Adaptive Immune Compartments Research Articles

Abstract 316: A Superantigen Whole Blood Challenge Test for the Quantification of Immuno-inflammation in Early Phase Drug Development

Development of immune-modulating drugs is challenging as assessment of intended pharmacology in healthy volunteers is difficult. Commonly, ex-vivo LPS challenges of whole blood or isolated cells are applied. However, the applicability of the LPS challenge is limited as LPS primarily stimulates germ-line encoded innate-immune TLR4-receptors, and thus does not allow monitoring of pharmacological modulation of the adaptive immune compartment. We explored an alternative whole blood challenge, inducing lymphocyte-driven inflammation by the superantigen staphylococcal enterotoxin B (SEB), and compared this to LPS. Further validation was performed by assessing the inhibitory effects of different immune-modulating compounds (MAP kinase inhibitors, calcitriol, methotrexate, protein kinase C, doxycycline, simvastatin), allowing discrimination between effects on monocytes and lymphocytes. SEB induction resulted in the release of a cytokine panel that well-resembled cytokine release observed after physiological lymphocyte stimulation, with clear induction of IL-2, IFN-γ, TNF- α, IL-10 and GM-CSF. Based on TNF-α and IL-2 release, SEB’s maximal effect was reached at 100 ng/mL. Whereas inter-individual variability in EC50 was relatively small for LPS (0.32±0.10 ng/mL), for SEB inter-subject variability was distinctly higher (62±54 ng/mL). MAP kinase inhibitors and calcitriol were effective in inhibiting SEB-induced TNF-α and IL-2 release, demonstrating an inhibitory effect on the monocyte and/or lymphocyte. Importantly, whereas methotrexate, doxycycline, and simvastatin did not affect SEB-induced TNF-α release, a clear inhibiting effect on SEB-induced IL-2 release was observed, implying a direct action of these compounds on the lymphocyte. These data demonstrate that the SEB challenge can be used to estimate the immune-modulating effects of compounds directly acting on the T-lymphocyte population. As such, the whole blood SEB challenge is a useful translational tool in early drug development, especially since immune-inflammation is a target in the development of new treatment modalities for currently poorly-controlled diseases such as rheumatoid arthritis and psoriasis, and cardiovascular disease.

Immunotherapy in HIV Infection

Substantial progress has been made in devising successful therapies against Human Immunodeficiency Virus (HIV) replication, and antiretroviral therapy (ART) can provide a sustained control of HIV replication. It is, however, associated with at best partial immune reconstitution, as well as lack of elimination of viral reservoirs. Both innate and adaptive immune cell compartments that suffer as a result of HIV replication, fail to recover completely under ART, hence, the need for lifelong therapy once infected. Novel therapeutic approaches are being tested at an encouraging rate and have the potential to improve the odds against the virus. Among them, immunotherapeutic approaches are one of the exciting areas and can be considered as adjunct to ART for improving immune competence. Cells of the innate immune system, including natural killer (NK) cells, gamma delta (γδ) T cells and natural killer T (NKT) cells have cytotoxic potential against viruses, and therapies including such effector cells will be useful. The fact that HIV infection results in a severe dysfunction in most of these effector subsets, warrants approaches that take this into consideration. One reason for the dysfunction of immune cells is the disruption of the common γ chain family of cytokines during HIV infection. These cytokines play vital role in regulating functional activities of such immune effector cells. Consequently, besides and along with cells, these cytokines including IL-2, IL-7, IL-15 and IL-21 have shown some promise against HIV infection. This review briefly summarizes the immune therapy options in HIV infection, with special focus on innate cells and cytokines.

Impact of protective killer inhibitory receptor/human leukocyte antigen genotypes on natural killer cell and T-cell function in HIV-1-infected controllers.

Both protective T-cell genotypes and natural killer (NK) cell genotypes have been associated with delayed progression to AIDS and shown to be co-inherited in HIV-1-infected individuals who limit viral replication in absence of antiretroviral therapy ('controllers'). However, a comparative analysis of the genotype and function of the innate and adaptive immune compartments in HIV-1-infected controller individuals has been understudied to date. Here, we simultaneously tested NK and T-cell function in controllers to investigate the mechanism(s) that might account for host immune control over viral replication. We measured CD8 T-cell responses against HIV-1 utilizing overlapping 15-mer peptides spanning the HIV-1 consensus clade B Gag protein and tested NK cell degranulation and cytokine secretion against tumor target cells following interferon-α (IFNα) stimulation. Among a cohort of 37 controllers, the presence of protective major histocompatibility complex class I human leukocyte antigen (HLA) alleles (such as HLA-B*57) was not correlated with HIV-specific CD8 responses. In contrast, the inheritance of a protective killer inhibitory receptor KIR3DL1*h/*y receptor genotype along with the corresponding HLA-Bw4*80I ligand was associated with significantly heightened target cell-induced NK degranulation and cytokine secretion following IFNα stimulation (P = 0.0201, n = 13). Interestingly, we observed a significant inverse association between the IFNα stimulated NK response to K562 cells and the HIV-specific CD8 T-cell response to Gag among elite controllers (rho = -0.8321, P = 0.0010, n = 12). Together, these results suggest that heightened NK responses can be evidenced independently of HIV-specific T-cell responses in HIV-1-infected elite controllers.

Expression of CD200 on AML blasts directly suppresses memory T-cell function

Previous studies have shown that immunosuppression in acute myeloid leukemia (AML) is associated with changes in the adaptive immune compartment. Such changes include the suppression of memory T-cell function1 and the suppression of Th1 cytokine (TNFα, IL-2 and IFNγ)-producing cells.2 A suppressed immune response in AML is associated with a worse patient outcome and increased risk of relapse,3 as well as increased risk of infection impairing patient recovery.4 The over-expression of the immunosuppressive ligand CD200 is also associated with an increased risk of relapse in AML (hazard ratio 1.7); an observation consistent with a hypothesis in which CD200 inhibits clearance of residual disease.5, 6 As memory T-cell responses are central for tumor immunosurveillance and contribute to prolonged molecular remission,7 we carried out this study to establish how these responses were affected in AML patients over-expressing CD200 (Supplemental Table S1). We initially investigated whether CD200 expression on AML blasts influenced CD8+ T-cell cytotoxic potential and the frequency of TNFα-, IL-2- and IFNγ-producing CD4+/CD8+ memory T-cells (Supplementary Materials and Methods and Supplemental Figure S1 for gating strategy). Using CD107a as a marker of cytotoxic function, AML cells were activated with PMA/ionomycin. We show that the frequency of CD107a+CD8+ memory T-cells was significantly reduced by ~50% for CD200hi patients when compared with CD200lo AML, demonstrating that cytotoxic memory T-cell activity was compromised in CD200hi patients (Figure 1a). Furthermore, the frequencies of TNFα-, IL-2- and IFNγ-producing CD4+ memory cells were also reduced by ~50% for CD200hi patients when compared with CD200lo AML (Figure 1b), significantly so in the case of IL-2 and IFNγ. Interestingly, CD200lo patients displayed a higher IFNγ response, not only with respect to CD200hi patients but also in comparison to healthy donors, suggesting a role for this cytokine in AML, which is attenuated by CD200. No difference was observed for TNFα-, IL-2- and IFNγ-producing CD8+ memory cells between CD200hi, CD200lo and healthy donors (data not shown). CD200 has also been reported to mediate suppression of the Th1 response in chronic lymphocytic leukemia as well as solid tumors,8, 9 suggesting that CD200-mediated Th1 suppression is a central mechanism in cancer immunomodulation.

Helminth Infection and Type 1 Diabetes

The increasing incidence of type 1 diabetes (T1D) and autoimmune diseases in industrialized countries cannot be exclusively explained by genetic factors. Human epidemiological studies and animal experimental data provide accumulating evidence for the role of environmental factors, such as infections, in the regulation of allergy and autoimmune diseases. The hygiene hypothesis has formally provided a rationale for these observations, suggesting that our co-evolution with pathogens has contributed to the shaping of the present-day human immune system. Therefore, improved sanitation, together with infection control, has removed immunoregulatory mechanisms on which our immune system may depend. Helminths are multicellular organisms that have developed a wide range of strategies to manipulate the host immune system to survive and complete their reproductive cycles successfully. Immunity to helminths involves profound changes in both the innate and adaptive immune compartments, which can have a protective effect in inflammation and autoimmunity. Recently, helminth-derived antigens and molecules have been tested in vitro and in vivo to explore possible applications in the treatment of inflammatory and autoimmune diseases, including T1D. This exciting approach presents numerous challenges that will need to be addressed before it can reach safe clinical application. This review outlines basic insight into the ability of helminths to modulate the onset and progression of T1D, and frames some of the challenges that helminth-derived therapies may face in the context of clinical translation.

Functional Alterations of Proinflammatory Monocytes by T Regulatory Cells: Implications for the Prevention and Reversal of Type 1 Diabetes

The onset and development of type 1 diabetes (T1D) occurs in genetically predisposed individuals, and is attributed to autoimmune destruction of pancreatic beta-cells involving a multitude of immune mechanisms. Defects in immune regulation may play a central role in T1D, involving impaired function and communication of both myeloid and lymphoid cells of the innate and adaptive immune compartments. Dendritic cells and regulatory T (Treg) cells are part of this network, which seem to be hampered in their quest to control and regulate tissue-destructive autoimmunity. Recent studies have shown that in vivo activated CD16- blood monocytes exhibiting proinflammatory features are present in diabetic subjects. These monocytes may govern T cell-mediated immune responses towards the development of tissue-destructive Th1 and Th17 subtypes, and give rise to inflammatory macrophages in tissues. Differential effects of cytokines IFN-gamma and IL-4 in the development of inflammatory macrophages, and the distinct developmental pathways of proinflammatory or tissue-repair-associated monocytes suggest that controlling the activity of these monocytes could be part of an immune intervention strategy to prevent T1D. Similarly, strategies to target autoantigens to immature, steady-state dendritic cells could guide the immune response away from Th1 and Th17 immune effectors. This review examines potential approaches to this goal by manipulation of myeloid and lymphoid cell regulatory networks in T1D.

Immune regulation of embryo implantation—all about quality control?

Conventional belief holds that an immune response to ejaculate antigens should interfere with fertilisation and establishment of pregnancy. However, emerging evidence now supports the opposing view—that insemination acts to activate maternal immune mechanisms exerting a positive effect on reproductive events. In a response well documented in rodents, semen triggers an influx of antigen-presenting cells into the female reproductive tract which process and present paternal ejaculate antigens to elicit activation of lymphocytes in the adaptive immune compartment. Transforming growth factor beta (TGFβ), a cytokine present in abundance in seminal plasma, initiates this inflammatory response by stimulating the synthesis of pro-inflammatory cytokines and chemokines in uterine tissues. Lymphocyte activation is evident in lymph nodes draining the uterus and leads to hypo-responsiveness in T-cells reactive with paternal alloantigens. TGFβ has potent immune-deviating effects and is likely to be the key agent in skewing the immune response against a Type-1 bias. Prior exposure to semen in the context of TGFβ can be shown to be associated with enhanced fetal–placental development late in gestation. In this paper, we review the experimental basis for these claims and propose the hypothesis that, in women, the partner-specific protective effect of insemination in pre-eclampsia might be explained by induction of immunological hypo-responsiveness conferring tolerance to histocompatibility antigens present in the ejaculate and shared by the conceptus.

B Lymphocytes from Patients with Tuberculosis Exhibit Hampered Antigen‐Specific Responses with Concomitant Overexpression of Interleukin‐8

To the Editor-Tuberculosis due to Mycobacterium tuberculosis is 1 of the 3 major killers among infectious diseases. Deciphering the interactions between M. tuberculosis and the innate and adaptive immune compartments of the host is critical for understanding the pathogenesis of tuberculosis and for designing effective immunotherapeutic interventions. By use of a juvenile rhesus monkey model, Qiu et al recently demonstrated that severe tuberculosis induces unbalanced up-regulation of the immune gene networks of inflammatory cytokines, chemokines, and their receptors, but low levels of T cell responses specific to purified protein derivative [1]. The authors conclude that the overexpression of immune genes after tuberculosis infection favors inflammation and suppression of antigen-specific T cells.

Platelet influence on T- and B-cell responses

Understanding the adaptive immune response is an area of research critically important in medicine. Several positive regulators of B- and T-cell activation exist to eliminate pathogens, in which CD40 ligand (CD154) plays a fundamental role. It is well documented that CD154 expressed by CD4 T helper cells can be critical in the proper activation of dendritic cells for the productive stimulation of CD8 T cells and is required for proper T-dependent B-cell immunity. However, platelets are an abundant and systemic source of CD154. While classically known to be important for hemostasis and inflammation, several lines of evidence suggest that platelet-derived ligands can modulate the adaptive immune compartment.

Prevention of Autoimmunity and Control of Recall Response to Exogenous Antigen by Fas Death Receptor Ligand Expression on T Cells

Mice with mutations in the gene encoding Fas ligand (FasL) develop lymphoproliferation and systemic autoimmune diseases. However, the cellular subset responsible for the prevention of autoimmunity in FasL-deficient mice remains undetermined. Here, we show that mice with FasL loss on either B or T cells had identical life span as littermates, and both genotypes developed signs of autoimmunity. In addition, we show that T cell-dependent death was vital for the elimination of aberrant T cells and for controlling the numbers of B cells and dendritic cells that dampen autoimmune responses. Furthermore, we show that the loss of FasL on T cells affected the follicular dentritic cell network in the germinal centers, leading to an impaired recall response to exogenous antigen. These results disclose the distinct roles of cellular subsets in FasL-dependent control of autoimmunity and provide further insight into the role of FasL in humoral immunity.

Late CD8+ T Cell-Dependent Xenoantibody Production in Innate Tolerant Nude Rats After Hamster Islet Grafting But Not After Hamster Heart Grafting

Xenograft rejection can be provoked by both the innate and adaptive immune compartments and close reciprocal interactions exist between these two systems. We investigated the interdependent roles of T and B lymphocytes in vascularized (heart) and cellular (islet) xenograft rejection in a model with established xeno-nonreactivity of the innate immune system. Specific innate xenotolerance was induced in nude rats bearing either a hamster heart or a hamster pancreatic islet graft by a tolerizing regimen consisting of donor antigen infusion, temporary natural killer cell depletion and a 4-week administration of leflunomide. One month after transplantation, syngeneic CD4 and CD8 T cells were adoptively transferred. Both vascular and cellular xenografts were rejected after CD4 T cell reconstitution, corresponding with production of high IgM and IgG xenoantibody titers. Deposition of xenoantibodies and complement was seen in the heart but not in the islet xenografts. After infusion of CD8 T cells, xenohearts underwent a delayed type of rejection without xenoantibody production and xenoislets were not rejected. In xenoislet recipients, CD8 dependent B cells were not tolerized, resulting in the production of IgG xenoantibodies belonging to Th2-dependent isotypes, known not to cause graft rejection, and deposited at the graft implantation site. We conclude that distinct mechanisms of immune activation underlie xenogeneic reactions against vascular and cellular grafts.

Benznidazole Treatment during Early‐indeterminate Chagas’ Disease Shifted the Cytokine Expression by Innate and Adaptive Immunity Cells toward a Type 1‐modulated Immune Profile

Trypanosoma cruzi-infected children was treated with benznidazole (Bz) during the early-indeterminate disease (E-IND) and the cytokine pattern of innate and adaptive immune compartments were evaluated prior to the treatment and 1 year after it. At first, we observed that the ex vivo cytokine profile of circulating leukocytes from E-IND (n = 6) resembled the one observed for healthy schoolchildren (n = 7). Additionally, in vitro stimulation with T. cruzi antigens drove the E-IND cytokine pattern toward a mixed immune profile with higher levels of IFN-gamma+, TNF-alpha+ and IL-4+ NK cells, increased numbers of IFN-gamma+, TNF-alpha+ and IL-10+ CD4+ T cells in addition to enhanced frequency of TNF-alpha+/IL-4+ CD19+ lymphocytes. Interestingly, upon T. cruzi antigen in vitro stimulation, E-IND CD8+ lymphocytes displayed a selective enhancement of IFN-gamma expression, accounting for a global type 1-modulated cytokine microenvironment. A shift toward a type 1-modulated profile was also the hallmark of Bz-treated children (E-IND(T)). In this context, despite the mixed overall ex vivo cytokine profile observed for NK and CD8+ T cells, increased ability of these leukocytes to produce IFN-gamma in response to T. cruzi antigens was reported. Most noteworthy was the IL-10 production evidenced at T lymphocytes, mainly CD4+ cells, as well as B lymphocytes, both ex vivo and upon antigen stimulation. Together, these findings gave evidence that NK cells and CD8+ T lymphocytes are the major sources of IFN-gamma, a pivotal cytokine for successful therapeutic response in human Chagas' disease. Moreover, our data have also brought additional information, pointing out IL-10 production by CD4+ cells and B lymphocytes, as the putative key element for parasite clearance in the absence of deleterious tissue damage.

Cooperation between platelet-derived CD154 and CD4+ T cells for enhanced germinal center formation

It has been demonstrated previously that platelet-derived CD154 communicates with the adaptive immune compartment, enhancing B and T cell responses in CD154(-/-) mice. The presence of platelets was also shown to be necessary for optimal production of immunoglobulin G (IgG) in normal C57BL/6 mice. These data led us to hypothesize that platelets perform a sentinel function, quickly relaying activating signals to the adaptive immune compartment. Here, we report that platelet-derived CD154 increases serum IgG levels and germinal center formation under conditions where antigen-specific CD4(+) T cell numbers are limiting. We propose that in the physiologic setting where antigen-specific B and T cells are rare, platelets function to enhance signals required for robust adaptive humoral immunity.

Platelet-Mediated Modulation of Adaptive Immunity: A Communication Link between Innate and Adaptive Immune Compartments

Platelets are highly reactive components of the circulatory system with well-documented hemostatic function. Recent studies extend platelet function to modulation of local inflammatory events through the release of chemokines, cytokines, and a number of immunomodulatory ligands, including CD154. We hypothesized that platelet-derived CD154 modulates adaptive immunity. The data reported herein demonstrate that platelets, via CD154, induce dendritic cell maturation, B cell isotype switching, and augment CD8 + T cell responses both in vitro and in vivo. Platelet transfusion studies demonstrate that platelet-derived CD154 alone is sufficient to induce isotype switching and augment T lymphocyte function during viral infection, leading to enhanced protection against viral rechallenge. Additionally, depletion of platelets in normal mice results in decreased antigen-specific antibody production.

Monokine induced by IFN-gamma is a dominant factor directing T cells into murine cardiac allografts during acute rejection.

The use of chemokine antagonism as a strategy to inhibit leukocyte trafficking into inflammatory sites requires identification of the dominant chemokines mediating recruitment. The chemokine(s) directing T cells into cardiac allografts during acute rejection remain(s) unidentified. The role of the CXC chemokines IFN-gamma inducible protein 10 (IP-10) and monokine induced by IFN-gamma (Mig) in acute rejection of A/J (H-2(a)) cardiac grafts by C57BL/6 (H-2(b)) recipients was tested. Intra-allograft expression of Mig was observed at day 2 posttransplant and increased to the time of rejection at day 7 posttransplant. IP-10 mRNA and protein production were 2.5- to 8-fold lower than Mig. Whereas allografts were rejected at day 7-9 in control recipients, treatment with rabbit antiserum to Mig, but not to IP-10, prolonged allograft survival up to day 19 posttransplant. At day 7 posttransplant, allografts from Mig antiserum-treated recipients had marked reduction in T cell infiltration. At the time of rejection in Mig antiserum-treated recipients (i.e., days 17-19), intra-allograft expression of macrophage-inflammatory protein-1alpha, -1beta, and their ligand CCR5 was high, whereas expression of CXCR3, the Mig receptor, was virtually absent. Mig was produced by the allograft endothelium as well as by recipient allograft-infiltrating macrophages and neutrophils, indicating the synergistic interactions between innate and adaptive immune compartments during acute rejection. Collectively, these results indicate that Mig is a dominant recruiting factor for alloantigen-primed T cells into cardiac allografts during acute rejection. Although Mig antagonism delays acute heart allograft rejection, the results also suggest that the alloimmune response circumvents Mig antagonism through alternative mechanisms.

Potential role of seminal plasma TGFβ, in the initiation of the post-coital inflammatory response in humans

Conventional belief holds that an immune response to ejaculate antigens should interfere with fertilisation and establishment of pregnancy. However, emerging evidence now supports the opposing view—that insemination acts to activate maternal immune mechanisms exerting a positive effect on reproductive events. In a response well documented in rodents, semen triggers an influx of antigen-presenting cells into the female reproductive tract which process and present paternal ejaculate antigens to elicit activation of lymphocytes in the adaptive immune compartment. Transforming growth factor beta (TGFβ), a cytokine present in abundance in seminal plasma, initiates this inflammatory response by stimulating the synthesis of pro-inflammatory cytokines and chemokines in uterine tissues. Lymphocyte activation is evident in lymph nodes draining the uterus and leads to hypo-responsiveness in T-cells reactive with paternal alloantigens. TGFβ has potent immune-deviating effects and is likely to be the key agent in skewing the immune response against a Type-1 bias. Prior exposure to semen in the context of TGFβ can be shown to be associated with enhanced fetal–placental development late in gestation. In this paper, we review the experimental basis for these claims and propose the hypothesis that, in women, the partner-specific protective effect of insemination in pre-eclampsia might be explained by induction of immunological hypo-responsiveness conferring tolerance to histocompatibility antigens present in the ejaculate and shared by the conceptus.