Scientific Resources

Accumulating evidence suggests elements within tumors induce exhaustion of effector T cells and infiltration of immunosuppressive regulatory T cells (Tregs),thus preventing the development of durable antitumor immunity. Therefore,the discovery of agents that simultaneously block Treg suppressive function and reinvigorate effector function of lymphocytes is key to the development of effective cancer immunotherapy. Previous studies have shown that TLR ligands (TLRLs) could modulate the function of these T cell targets; however,those studies relied on cell-free or accessory cell-based assay systems that do not accurately reflect in vivo responses. In contrast,we used a human PBMC-based proliferation assay system to simultaneously monitor the effect of TLRLs on T cells (CD4(+),CD8(+),Tregs),B cells,and NK cells,which gave different and even conflicting results. We found that the TLR7/8L:CL097 could simultaneously activate CD8(+) T cells,B cells,and NK cells plus block Treg suppression of T cells and B cells. The TLRLs TLR1/2L:Pam3CSK4,TLR5L:flagellin,TLR4L:LPS,and TLR8/7L:CL075 also blocked Treg suppression of CD4(+) or CD8(+) T cell proliferation,but not B cell proliferation. Besides CL097,TLR2L:PGN,CL075,and TLR9L:CpG-A,CpG-B,and CpG-C) were strong activators of NK cells. Importantly,we found that Pam3CSK4 could: 1) activate CD4(+) T cell proliferation,2) inhibit the expansion of IL-10(+) naturally occurring FOXP3(+) Tregs and induction of IL-10(+) CD4(+) Tregs (IL-10-producing type 1 Treg),and 3) block naturally occurring FOXP3(+) Tregs suppressive function. Our results suggest these agents could serve as adjuvants to enhance the efficacy of current immunotherapeutic strategies in cancer patients. View Publication

Multi-parameter flow cytometry analysis of T regulatory (Treg) cells is a widely used approach in basic and translational research studies. This approach has been complicated by a lack of specific markers for Treg cells and lack of uniformity in the quantification of Treg cells. Given the central role of Treg cells in the inception and perpetuation of diverse immune responses as well as its target as a therapeutic,it is imperative to have established methodologies for Treg cell analysis that are robust and usable for studies with multiple subjects as well as multicenter studies. In this study,we describe an optimized multi-parameter flow cytometry protocol for the quantification of human Treg cells from freshly obtained and viably frozen samples and correlations with epigenetic Treg cell analysis (TSDR demethylation). We apply these two methodologies to characterize Treg cell differences between cord blood and adult peripheral blood. In summary,the optimized protocol appears to be robust for Treg cell quantification from freshly isolated or viably frozen cells and the multi-parameter flow cytometry findings are strongly positively correlated with TSDR demethylation thus providing several options for the characterization of Treg cell frequency and function in large translational or clinical studies. View Publication

HIV elite controllers (EC) are a rare group of HIV-infected patients who are able to maintain undetectable viral loads during a long period of time in the absence of antiretroviral treatment. Adaptive immunity and host genetic factors,although implicated,do not entirely explain this phenomenon. On the other hand,plasmacytoid dendritic cells (pDCs) are the principal type I interferon (IFN) producers in response to viral infection,and it is unknown whether pDCs are involved in the control of HIV infection in EC. In our study,we analyzed peripheral pDC levels and IFN-α production by peripheral blood mononuclear cells (PBMCs) in EC compared to other groups of HIV-infected patients,the ability of pDCs to reduce HIV production in vitro,and the mechanisms potentially involved. We showed preserved pDC counts and IFN-α production in EC. We also observed a higher capacity of pDCs from EC to reduce HIV production and to induce T cell apoptosis,whereas pDCs from viremic patients barely responded without previous Toll-like receptor 9 (TLR-9) stimulus. The preserved functionality of pDCs from EC to reduce viral production may be one of the mechanisms involved in the control of HIV viremia in these subjects. These results demonstrate the importance of innate immunity in HIV pathogenesis,and an understanding of pDC mechanisms would be helpful for the design of new therapies. View Publication

How HIV controllers (HICs) maintain undetectable viremia without therapy is unknown. The strong CD8(+) T-cell HIV suppressive capacity found in many,but not all,HICs may contribute to long-lasting viral control. However,other earlier defense mechanisms may be involved. Here,we examined intrinsic HIC cell resistance to HIV-1 infection. After in vitro challenge,monocyte-derived macrophages and anti-CD3-activated CD4(+) T cells from HICs showed low HIV-1 susceptibility. CD4 T-cell resistance was independent of HIV-1 coreceptors and affected also SIVmac infection. CD4(+) T cells from HICs expressed ex vivo higher levels of p21(Waf1/Cip1),which has been involved in the control of HIV-1 replication,than cells from control subjects. However,HIV restriction in anti-CD3-activated CD4(+) T cells and macrophages was not associated with p21 expression. Restriction inhibited accumulation of reverse transcripts,leading to reduction of HIV-1 integrated proviruses. The block could be overcome by high viral inocula,suggesting the action of a saturable mechanism. Importantly,cell-associated HIV-1 DNA load was extremely low in HICs and correlated with CD4(+) T-cell permissiveness to infection. These results point to a contribution of intrinsic cell resistance to the control of infection and the containment of viral reservoir in HICs. View Publication

Recently,vaccines against the Wilms Tumor antigen 1 (WT1) have been tested in cancer patients. However,it is currently not known whether physiologic levels of WT1 expression in stem and progenitor cells of normal tissue result in the deletion or tolerance induction of WT1-specific T cells. Here,we used an human leukocyte antigen-transgenic murine model to study the fate of human leukocyte antigen class-I restricted,WT1-specific T cells in the thymus and in the periphery. Thymocytes expressing a WT1-specific T-cell receptor derived from high avidity human CD8 T cells were positively selected into the single-positive CD8 population. In the periphery,T cells specific for the WT1 antigen differentiated into CD44-high memory phenotype cells,whereas T cells specific for a non-self-viral antigen retained a CD44(low) naive phenotype. Only the WT1-specific T cells,but not the virus-specific T cells,displayed rapid antigen-specific effector function without prior vaccination. Despite long-term persistence of WT1-specific memory T cells,the animals did not develop autoimmunity,and the function of hematopoietic stem and progenitor cells was unimpaired. This is the first demonstration that specificity for a tumor-associated self-antigen may drive differentiation of functionally competent memory T cells. View Publication

The mechanisms of CD4(+) T-cell count decline,the hallmark of HIV disease progression,and its relationship to elevated levels of immune activation are not fully understood. Massive depletion of CD4(+) T cells occurs during the course of HIV-1 infection,so that maintenance of adequate CD4(+) T-cell levels probably depends primarily on the capacity to renew depleted lymphocytes,that is,the lymphopoiesis. We performed here a comprehensive study of quantitative and qualitative attributes of CD34(+) hematopoietic progenitor cells directly from the blood of a large set of HIV-infected persons compared with uninfected donors,in particular the elderly. Our analyses underline a marked impairment of primary immune resources with the failure to maintain adequate lymphocyte counts. Systemic immune activation emerges as a major correlate of altered lymphopoiesis,which can be partially reversed with prolonged antiretroviral therapy. Importantly,HIV disease progression despite elite control of HIV replication or virologic success on antiretroviral treatment is associated with persistent damage to the lymphopoietic system or exhaustion of lymphopoiesis. These findings highlight the importance of primary hematopoietic resources in HIV pathogenesis and the response to antiretroviral treatments. View Publication

CD4(+) T cells play a key role in host defense against Pneumocystis infection. To define the role of naïve CD4(+) T cell production through the thymopoietic response in host defense against Pneumocystis infection,Pneumocystis murina infection in the lung was induced in adult male C57BL/6 mice with and without prior thymectomy. Pneumocystis infection caused a significant increase in the number of CCR9(+) multipotent progenitor (MPP) cells in the bone marrow and peripheral circulation,an increase in populations of earliest thymic progenitors (ETPs) and double negative (DN) thymocytes in the thymus,and recruitment of naïve and total CD4(+) T cells into the alveolar space. The level of murine signal joint T cell receptor excision circles (msjTRECs) in spleen CD4(+) cells was increased at 5 weeks post-Pneumocystis infection. In thymectomized mice,the numbers of naïve,central memory,and total CD4(+) T cells in all tissues examined were markedly reduced following Pneumocystis infection. This deficiency of naïve and central memory CD4(+) T cells was associated with delayed pulmonary clearance of Pneumocystis. Extracts of Pneumocystis resulted in an increase in the number of CCR9(+) MPPs in the cultured bone marrow cells. Stimulation of cultured bone marrow cells with ligands to Toll-like receptor 2 ([TLR-2] zymosan) and TLR-9 (ODN M362) each caused a similar increase in CCR9(+) MPP cells via activation of the Jun N-terminal protein kinase (JNK) pathway. These results demonstrate that enhanced production of naïve CD4(+) T lymphocytes through the thymopoietic response and enhanced delivery of lymphopoietic precursors from the bone marrow play an important role in host defense against Pneumocystis infection. View Publication

B1 cells differ in many ways from conventional B cells,most prominently in the production of natural immunoglobulin,which is vitally important for protection against pathogens. B1 cells have also been implicated in the pathogenesis of autoimmune dyscrasias and malignant diseases. It has been impossible to accurately study B1 cells during health and illness because the nature of human B1 cells has not been successfully defined. This has produced controversy regarding the existence of human B1 cells. Here,we determined the phenotype of human B1 cells by testing sort-purified B cell fractions for three fundamental B1 cell functions based on mouse studies: spontaneous IgM secretion,efficient T cell stimulation,and tonic intracellular signaling. We found that a small population of CD20(+)CD27(+)CD43(+) cells present in both umbilical cord and adult peripheral blood fulfilled these criteria and expressed a skewed B cell receptor repertoire. These B cells express little or no surface CD69 and CD70,both of which are markedly up-regulated after activation of CD20(+)CD27(-)CD43(-) (naive) and CD20(+)CD27(+)CD43(-) (memory) B cells. This work identifies human B1 cells as CD20(+)CD27(+)CD43(+)CD70(-). We determined that the proportion of B1 cells declines with age,which may contribute to disease susceptibility. Identification of human B1 cells provides a foundation for future studies on the nature and role of these cells in human disease. View Publication

Infection with human immunodeficiency virus (HIV)-1 induces a progressive deterioration of the immune system that ultimately leads to acquired immune deficiency syndrome (AIDS). Murine models indicate that the common γ-chain (γ(c))-sharing cytokine interleukin (IL)-21 and its receptor (IL-21R) play a crucial role in maintaining polyfunctional T cell responses during chronic viral infections. Therefore,we analyzed the ability of this cytokine to modulate the properties of human CD8 T cells in comparison with other γ(c)-sharing cytokines (IL-2,IL-7,and IL-15). CD8 T cells from healthy volunteers were stimulated in vitro via T cell receptor signals to mimic the heightened status of immune activation of HIV-infected patients. The administration of IL-21 upregulated cytotoxic effector function and the expression of the costimulatory molecule CD28. Notably,this outcome was not accompanied by increased cellular proliferation or activation. Moreover,IL-21 promoted antiviral activity while not inducing HIV-1 replication in vitro. Thus,IL-21 may be a favorable molecule for immunotherapy and a suitable vaccine adjuvant in HIV-infected individuals. View Publication

It is unknown how dendritic cells (DCs) become specialized as mucosal DCs and maintain intestinal homeostasis. We report that a subset of bone marrow cells freshly isolated from C57BL/6 mice express the retinoic acid (RA)-synthesizing enzyme aldehyde dehydrogenase family 1,subfamily A2 (ALDH1a2) and are capable of providing RA to DC precursors in the bone marrow microenvironment. RA induced bone marrow-derived DCs to express CCR9 and ALDH1a2 and conferred upon them mucosal DC functions,including induction of Foxp3(+) regulatory T cells,IgA-secreting B cells,and gut-homing molecules. This response of DCs to RA was dependent on a narrow time window and stringent dose effect. RA promoted bone marrow-derived DC production of bioactive TGF-β by inhibiting suppressor of cytokine signaling 3 expression and thereby enhancing STAT3 activation. These RA effects were evident in vivo,in that mucosal DCs from vitamin A-deficient mice had reduced mucosal DC function,namely failure to induce Foxp3(+) regulatory T cells. Furthermore,MyD88 signaling enhanced RA-educated DC ALDH1a2 expression and was required for optimal TGF-β production. These data indicate that RA plays a critical role in the generation of mucosal DCs from bone marrow and in their functional activity. View Publication

To advance T cell-based HIV vaccine development,it is necessary to evaluate the immune correlates of a protective CD8(+) T-cell response. We have developed an assay that assesses the capacity ex vivo of HIV-specific CD8(+) T cells to suppress HIV-1 infection of autologous CD4(+) T cells. This assay directly reflects the ultimate effector function of CD8(+) T cells,the elimination of infected cells,and accurately differentiates the effective CD8(+) T-cell response in spontaneous HIV controllers from ineffective responses in other patients. In this article,we describe all the steps from cell purification to assessment of viral replication by HIV-p24 ELISA and analysis,along with conditions for cell culturing,and how to choose the viral infectious dose that gives the most reliable results. We also depict the conditions of a rapid assay on the basis of flow cytometry analysis of intracellular HIV-Gag products. These procedures take 14-17 d when the p24 ELISA assay is used,or 6 d with the intracellular Gag assay. View Publication

A key event in the successful induction of adaptive immune responses is the antigen-specific activation of T cells by dendritic cells (DCs). Although LFA-1 (lymphocyte function-associated antigen 1) on T cells is considered to be important for antigen-specific T-cell activation,the role for LFA-1 on DCs remains elusive. Using 2 different approaches to activate LFA-1 on DCs,either by deletion of the αL-integrin cytoplasmic GFFKR sequence or by silencing cytohesin-1-interacting protein,we now provide evidence that DCs are able to make use of active LFA-1 and can thereby control the contact duration with naive T cells. Enhanced duration of DC/T-cell interaction correlates inversely with antigen-specific T-cell proliferation,generation of T-helper 1 cells,and immune responses leading to delayed-type hypersensitivity. We could revert normal interaction time and T-cell proliferation to wild-type levels by inhibition of active LFA-1 on DCs. Our data further suggest that cytohesin-1-interacting protein might be responsible for controlling LFA-1 deactivation on mature DCs. In summary,our findings indicate that LFA-1 on DCs needs to be in an inactive state to ensure optimal T-cell activation and suggest that regulation of LFA-1 activity allows DCs to actively control antigen-driven T-cell proliferation and effective immune responses. View Publication