scholarly journals Oral immune dysfunction is associated with the expansion of FOXP3+PD-1+Amphiregulin+ T cells during HIV infection

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
N. Bhaskaran ◽  
E. Schneider ◽  
F. Faddoul ◽  
A. Paes da Silva ◽  
R. Asaad ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Hiv Infection ◽  
Immune Dysfunction ◽  
Hiv Positive ◽  
People Living With Hiv ◽  
Asparaginyl Endopeptidase ◽  
Ifn Γ ◽  
Living With Hiv

AbstractResidual systemic inflammation and mucosal immune dysfunction persist in people living with HIV, despite treatment with combined anti-retroviral therapy, but the underlying immune mechanisms are poorly understood. Here we report that the altered immune landscape of the oral mucosa of HIV-positive patients on therapy involves increased TLR and inflammasome signaling, localized CD4+ T cell hyperactivation, and, counterintuitively, enrichment of FOXP3+ T cells. HIV infection of oral tonsil cultures in vitro causes an increase in FOXP3+ T cells expressing PD-1, IFN-γ, Amphiregulin and IL-10. These cells persist even in the presence of anti-retroviral drugs, and further expand when stimulated by TLR2 ligands and IL-1β. Mechanistically, IL-1β upregulates PD-1 expression via AKT signaling, and PD-1 stabilizes FOXP3 and Amphiregulin through a mechanism involving asparaginyl endopeptidase, resulting in FOXP3+ cells that are incapable of suppressing CD4+ T cells in vitro. The FOXP3+ T cells that are abundant in HIV-positive patients are phenotypically similar to the in vitro cultured, HIV-responsive FOXP3+ T cells, and their presence strongly correlates with CD4+ T cell hyper-activation. This suggests that FOXP3+ T cell dysregulation might play a role in the mucosal immune dysfunction of HIV patients on therapy.

scholarly journals PD-1 dependent expansion of Amphregulin+FOXP3+ cells is associated with oral immune dysfunction in HIV patients on therapy

2021 ◽  
Author(s):  
N Bhaskaran ◽  
E Schneider ◽  
F Faddoul ◽  
A Paes da Silva ◽  
R Asaad ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Oral Mucosa ◽  
Immune Dysfunction ◽  
People Living With Hiv ◽  
Dependent Manner ◽  
Mucosal Tissues ◽  
T Cell Regulation ◽  
Ifn Γ

AbstractResidual systemic inflammation and mucosal immune dysfunction persist in people living with HIV (PLWH) despite treatment with combined anti-retroviral therapy (cART), but the underlying immune mechanisms are poorly understood. Here we report an altered immune landscape involving upregulation of TLR- and inflammasome signaling, localized CD4+ T cell hyperactivation, and counterintuitively, an enrichment of CD4+CD25+FOXP3+ regulatory T cells (Tregs) in the oral mucosa of HIV+ patients on therapy. Using human oral tonsil cultures, we found that HIV infection causes an increase in a unique population of FOXP3+ cells expressing PD-1, IFN-γ, Amphiregulin (AREG), and IL-10. These cells persisted even in the presence of the anti-retroviral drug and underwent further expansion driven by TLR-2 ligands and IL-1β. IL-1β also promoted PD-1 upregulation in AKT1 dependent manner. PD-1 stabilized FOXP3 and AREG expression in these cells through a mechanism requiring the activation of Asparaginyl Endopeptidase (AEP). Importantly, these FOXP3+ cells were incapable of suppressing CD4+ T cells in vitro. Concurrently, HIV+ patients harbored higher levels of PD-1, IFN-γ, Amphiregulin (AREG), and IL-10 expressing FOXP3+ cells, which strongly correlated with CD4+ T cell hyperactivation, suggesting an absence of CD4+ T cell regulation in the oral mucosa. Taken together, this study provides insights into a novel mechanism of FOXP3+ cell dysregulation and reveals a critical link in the positive feedback loop of oral mucosal immune activation events in HIV+ patients on therapy.One Sentence SummaryHIV-induced immune dysfunction in lymphoid and mucosal tissues

scholarly journals T cell-tropic HIV efficiently infects alveolar macrophages through contact with infected CD4+ T cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Abigail E. Schiff ◽  
Alice H. Linder ◽  
Shillah N. Luhembo ◽  
Stephanie Banning ◽  
Martin J. Deymier ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Alveolar Macrophages ◽  
Cd4 T Cells ◽  
Cd4 T Cell ◽  
Productive Infection ◽  
People Living With Hiv ◽  
Living With Hiv ◽  
Hiv Envelope

AbstractAlveolar macrophages (AMs) are critical for defense against airborne pathogens and AM dysfunction is thought to contribute to the increased burden of pulmonary infections observed in individuals living with HIV-1 (HIV). While HIV nucleic acids have been detected in AMs early in infection, circulating HIV during acute and chronic infection is usually CCR5 T cell-tropic (T-tropic) and enters macrophages inefficiently in vitro. The mechanism by which T-tropic viruses infect AMs remains unknown. We collected AMs by bronchoscopy performed in HIV-infected, antiretroviral therapy (ART)-naive and uninfected subjects. We found that viral constructs made with primary HIV envelope sequences isolated from both AMs and plasma were T-tropic and inefficiently infected macrophages. However, these isolates productively infected macrophages when co-cultured with HIV-infected CD4+ T cells. In addition, we provide evidence that T-tropic HIV is transmitted from infected CD4+ T cells to the AM cytosol. We conclude that AM-derived HIV isolates are T-tropic and can enter macrophages through contact with an infected CD4+ T cell, which results in productive infection of AMs. CD4+ T cell-dependent entry of HIV into AMs helps explain the presence of HIV in AMs despite inefficient cell-free infection, and may contribute to AM dysfunction in people living with HIV.

scholarly journals Long Non-coding RNA GAS5 Regulates T Cell Functions via miR21-Mediated Signaling in People Living With HIV

2021 ◽  
Vol 12 ◽  
Author(s):  
Lam Ngoc Thao Nguyen ◽  
Lam Nhat Nguyen ◽  
Juan Zhao ◽  
Madison Schank ◽  
Xindi Dang ◽  
...  
Keyword(s):  
Dna Damage ◽  
T Cells ◽  
T Cell ◽  
Hiv Infection ◽  
Cd4 T Cells ◽  
People Living With Hiv ◽  
Low Grade ◽  
Cell Functions ◽  
Non Coding Rnas ◽  
Living With Hiv

T cells are critical for the control of viral infections and T cell responses are regulated by a dynamic network of non-coding RNAs, including microRNAs (miR) and long non-coding RNAs (lncRNA). Here we show that an activation-induced decline of lncRNA growth arrest-specific transcript 5 (GAS5) activates DNA damage response (DDR), and regulates cellular functions and apoptosis in CD4 T cells derived from people living with HIV (PLHIV) via upregulation of miR-21. Notably, GAS5-miR21-mediated DDR and T cell dysfunction are observed in PLHIV on antiretroviral therapy (ART), who often exhibit immune activation due to low-grade inflammation despite robust virologic control. We found that GAS5 negatively regulates miR-21 expression, which in turn controls critical signaling pathways involved in DNA damage and cellular response. The sustained stimulation of T cells decreased GAS5, increased miR-21 and, as a result, caused dysfunction and apoptosis in CD4 T cells. Importantly, this inflammation-driven T cell over-activation and aberrant apoptosis in ART-controlled PLHIV and healthy subjects (HS) could be reversed by antagonizing the GAS5-miR-21 axis. Also, mutation of the miR-21 binding site on exon 4 of GAS5 gene to generate a GAS5 mutant abolished its ability to regulate miR-21 expression as well as T cell activation and apoptosis markers compared to the wild-type GAS5 transcript. Our data suggest that GAS5 regulates TCR-mediated activation and apoptosis in CD4 T cells during HIV infection through miR-21-mediated signaling. However, GAS5 effects on T cell exhaustion during HIV infection may be mediated by a mechanism beyond the GAS5-miR-21-mediated signaling. These results indicate that targeting the GAS5-miR-21 axis may improve activity and longevity of CD4 T cells in ART-treated PLHIV. This approach may also be useful for targeting other infectious or inflammatory diseases associated with T cell over-activation, exhaustion, and premature immune aging.

scholarly journals Gamma Interferon Production, but Not Perforin-Mediated Cytolytic Activity, of T Cells Is Required for Prevention of Toxoplasmic Encephalitis in BALB/c Mice Genetically Resistant to the Disease

2004 ◽  
Vol 72 (8) ◽  
pp. 4432-4438 ◽  
Author(s):  
Xisheng Wang ◽  
Hoil Kang ◽  
Takane Kikuchi ◽  
Yasuhiro Suzuki
Keyword(s):  
T Cells ◽  
T Cell ◽  
Nude Mice ◽  
Genetic Resistance ◽  
Cytolytic Activity ◽  
Gamma Interferon ◽  
Toxoplasmic Encephalitis ◽  
Wild Type ◽  
Ifn Γ

ABSTRACT We previously showed the requirement of both T cells and gamma interferon (IFN-γ)-producing non-T cells for the genetic resistance of BALB/c mice to the development of toxoplasmic encephalitis (TE). In order to define the role of IFN-γ production and the perforin-mediated cytotoxicity of T cells in this resistance, we obtained immune T cells from spleens of infected IFN-γ knockout (IFN-γ−/−), perforin knockout (PO), and wild-type BALB/c mice and transferred them into infected and sulfadiazine-treated athymic nude mice, which lack T cells but have IFN-γ-producing non-T cells. Control nude mice that had not received any T cells developed severe TE and died after discontinuation of sulfadiazine treatment due to the reactivation of infection. Animals that had received immune T cells from either wild-type or PO mice did not develop TE and survived. In contrast, nude mice that had received immune T cells from IFN-γ−/− mice developed severe TE and died as early as control nude mice. T cells obtained from the spleens of animals that had received either PO or wild-type T cells produced large amounts of IFN-γ after stimulation with Toxoplasma gondii antigens in vitro. In addition, the amounts of IFN-γ mRNA expressed in the brains of PO T-cell recipients did not differ from those in wild-type T-cell recipients. Furthermore, PO mice did not develop TE after infection, and their IFN-γ production was equivalent to or higher than that of wild-type animals. These results indicate that IFN-γ production, but not perforin-mediated cytotoxic activity, by T cells is required for the prevention of TE in genetically resistant BALB/c mice.

scholarly journals 323 Immunogenic syngeneic model MC38-OVA for the preclinical evaluation of immune evasion and checkpoint blockade

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A348-A348
Author(s):  
Jessie Wang ◽  
Kaixia Lian ◽  
Jia Zheng ◽  
Chenpan Nie ◽  
Annie An ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Immunity ◽  
Immune Evasion ◽  
Syngeneic Mouse ◽  
Ifn Γ ◽  
Draining Lymph Nodes ◽  
Syngeneic Tumors

BackgroundThe development of immuno-oncology (I/O) therapeutics has revolutionized the cancer treatment landscape. Despite this achievement, the mechanism behind limited responses is poorly understood. Tumor immune evasion has been reported to arise through the loss of tumor necrosis factor (TNF) signaling, interferon-γ (IFN-γ) signaling, and antigen presentation pathways, which are crucial to CD8+ T cell-mediated killing. Syngeneic mouse models have been widely used as they have an intact immune system, are easily accessible, and have a vast array of historical data for comparison. However, limited syngeneic models respond to immune checkpoint inhibitors, possibly due to low intrinsic immunogenicity. The expression of ovalbumin (OVA) has previously shown to sufficiently alter the susceptibility of syngeneic tumors to host T cell-mediated responses. In this study, the newly developed OVA-expressing MC38 syngeneic line was characterized for tumor immunity, checkpoint blockade response and response durability.MethodsMurine colon cancer MC38 cells were transduced by lentiviral vector with chicken OVA coding cDNA. A single clone was selected, and OVA expression was confirmed by western blot. The MC38-OVA cells were subcutaneously implanted into immunocompetent mice to evaluate the tumorigenicity and in vivo response to anti-PD-1 antibody treatment. Blood was collected 2 days post final dose of anti-PD-1 treatment for phenotypic analysis by FACS. Spleen and tumor draining lymph nodes were collected at termination for FACS analysis of IFN-γ+ T cells and OVA specific CD8+ T cells. Adoptive transfer was evaluated by challenge studies in both MC38-OVA and MC38 tumor-bearing mice with T cells derived from MC38-OVA mice, anti-PD-1 cured mice and OT-I mice. In vitro killing assays were performed to evaluate the function of adoptive CD3+ T cells transfer.ResultsOVA-expressing MC38 presented complete regression under anti-PD-1 treatment in vivo. T cell expansion was observed after anti-PD-1 treatment in peripheral blood with increased IFN-γ+ T cells in both tumor-draining lymph nodes and spleen. Additionally, anti-PD-1 cured mice generated robust tumor specific memory T cell, which successfully inhibited MC38-OVA and MC38 tumor growth following adoptive transfer. CD3+ T cells from MC38-OVA-bearing mice and OT-I mice showed anti-tumor immunity in vivo. In vitro killing assay demonstrated increased immunity.ConclusionsSyngeneic mouse tumor models are preferred preclinical models for I/O research, despite limited intrinsic immunogenicity. OVA expression in syngeneic tumors largely increased T cell-mediated immunity to enhance antigen-specific T cell responses during tumorigenesis, providing novel immunogenic models for preclinical immunotherapy evaluation.

scholarly journals Sunitinib Exerts In Vitro Immunomodulatory Activity on Sarcomas via Dendritic Cells and Synergizes With PD-1 Blockade

2021 ◽  
Vol 12 ◽  
Author(s):  
Darina Ocadlikova ◽  
Mariangela Lecciso ◽  
Javier Martin Broto ◽  
Katia Scotlandi ◽  
Michele Cavo ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Cell Lines ◽  
Effector T Cells ◽  
Sarcoma Cell ◽  
Ifn Γ ◽  
Sarcoma Cells

BackgroundHigh-grade sarcomas are a heterogeneous group of aggressive tumors arising in bone and soft tissues. After relapse, treatment options are limited. The multi-targeted receptor tyrosine kinase inhibitors (TKIs) sunitinib and inhibitor of PD-1 (anti-PD-1) nivolumab have shown antitumor activity in selected subtypes. In this study, we examine the role of TKIs and PD-1 based therapy in in vitro cocultures of sarcoma.MethodsThe human osteosarcoma (SaOS-2) and synovial sarcoma (SYO-1) cell lines were treated with sunitinib. After cell death and proliferation assessment, expression of PD-L1 was analyzed by flow cytometry. Sunitinib-treated sarcoma cells were cocultured with dendritic cells (DCs), and the phenotype of mature DCs was determined by flow cytometry. Mature DCs were cultured with autologous T cells. PD-1 expression on T cells, their proliferation, T regulatory cell (Tregs) induction and IFN-γ production, before and after nivolumab exposure, were analyzed.ResultsAlong with its anti-proliferative and direct pro-apoptotic effect on sarcoma cell lines, sunitinib prompted PD-L1 upregulation on sarcoma cells. Interestingly, sunitinib-treated sarcoma cells drive DCs to full maturation and increase their capacity to induce sarcoma-reactive T cells to produce IFN-γ. Conversely, no effect on T cell proliferation and T cell subpopulation composition was observed. Moreover, both bone and synovial sarcoma cell lines induced Tregs through DCs but sunitinib treatment completely abrogated Treg induction. Finally, sarcoma cell lines induced PD-1 upregulation on both effector T cells and Tregs when loaded into DCs, providing a rationale for using PD-1 blockade. Indeed, PD-1 blockade by nivolumab synergized with sunitinib in inducing IFN-γ-producing effector T cells.ConclusionsTaken together, our in vitro data indicate that the treatment of sarcoma cells with sunitinib can exert significant changes on immune cell subsets toward immune activation, leading to DC-based cross-priming of IFN-γ-producing effector T cells and reduced Treg induction. PD-1 blockade with nivolumab has a synergistic effect with sunitinib, supporting the use of TKI and anti-PD-1 approach in sarcomas, and perhaps in other cancers. DC-targeted drugs, including toll-like receptor 3 inhibitors and CD47 inhibitors, are under development and our preclinical model might help to better design their clinical application.

scholarly journals An Ex Vivo Study of T Lymphocytes Recovered from the Lungs of I/St Mice Infected with and Susceptible toMycobacterium tuberculosis

1998 ◽  
Vol 66 (10) ◽  
pp. 4981-4988 ◽  
Author(s):  
Irina Lyadova ◽  
Vladimir Yeremeev ◽  
Konstantin Majorov ◽  
Boris Nikonenko ◽  
Sergei Khaidukov ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Ex Vivo ◽  
Antimycobacterial Activity ◽  
Enzymatic Digestion ◽  
Interleukin 4 ◽  
T Cell Clones ◽  
Cell Clones ◽  
Ifn Γ

ABSTRACT I/St mice, previously characterized as susceptible toMycobacterium tuberculosis H37Rv, were given 103 or 105 CFU intravenously. At two time points postinoculation, the cell suspensions that resulted from enzymatic digestion of lungs were enumerated and further characterized phenotypically and functionally. Regarding the T-cell populations recovered at 2 and 5 weeks postinfection, two main results were obtained: (i) the population of CD44− CD45RB+cells disappeared within 2 weeks postinfection, while the number of CD44+ CD45RB−/low cells slowly increased between weeks 2 and 5; (ii) when cocultured with irradiated syngeneic splenocytes, these lung T cells proliferated in the presence of H37Rv sonicate. Using H37Rv sonicate and irradiated syngeneic splenocytes to reactivate lung T cells, we selected five CD3+CD4+ CD8− T-cell clones. In addition to the H37Rv sonicate, the five clones react to both a short-term culture filtrate and an affinity-purified 15- to 18-kDa mycobacterial molecule as assessed by the proliferative assay. However, there was a clear difference between T-cell clones with respect to cytokine (gamma interferon [IFN-γ] and interleukin-4 [IL-4] and IL-10) profiles: besides one Th1-like (IFN-γ+ IL-4−) clone and one Th0-like (IFN-γ+ IL-4+IL-10+) clone, three clones produced predominantly IL-10, with only marginal or no IL-4 and IFN-γ responses. Inhibition of mycobacterial growth by macrophages in the presence of T cells was studied in a coculture in vitro system. It was found that the capacity to enhance antimycobacterial activity of macrophages fully correlated with INF-γ production by individual T-cell clones following genetically restricted recognition of infected macrophages. The possible functional significance of cytokine diversity among T-cell clones is discussed.

scholarly journals Mapping evidence on adolescents’ HIV-positive status disclosure in sub-Saharan Africa: a protocol for a scoping review

2020 ◽  
Vol 9 (1) ◽  
Author(s):  
Patience Adzordor ◽  
Clement Avoka ◽  
Vitalis Bawontuo ◽  
Silas Agbesi ◽  
Desmond Kuupiel
Keyword(s):  
Hiv Infection ◽  
Scoping Review ◽  
Keyword Search ◽  
Sub Saharan Africa ◽  
Hiv Positive ◽  
People Living With Hiv ◽  
Sub Saharan ◽  
Living With Hiv ◽  
Status Disclosure ◽  
Hiv Aids

Abstract Background Sub-Saharan Africa (SSA) homes most of the people living with HIV/AIDS in the world. Adolescents/young people are a vulnerable population and at high risk of HIV infection. Identifying and bridging the research gaps on the disclosure of HIV-positive status among adolescents, particularly to their sexual partners, is essential to inform appropriate policy planning and implementation towards preventing HIV transmission. This study will aim to explore literature and describe the evidence on HIV-positive status disclosure among adolescents in SSA. Methods The framework provided by Arksey and O’Malley’s framework and improved by Levac and colleagues will be used to conduct a scoping review. A keyword search for relevant literature presenting evidence on HIV-positive status disclosure among adolescents in SSA will be conducted in CINAHL, PubMed, Science Direct, Google Scholar, and SCOPUS. Date limitations will be removed, but Boolean terms “AND” and “OR” as well as Medical Subject Headings terms will be included where possible and syntax modified to suit the database during the search. Additional relevant articles will be sought from the reference lists of all included studies using a snowballing method. Two reviewers will independently screen the articles at the abstract and full-text screening phases in order to reduce bias and improve the reliability of this study’s findings. A tabular form will be developed using Microsoft Word and piloted for data extraction. Thematic content analysis will be conducted, and a narrative summary of all relevant outcomes reported. Quality appraisal of the included studies for this proposed study will be performed utilizing the recent mixed methods appraisal tool. Discussion The evidence produced by this review may help inform policy and strategies to reduce the incidence of HIV infection among adolescents and improve social support for adolescents living with HIV/AIDS in SSA. It may also reveal literature gaps to guide future researches to further inform HIV policies for adolescents in SSA. Platforms such as peer review journals, policy briefs, and conferences will be used to disseminate this study’s findings.

scholarly journals Phosphoantigen-activated Vγ2Vδ2 T cells antagonize IL-2–induced CD4+CD25+Foxp3+ T regulatory cells in mycobacterial infection

Blood ◽  
2009 ◽  
Vol 113 (4) ◽  
pp. 837-845 ◽  
Author(s):  
Guangming Gong ◽  
Lingyun Shao ◽  
Yunqi Wang ◽  
Crystal Y. Chen ◽  
Dan Huang ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Treatment Regimen ◽  
T Regulatory Cells ◽  
Mycobacterial Infection ◽  
T Cell Subsets ◽  
Regulatory Cells ◽  
Ifn Γ ◽  
Vγ2vδ2 T Cells

Abstract Although Foxp3+ T regulatory cells (Tregs) are well documented for their ability to suppress various immune cells, T-cell subsets capable of counteracting Tregs have not been demonstrated. Here, we assessed phosphoantigen-activated Vγ2Vδ2 T cells for the ability to interplay with Tregs in the context of mycobacterial infection. A short-term IL-2 treatment regimen induced marked expansion of CD4+CD25+Foxp3+ T cells and subsequent suppression of mycobacterium-driven increases in numbers of Vγ2Vδ2 T cells. Surprisingly, activation of Vγ2Vδ2 T cells by adding phosphoantigen Picostim to the IL-2 treatment regimen down-regulated IL-2–induced expansion of CD4+CD25+Foxp3+ T cells. Consistently, in vitro activation of Vγ2Vδ2 T cells by phosphoantigen plus IL-2 down-regulated IL-2–induced expansion of CD4+CD25+Foxp3+ T cells. Interestingly, anti–IFN-γ–neutralizing antibody, not anti–TGF-β or anti–IL-4, reduced the ability of activated Vγ2Vδ2 T cells to down-regulate Tregs, suggesting that autocrine IFN-γ and its network contributed to Vγ2Vδ2 T cells' antagonizing effects. Furthermore, activation of Vγ2Vδ2 T cells by Picostim plus IL-2 treatment appeared to reverse Treg-driven suppression of immune responses of phosphoantigen-specific IFNγ+ or perforin+ Vγ2Vδ2 T cells and PPD-specific IFNγ+αβ T cells. Thus, phos-phoantigen activation of Vγ2Vδ2 T cells antagonizes IL-2–induced expansion of Tregs and subsequent suppression of Ag-specific antimicrobial T-cell responses in mycobacterial infection.

Cultured Human B Cell APCs Expanded Using Il-4 and CD40 Ligand Preferentially Promote a Type 2 T Cell Response to Alloimmune Stimulation.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2668-2668
Author(s):  
Abdul Tawab ◽  
Yoshiyuki Takahashi ◽  
Childs Richard ◽  
Kurlander J. Roger
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cell Response ◽  
Cd40 Ligand ◽  
T Cell Response ◽  
Ifn Γ

Abstract In vitro stimulation of human peripheral blood B cells with recombinant IL-4 and CD40 ligand (CD40L) markedly increases their expression of MHC and costimulatory molecules, thus enhancing antigenic peptide presentation to T cells. Because these cells proliferate extensively in vitro (unlike monocytes or dendritic cells), they represent a promising and convenient reagent for the generation and maintenance of antigen-specific T cells for use in a variety of experimental or therapeutic settings. However, the impact of this type of B cell APC on cytokine production by responder T cells has hitherto not been examined. To address this issue, we stimulated normal human T cells with either allogeneic B cells (generated in vitro) or with MNCs obtained from the same donor. After 7 days, T cells were washed and re-challenged with the same APCs. The resulting alloreactive cytokine response was measured using quantitative ELISPOT methods and expressed as the frequencies of IFN-γ, IL-4, and IL-5 producing cells per thousand responder cells added. B cell- and MNC-primed cell lines both produced vigorous lymphokine responses, but B cell-stimulated T cells consistently produced more IL-5 spots (mean of 265 vs. 98/1000 responders, p<0.002) and fewer IFN-γ spots (163 vs 386/1000 cells, p<0.005) than MNC-stimulated cells. Further, the ratio of IFN-γ to IL-5 spots was almost ten-fold lower in B cell-stimulated cultures compared to MNC-induced cultures (0.67 vs. 5.2, p<0.001). ELISPOT studies assessing the ratio of IFN-γ to IL-4 spots and ELISA assays comparing IFN-γ and IL-5 levels from culture supernatants demonstrated the same pattern of marked type 2 skewing by B cells. This pattern was unaffected by the presence of anti-IL-4 antibody suggesting type 2 skewing was not mediated by IL-4. Cytokine skewing produced by B cells or MNC could be partially reversed by swapping MNC and B cells during re-stimulation on day 7, but this plasticity was markedly reduced after 3 (weekly) cycles of B cell or MNC re-stimulation in vitro. Type 2 skewing by B cells was enhanced when monocytes were removed from responder T cell populations by either depleting CD14+ positive cells or by positive selection of T cells prior to stimulation. In contrast, type 2 polarization could be prevented using recombinant IL-12. Not all cells of B-cell origin share the same propensity to type 2 skewing observed with IL-4/CD40L-stimulated B cells; under identical conditions, EBV-transformed B cells stimulated alloimmune T cells to produce a strong type 1 cytokine response comparable to that produced by MNCs. In summary, IL-4/CD40L-stimulated B cells strongly promote a type 2 T cell response during primary alloimmune challenge; this skewing can become fixed after repeated B cell stimulation. Investigators using these cells as APC should be aware of this potential phenomenon, particularly during primary T cell responses. It is also important to consider the factors described above that may exacerbate or ameliorate this effect.

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