Loss Of CD4 Research Articles

Loss of CD28 within CD4+ T cell subsets from cervical cancer patients is accompanied by the acquisition of intracellular perforin, and is further enhanced by NKG2D expression

CD28 is well characterized as an essential co-stimulatory receptor critical for activation, proliferation and survival processes in CD4+ T cells. Populations of CD4+CD28null T cells, with apparently contradictory physiological roles, have recently been reported, along with the co-expression of the NK activating receptor NKG2D, in autoimmune diseases and chronic viral inflammation. Paradoxically, studies in cancer suggest that an expanded CD4+NKG2D+ population may be armed with immunosuppressive properties. We have recently reported the existence of two separate CD4+NKG2D+ T cell populations, which were defined by the presence or absence of the co-stimulatory molecule CD28, with the CD4+CD28nullNKG2D+ population more frequently observed in women with cervical cancer. This has led to the present effort to further characterize this population and to determine if the loss of CD28 influences the acquisition of cytotoxic or regulatory markers. In the present work, a multicolor flow cytometry protocol was used to analyze the expression of cytotoxic and immunoregulatory markers on circulating CD4+ T cells characterized by the presence or absence of CD28 and NKG2D in patients with invasive cervical carcinoma and age/gender-matched healthy controls. A noticeable expansion of CD4+CD28null cells, many of them NKG2D+, were observed in selected cervical cancer samples. This CD4+CD28null T cell population was characterized by a lack of immunoregulatory markers, as well as very low basal levels of intracellular IFN-γ, TNF-α, TGF-β, and IL-10. Intracellular perforin, however, was found to be significantly increased in this CD4+CD28null population, and increases in the mean fluorescence intensity of perforin were found to be enhanced by the presence of NKG2D. In conclusion, our data provide the first evidence of a strict link between the absence of CD28 and the expression of perforin, which is likewise enhanced by the expression of NKG2D, within selected CD4+ T cells from cervical cancer patients.

A Clinicopathological Study of cd34 Antigen Expression in Benign and Malignant Breast Lesions

Objectives: To study stromal CD34 expression in benign and malignant breast lesions and evaluate whether loss of CD34 is specific for invasive disease. Material and methods: 317 cases of benign and malignant breast lesions were studied over a period of 2 years (July 2012 to June 2014). All the cases underwent routine processing followed by H and E staining. 178 cases underwent CD34 immunohistochemical staining and semi quantitative assessment. Further, 74 cases of infiltrating ductal carcinoma were evaluated to determine relation of stromal CD34 expression with histological grade and lymph node metastasis. Results: Normal breast showed retained CD34 expression in 90.9% cases. Among benign lesions, 83% cases showed retained stromal CD34 expression (except cases of apocrine metaplasia, intraductal papilloma and fibroadenoma with focal atypia which showed reduced expression in 46.67%, 100% and 33.3% cases respectively). Both DCIS and LCIS showed reduced expression. Malignant tumors showed grade 0 staining in 97.9% cases, irrespective of the histological type. Statistically significant differences in stromal CD34 expression were observed between: Malignant phyllode`s tumor compared to its benign counterpart Papilloma and papillary carcinoma Benign and malignant lesions overall No significant correlation was found between CD34 expression and histological grade or lymph node metastasis. The metastatic foci in node positive patients showed a similar pattern of CD34 expression in the lymph nodes as those seen in the breast. Conclusion: Owing to the significant differences in CD34 expression between benign and malignant breast lesions, it can potentially be used to differentiate between the two and can serve as an important diagnostic marker. Further studies can also be undertaken to establish its role as a therapeutic target in cases of breast cancer.

The Role of Defensins in HIV Pathogenesis.

Profound loss of CD4+ T cells, progressive impairment of the immune system, inflammation, and sustained immune activation are the characteristics of human immunodeficiency virus-1 (HIV-1) infection. Innate immune responses respond immediately from the day of HIV infection, and a thorough understanding of the interaction between several innate immune cells and HIV-1 is essential to determine to what extent those cells play a crucial role in controlling HIV-1 in vivo. Defensins, divided into the three subfamilies α-, β-, and θ-defensins based on structure and disulfide linkages, comprise a critical component of the innate immune response and exhibit anti-HIV-1 activities and immunomodulatory capabilities. In humans, only α- and β-defensins are expressed in various tissues and have broad impacts on HIV-1 transmission, replication, and disease progression. θ-defensins have been identified as functional peptides in Old World monkeys, but not in humans. Instead, θ-defensins exist only as pseudogenes in humans, chimpanzees, and gorillas. The use of the synthetic θ-defensin peptide “retrocyclin” as an antiviral therapy was shown to be promising, and further research into the development of defensin-based HIV-1 therapeutics is needed. This review focuses on the role of defensins in HIV-1 pathogenesis and highlights future research efforts that warrant investigation.

Cellular Mechanisms Regulating CD20 As a Target of Monoclonal Antibody Therapy in B-Lymphoid Malignancies

Standard treatment of B-lymphoid malignancies nowadays still relies on the administration of monoclonal antibodies (mAbs), with CD20 antigen being the prime target. Although initially effective, repeated cycles of anti-CD20 monoclonal antibody therapy often result in the loss of CD20 on the surface of malignant B cells and consequently in therapy resistance. In spite of the widespread use of CD20 monoclonal antibodies, the exact mechanisms regulating CD20 expression stay largely unrevealed and it mainly remains unclear whether they can be exploited pharmacologically to modulate expression of CD20 in the clinic.Interestingly, application of CD20 mAbs on various B-cell lines in vitro triggers active CD20 signaling within the cells. This signal transmission results in rapid CD20 downregulation at the surface accompanied by dramatically reduced CD20 transcription. Contrary to the prompt initial downmodulation of CD20 transcription, it takes several days upon mAb removal until CD20 transcriptional gene activity and thereafter CD20 surface levels are restored back to normal. To further study cellular mechanisms responsible for regulating CD20 expression, we have mimicked the situation in patients in an in vitro setting by repeated chronic treatments of diverse B-cell lines with gradually increasing doses of clinically used anti-CD20 monoclonal antibodies Rituximab, Ofatumumab and Obinutuzumab. Thereby, we managed to generate permanently resistant lines that no more respond to additional administration of any of the anti-CD20 mAbs. Notably, these cells have permanently strongly downregulated CD20 expression, both on the cell surface as well as already on the level of mRNA gene transcription. In addition, we have utilized the state-of-the-art CRISPR/Cas9 system to fully knock out CD20 gene in B-cell lymphoma (Ramos) and CLL (MEC1) cell lines. As expected, these cells are totally resistant to CD20 monoclonal antibodies. CD20 was originally proposed to function as a calcium channel and to contribute to B-cell receptor signaling, however the exact function of CD20 remains largely elusive yet. Using both experimental systems, we demonstrate that B cells with the loss of CD20 have fairly normal B-cell receptor signaling with no signs of any large defect. Specifically, activation of major signaling proteins like SYK, AKT, ERK, PLCgamma or p38 upon BCR stimulation was equal in CD20 depleted cells when compared to normal cells. Also calcium flux in response to BCR triggering seems to be normal in CD20-deficient cells, thus suggesting that CD20 is dispensable for proper B-cell receptor signaling. Next, we have performed RNA sequencing on these cells in order to better understand intracellular changes on a more global level that are imposed by the deletion or chronic downmodulation of CD20. We have compared the expression of individual genes and gene sets in cells upon CD20 removal and have performed the differential gene set enrichment analysis that will be presented.In summary, analysis of mechanisms regulating CD20 expression and/or function is critically needed for identification of potential novel drug targets that might be applied in the clinic to control CD20 persistence and thereby improve the outcome of anti-CD20 monoclonal antibody therapy.This research has been financially supported by the Ministry of Education, Youth and Sports of the Czech Republic under the project CEITEC 2020 (LQ1601) and by the research grant AZV-MZ-CR 15-33561AA-4/2015 and grant MUNI/A/1028/2015. DisclosuresNo relevant conflicts of interest to declare.

Safety and Tolerability of Donor Type Red Blood Cell Transfusion before Allogeneic Stem Cell Transplantation in Children with Major ABO Mismatch

BackgroundMajor ABO mismatch in allogeneic bone marrow transplant (BMT) can cause clinical problems like severe transfusion reactions, acute haemolysis, and delay of red blood cell (RBC) engraftment or manifestation of pure red cell aplasia, related to circulating isoagglutinin titers. Even there is no increased incidence of graft failure or slower engraftment of neutrophils and platelets ABO mismatch leads to an increase of transplant related mortality. To reduce circulating isoagglutinin titers pre transplant plasma exchange during conditioning period can be performed. This procedure can be wearing for the patient and rebound effects are observed. RBC depletion can cause a loss of CD34+stem cells in bone marrow. A further strategy to reduce circulating isoagglutinin titers is in vivo immunoadsorption due to donor type RBC transfusion pre transplant.Aim of the studyThe Aim of the retrospective single center study was to investigate the safety and tolerability of donor-type red blood cell transfusion prior to allogeneic stem cell transplantation in children with major ABO mismatch.MethodsFrom 2007-2015 37 children (median age 7 years, range 0.6-18.4) received an ABO mismatched RBC transfusion (2-3 ml KG BW) pre transplant under antihistaminic and steroid cover. Reaction to donor type RBC and graft transfusion, number of donor type RBC transfusions, haemolysis parameters, and trend of isoagglutinin titers, and engraftment data were observed.Wilcoxon signed-rank test was used to examine the difference between the paired values of titers (Conventional test tube, CTT; titration method using nonspecific anti-IgG antibody; Anti-IgG gel titer) before and after donor type RBC transfusion. Age and time until engraftment were reported using median and ranges. The quantitative values were reported using frequency and percentage. All statistical tests were two-sided with a significance level of 5%. Data analysis was performed using commercial software R.Statistical methods.ResultsSafety of mismatched RBC transfusionsCompared with accidentally transfused RBC no severe complications are observed in our cohort. 34 (92%) children presented no reactions (Grade 0), 1 child had a minimal oxygen demand,1 child showed a mild hypertension (Grade 1) and 1 child a moderate hypertension, requiring treatment. Neither severe reactions nor anaphylactic shock were observed.Efficiency of mismatched RBC transfusionA significant reduction of isoagglutinin titers was defined as a final titer of 1:4 or at least a reduction of 3 titer steps was achieved. 23 of 37 (64%) required 1 donor type RBC transfusion to reduce significant the isoagglutinin titers, 8 patients (22%) needed a second and 5 patients (14%) a third transfusion. Figure 1 showed the significant reduction of isoagglutinin titers in the different patients groups after donor type RBC transfusion. The pairwise analysis of isoagglutinin titers indicates that the initial titer of patients required one donor type transfusion of RBC was significant lower than in the patient group required 2 or 3 transfusions.Engraftment dataThe median time to WBC and platelet engraftment was 21.5 (range 19-24) and 28 (range 26-60) days respectively.Haemolysis parameter post mismatched RBC transfusionHaemolysis parameter lactat dehydrogenase (LDH) and bilirubin was determined on a daily base. In all patients no significant increase of bilirubin levels was observed.ConclusionDonor type RBC transfusion is a safe, tolerable and effective procedure to reduce the isoagglutinin titers prior to allogeneic ABO mismatched bone marrow transplantation in children. In our cohort no severe transfusion reactions, acute haemolysis, delay of engraftment or manifestation of pure red cell aplasia was observed. [Display omitted] DisclosuresJarisch:Novartis: Consultancy. Bader:Novartis: Consultancy, Honoraria; Servier: Consultancy, Honoraria; Medac: Research Funding; Riemser: Research Funding; Neovii: Research Funding.

Monocytes Are Required for Both Optimal Anti-Leukemic Efficacy and the Cytokine Release Syndrome By CAR-T Cells: Lessons from an Innovative Xenotolerant Mouse Model

Background: Chimeric antigen-receptor (CAR)-engineered T cells promise to cure chronic and acute leukemias refractory to standard treatments. Before this promise is fulfilled, however, two crucial issues need to be solved: i) how to circumvent the emergence of secondary resistance (e.g. due totarget-antigen loss; leukemic lineage switch); ii) how to manage associated toxicities (e.g. the cytokine release syndrome, CRS; lineage aplasias). Unfortunately, all these issues cannot be addressed pre-clinically in currently available NSG mouse models, because they lack human hematopoiesis and, furthermore, ultimately develop xenograft-versus-host disease (X-GVHD), preventing the evaluation of long-term effects.Methods: We have developed an innovative xenotolerant model by transplanting human hematopoietic stem cells (HSCs) intraliver in newborn NSG mice triple transgenic for human SCF, GM-SCF and IL-3 (SGM3). Differently from "classical" NSG, SGM3 mice reconstituted high levels of human T cells (>1000 cells per microL at week 8), which, once transferred in secondary recipients, persisted up to 200d without causing X-GVHD, even after irradiation. Robust and specific xenotolerance was confirmed by in vitrohyporesponsiveness to NSG, bot not to C57/Bl6 antigens (irradiated splenocytes) or human HLAs (PBMCs). Secondary transfer experiments in leukemic and/or HSC-humanized SGM-3 mice have been then designed for studying the determinants of CAR-T cell efficacy and associated toxicities in the absence of confounding xenoreactivity.Results: SGM3-derived T cells were transduced ex vivo with either a CD19 or a CD44v6 CAR (both having a CD28 2G design) after activation with CD3/CD28-beads and IL-7/IL-15, resulting in a preferential and functional CD45RA+/CD62L+/CD95+ stem memory T cell (TSCM) phenotype. Once transferred in secondary recipients previously engrafted with a CD19+/CD44v6 leukemic cell line, CD19 or CD44v6 CAR-T cells equally mediated rapid tumor clearance both in low and high tumor-burden settings, in the absence of malaise or elevated human IL-6 levels in vivo. At later time points (after 100d), however, approximately 50% of responding mice relapsed despite significant CAR-T cell persistence in vivo (>50 cells per microL). A significant fraction of leukemia relapses were characterized by post-transcriptional down-regulation of CD44v6 expression or CD19 loss, respectively. Conversely, secondary transfer of SGM3-derived CAR-T cells in leukemic SGM3 mice that had been previously humanized with HSCs resulted in the development of a clinical syndrome similar to the CRS observed in clinical trials (high fevers, elevated IL-6, TNF-alpha and serum amyloid A levels - mouse analog of C-reactive protein in humans), resulting in 30% lethality. This CRS was anticipated and shortened for CD44v6 compared with CD19 CAR-T cells and worse in the case of 4-1BB compared with the original CD28 2G CAR designs. Strikingly, mice recovering from the CRS benefited from durable leukemic remissions, yet experienced long-lasting CD19+ B-cell or CD44v6+ monocyte aplasias. Deepness of remission was confirmed in "tertiary" recipients, which did not develop leukemia after the infusion of bone-marrow cells from mice in remission 150d since CAR-T cell infusion. Interestingly, in this model, tocilizumab administration at the time of either CD19 or CD44v6 CAR-T cell infusion efficiently prevented the CRS, but did not interfere with their comparable and long-term anti-leukemic effects. Conversely, depleting monocytes/macrophages before therapeutic CAR-T cell infusion by either lyposomal clodronate or by the prophylactic CD44v6 CAR-T cells inhibited CRS development, but also resulted in significantly worse leukemia-free survival (at 250d, 0% vs 80%, P<0.0001).Conclusions: A number of lessons can be learned from this innovative xenotolerant mouse model of CAR-T cell immunotherapy: monocytes are required for both i) optimal anti-leukemic efficacy, and ii) the occurrence of CRS; iii) tocilizumab prevents the CRS without interfering with efficacy; iv) monocyte aplasia induced by CD44v6 CAR-T cells does not impact on their efficacy, at least in the theraeputic setting, and may ameliorate CRS toxicity. As for CD44v6 CAR-T cells, this model could be used for effectively predicting the efficacy and associated toxicities of new CAR-T cell therapies, speeding up their clinical development. DisclosuresTraversari:MolMed SpA: Employment. Bordignon:MolMed SpA: Employment. Ciceri:MolMed SpA: Consultancy. Bonini:TxCell: Membership on an entity's Board of Directors or advisory committees; Molmed SpA: Consultancy. Bondanza:Formula Pharmaceuticals: Honoraria; TxCell: Research Funding; MolMed SpA: Research Funding.

CD19 Epitope Escape after 4SCAR19 T Cell Therapy Resulted in Re-Establishment of Chemo-Sensitivity in Adult B-Cell Acute Lymphocytic Leukemia Patients

Background: CD19-targeted chimeric antigen receptor (CAR) T cell treatment has emerged as a highly effective therapy in patients with refractory B cell acute lymphoblastic leukemia (B-ALL). Despite obvious successes, there have been documented relapses in which CAR T cells are still present but the leukemia cells have lost CD19 surface epitopes. In this study, we aim to investigate these patients and characterize their leukemic cell clones.Methods: As part of a multi-center trial, this single-arm, phase I trial enrolled 10 patients between March 2015 and June 2016 based on the following criteria: poor physical condition, chemotherapy resistance, and lack of suitable donor. Following salvage chemotherapy, peripheral lymphocytes were collected from leukapheresis, and T cells were transduced with a 4th generation, safety-engineered, CD19scFv/CD28/CD137/CD27/ CD3zeta lentivector. The pre-infusion lymphodepleting chemotherapy conditioning is cyclophosphamide 800mg/m2 x 3 days, followed by infusions at a dose of 2.13 (range from 0.42 - 5.9) x106 4SCAR19 T cells per kg body weight . Patients who achieved complete remission (CR) but later relapsed received chemotherapy of VDP (vincristine 1.4mg/m2 d1,8,15,22, daunomycin 40mg/m2 d1,8,15,22, prednison 40mg/m2d1-28) regimen. CD19-negative relapsed patients were assessed by surface immunophenotyping, cytoplasm immunophenotyping ,bone marrow biopsy and immunohistochemistry staining, T cell receptor(TCR) and immunoglobulin heavy chain (IgH) rearrangement analysis.Results and Discussion: Total 10 patients received 4SCAR19 treatment and 7 achieved CR. Following CR, 5 relapsed, including 3 CD19 positive and 2 CD19 negative relapses. Surface marker analysis showed that the CD19-negative relapsed leukemia cells displayed altered surface immunophenotype different from the original CD19-positive leukemia cells. Comparing the leukemia cells of the two CD19-negative relapsed patients by immunophenotype analysis for CD19,CD10,CD34,and CD79a, the CD10 surface expression in one of the two CD19-negative leukemia cells was down-regulated from 90% to 10%, but cytoplasmic CD10 expression remained at 90% which was confirmed by immunohistochemical staining of bone marrow biopsy. The CD10 expression profile, however, did not alter in the other CD19-negative leukemia cells. The loss of surface CD19 expression was coupled with the loss of cytoplasm CD19 as determined by intracellular flow cytometry and immunohistochemical staining; however, the nuclear PAX-5 expression was positive. Another B-ALL index ,the IgH rearrangement was examined by multiplex PCR and their original clonal identify was confirmed. It is thus conceivable that the CD19 negative relapsed leukemia cells were associated with escape variants rather than selection of de novo leukemia clones following 4SCAR19 therapy. From the diverse immune phenotype of the CD19 negative relapsed leukemia cells, it should be feasible to select another surface epitope(s) as target(s) for CAR T therapy. Importantly, after CAR T therapy, the two CD19-negative relapsed patients responded to chemotherapy and achieved CR again. However, the three CD19-positive relapsed patients experienced inferior chemotherapy outcomes. These results suggest that chemo-sensitivity could be re-established after CD19-negative relapse following 4SCAR19 therapy. Other studies have found that CD19 mutations in CAR T-treated leukemic cells could result in the loss of PI3K and SFTK signaling, and further, altered signaling pathways such as p53 and PTEN mutations have been shown to contribute to chemo-resistance of leukemic cells. The forced pathway alteration in the CD19-negative leukemic clones after CAR T treatment could involve one of these pathways, and further investigation is warranted. DisclosuresNo relevant conflicts of interest to declare.

Analysis of the intestinal microbial community and inferred functional capacities during the host response to Pneumocystis pneumonia

ABSTRACTBackground: Pneumocystis pneumonia is a major cause of morbidity and mortality in patients infected with HIV/AIDS. In this study, we evaluated the intestinal microbial communities associated with the development of experimental Pneumocystis pneumonia, as there is growing evidence that the intestinal microbiota is critical for host defense against fungal pathogens. Methods: C57BL/6 mice were infected with live Pneumocystis murina (P. murina) via intratracheal inoculation and sacrificed 7 and 14 days postinfection for microbiota analysis. In addition, we evaluated the intestinal microbiota from CD4+ T cell depleted mice infected with P. murina. Results: We found that the diversity of the intestinal microbial community was significantly altered by respiratory infection with P. murina. Specifically, mice infected with P. murina had altered microbial populations, as judged by changes in diversity metrics and relative taxa abundances. We also found that CD4+ T cell depleted mice infected with P. murina exhibited significantly altered intestinal microbiota that was distinct from immunocompetent mice infected with P. murina, suggesting that loss of CD4+ T cells may also affects the intestinal microbiota in the setting of Pneumocystis pneumonia. Finally, we employed a predictive metagenomics approach to evaluate various microbial features. We found that Pneumocystis pneumonia significantly alters the intestinal microbiota's inferred functional potential for carbohydrate, energy, and xenobiotic metabolism, as well as signal transduction pathways. Conclusions: Our study provides insight into specific-microbial clades and inferred microbial functional pathways associated with Pneumocystis pneumonia. Our data also suggest a role for the gut-lung axis in host defense in the lung.

A transcriptome-based model of central memory CD4 T cell death in HIV infection.

BackgroundHuman central memory CD4 T cells are characterized by their capacity of proliferation and differentiation into effector memory CD4 T cells. Homeostasis of central memory CD4 T cells is considered a key factor sustaining the asymptomatic stage of Human Immunodeficiency Virus type 1 (HIV-1) infection, while progression to acquired immunodeficiency syndrome is imputed to central memory CD4 T cells homeostatic failure. We investigated if central memory CD4 T cells from patients with HIV-1 infection have a gene expression profile impeding proliferation and survival, despite their activated state.MethodsUsing gene expression microarrays, we analyzed mRNA expression patterns in naive, central memory, and effector memory CD4 T cells from healthy controls, and naive and central memory CD4 T cells from patients with HIV-1 infection. Differentially expressed genes, defined by Log2 Fold Change (FC) ≥ |0.5| and Log (odds) > 0, were used in pathway enrichment analyses.ResultsCentral memory CD4 T cells from patients and controls showed comparable expression of differentiation-related genes, ruling out an effector-like differentiation of central memory CD4 T cells in HIV infection. However, 210 genes were differentially expressed in central memory CD4 T cells from patients compared with those from controls. Expression of 75 of these genes was validated by semi quantitative RT-PCR, and independently reproduced enrichment results from this gene expression signature. The results of functional enrichment analysis indicated movement to cell cycle phases G1 and S (increased CCNE1, MKI67, IL12RB2, ADAM9, decreased FGF9, etc.), but also arrest in G2/M (increased CHK1, RBBP8, KIF11, etc.). Unexpectedly, the results also suggested decreased apoptosis (increased CSTA, NFKBIA, decreased RNASEL, etc.). Results also suggested increased IL-1β, IFN-γ, TNF, and RANTES (CCR5) activity upstream of the central memory CD4 T cells signature, consistent with the demonstrated milieu in HIV infection.ConclusionsOur findings support a model where progressive loss of central memory CD4 T cells in chronic HIV-1 infection is driven by increased cell cycle entry followed by mitotic arrest, leading to a non-apoptotic death pathway without actual proliferation, possibly contributing to increased turnover.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3308-8) contains supplementary material, which is available to authorized users.

The MicroRNA miR-191 Supports T Cell Survival Following Common γ Chain Signaling

To ensure lifelong immunocompetency, naïve and memory T cells must be adequately maintained in the peripheral lymphoid tissues. Homeostatic maintenance of T cells is controlled by tonic signaling through T cell antigen receptors and common γ chain cytokine receptors. In this study, we identify the highly expressed microRNA miR-191 as a key regulator of naïve, memory, and regulatory T cell homeostasis. Conditional deletion of miR-191 using LckCre resulted in preferential loss of peripheral CD4+ regulatory T cells as well as naïve and memory CD8+ T cells. This preferential loss stemmed from reduced survival following deficient cytokine signaling and STAT5 activation. Mechanistically, insulin receptor substrate 1 (Irs1) is a direct target of miR-191, and dysregulated IRS1 expression antagonizes STAT5 activation. Our study identifies a novel role for microRNAs in fine-tuning immune homeostasis and thereby maintaining the lymphocyte reservoir necessary to mount productive immune responses.

CD4+ T-cell–independent mechanisms suppress reactivation of latent tuberculosis in a macaque model of HIV coinfection

The synergy between Mycobacterium tuberculosis (Mtb) and HIV in coinfected patients has profoundly impacted global mortality because of tuberculosis (TB) and AIDS. HIV significantly increases rates of reactivation of latent TB infection (LTBI) to active disease, with the decline in CD4(+) T cells believed to be the major causality. In this study, nonhuman primates were coinfected with Mtb and simian immunodeficiency virus (SIV), recapitulating human coinfection. A majority of animals exhibited rapid reactivation of Mtb replication, progressing to disseminated TB and increased SIV-associated pathology. Although a severe loss of pulmonary CD4(+) T cells was observed in all coinfected macaques, a subpopulation of the animals was still able to prevent reactivation and maintain LTBI. Investigation of pulmonary immune responses and pathology in this cohort demonstrated that increased CD8(+) memory T-cell proliferation, higher granzyme B production, and expanded B-cell follicles correlated with protection from reactivation. Our findings reveal mechanisms that control SIV- and TB-associated pathology. These CD4-independent protective immune responses warrant further studies in HIV coinfected humans able to control their TB infection. Moreover, these findings will provide insight into natural immunity to Mtb and will guide development of novel vaccine strategies and immunotherapies.

Abstract 530: Location of oncogene expression within a stratified squamous epithelium drives distinct B and CD4 T-cell crosstalk to dictate the tumor immune response

Abstract Tumors from stratified epithelium contain proliferating and differentiating compartments, but it is not clear how tumor cells in different layers alternatively engage the immune system. We have established two doxycycline inducible Ras models in which oncogenic RasV12G is targeted to either the epidermal basal/stem cell layer with a Keratin14-rtTA transgene (K14Ras), or suprabasal differentiating cells with an Involucrin-tTA transgene (InvRas). On threshold doxycycline levels yielding similar tumor numbers and Ras expression over 30 days, mice with basal cell targeted Ras developed focal squamous cell carcinoma while suprabasal targeting caused benign squamous papilloma formation. On a Rag1-/- background InvRas mice developed fewer tumors that regressed over time while K14Ras mice developed more tumors with shorter latency than Rag1+/+ controls. Depletion and adoptive transfer studies revealed that naïve B and CD4 T cells together, but not alone, suppressed tumor formation in K14Ras mice but restored tumor numbers in InvRas mice. Tumors developing in K14Ras mice showed a loss of proinflammatory CD4 T and B cells and increased percentages of regulatory cells infiltrating the tumor tissue. In vivo cotransfers show that B and CD4 T cells reciprocally prime each other towards a regulatory phenotype in the K14Ras tumor microenvironment, but in the InvRas tumor microenvironment proinflammatory CD4 T and B cell phenotypes result. Coculture of tumor-conditioned B cells with stimulated naïve CD4 T cells showed an importance of direct contact and CD40/CD40L interactions for the generation of regulatory T cells (Tregs) by B cells in K14Ras mice. In contrast, tumor-conditioned B cells from InvRas mice support generation of proinflammatory CD4 T cells, and antagonize Treg development. This function is restricted to tumor-conditioned B cells, as splenic B cells from tumor-bearing mice had no effect on CD4 T cell phenotype. In vivo blockade of CD40L in K14Ras mice resulted in significantly increased tumor counts as well as reduced B and Treg prevalence. Blockade of CD40L in InvRas mice significantly reduced tumor number, increasing Treg cell count and decreasing neutrophil infiltration into the tumor tissue. Time-course experiments suggest a protective role of Tregs in late stages of K14Ras tumors, and a tumor-promoting role of proinflammatory CD4 T cells in InvRas tumors. Thus, basal/stem cell expression of Ras provokes a regulatory cell inducing microenvironment, suppressing tumor-promoting inflammation in late-stage tumors. Ras expression in differentiating cells activates a tumor promoting proinflammatory phenotype in B and CD4 T cells without provoking immunosurveillance. Taken together, these data show that tumor cell position within a stratified epithelium differentiation hierarchy provokes distinct B and CD4 T cell interactions with opposing effects on tumor development. Citation Format: Michael A. Podolsky, Carrie J. Oakes, Andrew Gunderson, Kyle Breech, Jacob Bailey, Adam B. Glick. Location of oncogene expression within a stratified squamous epithelium drives distinct B and CD4 T-cell crosstalk to dictate the tumor immune response. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 530.

A Mouse Model of Latent Tuberculosis Infection to Study Intervention Strategies to Prevent Reactivation.

Infection with Mycobacterium tuberculosis (Mtb) is the leading cause of death in human immunodeficiency virus (HIV)+ individuals, particularly in Sub-Saharan Africa. Management of this deadly co-infection is a significant global health challenge that is exacerbated by the lack of efficient vaccines against both Mtb and HIV, as well as the lack of reliable and robust animal models for Mtb/HIV co-infection. Here we describe a tractable and reproducible mouse model to study the reactivation dynamics of latent Mtb infection following the loss of CD4+ T cells as it occurs in HIV-co-infected individuals. Whereas intradermally (i.d.) infected C57BL/6 mice contained Mtb within the local draining lymph nodes, depletion of CD4+ cells led to progressive systemic spread of the bacteria and induction of lung pathology. To interrogate whether reactivation of Mtb after CD4+ T cell depletion can be reversed, we employed interleukin (IL)-2/anti-IL-2 complex-mediated cell boost approaches. Although populations of non-CD4 lymphocytes, such as CD8+ memory T cells, natural killer (NK) cells and double-negative (DN) T cells significantly expanded after IL-2/anti-IL-2 complex treatment, progressive development of bacteremia and pathologic lung alterations could not be prevented. These data suggest that the failure to reverse Mtb reactivation is likely not due to anergy of the expanded cell subsets and rather indicates a limited potential for IL-2-complex-based therapies in the management of Mtb/HIV co-infection.

Splenic Damage during SIV Infection: Role of T-Cell Depletion and Macrophage Polarization and Infection

The effects of HIV infection on spleen and its cellular subsets have not been fully characterized, particularly for macrophages in which diverse populations exist. We used an accelerated SIV-infected macaque model to examine longitudinal effects on T-cell and macrophage populations and their susceptibilities to infection. Substantial lymphoid depletion occurred, characterized by follicular burn out and a loss of CD3 T lymphocytes, which was associated with cellular activation and transient dysregulations in CD4/CD8 ratios and memory effector populations. In contrast, the loss of CD68 and CD163(+)CD68(+) macrophages and increase in CD163 cells was irreversible, which began during acute infection and persisted until terminal disease. Mac387 macrophages and monocytes were transiently recruited into spleen, but were not sufficient to mitigate the changes in macrophage subsets. Type I interferon, M2 polarizing genes, and chemokine-chemokine receptor signaling were up-regulated in spleen and drove macrophage alterations. SIV-infected T cells were numerous within the white pulp during acute infection, but were rarely observed thereafter. CD68, CD163, and Mac387 macrophages were highly infected, which primarily occurred in the red pulp independent of T cells. Few macrophages underwent apoptosis, indicating that they are a long-lasting target for HIV/SIV. Our results identify macrophages as an important contributor to HIV/SIV infection in spleen and in promoting morphologic changes through the loss of specific macrophage subsets that mediate splenic organization.

CD4(+) T-Cell-Independent Secondary Immune Responses to Pneumocystis Pneumonia.

Pneumocystis pneumonia is a major cause of morbidity and mortality among immunocompromised patients, especially in the context of HIV/AIDS. In the murine model of Pneumocystis pneumonia, CD4+ T-cells are required for clearance of a primary infection of Pneumocystis, but not the memory recall response. We hypothesized that the memory recall response in the absence of CD4+ T-cells is mediated by a robust memory humoral response, CD8+ T-cells, and IgG-mediated phagocytosis by alveolar macrophages. To investigate the role of CD8+ T-cells and alveolar macrophages in the immune memory response to Pneumocystis, mice previously challenged with Pneumocystis were depleted of CD8+ T-cells or alveolar macrophages prior to re-infection. Mice depleted of CD4+ T-cells prior to secondary challenge cleared Pneumocystis infection within 48 h identical to immunocompetent mice during a secondary memory recall response. However, loss of CD8+ T-cells or macrophages prior to the memory recall response significantly impaired Pneumocystis clearance. Specifically, mice depleted of CD8+ T-cells or alveolar macrophages had significantly higher fungal burden in the lungs. Furthermore, loss of alveolar macrophages significantly skewed the lung CD8+ T-cell response toward a terminally differentiated effector memory population and increased the percentage of IFN-γ+ CD8+ T-cells. Finally, Pneumocystis-infected animals produced significantly more bone marrow plasma cells and Pneumocystis-specific IgG significantly increased macrophage-mediated killing of Pneumocystis in vitro. These data suggest that secondary immune memory responses to Pneumocystis are mediated, in part, by CD8+ T-cells, alveolar macrophages, and the production of Pneumocystis-specific IgG.

A unique subset of CD11c+ B cells upregulate FasL expression in response to IL-12p40 during lethal dose influenza virus (IAV) infections.

Abstract Our recent work has detailed a novel regulation of the IAV-specific CD8 T cell response during lethal dose IAV infections. During such infections, FasL+ plasmacytoid dendritic cells (pDC) within the regional lymph nodes (LN) target and eliminate the developing Fas+ effector CD8 T cell response limiting immunity and driving severe disease. While our studies have shown that pDC are sufficient to drive this mechanism (i.e. transfer of wild type pDC into gld mice results in elimination of activated CD8 T cells), antibody targeted elimination of pDC in wild type mice does not completely reverse CD8 T cell apoptosis. This suggested that an additional cell type might contribute to the overall loss of CD8 T cells during lethal/high pathogenic IAV infections. Here we describe a CD11c+B cell population that shares some common markers with both pDC and several defined B cell subsets, but can be distinguished from both based on unique marker expression. Our data suggests that these CD11c+ B cells are recruited to the LN in greater numbers compared to pDCs and appear to express FasL upon exposure to IL-12p40 homodimer in lethal dose IAV infections. Further our results suggest that FasL+ expression with BST2+ expression within CD11c+ B cells. Our current studies are focused on further determining the factors regulating this CD11c+B cell population and its relative contribution to the overall FasL-dependent control of the magnitude of the CD8 T cell response during lethal dose IAV infections.

Fatty Acid-Induced T Cell Loss Greases Liver Carcinogenesis

A new study has added loss of CD4(+) Tcells caused by aberrant lipid metabolism to the list of mechanisms promoting nonalcoholic steatohepatitis progression to liver cancer (Ma etal., 2016). Exposure of CD4(+) Tcells to free linoleic acid causes their ROS-mediated depletion, thereby favoring liver cancer growth.

Chronic Δ(9)-Tetrahydrocannabinol Administration Reduces IgE(+)B Cells but Unlikely Enhances Pathogenic SIVmac251 Infection in Male Rhesus Macaques of Chinese Origin.

Delta9-tetrahydrocannabinol (Δ(9)-THC) is the major psychoactive component of the cannabis plant. Δ(9)-THC has been used in the active ingredient of Marinol as an appetite stimulant for AIDS patients. Its impact on progression of HIV-1 infection, however, remains debatable. Previous studies indicated that Δ(9)-THC administration enhanced HIV-1 infection in huPBL-SCID mice but seemingly decreased early mortality in simian immunodeficiency virus (SIV) infected male Indian-derived rhesus macaques. Here, we determine the chronic effect of Δ(9)-THC administration using0.32mg/kg or placebo (PBO), i.m., twice daily for 428days on SIVmac251 infected male Chinese-derived rhesus macaques. Sixteen animals were divided into four study groups: Δ(9)-THC(+)SIV(+), Δ(9)-THC(+)SIV(-), PBO/SIV(+) and PBO/SIV(-) (n=4/group). One-month after daily Δ(9)-THC or PBO administrations, macaques in groups one and three were challenged intravenously with pathogenic SIVmac251/CNS, which was isolated from the brain of a Chinese macaque with end-staged neuroAIDS. No significant differences in peak and steady state plasma viral loads were seen between Δ(9)-THC(+)SIV(+) and PBO/SIV(+) macaques. Regardless of Δ(9)-THC, all infected macaques displayed significant drop of CD4/CD8 T cell ratio, loss of CD4(+) T cells and higher persistent levels of Ki67(+)CD8(+) T cells compared with uninfected animals. Moreover, long-term Δ(9)-THC treatment reduced significantly the frequency of circulating IgE(+)B cells. Only one Δ(9)-THC(+)SIV(+) macaque died of simian AIDS with paralyzed limbs compared with two deaths in the PBO/SIV(+) group during the study period. These findings indicate that chronic Δ(9)-THC administration resulted in reduction of IgE(+)B cells, yet it unlikely enhanced pathogenic SIVmac251/CNS infection in male Rhesus macaques of Chinese origin.

The Role of HIV-1 in Affecting the Proliferation Ability of HPCs Derived From BM.

HIV-1 causes chronic infection characterized by the depletion of CD4+ T lymphocytes and the development of AIDS. Current antiretroviral drugs inhibit viral spread, but they do not lead to a full immune recovery. Hematopoietic stem cells (HSCs) and multipotent hematopoietic progenitor cells (HPCs) give rise to all blood and immune cells, and in HIV infection, hematological abnormalities frequently occur in patients. Here, we used bone marrow samples from HIV-1-infected people to study the relationship between the proliferation ability of HSCs/HPCs and peripheral CD4+ T lymphocytes. Three indexes were used to reflect the proliferation ability of HSCs and HPCs: (1) colony-forming units of bone marrow mononuclear cells (BMMCs), (2) amplification of CD34+ cells purified from bone marrow mononuclear cells, (3) expression of HOXB4 and HOXA9 in CD34+ cells. We observed a direct correlation between peripheral number of CD4+ T lymphocytes and the HSCs/HPCs proliferation ability in our study. We also compared HIV-infected patients with or without antiretroviral therapy (ART). Our results demonstrated that after antiretroviral therapy, CD4+ T-cell recovery and HPCs proliferation ability are correlated. Our findings have implications in understanding whether bone marrow-derived HPCs can supplement for the loss of CD4+ T lymphocytes during HIV-1 infection.

HIV Skews the Lineage-Defining Transcriptional Profile of Mycobacterium tuberculosis-Specific CD4+ T Cells.

HIV-infected persons are at greater risk of developing tuberculosis (TB) even before profound CD4 loss occurs, suggesting that HIV alters CD4(+) T cell functions capable of containing bacterial replication. An effective immune response to Mycobacterium tuberculosis most likely relies on the development of a balanced CD4 response, in which distinct CD4(+) Th subsets act in synergy to control the infection. To define the diversity of M. tuberculosis-specific CD4(+) Th subsets and determine whether HIV infection impacts such responses, the expression of lineage-defining transcription factors T-bet, Gata3, RORγt, and Foxp3 was measured in M. tuberculosis-specific CD4(+) T cells in HIV-uninfected (n = 20) and HIV-infected individuals (n = 20) with latent TB infection. Our results show that, upon 5-d restimulation in vitro, M. tuberculosis-specific CD4(+) T cells from healthy individuals have the ability to exhibit a broad spectrum of Th subsets, defined by specific patterns of transcription factor coexpression. These transcription factor profiles were skewed in HIV-infected individuals where the proportion of T-bet(high)Foxp3(+) M. tuberculosis-specific CD4(+) T cells was significantly decreased (p = 0.002) compared with HIV-uninfected individuals, a change that correlated inversely with HIV viral load (p = 0.0007) and plasma TNF-α (p = 0.027). Our data demonstrate an important balance in Th subset diversity defined by lineage-defining transcription factor coexpression profiles that is disrupted by HIV infection and suggest a role for HIV in impairing TB immunity by altering the equilibrium of M. tuberculosis-specific CD4(+) Th subsets.