Expression Of Activation Antigens Research Articles

Bivalent BCMA Binding and Low Affinity CD3 T-Cell Engagement By Abbv-383 Drives Sustained Activation with Reduced T-Cell Exhaustion in Preclinical Models of Multiple Myeloma

Introduction: ABBV-383 is an IgG4-based CD3-bispecific T-cell engager (TCE) that has shown promising efficacy and a favorable safety profile in heavily pre-treated patients with relapsed/refractory multiple myeloma (MM). ABBV-383 possesses a unique combination of a low-affinity CD3δε antibody coupled to a bivalent, high-affinity BCMA engager arm in a 2+1 format. Prior pre-clinical studies demonstrated that ABBV-383 induces potent redirected T-cell killing of MM tumors with reduced cytokine release. Here, we provide additional evidence that bivalent BCMA binding by ABBV-383 reduces the negative impact of soluble BCMA and low affinity CD3 T-cell engagement drives sustained T-cell activation with reduced immune exhaustion. Methods: Structural variants of ABBV-383 were created, including 1) the bivalent BCMA targeting antibody of ABBV-383 replaced by proximal and distal monovalent VH targeting molecules, and 2) BCMA antibody structures coupled to high-and low affinity CD3 variants ( Figure). For functional testing, ABBV-383 and the structural variants were added to PBMCs or T-cells from healthy donors to serve as effector cells in co-culture with BCMA-positive MM tumor cell lines. Tumor cell killing and T-cell activation were determined by flow cytometry, and cytokine production was assessed using Luminex instrumentation. Recombinant soluble BCMA (sBCMA) was utilized in the experimental models to evaluate the extent of BCMA TCE suppression. To define the relationship between CD3-affinity and T-cell exhaustion, multi-dimensional flow cytometry was performed in long-term, 28-day co-cultures with irradiated tumor cells replated every 7 days. Results: T-cell binding was shifted by >100x with substitution of the high affinity CD3 antibody compared to ABBV-383. While all antibody variants tested induced T-cell specific MM cell killing in a BCMA-dependent manner, the negative impact of soluble BCMA on tumor killing was reduced with ABBV-383 due to its bivalent interaction with tumor cells compared to the monovalent TCE formats. In addition, the low CD3 binding affinity of ABBV-383 lowered T-cell activation antigen expression and reduced inflammatory cytokine production but resulted in similar maximal cytotoxicity of tumor cells in co-cultures of healthy and MM patient samples compared to high-affinity CD3 targeting. While maximal tumor cytotoxicity was similar, ABBV-383 was found to significantly delay the induction of a critical exhaustion-inducing transcription factor, TOX, in long-term assays. T-cell population changes were observed as early as 3 days with decreased effector memory cells (CD45RA-CD62L-) and increased naïve T-cell populations (CD62L+CD45RA+) compared to high-affinity CD3 targeting. On day 21, the high-affinity CD3 x bivalent BCMA molecule resulted in 85% of the CD8+ T-cell population expressing the transcription factor TOX compared to only 15% for ABBV-383 co-cultures. TOX induces several gene families with chromatin-modulating capacity that drive the distinct transcriptional trajectory leading to T-cell dysfunction during chronic stimulation. Simultaneous expression of multiple checkpoint inhibitory proteins is indicative of T-cell exhaustion. Triple positive CD8+ T-cells expressing LAG3, PD-1 and CTLA4 were significantly reduced by ABBV-383 versus the high-affinity CD3 variant ( Figure). Conclusions: High avidity BCMA binding coupled to low affinity T-cell engagement distinguishes the resulting mechanism of action for ABBV-383 in MM. Here, we show that the presence of bivalent-BCMA reduced the negative impact of soluble BCMA, reduced inflammatory cytokine production, and dampened the emergence of TOX+ CD8+ T-cells indicating that ABBV-383 structure maximizes its clinical potential as a safe and effective MM therapy.

Immunological markers of arthroplasty failure

Periprosthetic joint infection still remains a clinical challenge since accurate definition of this condition and reliable laboratory markers have not been established yet. This study aimed to evaluate the benefit of some lymphocyte and monocyte subset determination in patients with periprosthetic joint infection and non-infectious arthroplasty failure. Thirty-four patients with chronic periprosthetic joint infection, 12 patients with non-infectious arthroplasty and 30 healthy persons were included in the study. The counts of CD3+, CD3+CD4+, CD3+CD8+, CD19+, CD3-CD16+CD56+, CD3+HLA-DR+, CD4+CD45RACD45RО+, CD4+CD45RA+ CD45RО- and CD14+ HLA-DR+ subsets in peripheral blood were assessed by flow cytometry. The assessment of the intensity of antigen expression was carried out according to mean fluorescence intensity. A significant increase in CD3+CD4+ subsets (p < 0,01) and a significant decrease in CD3-CD16+CD56+ subsets (p < 0,005) were revealed in patients with periprosthetic joint infection compared to the healthy controls. The content of CD19+ lymphocytes in these patients was significantly higher than in aseptic ones (p < 0,005); the latter group was also characterized by more pronounced increase in the number of activated T lymphocytes (CD3+HLA-DR+) compared to controls (p < 0,001). Patients with periprosthetic joint infection showed decreased “naïve” T lymphocytes (CD4+CD45RA+CD45RO-) count compared to aseptic ones (p < 0,05), and both groups showed a decrease counts compared to controls (p < 0,001). On the contrary, memory T lymphocyte (CD4+CD45RACD45RO+) count was significantly increased in both compared groups (p < 0,05). Patients with periprosthetic joint infection compared with other two groups demonstrated a significant decrease in the number of activated monocytes (CD14+HLA-DR+) and pronounced decrease in the expression intensity of this marker on cell membrane (p < 0,05 and p < 0,001, respectively). Thus, evaluation of lymphocyte and monocyte subsets, including expression of cell activation antigens could be useful as additional laboratory test in combination with other conventional methods for differentiation between periprosthetic joint infection and aseptic arthroplasty failure.

Changes in T-Cell Subpopulations and Cytokine Levels in Patients with Treatment-Resistant Depression—A Preliminary Study

Although there is some evidence for the involvement of cytokines and T cells in the pathophysiology of treatment-resistant depression (TRD), the nature of this relationship is not entirely clear. Therefore, we compared T-cell subpopulations and serum cytokine levels in TRD patients to find relationships between their immunological profiles, clinical presentation, and episode severity. Blood samples from TRD patients (n = 20) and healthy people (n = 13) were collected and analyzed by flow cytometry. We analyzed the percentages of helper and cytotoxic T cells according to the expression of selected activation markers, including CD28, CD69, CD25, CD95, and HLA-DR. The serum levels of inflammatory cytokines IL12p70, TNF-α, IL-10, IL-6, IL-1β, and IL-8 were also determined. TRD patients had a lower percentage of CD3+CD4+CD25+ and CD3+CD8+CD95+ cells than healthy people. They also had lower serum levels of IL-12p70 and TNF-α, whereas IL-8 levels were significantly higher. Receiver operating characteristic (ROC) analysis demonstrated that serum IL-8 values above 19.55 pg/mL were associated with a 10.26 likelihood ratio of developing TRD. No connections were found between the MADRS score and immunological parameters. These results show that TRD patients have reduced percentages of T cells expressing activation antigens (CD25 and CD95) and higher serum concentrations of proinflammatory and chemotactic IL-8. These changes may indicate reduced activity of the immune system and the important role of IL-8 in maintaining chronic inflammation in the course of depression.

The pattern and magnitude of T cell subsets reconstitution during ten years of ART with viral suppression in HIV-infected patients

The extent of immune reconstitution in human immunodeficiency virus (HIV) infected persons receiving long-term antiretroviral therapy (ART) with controlled viral load has been controversial. We studied the extent and speed of T cell subsets retrieval after long-term antiretroviral treatment. 662 HIV-infected patients followed at least 2 years whose plasma HIV-1 RNA load <50 copies/mL were evaluated for longitudinal and functional phenotypic indices of immune restoration. Determinants of change in magnitude and importance of recovery have been evaluated using mixed linear regression models. Almost all robust immune restorations achieved occurred after 2-3 years of ART. The median CD4 lymphocyte count increased 449 cells/μl (IQR 303-604) from 226 cells/μl (IQR 83-336) at baseline during the third year (P < 0.001); CD4+T lymphocyte rises during the sixth and tenth years were not significant. Naive and memory CD4+T cells'reconstitution occurred in the sixth and eighth years of ART but no significant change thereafter. The change of CD45RA+Naïve and CD45RA-memory CD4+T cell reconstitution is different in baseline CD4+T cell counts <100 cells/μl group and in baseline CD4+T cell counts >100 cells/μl group. Activation antigen expression (CD38 or HLA-DR) on CD8 lymphocytes declined mostly during the first till second year, and after 4 years, activation antigen expression on patient lymphocytes showed no significant change. The proportion of CD4 cells expressing CD28 climbed during the first years and reached normal levels in the second year. Immune restoration was dependent on the capacity of immune system during the first 2-3 year of ART. But the significant change of CD4 and compartments of CD4+T cells could persist until 6-8 years. The pattern of CD38+CD8+, HLA-DR+CD8+, CD28+CD4+ T cells could quickly return to normal level and no significant change after sufficient time of ART. In general, the immune response compared to the baseline status may be the overall effect from the age and time of antiretroviral treatment.

STATUS OF THE CELLULAR IMMUNITY IN CLEAN-UP WORKERS OF THE CHORNOBYL ACCIDENT AND PATIENTS WITH CANCER OF THE MOUTH CAVITY, ORAL AND LARYNGEAL PART OF THE PHARYNX.

to establish cellular immunity in clean-up workers of the Chornobyl accident compared to patients with malignant neoplasms of the oral cavity, oral and laryngeal parts of the pharynx according to the subpopulation organization of peripheral blood leukocytes. We examined 112 males, age (56,92 ± 6,17) years (M ± SD), including 26 male clean-up workers exposed at the radiation dose 10-500 mSv; 20 male clean-up workers exposed at the dose range 504-990mSv; 33 patients with malignant neoplasms of the oral cavity, oral and laryngeal parts of the pharynx and 33 non-exposed subjects of the control group. Immune cell subsets analysis was performed by the expression of differential and activation antigens of peripheral blood leukocytes using flow cytometry. In assessing each group's cellular immunity, there was a decrease in the number of CD19+ B-lymphocytes, CD3+ HLA-DR+ T- and CD3- HLA-DR+ B-lymphocytes, CD3- 16+ 56+ natural killer cells, combined with an increase in the number of CD8+ T-lymphocytes. An increase in the relative number of CD4+ CD8+ T-lymphocytes was determined in clean-up workers (D < 500 mSv) and cancer patients. An increase in the number of CD8+ T-lymphocytes and a decrease in the number of CD4+ T-lymphocytes were observed in clean-up workers (D > 500 mSv), as well as in cancer patients. In addition, a decrease of monocytes, CD3+ 16+ 56+ , and CD3+ TCRαβ+ T-lymphocytes was determined in patients with oncological pathology. The obtained results show the unidirectionality of changes in cellular immunity in the participants of the liquidation of the consequences of the accident at the Chornobyl nuclear power plant and patients with the investigated oncological pathology, which indicates the formation of persistent violations of antitumor protection in the participants of the liquidation of the consequences of the accident as the basis of oncogenesis. Determining changes in the number of lymphocytes, monocytes, granulocytes, CD4+ T-lymphocytes, CD4+ CD8+ immature T-lymphocytes, the immunoregulatory ratio CD4+ / CD8+, CD3+TCRαβ+ T-lymphocytes and CD3+ 16+ 56+ CTL can have an additional effect on the effectiveness antitumor protection and the probability of oncogenesis risk in immunocompromised individuals.

STATUS OF THE CELLULAR IMMUNITY IN CLEAN-UP WORKERS OF THE CHORNOBYL ACCIDENT AND PATIENTS WITH CANCER OF THE MOUTH CAVITY, ORAL AND LARYNGEAL PART OF THE PHARYNX

Objective: to establish cellular immunity in clean-up workers of the Chornobyl accident compared to patients with malignant neoplasms of the oral cavity, oral and laryngeal parts of the pharynx according to the subpopulation organization of peripheral blood leukocytes. Materials and methods. We examined 112 males, age (56,92 ± 6,17) years (M ± SD), including 26 male clean-up workers exposed at the radiation dose 10–500 mSv; 20 male clean-up workers exposed at the dose range 504–990 mSv; 33 patients with malignant neoplasms of the oral cavity, oral and laryngeal parts of the pharynx and 33 nonexposed subjects of the control group. Immune cell subsets analysis was performed by the expression of differential and activation antigens of peripheral blood leukocytes using flow cytometry. Results. In assessing each group's cellular immunity, there was a decrease in the number of CD19+ B-lymphocytes, CD3+HLA-DR+ T- and CD3-HLA-DR+ B-lymphocytes, CD3-16+56+ natural killer cells, combined with an increase in the number of CD8+ T-lymphocytes. An increase in the relative number of CD4+CD8+ T-lymphocytes was determined in clean-up workers (D ≤ 500 mSv) and cancer patients. An increase in the number of CD8+ T-lymphocytes and a decrease in the number of CD4+ T-lymphocytes were observed in clean-up workers (D &gt; 500 mSv), as well as in cancer patients. In addition, a decrease of monocytes, CD3+16+56+, and CD3+TCRαβ+ T-lymphocytes was determined in patients with oncological pathology. Conclusions. The obtained results show the unidirectionality of changes in cellular immunity in the participants of the liquidation of the consequences of the accident at the Chornobyl nuclear power plant and patients with the investigated oncological pathology, which indicates the formation of persistent violations of antitumor protection in the participants of the liquidation of the consequences of the accident as the basis of oncogenesis. Determining changes in the number of lymphocytes, monocytes, granulocytes, CD4+ T-lymphocytes, CD4+CD8+ immature T-lymphocytes, the immunoregulatory ratio CD4+ / CD8+, CD3+TCRαβ+ T-lymphocytes and CD3+16+56+ CTL can have an additional effect on the effectiveness antitumor protection and the probability of oncogenesis risk in immunocompromised individuals. Key words: ionizing radiation, Chornobyl, clean-up workers of the Chornobyl accident, neoplasms of the oral cavity, oral and laryngeal parts of the pharynx, immune system.

Effective chimeric antigen receptor T cells against SARS-CoV-2

SummaryCurrent therapies to treat coronavirus disease 2019 (COVID-19) involve vaccines against the spike protein S1 of SARS-CoV-2. Here, we outline an alternative approach involving chimeric antigen receptors (CARs) in T cells (CAR-Ts). CAR-T recognition of the SARS-CoV-2 receptor-binding domain (RBD) peptide induced ribosomal protein S6 phosphorylation, the increased expression of activation antigen, CD69 and effectors, interferon-γ, granzyme B, perforin, and Fas-ligand on overlapping subsets of CAR-Ts. CAR-Ts further showed potent in vitro killing of target cells loaded with RBD, S1 peptide, or expressing the S1 protein. The efficacy of killing varied with different sized hinge regions, whereas time-lapse microscopy showed CAR-T cluster formation around RBD-expressing targets. Cytolysis of targets was mediated primarily by the GZMB/perforin pathway. Lastly, we showed in vivo killing of S1-expressing cells by our SARS-CoV-2 CAR-Ts in mice. The successful generation of SARS-CoV-2 CAR-Ts represents a living vaccine approach for the treatment of COVID-19.

Characterization of novel CD25 antibodies that deplete human regulatory T cells in humanized mouse model

Abstract Regulatory T cells (Tregs) are key players in the suppression of immune responses and in the maintenance of immune homeostasis. Due to their central role in pathogenesis of multiple conditions, Tregs are an attractive target for immunomodulatory drug development. Tregs exhibit an activated phenotype and share the expression of many activation antigens with activated conventional T cells (Tconv). Our goal in this project was to generate and characterize monoclonal antibodies (mAbs) that would specifically react with human Tregs. mAbs were raised in mice by immunization with purified, in vitro expanded human Tregs. In contrast to widely available anti-CD25 mAbs that equally bound both activated Tregs and activated Tconv, we identified several mAbs that bound CD25 on expanded Tregs, but failed to bind to CD25 on expanded Tconv. To determine whether a similar selective binding could be demonstrated in vivo, we used the xeno-GVHD model in which NOD-SCID-γc−/− (NSG) mice are engrafted with human peripheral blood mononuclear cells (PBMCs). Several of the mAbs bound to CD25 expressed on Tregs, but not to CD25 expressed on activated Tconv. To test the effect of in vivo administration of the Treg specific mAbs, one of the clones, 2B010, was selected and injected intravenously (i.v) 2 weeks after PBMCs engraftment. Injection of 2B010 resulted in markedly reduced Tregs frequencies and numbers, much less depletion of CD25+ activated Tconv, and no reduction of CD25+CD8+ T cells. Further studies will address the effect of these mAbs at reversing the Treg-mediated protection of xeno-GVHD and enhancing tumor immunity in reconstituted NSG recipients bearing transplanted tumors.

Type I interferon signaling attenuates Regulatory T cells function in LCMV infection

Abstract Regulatory T cells (Tregs) play a cardinal role in the immune system by suppressing detrimental autoimmune responses, but their role in acute and chronic infectious diseases remains unclear. We recently demonstrated that IFN-α/β receptor (IFNAR) signaling promotes Treg function in autoimmunity. To dissect the functional role of IFNAR-signaling in Tregs during acute and chronic viral infection, we infected Treg-specific IFNAR deficient (IFNARfl/flxFoxp3YFP-Cre) mice with LCMV Armstrong and Clone-13. In both models, IFNARfl/flxFoxp3YFP-Cre mice Tregs expressed enhanced expression of Treg associated activation antigens. The enhanced activated phenotype was also seen when we compared the transcriptomes of IFNARfl/flxFoxp3YFP-Cre and wild type (WT) Tregs by RNA-Seq on day 25-post Clone-13 infection. LCMV-specific CD8+ T cells from IFNARfl/flxFoxp3YFP-Cre mice produced less antiviral IFNγ and TNFα in both acute and chronic LCMV. In the chronic model, the numbers of anti-viral effector and memory CD8+ T cells were decreased in IFNARfl/flxFoxp3YFP-Cre mice and the effector CD4+ and CD8+ T cells exhibited a phenotype compatible with enhanced exhaustion. IFNARfl/flxFoxp3YFP-Cre mice cleared Armstrong infection normally, but had higher viral titers in sera, kidneys and lungs than WT mice during chronic infection. Thus, type I IFN signaling in Tregs is context-dependent, resulting in enhanced suppressor function in some models of autoimmunity, but decreased suppressor function in acute and chronic viral infection.

SUCCESSFUL USE OF BRENTUXIMAB VEDOTIN IN THE TREATMENT OF PROGRESSIVE PERIPHERAL UNSPECIFIED T-CELL LYMPHOMA IN AN ELDERLY FEMALE PATIENT

Peripheral T-cell lymphoma not otherwise specified (PTCL-NOS) is a rare subgroup of lymphomas. This disease is predominantly found in older people and often has extranodal involvement including skin and soft tissues. To verify the diagnosis, it is necessary to perform histological and immunohistochemical tests, which show the expression of CD30 activation antigen in different proportions (less than 10 %, not more than 80 %) in addition to the expression of other T-cell antigens (CD2, CD4, CD5, CD7). When choosing the induction regimen in older people with relevant cardiac history, it is preferable to use non-anthracycline-containing chemotherapy courses. However, despite the low intensity of such regimens, treatment of elderly patients is associated with high risk of side effects. Using new drugs (monoclonal antibodies, epigenetic agents) in combination with polychemotherapy is a promising direction opening a possibility of successful management for all patients including those older than 65 years. In the described clinical case of PTCL-NOS, the patient had primary refractory disease. Significant clinical response (complete remission) was achieved as a result of targeted therapy with brentuximab vedotin containing anti-CD30 chimeric antibody conjugated to a microtubuledisrupting agent (monomethyl auristatin E) without toxicity and any infections. In case of ambiguous situation (an increase in the size of one of the tumor sites with regression of others, signs of infection – hyperemia, perifocal edema, the spread of the necrosis zone), histological verification is necessary to confirm the disease progression.

Pathogen Inactivated Platelet Components Used without Leukofiltration, or Gamma Irradiation Show Comparable Therapeutic Efficacy and Safety to Gamma Irradiated and Non-Leukofiltered Conventional Products for the Support of HSCT Patients in Hong Kong

▪Introduction: Depending on the conditioning intensity and graft source, mosthematopoietic stem cell transplant(HSCT) patients invariably experience a cytopenic phase of at least 2-3 weeks and require transfusion support until engraftment. All transfused blood components post-transplant need to be gamma irradiated to prevent transfusion associated graft versus host disease (TA-GVHD). Platelet component (PC) manufacturing methodologies differ around the globe. Currently, Hong Kong HSCT patients are supported with individual platelet rich plasma (PRP) PCs without leukocyte filtration (NLF), tested by short-term aerobic cultures for bacterial contamination (STABC), and gamma irradiated. When new PC manufacturing methodologies are considered for adoption, it is important to assess the immediate and longer term effects on transfused patients. Amotosalen+UVA light (A-UVA) pathogen reduction technology (INTERCEPT™ Blood System, Cerus Corporation, Concord, CA) inactivates bacteria, viruses, parasites and leukocytes by efficient covalent adduct modification of DNA and RNA. Treatment with A-UVA prevents TA- GVHD in an animal model, inhibits clonal T cell proliferation, prevents allogeneic antigen stimulation in mixed lymphocyte reactions, and inhibits transcription mediated cytokine production and early activation antigen expression (Corash et al, BMT 2004). The common strategy of prescribing gamma irradiated PC only for suspected "high risk" patients is sub-optimal, as failure to identify recipients at risk for TA-GVHD was responsible for 50% of reported cases (Kopolovic et al. Blood 2016). Treatment of PC with A-UVA addresses two key issues for HSCT patients: bacterial contamination, and universal prevention of TA-GVHD. Here, we compared the clinical support of HSCT patients with conventional PCs (C-PC) vs. an equivalent HSCT group supported with A-UVA PCs (I-PC) using a sequential patient cohort design within a single HSCT clinical center.Methods: I-PCs were prepared from 5 pooled ABO-matched, NLF buffy coat PCs in platelet additive solution and treated with A-UVA replacing gamma irradiation and bacterial detection. C-PCs were prepared from 5 ABO-matched, STABC-negative, NLF PRP PCs, and treated with gamma irradiation. Each patient could receive up to 5 PC transfusions. The primary efficacy endpoint was the 1-hour corrected count increment (CCI) and the primary safety endpoint was the proportion of patients (P) with acute transfusion reactions (ATR). Follow-up data on mortality, hematopoiesis engraftment, and immune status were collected up to 100 days post HSCT.Results: Patient demographics and type of HSCT were similar between patient cohorts. 33 patients received 76 A-UVA PCs and 31 patients received 89 C-PCs. The mean days of platelet support (p=0.618) and mean 1-hour CCIs per patient averaged for all transfusions were comparable (p=0.296) between the I- PC and conventional C-PC cohorts (Table). The proportion of patients with AEs was lower (p=0.021) for the I-PC group (Table), and no related SAEs were observed during the entirety of trial. Survival at 100 days post HSCT and rates of remission were similar between the cohorts (Table). The ATR rate trended lower, although not significantly different (p=0.296), in the I- PC group (Table). Follow-up data showed that the patients had comparable neutrophil and platelet engraftment (Table) with comparable immune system reconstitution by 100 days post HSCT. No cases of TA-GVHD were observed in either cohort.Conclusions: A-UVA-treated PCs prepared without LF, gamma irradiation, and bacterial detection can replace C-PCs for support of HSCT patients resulting in comparable post transfusion CCI responses and short and intermediate term clinical outcomes, while offering additional protection against transfusion transmitted bacteria and emerging or untested pathogens. [Display omitted] DisclosuresSim Pui Yin:Cerus Corporation: Other: Investigator sponsored trial. Leung Yuk Yan:Cerus Corporation: Other: Investigator sponsored trial. Lam Chun Kit:Cerus Corporation: Other: Investigator sponsored trial. Tsoi Wai:Cerus Corporation: Other: Investigator sponsored trial. Lee Cheuk:Cerus Corporation: Other: Investigator sponsored trial. Lie Kwok Wai:Cerus Corporation: Other: Investigator sponsored trial. Huang:Cerus Corporation: Employment. Rico:Cerus Corporation: Employment. Lin:Cerus corp: Employment. Corash:Cerus Corporation: Employment. Stassinopoulos:Cerus Corporation: Employment.

Treatment Use Study of Platelet Components Treated with Amotosalen and Ultraviolet a Light in Response to Emerging Arboviruses in Puerto Rico - True Study Final Results

▪Background: In 2014, epidemic chikungunya virus (CHIKV) emerged in Puerto Rico, concurrent with endemic dengue (DENV). The Department of Health mandated blood donation quarantine for 72h pending follow-up donor contact for infectious symptoms. The American Red Cross (ARC) halted local platelet collections and began importing blood components. Imported platelet components (PCs) from the continental U.S. were used to supply hospitals in the Caribbean. An alternative strategy is local PC production with amotosalen +UVA (A-UVA) pathogen reduction (PR) technology (INTERCEPT™ Blood System, Cerus Corporation, Concord, CA) as utilized by the French Transfusion Service in the Overseas Territories, including the Caribbean since 2007. The major logistical advantage of such a strategy is earlier PC availability to hospitals since there is no need to wait for the results of bacterial culture, or transport of PCs from the continental U.S. A-UVA technology inactivates bacteria, viruses, parasites and leukocytes by efficient covalent adduct modification of DNA and RNA, thus addressing risks of bacterial contamination and reducing the risk of transfusion-transmitted infection (TTI). Treatment with A-UVA also prevents transfusion associated graft vs. host disease (TA-GVHD) in an animal model, inhibits clonal T cell proliferation, prevents allogeneic antigen stimulation in mixed lymphocyte reactions, and inhibits transcription mediated cytokine production and early activation antigen expression (Corash et al, BMT 2004) and is recognized by the AABB as a replacement for gamma irradiation in the prevention of TA-GVHD.Aims: A treatment use clinical study (TRUE) was conducted in Puerto Rico under the Investigational Device Exemption (IDE) early/expanded access regulations while the INTERCEPT Premarket approval (PMA) application was under review by the Food and Drug Administration (FDA). The objective of the TRUE study was to provide early access to INTERCEPT to reduce the risk of TTIs during the ongoing CHIKV/DENV epidemic.Methods: A prospective, open label, single arm, treatment use, multi-center study was conducted involving 7 hospitals and the ARC. Apheresis (Trima® or Amicus® collectors) leukocyte-reduced PR PCs, suspended in 100% plasma without gamma irradiation, bacterial contamination testing, or the need for CMV serology were produced. Safety outcome measures included all adverse events (AEs), serious adverse events (SAEs) and acute transfusion reactions. Patients were transfused with study PCs according to local practices for as long as clinically indicated.Results: 90 patients (55.6% male, mean age 67.2 y) were transfused with 256 PR PCs (2.8 ± 3.5/patient). The vast majority of the patients were hematology oncology patients (76.7%). Most patients (81.1%) received 1 transfusion cycle (transfusion support without > 5 days between transfusions) with 2 PR PCs (40.0%), while 32.2% received 1 PR PC, and 25.6% received between 3-10 PR PCs. Mean duration of platelet support was 14.7 days. Thirty-three unrelated AEs were reported in 26 patients. Nineteen patients (21.1%) had unrelated SAEs (including 17 deaths [18.9%]). Two febrile non-hemolytic reactions and one allergic transfusion reaction were reported and deemed unrelated to INTERCEPT by the investigators. No suspected cases of CHIKV or DENV infections or other TTIs were reported. Reported AEs were within the expected spectrum of co-morbidity and mortality for patients of similar age with advanced hematology-oncology diseases.Conclusions: The INTERCEPT Blood System was rapidly deployed in response to the CHIKV and DENV epidemic with replacement of bacterial detection, gamma irradiation and the need for CMV serology. No PR PC related AEs were reported. INTERCEPT was approved in late 2014 by the FDA to treat Amicus apheresis PCs suspended in platelet additive solution and in 2016 for Trima PCs suspended in 100% plasma. The importance of this proactive technology is highlighted by the recent emergence of Zika virus (ZIKV). FDA has recently requested that all blood establishments in Puerto Rico and in certain counties in Florida cease collecting blood immediately until investigational donor screening test for ZIKV RNA is implemented or until they implement the use of an approved or investigational pathogen reduction technology. DisclosuresRico:Cerus Corporation: Employment. Stramer:Cerus Corporation: Other: fee for service study with Cerus Corporation. Benjamin:Cerus corp.: Employment. Cartagena:Cerus Corporation: Other: fee for service study with Cerus Corporation. Arroyo:Cerus Corporation: Other: fee for service study with Cerus Corporation. Robles:Cerus Corporation: Other: fee for service study with Cerus Corporation. Carlo:Cerus Corporation: Other: fee for service study with Cerus Corporation. Vera:Cerus Corporation: Other: fee for service study with Cerus Corporation. Solivan-Ortiz:Cerus Corporation: Other: fee for service study with Cerus Corporation. Huang:Cerus Corporation: Employment. Koontz:Cerus Corporation: Employment. Berry:Cerus Corporation: Employment. Corash:Cerus Corporation: Employment.

Resistance of novel mouse strains different in MHC class I and the NKC domain to the development of experimental tumors.

To elucidate the immunological mechanisms critical for tumor progression, we bred novel mouse strains, different in the NKC and H-2D domains. We used inbreeding to generate hybrids of Balb/c and C57BL/6 of stable H-2Db+d-NK1.1neg and H-2Db-d+NK1.1high phenotypes. We analyzed the growth of three established MHC class I-deficient tumor cell lines: TC-1/A9 tumor (HPV-associated) and B16F10 melanoma, both syngeneic to C57BL/6, and the MCB8 (3-methycholanthrene-induced tumor) syngeneic to Balb/c. Furthermore, we induced colorectal carcinoma by azoxymethane-DSS treatment to test the susceptibility to chemically-induced primary cancer. We found that the novel strains spontaneously regressed the tumor transplants syngeneic to both Balb/c (MCB8) and C57BL/6 (B16F10 and TC-1/A9) mice. The H2-Db+d-NK1.1neg, but not the H2-Db-d+NK1.1high strain was also highly resistant to chemically-induced colorectal cancer in comparison to the parental mice. The immune changes during TC-1/A9 cancer development involved an increase of the NK cell distribution in the peripheral blood and spleen along with higher expression of NKG2D activation antigen; this was in correlation with the time-dependent rise of cytotoxic activity in comparison to C57BL/6 mice. The TC-1/A9 cancer regression was accompanied by higher proportion of B cells in the spleen and B220+/CD86+ activated antigen-presenting B cells distributed in the lymphoid organs, as well as in the periphery. The changes in the T-cell population were represented mainly by the prevalence of T helper cells reflected by grown CD4/CD8 ratio, most prominent in the b+d-NK1.1neg strain. The results of the present study imply usefulness of the two novel mouse strains as an experimental model for further studies of tumor resistance mechanisms.

Glioma-derived extracellular vesicles selectively suppress immune responses.

Glioma-related immunosuppression is well documented; however, the mechanisms of suppression are not fully understood. Here we explore a role for glioma extracellular vesicles (EVs) as a means of immune modulation. Healthy donor peripheral blood mononuclear cells (PBMCs) were incubated with mitogenic stimuli and various concentrations of glioma-derived EVs. Intracellular signaling and cytokine output were determined by protein microarrays, and phenotypic changes were assessed by flow cytometry. Recall antigen testing, mixed lymphocyte reactions, and migration assays analyzed PBMC functional capacity. Protein microarray data revealed induction of an immunosuppressive phenotype and cytokine output at high tumor-vesicle concentrations but an activated phenotype at low concentrations. T cell activation antigen expression confirmed differential activation profiles. Functional analyses revealed decreased migratory capacity of PBMCs after incubation with EVs; however, recall antigen and mixed lymphocyte tests indicated that activation capacity is still retained in EV-treated cells. The differential effects of high and low EV concentrations dictate modulatory effects on PBMCs. These data provide a role for EVs at high concentrations for inducing selective tolerance of an immune response in a tumor setting. This suggests that lymphocytes in patients' circulation are not irreparably impaired, as previously thought, but can be rescued to augment antitumor responses.

In-vitro activation of natural killer cells from regional lymph nodes of melanoma patients with interleukin-2 and interleukin-15.

Regional lymph nodes (LNs) represent the first barrier in lymphogenic tumor dissemination in melanoma. Natural killer (NK) cells, the effector cell subpopulation of the innate immune system, are in the first line of antitumor immune defense. Therefore, the aim of this study was to investigate the effect of interleukin (IL)-2 and IL-15, two cytokines with similar immune-enhancing effects, on antitumor cytotoxic function and immunophenotype of NK cells from regional LNs of melanoma patients. Mononuclear cells purified from regional LNs of 50 melanoma patients in clinical stage II-IV were treated in vitro for 72 h and 7 days with 200 IU/ml rhIL-2 and 25 ng/ml IL-15 at 37°C in 5% CO2. Both cytokines significantly augmented NK cell cytotoxic activity, transcription of the cytotoxic molecule perforin, and the level of functionally mature perforin in both nonmetastatic and metastatic regional LNs. IL-2 treatment increased the percentage of CD3CD56 NK cells by increasing the CD56 NK cell subset in both nonmetastatic and metastatic LNs, whereas IL-15 treatment did not affect the percentage of NK cells and their subsets. Both cytokines increased on NK cells from nonmetastatic and metastatic LNs the expression of CD69 early activation antigen, the NKG2D activating receptor, as well as CD16 and inhibitory killer-cell immunoglobulin-like receptor CD158b, both inherent to the mature and the cytotoxic NK cell phenotype. In conclusion, our data may indicate the therapeutic potential of the NK cell population from regional LNs either as immunotherapeutic targets or as adoptively transferred after activation with IL-2 or IL-15.

A Dose-Escalation Study of Recombinant Human Interleukin-18 in Combination With Rituximab in Patients With Non-Hodgkin Lymphoma

Interleukin-18 (IL-18) is an immunostimulatory cytokine with antitumor activity in preclinical models. Rituximab is a CD20 monoclonal antibody with activity against human B-cell lymphomas. A phase I study of recombinant human (rh) IL-18 given with rituximab was performed in patients with CD20+ lymphoma. Cohorts of 3-4 patients were given infusions of rituximab (375 mg/m2) weekly for 4 weeks with escalating doses of rhIL-18 as a 2-hour intravenous infusion weekly for 12 consecutive weeks. Toxicities were graded using standard criteria. Blood samples were obtained for safety, pharmacokinetic, and pharmacodynamic studies. Nineteen patients with CD20+ B-cell non-Hodgkin lymphoma were given rituximab in combination with rhIL-18 at doses of 1, 3, 10, 20, 30, and 100 μg/kg. Common side effects included chills, fever, headache, and nausea. Common laboratory abnormalities included transient, asymptomatic lymphopenia, hyperglycemia, anemia, hypoalbuminemia, and bilirubin and liver enzyme elevations. No dose-limiting toxicities were observed. Biologic effects of rhIL-18 included transient lymphopenia and increased expression of activation antigens on lymphocytes. Increases in serum concentrations of IFN-γ, GM-CSF, and chemokines were observed after dosing. Objective tumor responses were seen in 5 patients, including 2 complete and 3 partial responses. rhIL-18 can be given in biologically active doses by weekly infusions in combination with rituximab to patients with lymphoma. A maximum tolerated dose of rhIL-18 plus rituximab was not determined. Further studies of rhIL-18 and CD20 monoclonal antibodies in B-cell malignancies are warranted.

Monocyte derived dendritic cells have reduced expression of co-stimulatory molecules but are able to stimulate autologous T-cells in patients with MDS

Research has implied that the immune system plays a role in the pathogenesis of MDS and that T-cells are reacting to tumour antigen present on the surface of the malignant cells. This could imply that the immune system could be utilized to generate immune based therapy. The aim of this pilot study was to examine the feasibility of studying this further by analysing the interaction of dendritic cells with T-cells in a small cohort of MDS patients. Dendritic cells were generated in 6 MDS patients and 9 controls by culturing monocytes with GM-CSF and IL-4. After activation with LPS and TNFα, the dendritic cells were analyzed for expression of co-stimulatory and activation antigens. Thereafter, they were co-cultured with T-cells and the T-cell response was examined by measuring the % change in expression of the activation antigen CD69. MDS MoDC had reduced expression of HLA-DR (p=0.006), CD11c (p=0.0004), CD80 (p=0.03) and CD86 (p=0.003), while resting T-cells from MDS patients had higher expression of the activation antigen CD69 on all subsets. The % change in CD69 expression increased significantly for both the control and MDS T-cells after co-culture with allogeneic dendritic cells, however this change was lower in the MDS group. Despite the increased CD69 expression prior to culture, MDS MoDC significantly up-regulated CD69 expression on autologous T-cells to values that were statistically higher than control cells. This initial study suggests that the T-cells in MDS are able to respond to dendritic cells and are therefore probably not part of the malignant clone. It further implies that the dendritic cell population could be capable of presenting antigen and initiating an immune response and therefore further study is both feasible and warranted.

Distribution of several activating and inhibitory receptors on CD3−CD56+ NK cells in regional lymph nodes of melanoma patients

BackgroundNatural killer (NK) cells, as the main effector subpopulation of the innate immune system, play an important role in the control of the rise and spread of malignant tumors. Regional lymph nodes (LN) represent the first immunologic barrier to tumor metastasis. Since there are scarce data on NK cells from regional LN of cancer patients, the aim of this study was to investigate the expression of several activating and inhibitory receptors on the entire NK cell population as well as their CD3−CD56dim and CD3−CD56bright functional NK subsets from regional LN of melanoma patients. Materials and methodsMononuclear cells were isolated from 50 regional LN of melanoma patients. The expression of several receptors on NK cells and their functional subsets was analyzed by flow cytometry. ResultsWe show increased percentages of CD3−CD56+ NK cells in involved LN compared with uninvolved LN, mostly in favor of the CD56dim NK cell subset. NK cells in involved LN express similar levels of activating receptor NKG2D, while the level of another activating receptor, CD16, is increased compared with uninvolved LN. Regarding the expression of inhibitory NK cell receptors, we show increased CD158b, but similar low CD158a, inhibitory killer Ig-like cell receptor expression in involved LN compared with uninvolved LN. Furthermore, NK cells in involved compared with uninvolved LN displayed increased CD69 early activation antigen expression. ConclusionsOur results indicate that with tumor infiltration into regional LN of melanoma patients, NK cells, mostly of the CD56dim subset, are recruited into draining LN. The invading NK cells show counterbalance of the increased expression of CD16 activating receptor and increased CD158b inhibitory killer Ig-like cell receptor.

The Influence of Recombinant Human Erythropoietin on Apoptosis and Cytokine Production of CD4+ lymphocytes from Hemodialyzed Patients

Recombinant human erythropoietin (rhEPO) treatment of hemodialyzed (HD) patients normalizes the altered phenotype of CD4+ lymphocytes and restores the balance of Th1/Th2 cytokines. We decided to test how the presence of rhEPO in cell culture modulates cytokine production of CD4+ lymphocytes in HD patients with stable hemoglobin level and expression of activation antigens of stimulated CD4+ lymphocytes similar to those observed in healthy individuals. We also tested whether the presence of rhEPO in cell culture protects stimulated CD4+ lymphocytes of HD patients from apoptosis. Peripheral blood mononuclear cells (PBMC) of HD patients were stimulated with an immobilized anti-CD3 antibody with or without addition of rhEPO. The percentage of apoptotic CD4+ lymphocytes and the level of Th1/Th2 cytokines in culture supernatants were measured with flow cytometry. HD patients showed a decrease in the percentage of apoptotic CD4+ cells after stimulation with the anti-CD3 antibody combined with rhEPO. The level of IFN-γ and IL-10 was increased while the level of TNF-α was decreased in the presence of rhEPO in cell culture from HD patients. These results confirm the role of rhEPO signaling in T lymphocytes of HD patients.

Long-term culture in vitro impairs the immunosuppressive activity of mesenchymal stem cells on T cells

Improved knowledge of the immunological properties of mesenchymal stem cells (MSCs) creates a potential cell-mediated immunotherapeutic approach for arthritic diseases. The low frequency of MSCs necessitates their invitro expansion prior to clinical use. As sequential passage has been used as the most popular strategy for expansion of MSCs, the effect of long-term culture on the immunological properties of MSCs is not clear. In this study, we observed that the morphology of MSCs showed the typical characteristics of the Hayflick model of cellular aging during sequential expansion. The growth kinetics of MSCs decreased while the number of MSCs staining positive for SA β-gal (senescence marker) increased in long-term culture. Although long-term culture exerts less of an effect on the immunophenotype of MSCs, the immunosuppressive effects of MSCs on the allogeneic T-cell proliferation, activation-antigen expression (CD69 and CD25) and cytokine production (IFN-γ, TNF-α, IL-10) were significantly impaired following stimulation with phytohemagglutinin (PHA).