Autologous Mixed Leukocyte Reaction Research Articles

T Cell Exhaustion/Senescence in Relapsed Multiple Myeloma after Autologous Stem Cell Transplantation

▪BACKGROUND: Multiple myeloma (MM) is the most common indication for high-dose chemotherapy and autologous stem cell transplantation (ASCT). Post-transplant lenalidomide maintenance therapy doubles progression-free survival, but almost all patients eventually relapse. The immune system participates in the control of MM, whereas compromised immunity contributes to its evolution. Post-transplant immunotherapy to induce or restore antitumor immunity offers a promising approach to target residual MM and improve patient outcomes. The rational development of immunotherapeutic interventions after ASCT, however, requires a comprehensive understanding of the immunologic milieu. We therefore evaluated lymphocyte composition and function after ASCT to guide optimal timing of immunotherapy and to identify potential markers of relapse.METHODS: Fifty-five MM patients undergoing ASCT were evaluated for at least one year. Peripheral blood from patients was obtained before ASCT and on d +12, +30, +90, +180, and +365 after ASCT, and at the time of relapse where applicable. Leukocyte concentrates were used as a source of healthy donor cells. Mononuclear cells were analyzed by flow cytometry for phenotypic assessment of lymphocyte subset composition. Functional assessment of dendritic cell and T cell activity in vitro was assayed in autologous and allogeneic mixed leukocyte reactions, cytotoxic T lymphocyte (CTL) lysis assays, and PD-1 blockade experiments.RESULTS: CD3+ CD4+ CD25bright CD127neg regulatory T cells (Tregs) decline as CD8+ T cells expand during early lymphocyte recovery after ASCT, markedly reducing the Treg:CD8+ effector T-cell ratio (Fig 1A) and providing a critical early window for the introduction of immune-based post-transplant consolidation therapies. CD8+ T cells can respond to autologous dendritic cells presenting tumor antigen in vitro as early as day +12 post-transplant, becoming antigen-specific CTL effectors and thereby demonstrating preservation of cellular reactivity (Fig 1B). CD4+ and CD8+ T cells express the negative regulatory molecules, CTLA-4, PD-1, LAG-3, and TIM-3, before and after ASCT (data not shown). A subpopulation of exhausted/senescent CD8+ T cells, however, down-regulates CD28 (Fig 2A) and up-regulates CD57 (Fig 2B) and PD-1 (Fig 2C), characterizing immune impairment and relapse after ASCT. CD4+ T cells show the same trends in the frequencies of CD28neg, CD28neg CD57+, and CD28neg PD-1+ cells, albeit at lower levels of expression (Fig 2D). Relapsing patients have higher numbers of CD8+ CD28neg PD-1+ T cells at +3 months after transplant (Fig 2E), but before detection of clinical disease, indicating their applicability in identifying patients at higher risk of relapse. PD-1 blockade revives the proliferation and cytokine secretion of the hyporesponsive, CD8+ CD28neg PD-1+ T cells in vitro (Fig 3).CONCLUSION: These results identify T cell exhaustion/senescence as a distinguishing feature of relapse and support early introduction of immunotherapy to stimulate antitumor immunity after ASCT. [Display omitted] [Display omitted] DisclosuresLesokhin:Genentech: Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Aduro: Consultancy; Janssen: Consultancy, Research Funding; Efranat: Consultancy. Giralt:CELGENE: Consultancy, Honoraria, Research Funding; SANOFI: Consultancy, Honoraria, Research Funding; AMGEN: Consultancy, Research Funding; JAZZ: Consultancy, Honoraria, Research Funding, Speakers Bureau; TAKEDA: Consultancy, Honoraria, Research Funding.

A human dendritic cell-based in vitro model to assess Mycobacterium tuberculosis SO2 vaccine immunogenicity.

Among the tuberculosis (TB) vaccine candidates, SO2 is the prototype of the first live-attenuated vaccine that recently entered into clinical trials. To investigate the capacity of SO2 to stimulate an appropriate immune response in vitro within a human immunological context, a comparative analysis of the effects promoted by SO2, the current Bacille Calmette-Guerin (BCG) vaccine and Mycobacterium tuberculosis (Mtb) was conducted in human primary dendritic cells (DC), which are critical modulators of vaccine-induced immunity. In particular, we found that SO2 promotes the expression of maturation markers similarly to BCG but at a lower extent than Mtb. Moreover, SO2-infected DC released higher levels of interleukin (IL)-23 than BCG-infected cells, which account for the expansion of interferon (IFN)-γ-producing T cells in an IL-12-independent manner. In the autologous mixed leukocyte reaction setting, the expansion of IL-17-producing T cells was also observed in response to SO2 infection. Interestingly, apoptosis and autophagic flux, events required for the antigen presentation within MHC class II complex, were not affected in DC infected with SO2, conversely to what observed upon Mtb stimulation. Collectively, our results indicate that SO2 represents a promising TB vaccine candidate, which displays an attenuated phenotype and promotes in DC a stronger capacity to stimulate the Th response than BCG vaccine. Interestingly, the data obtained by using the human DC-based experimental setting mirrored the results derived from studies in animal models, suggesting that this system could be used for an efficient and rapid down-selection of new TB vaccine candidates, contributing to achieve the "3Rs" objective.

Activation-Induced FoxP3 Expression Regulates Cytokine Production in Conventional T Cells Stimulated with Autologous Dendritic Cells

A defining feature of dendritic cells (DCs) is their ability to induce the proliferation of autologous T cells in the absence of foreign antigen-a process termed the "autologous mixed leukocyte reaction" (AMLR). We report that equine monocyte-derived DCs, but not macrophages, are potent inducers of the AMLR. The response is contact dependent and major histocompatibility complex class II dependent and primarily involves CD3(+) CD4(+) CD8(-) T cells. Upon stimulation with DCs or the mitogen concanavalin A, a subset of the proliferating T cells expresses the regulatory T-cell (Treg) transcription factor FoxP3. Although many of these FoxP3(+) T cells are capable of producing the effector cytokines interleukin-4 (IL-4) and gamma interferon (IFN-γ), they are more likely to produce IL-10 and less likely to produce IFN-γ than equivalent FoxP3(-) cells. Therefore, FoxP3 expression is an inherent component of equine T cell activation and is associated with a more immunosuppressive cytokine profile. These results confirm that FoxP3 expression in the horse, in contrast to the mouse, is regulated similarly to FOXP3 expression in humans and provide evidence that FoxP3 expression by conventional T cells may help regulate the developing immune response.

The evaluation of survival and proliferation of lymphocytes in autologous mixed leukocyte reaction with dendritic cells. The comparison of incorporation of 3H-thymidine and differential gating method

Dendritic cells (DCs) play the key role in T-lymphocyte proliferation and induction of antitumour response. The mixed leukocyte reaction (MLR) of T-lymphocytes and DCs is essential instrument for immunological mechanisms studies. Conventionally used method for determination of T-lymphocytes proliferation, 3H-thymidine incorporation, provides only general information. The method of flow cytometry and differential gating seems to be more suitable for quantitative and qualitative analysis of T-lymphocyte proliferation. It is based on time limited acquisition of events and on its distribution according to forward and side scatter values. We decided to compare these two methods and determine mutual correlation and compatibility. Eleven patients were studied and in all cases DCs promoted the survival and proliferation of both CD4 and CD8 lymphocytes. Both methods retained consistency with regard to survival and proliferation of CD4/CD8 lymphocytes. However, the correlation of these methods was not convincing. Therefore, both these methods might be used for evaluation of MLR, but each of them gives specific and complementary information.

Functional analysis of dendritic cell–T cell interaction in sarcoidosis

The primary cause of the intense immune response in sarcoidosis is unclear. Potentially, a functional abnormality in dendritic cells (DCs) could cause a reduction in clearance of antigen and downstream persistence in immune activity. In this study, we investigate the interaction between monocyte-derived dendritic cells and T cells in patients with sarcoidosis compared to normal controls (n = 8 each) by examining the kinetics of autologous and allogeneic mixed leucocyte reactions over 9-10 days. We found markedly depressed proliferation kinetics in autologous DC-peripheral blood mononuclear cell (PBMC) co-cultures from sarcoid patients compared to normal subjects. In allogeneic experiments PBMCs from patients showed a reduced response to allogeneic DCs from a single donor, but no difference was observed in the ability of patients and control DCs to stimulate proliferation of allogeneic PBMC from a single donor. We conclude that there is a markedly impaired autologous mixed leucocyte reaction (MLR) in sarcoidosis patients. In allogeneic MLR, monocyte-derived DCs in sarcoidosis were able to stimulate T cells normally, but PBMCs responses were reduced. This contradicts recent published studies on ex vivo isolated myeloid DCs from sarcoidosis patients although, potentially, an in vivo conditioning factor, which reduces DC function in sarcoidosis, could be a unifying explanation for the contrasting findings.

The distribution and functional properties of dendritic cells in patients with seronegative arthritis

Dendritic cells (DC), potent antigen-presenting cells, are known to be increased in numbers in inflammatory lesions in rheumatoid arthritis and juvenile chronic arthritis. In this study, patients with seronegative arthritis were studied; the distribution and functional properties of DC enriched low density cells (LDC) from peripheral blood (PB) and synovial fluid (SF) were compared. The composition of LDC from both sources was similar, comprising approximately 30% DC, 60% monocytes with few T lymphocytes. SF was significantly enriched for LDC compared with paired peripheral blood (P less than 0.0001) or peripheral blood from healthy controls (P less than 0.001). In contrast, patient PB contained fewer LDC (P less than 0.05) overall than healthy controls. LDC from both sources were potent simulators of allogeneic PB T cells in a mixed leucocyte reaction (MLR), but in four out of 10 patients SF LDC were significantly more stimulatory. In autologous MLRs (AMLRs) SF T cells were not stimulated by either LDC population. This anergy of T cells was confined to the joint as patient PB T cells showed an AMLR response to PB LDC which was similar to that seen in cells from healthy controls. PB T cells also responded to SF LDC; in a minority of patients SF LDC caused significantly greater stimulation in AMLR than PB LDC and the possibility is discussed that this may represent presentation of antigen acquired in vivo.

Characterization of the binucleated giant cells generated in the autologous mixed leucocyte reaction from patients with rheumatoid arthritis

Binucleated giant cells several times larger than lymphocytes or monocytes were generated in an autologous mixed leucocyte reaction (AMLR) independent of DNA synthesis in patients with rheumatoid arthritis (RA). The AMLR giant cells with multiple cytoplasmic granules were non-specific esterase-staining positive, phagocytic, non-adherent, HLA-DR+, CD11b+, CD14+, 4F2+, CDW29+, and anti-transferrin receptor positive, but negative for T, B, or NK markers. RA patients aged less than 60 years from more giant cells: 12.6 +/- 13.5% (n = 33) as compared with 0.4 +/- 1.5% in age- and sex-matched normals (n = 38, (P less than 0.001). More giant cells were seen over age 60 in both groups: RA 20.1 +/- 15.5% (n = 5) and healthy controls 3.0 +/- 3.2% (n = 8) (P less than 0.01). Neither disease activity nor treatment appear to influence the result in RA. The giant cells that are probably derived from monocytes in AMLR may explain the formation of the giant cells in rheumatoid granulation tissues.

Dendritic cells infected in vitro with human T cell leukaemia/lymphoma virus type-1 (HTLV-1); enhanced lymphocytic proliferation and tropical spastic paraparesis.

Evidence supporting a role of the dendritic cell (DC) in stimulating autologous T cell activity in tropical spastic paraparesis (TSP) was sought by studies of cells taken from healthy volunteers and exposed to HTLV-1 in vitro. DC were co-cultured with an HTLV-1-producing cell line (MT-2) at 1:1 or 10:1 ratios. These DC stimulated high levels of proliferation in autologous T cells. This was similar to that seen in an autologous mixed leucocyte reaction (AMLR) using cells from TSP patients. The requirement for both DC and virus was confirmed, since neither DC co-cultured with uninfected MT-2 cells nor addition of infected MT-2 cells directly to T cells caused significant stimulation. DC exposed to the highest dose of HTLV-1 (1:1) for 24 h before addition of T cells caused strong stimulation that increased after 8 h but almost disappeared by 72 h. In situ hybridization showed that approximately 25% of DC became infected in cultured cells after preincubation for 24 h, and over 50% were infected with a 72-h preincubation. We suggest that infection of DC by HTLV-1 may be an initial step in altering the immune system in seronegative patients, and that persistent T cell stimulation in those with genetic susceptibility may underlie the production of neurological disease.

Mature Conventional Human Dendritic Cells Express Indoleamine 2,3-Dioxygenase and Support the Relative Expansion of Autologous Regulatory T Cells.

Effective immunotherapy must overcome tolerance toward tumor antigens and avoid subsequent inhibition of stimulated antitumor immunity. The specific contribution of immune regulatory mechanisms intrinsic to dendritic cells (DCs), especially with regard to regulatory T cells (T regs), is of emerging importance. We have found that all conventional, immunogenic human DCs express the immunomodulatory enzyme indoleamine 2,3-dioxygenase (IDO) and that IDO protein expression and activity are markedly increased in mature compared with immature DCs. Priming of resting T cells with mature IDO+ DCs in an autologous mixed leukocyte reaction (MLR) increases the proportion of CD4+CD25+ T cells capable of suppressing allogeneic T cell proliferation in secondary MLRs as much as 10-fold above baseline (Figure 1A). Conversely, 1-methyl-tryptophan, a competitive inhibitor of IDO, dampens the inhibitory activity (Figure 1A). Further characterization of the suppressor T cells was performed after cytofluorographic sorting into CD4+CD25hi, CD4+CD25int, and CD4+CD25low/− subpopulations. Post-sort analysis revealed that the majority (>60%) of the CD4+CD25hi cells coexpressed Foxp3, which was absent in the CD4+CD25low/− cells. Separate studies showed that these Foxp3+ cells express little or no CD127 (IL-7R-alpha). Candidate CD4+CD25hi T regs inhibited DC-stimulated allogeneic T cell proliferation in a dose dependent manner, with >90% inhibition at a suppressor to responder T cell ratio of 1:1 and ∼50% inhibition at a ratio as low as 1:25 (Figure 1B). CD4+CD25low/− cells were not inhibitory, and CD4+CD25int cells exerted intermediate suppression depending on dose (Figure 1B). CD4+CD25hi T regs exert similar inhibition of autologous T cell responses to stimulation de novo by DCs. Both the priming and effector phases of T reg suppression were contact dependent. Moreover, depletion of the trace population of CD4+CD25hi T cells at the outset of autologous priming largely abolished the relative expansion of this population. These results clearly demonstrate that mature conventional human DCs support relative but significant expansion of autologous, constitutive CD4+CD25hi T cells, which coexpress Foxp3, express little or no CD127, and exert significant suppression of both allogeneic and autologous T cells stimulated de novo by DCs. Although contrary to the anticipated enrichment of IDO in immature DCs because of their expected tolerogenicity, these findings underscore the importance of regulatory mechanisms exerted by immunogenic cells like mature conventional DCs. While this may provide a physiologic means of turning off an otherwise unchecked immune response, this IDO-mediated pathway in DCs provides a rational target for optimizing host immune responses against tumor antigens. This should result in more sustained benefit from active immunotherapy with DC-based vaccines. [Display omitted]

Phenotypic and functional characteristics of dendritic cells derived from human peripheral blood monocytes

This study is aimed at developing a simple and easy way to generate dendritic cells (DCs) from human peripheral blood monocytes (PBMCs) in vitro. PBMCs were isolated directly from white blood cell rather than whole blood and purified by patching methods (collecting the attached cell and removing the suspension cell). DCs were then generated by culturing PBMCs for six days with 30 ng/ml recombinant human granulocyte-macrophage stimulating factor (rhGM-CSF) and 20 ng/ml recombinant human interleukin-4 (rhIL-4) in vitro. On the sixth day, TNF-alpha (TNFalpha) 30 ng/ml was added into some DC cultures, which were then incubated for two additional days. The morphology was monitored by light microscopy and transmission electronic microscopy, and the phenotypes were determined by flow cytometry. Autologous mixed leukocyte reactions (MLR) were used to characterize DC function after TNFalpha or lipopolysaccharide (LPS) stimulations for 24 h. After six days of culture, the monocytes developed significant dendritic morphology and a portion of cells expressed CD1a, CD80 and CD86, features of DCs. TNFalpha treatment induced DCs maturation and up-regulation of CD80, CD86 and CD83. Autologous MLR demonstrated that these DCs possess potent T-cell stimulatory capacity. This study developed a simple and easy way to generate DCs from PBMCs exposed to rhGM-CSF and rhIL-4. The DCs produced by this method acquired morphologic and antigenic characteristics of DCs.

Immunological Responses Are Not Abnormal in Symptomatic Gulf War Veterans

The underlying etiology and pathogenesis of Gulf War veterans' illnesses continue to be under intense investigation. Reports have suggested the basis for these illnesses may be an altered immune system, but compelling evidence is lacking. We sought to determine whether in vitro immune responses were abnormal in symptomatic Gulf War veterans relative to matched controls. A randomized case-control study was conducted by blinded comparison of laboratory measures of in vitro immune responses in blood samples obtained from veterans in an outpatient facility of a Veterans Affairs medical center. Symptomatic Gulf War veterans with otherwise undefined illnesses (52 symptomatic subjects), asymptomatic Gulf War veterans (31 asymptomatic controls), and veterans who had applied for disability compensation and had not participated in the Gulf War (21 disability controls) represented the volunteer sample. In vitro cellular and humoral immune responses were measured to detect functional abnormalities in antigen presenting cells (autologous mixed leukocyte reactions and expression of interleukin (IL)-1beta, IL-6, IL-10, and tumor necrosis factor-alpha); T cells (lymphocyte proliferation using the polyclonal T-cell activators phytohemagglutinin and Concanavalin A; primary immune responses in allogeneic mixed leukocyte reactions; secondary immune response using the recall antigens tetanus toxoid, Candida albicans, and anthrax vaccine; and soluble IL-2 receptor expression); type-1 T-helper cells (gamma interferon expression); type-2 T-helper cells (IL-4 and IL-10 expression); and B cells (polyclonal B-cell activator pokeweed mitogen-induced immunoglobulin production). In general, immune response measures did not differ significantly between groups. Heightened responses observed in the disability control group (sporadically greater responses to one mitogen and two antigens) and the Gulf War participation control group (greater recall responses to anthrax vaccine) did not suggest impaired immune cell function in symptomatic veterans when compared with controls. We conclude that in vitro immunological responses are not abnormal in symptomatic Gulf War veterans.

Monocyte differentiation in localized juvenile periodontitis is skewed toward the dendritic cell phenotype.

Several lines of evidence indicate that the monocytes of subjects with localized juvenile periodontitis (LJP) are functionally distinct from cells of age- and race-matched nonperiodontitis (NP) subjects. Among the abnormalities are the propensity to secrete large amounts of prostaglandin E(2) and the induction of immunoglobulin G2 (IgG2) antibodies. The experiments described here were performed to further characterize the LJP monocytes and to determine if these cells mature differently than NP monocytes. When adherent monocytes from LJP subjects were cultured in the presence of human serum, both macrophages and cells with the morphology of immature monocyte-derived dendritic cells (MDDC) were observed. Within 4 days the prevalence of the immature MDDC was approximately twofold higher in LJP cultures than in NP cultures. In addition to their dendritic morphology, these cells were CD11c(+) and CD14(-) or CD14(low) and stimulated potent autologous mixed leukocyte reactions, consistent with differentiation to the MDDC phenotype. Like LJP monocytes, cultures of MDDC generated with interleukin-4 and granulocyte-macrophage colony-stimulating factor selectively induced IgG2 in cultures of pokeweed mitogen-stimulated NP leukocytes. Together, these data suggest that the monocytes of LJP subjects have a propensity to differentiate into MDDC and that this differentiation may be related to the high levels of IgG2 that are observed in the sera of LJP subjects. As high levels of circulating IgG2 are correlated with less severe disease, the propensity of LJP monocytes to differentiate into MDDC may have important implications for both the host response against oral pathogens and the progression of LJP.

High levels of transduction of human dendritic cells with optimized SIV vectors.

As major antigen-presenting cells and effectors in the maintenance of tolerance, dendritic cells (DCs) are key cells of the immune system and can thus be envisioned to have roles in immunotherapy strategies. We, and others, previously showed that simian immunodeficiency virus (SIV)-derived lentiviral vectors were able to deliver a gene into human differentiated DCs. We describe here the upgrading of the SIV vector system and the improvements of the transduction protocol, which allowed us to transduce more than 90% of human monocyte-derived DCs. We developed new SIV lentiviral vectors carrying SIV splice regulatory elements and either the woodchuck hepatitis virus regulatory element (WPRE) or the murine phosphoglycerate-kinase 1 (PGK) promoter. We show that insertion of the WPRE in the SIV vector is detrimental to gene transfer in DCs, while this sequence increases transgene expression in 293T cells. Using an optimized SIV vector, high levels of transgene expression were obtained in more than 30% of human DCs at a multiplicity of infection (MOI) of 1, and close to 100% using a MOI of 20. VSV-G pseudotyped vectors generated with only gag, pol, tat, and rev helper functions failed to transduce DCs. This defect was completely rescued when the SIV accessory gene vpx was provided in trans in vector-producing cells. Genetically modified DCs were shown to behave as bona fide DCs in both allogenic and autologous mixed leukocyte reactions. These findings allow us to propose an optimal system for efficient and safe DC transduction with improved SIV vectors.

Immunological characterization of a gammadelta T-cell stimulatory ligand on autologous monocytes.

Bovine gammadelta T cells are stimulated to proliferate by autologous monocytes. This is referred to as the autologous mixed leucocyte reaction (AMLR). It has been shown previously that the stimulatory component is constitutively expressed on the monocyte plasma membrane and is a protein or has a protein moiety. Here we showed that gammadelta T-cell responses to the monocytes requires interaction with the T-cell receptor because Fab1 fragments of a monoclonal antibody (mAb) that reacts with the delta chain of the T-cell receptor blocked proliferation in the AMLR. Monocyte molecules involved in stimulation were also characterized further by biochemical and immunological methods. A mAb, named M5, was generated by immunizing mice with bovine monocytes and shown to block the ability of monocytes to stimulate in the AMLR. Treatment of monocytes or monocyte membranes with high salt, chelating agents or phospholipase C did not affect their ability to stimulate gammadelta T-cell proliferation or reactivity with mAb M5 indicating the ability of monocytes to stimulate does not involve peripheral membrane components or a glycosyl-phosphatidylinsositol (GPI)-anchored components. Hence it was concluded that the stimulation occurred as a result of intergral membrane proteins including that recognized by mAb M5. The ligand for mAb M5 was on all bovine monocytes and to a lower level on granulocytes but not on lymphocytes. MAb M5 also reacted with sheep monocytes but not with human monocytes or murine macrophages, in agreement with a previous reports that sheep monocytes but not human or mouse mononuclear phagocytes have the capacity to stimulate bovine gammadelta T cells in in vitro cultures. The level of expression of the M5 ligand was not altered by gamma-irradiation or culture of monocytes with lipopolysaccharide but it was decreased following culture with interferon-gamma-containing cell culture supernatants.

Transgenic interleukin 2 secreted by CML dendritic cells stimulates autologous TH1 T cells

We investigated if dendritic cells (DC), derived from patients suffering from chronic myeloid leukemia (CML) could be modified by recombinant replication-defective adenoviruses to express functional interleukin 2 (IL-2). Such modification might confer onto antigen-presenting cells the ability to stimulate expansion of effector cells. To quantify the infection efficiency of CML dendritic cells (CML-DC) by recombinant adenovirus, we measured the expression of green fluorescent protein (GFP) gene contained in the virus. In CML-DC infected with an adenovirus containing the IL-2 gene, we evaluated their ability to secrete IL-2 and stimulate proliferation of autologous T cells. Uninfected CML-DC and normal DC secreted similar amounts of IL-12 and stimulated similarly efficient autologous mixed leukocyte reaction. Immature CML-DC infected by an adenovirus containing the gene for IL-2 secreted large amounts of IL-2 and stimulated proliferation of autologous T cells more efficiently than the corresponding CML-DC alone. High levels of interferon eta, but not of IL-4, in cell culture supernates indicated that the proliferating cells were T(H)1. Infected mature CML-DC were more effective than infected immature CML-DC, showing that T cell stimulation by mature DC and by IL-2 was additive. CML-DC can be modified genetically and functionally by recombinant replication-defective adenoviruses, providing new possibilities for clinical trials in dendritic cell-based immunotherapy.

Rapid changes occur in the percentage of circulating bovine WC1+γδ Th1 cells

γδ T cells found in the peripheral blood of cattle include a major subpopulation distinguished by expression of WC1. These cells are distinct from the WC1–γδ T cell population based on T cell receptor gene usage. We documented that a group of 6-month-old calves allowed free-range grazing and access to their mothers had a significantly greater proportion of total γδ T cells in their blood, attributable to the WC1+γδ T cell subpopulation, compared to age and breed-matched calves held in conventional housing. When the animals with the greater proportion of γδ T cells were transferred to conventional housing there was a decrease in the WC1+population so that by 3 weeks after transfer there was no longer a significant difference between the two groups. To investigate the biological activities of WC1+γδ T cells, the cells were purified by flow cytometric sorting. In vitro, they responded to stimulation by irradiated monocytes in autologous mixed leukocyte reaction (AMLR) cultures but not to direct stimulation through the T cell receptor (T c R) by anti-δ monoclonal antibody. After stimulation in the AMLR, WC1+γδ T cells had a Th1 cytokine profile characterised by production of IFN -γ and lack ofIL -4. Thus we propose that higher levels of the WC1+γδ T cells may provide calves with a mechanism to produce Th1 cytokines and that the level of these cells may be modulated according to environment or stress since both groups of calves were apparently disease-free.

Multiple sclerosis is associated with high levels of circulating dendritic cells secreting pro-inflammatory cytokines

Recent evidence emphasises a pivotal role for dendritic cells (DC) in the control of immunity by priming and tolerising T cells. DC capture and process antigens, express co-stimulatory molecules, migrate to lymphoid organs and secrete cytokines to initiate immune responses. In multiple sclerosis (MS), autoreactive T cells are proposed to play a pathogenic role by secreting pro-inflammatory cytokines, but studies on DC are lacking. To evaluate the involvement of DC in patients with MS, a modified procedure was used to prepare DC from blood of patients with MS and healthy subjects. DC were found to be potent stimulators of T cells in allogeneic and, to a lesser extent, in autologous mixed leukocyte reaction (MLR). Enzyme-linked immunospot (ELISPOT) assays were adopted to determine levels of IFN-γ, TNF-α, IL-6 and IL-10 secreting DC vs. mononuclear cells (MNC). Proportionally more DC than MNC secreted IFN-γ and IL-10 in both MS and healthy subjects. Patients with MS had higher levels of IFN-γ, TNF-α and IL-6 secreting DC than healthy subjects. The differences for IFN-γ and TNF-α secreting cells were confined to the subgroup of untreated MS patients and not observed in the subgroup examined during ongoing treatment with IFN-β. Circulating DC secreting pro-inflammatory cytokines may represent another focus for the study of both immuno-pathogenesis and therapeutic interventions in MS.

Activation of human Vα24NKT cells by α-glycosylceramide in a CD1d-restricted and Vα24TCR-mediated manner

Vα14NK(natural killer) T cells play an important role in controlling tumors or in preventing autoimmunity in the murine system. Vα24NKT cells, the human counterpart of Vα14NKT cells, may contribute to controlling the progression of autoimmune diseases in humans. These findings show the possibility that ligand(s) for these NKT cells can control the above-mentioned pathological conditions. Specific glycolipids such as α-galactosylceramide (α-GalCer) and α-glucosylceramide (α-GlcCer) have been identified as ligand(s) recognized by murine Vα14NKT cells in a CD1d-restricted manner, but it remains unclear whether these glycolipids are ligand(s) for Vα24NKT cells in humans. To determine whether α-glycosylceramide is presented by CD1d molecules in humans, we initially established a Vα24NKT cell line specific for α-glycosylceramide using dendritic cell (DC) like cells from normal peripheral blood mononuclear cells (PBMC) in an autologous mixed leukocyte reaction (auto-MLR) system, and characterized the Vα24NKT cell line. The Vα24NKT cells were CD3+CD4−CD8−Vα24+Vβ11+NKRP1A+ and specifically proliferated in response to α-glycosylceramide in CD1d-restricted and Vα24TCR-mediated manner. The phenotypic and functional similarities between murine Vα14NKT cells and human Vα24NKT cells suggest that Vα24NKT cells may play an important role in controlling tumors or in preventing autoimmunity as observed with Vα14NKT cells.

Autologous mixed leucocyte reaction and the polyclonal activation of bovine γ/δ T cells

Bovine γ/δ T cells proliferate in response to stimulation with γ-irradiated autologous monocytes in the autologous mixed leukocyte reaction (AMLR). Flow cytometric analyses indicated that the proliferating cells included three major subpopulations of bovine γ/δ T cells, distinguished by the differential expression of the γ/δ T cell receptor epitopes N6 and N7. Interleukin-2 and acid-labile interferon were produced in amlr cultures but the cultured cells did not lyse any of a large variety of target cells, including monocytes, allogeneic lymphoblasts, transformed bovine B cells (BL3), bovine fibroblast and the natural killer cell targets D17 and K562, even in the presence of lectins or after co-stimulation in the amlr with antibodies to WC1, the γ/δ T cell lineage-specific cell-surface differentiation antigen. Ex vivo γ/δ T cells did not display lymphokine-activated killing whereas populations of peripheral blood mononuclear cells containing α/β T cells did.

Modulation of WC1, a lineage-specific cell surface molecule of gamma/delta T cells augments cellular proliferation.

WC1, also known as T19, is the only unique gamma/delta T-cell differentiation antigen described to date other than the gamma/delta T-cell receptor. We present evidence that modulation of WC1 results in augmented proliferation of gamma/delta T cells. Immobilized IL-A29, a monoclonal antibody (mAb) specific for WC1, augmented proliferation of gamma/delta T cells in the autologous mixed leucocyte reaction (AMLR) as well as proliferation induced by either anti-CD3 or anti-CD5 mAb. In contrast, anti-CD5 mAb did not increase proliferation in the AMLR even though both CD5 and WC1 are members of the scavenger receptor cysteine-rich family of proteins and are expressed by bovine peripheral blood gamma/delta T cells. IL-A29 did not induce proliferation when assessed alone or in the presence of either phorbol myristate acetate (PMA) or interleukin-2. IL-A29 also did not induce detectable calcium mobilization when evaluated in the presence of monocytes, PMA, or following cross-linking of IL-A29 with anti-immunoglobulin antibody. We conclude that WC1 is a gamma/delta T-cell lineage-specific cell-surface differentiation antigen which is involved in activation of gamma/delta T cells using an as yet unidentified pathway.