can be performed outside a dedicated GMP facility. The cells are directly transfused to the patient without the requirement for ex vivo expansion. The use of allogeneic third party T-cell donors offers the option of selecting donors with high viral-specific T cell precursor frequencies and may serve as an alternative for patients receiving an allogeneic cord blood transplant or a transplant from a virus-seronegative donor.Here, we report the interim results of a Phase 2 study in 11 patients with persistent CMV infection after HSCT, despite optimum anti-viral therapy for at least 14 days. We established a bank of cryopreserved peripheral blood mononuclear cells (PBMCs) from 36 healthy donors. Eligible patients were treated with small numbers of CMV-specific T cells selected by cytokine capture from the most closely HLA-matched third party donor. Manufacture of third party derived CMV specific CTLs was performed at the MDACC Core Facility. Cryopreserved PBMCs were thawed, washed and diluted at 1 × 109 cells in 100 mL MACS GMP TexMACS Reagent (Miltenyi Biotec) for approximately 16 hours at 37°C. The product was stimulated with MACS® GMP Peptivator® HCMV pp65 for approximately 4 hours at 37°C, CO2. Enrichment of cytokine-secreting cells was performed using the cytokine secretion system and the CliniMACS device for immunomagnetic separation. CMV-specific T cells were transfused directly after isolation without any further in vitro expansion.To date, 11 HSCT recipients have received CMV-specific T-cells generated by gamma-catch. Of 11 patients, 2 had failed to respond to foscarnet, 12 to ganciclovir and 8 to both agents. One patient who had a complete response (CR) to his first CMV-specific T-cell infusion received a second infusion 166 days after the first dose for the treatment of CMV retinitis. The median number of infused CD3+IFN-gamma+ T-cells was 2598/kg (range, 61/kg-12717/kg). There were no immediate infusion-related adverse events. CMV-specific CTLs controlled infection in 9 of 11 evaluable infusions; 6 CR and 3 partial response (PR). The overall response and CR rates at 4 weeks were 77% (95%CI; 51%, 95%) and 54.5% (95%CI; 25.4%, 78.6%). Interestingly, the success of adoptive T-cell transfer was not related to the dose of infused T-cells. The median ratio of CD8+ IFN-gamma+ to CD4+ IFN-gamma+ T-cells was 2.7 (range, 0.17-13.4). Among 6 CR patients, 4 had a second episode of CMV infection at a median of 71 days (range, 61-355 days) after the first CTL infusion. Following infusion, there was expansion of both CD8+ and/or CD4+ CMV-specific T-cells, which persisted for a minimum of 1 month following infusion. Recurrence of CMV PCR after CTL infusion was associated with loss of CMV CTL response.Despite the use of third-party donors with HLA disparity, only one patient developed de novo graft versus host disease (GVHD) of the gastrointestinal tract (grade 1) 3 months after CTL infusion. Three patients with a history of acute GVHD prior to CTL infusion had flares 5-6 months after CTL infusions and responded to systemic steroid use.In summary, these interim results demonstrate the safety, feasibility and efficacy of immunotherapy with the most closely HLA-matched CMV specific CTLs generated by cytokine capture for the treatment of drug resistant CMV infection after HSCT patients. The clinical trial continues to enroll and an updated report of the study will be presented at the meeting. DisclosuresCiurea:Spectrum Pharmaceuticals: Other: Advisory Board; Cyto-Sen Therapeutics: Equity Ownership.

The Breadth of Synthetic Long Peptide Vaccine-Induced CD8+ T Cell Responses Determines the Efficacy against Mouse Cytomegalovirus Infection.

There is an ultimate need for efficacious vaccines against human cytomegalovirus (HCMV), which causes severe morbidity and mortality among neonates and immunocompromised individuals. In this study we explored synthetic long peptide (SLP) vaccination as a platform modality to protect against mouse CMV (MCMV) infection in preclinical mouse models. In both C57BL/6 and BALB/c mouse strains, prime-booster vaccination with SLPs containing MHC class I restricted epitopes of MCMV resulted in the induction of strong and polyfunctional (i.e., IFN-γ+, TNF+, IL-2+) CD8+ T cell responses, equivalent in magnitude to those induced by the virus itself. SLP vaccination initially led to the formation of effector CD8+ T cells (KLRG1hi, CD44hi, CD127lo, CD62Llo), which eventually converted to a mixed central and effector-memory T cell phenotype. Markedly, the magnitude of the SLP vaccine-induced CD8+ T cell response was unrelated to the T cell functional avidity but correlated to the naive CD8+ T cell precursor frequency of each epitope. Vaccination with single SLPs displayed various levels of long-term protection against acute MCMV infection, but superior protection occurred after vaccination with a combination of SLPs. This finding underlines the importance of the breadth of the vaccine-induced CD8+ T cell response. Thus, SLP-based vaccines could be a potential strategy to prevent CMV-associated disease.

Tracking of TCR-Transgenic T Cells Reveals That Multiple Mechanisms Maintain Cardiac Transplant Tolerance in Mice.

Solid organ transplantation tolerance can be achieved following select transient immunosuppressive regimens that result in long-lasting restraint of alloimmunity without affecting responses to other antigens. Transplantation tolerance has been observed in animal models following costimulation or coreceptor blockade therapies, and in a subset of patients through induction protocols that include donor bone marrow transplantation, or following withdrawal of immunosuppression. Previous data from our lab and others have shown that proinflammatory interventions that successfully prevent the induction of transplantation tolerance in mice often fail to break tolerance once it has been stably established. This suggests that established tolerance acquires resilience to proinflammatory insults, and prompted us to investigate the mechanisms that maintain a stable state of robust tolerance. Our results demonstrate that only a triple intervention of depleting CD25+ regulatory T cells (Tregs), blocking programmed death ligand-1 (PD-L1) signals, and transferring low numbers of alloreactive T cells was sufficient to break established tolerance. We infer from these observations that Tregs and PD-1/PD-L1 signals cooperate to preserve a low alloreactive T cell frequency to maintain tolerance. Thus, therapeutic protocols designed to induce multiple parallel mechanisms of peripheral tolerance may be necessary to achieve robust transplantation tolerance capable of maintaining one allograft for life in the clinic.