Tumor-reactive CD4+ T cells develop cytotoxic activity and eradicate large established melanoma after transfer into lymphopenic hosts

Adoptive transfer of large numbers of tumor-reactive CD8+ cytotoxic T lymphocytes (CTLs) expanded and differentiated in vitro has shown promising clinical activity against cancer. However, such protocols are complicated by extensive ex vivo manipulations of tumor-reactive cells and have largely focused on CD8+ CTLs, with much less emphasis on the role and contribution of CD4+ T cells. Using a mouse model of advanced melanoma, we found that transfer of small numbers of naive tumor-reactive CD4+ T cells into lymphopenic recipients induces substantial T cell expansion, differentiation, and regression of large established tumors without the need for in vitro manipulation. Surprisingly, CD4+ T cells developed cytotoxic activity, and tumor rejection was dependent on class II–restricted recognition of tumors by tumor-reactive CD4+ T cells. Furthermore, blockade of the coinhibitory receptor CTL-associated antigen 4 (CTLA-4) on the transferred CD4+ T cells resulted in greater expansion of effector T cells, diminished accumulation of tumor-reactive regulatory T cells, and superior antitumor activity capable of inducing regression of spontaneous mouse melanoma. These findings suggest a novel potential therapeutic role for cytotoxic CD4+ T cells and CTLA-4 blockade in cancer immunotherapy, and demonstrate the potential advantages of differentiating tumor-reactive CD4+ cells in vivo over current protocols favoring in vitro expansion and differentiation

Enhancement of Tumour-Specific Immune Responses In Vivo by ‘MHC Loading-Enhancer’ (MLE)

BACKGROUND:Class II MHC molecules (MHC II) are cell surface receptors displaying short protein fragments for the surveillance by CD4+ T cells. Antigens therefore have to be loaded onto this receptor in order to induce productive immune responses. On the cell surface, most MHC II molecules are either occupied by ligands or their binding cleft has been blocked by the acquisition of a non-receptive state. Direct loading with antigens, as required during peptide vaccinations, is therefore hindered. PRINCIPAL FINDINGS:Here we show, that the in vivo response of CD4+ T cells can be improved, when the antigens are administered together with 'MHC-loading enhancer' (MLE). MLE are small catalytic compounds able to open up the MHC binding site by triggering ligand-release and stabilizing the receptive state. Their enhancing effect on the immune response was demonstrated here with an antigen from the influenza virus and tumour associated antigens (TAA) derived from the NY-ESO-1 protein. The application of these antigens in combination with adamantane ethanol (AdEtOH), an MLE compound active on human HLA-DR molecules, significantly increased the frequency of antigen-specific CD4+ T cells in mice transgenic for the human MHC II molecule. Notably, the effect was evident only with the MLE-susceptible HLA-DR molecule and not with murine MHC II molecules non-susceptible for the catalytic effect of the MLE. CONCLUSION:MLE can specifically increase the potency of a vaccine by facilitating the efficient transfer of the antigen onto the MHC molecule. They may therefore open a new way to improve vaccination efficacy and tumour-immunotherapy

Aqueous extracts from dietary supplements influence the production of inflammatory cytokines in immortalized and primary T lymphocytes

<p>Abstract</p> <p>Background</p> <p>Congaplex^®and Immuplex^®are dietary supplements that have been traditionally used to support immune system function. The purpose of these experiments was to determine whether Congaplex^®and Immuplex^®affect immune function using primary and immortalized T lymphocytes.</p> <p>Methods</p> <p>Immortalized CEM and Jurkat T lymphocytes and primary peripheral mononuclear blood cells (PBMCs) were treated with the aqueous extracts from Congaplex^®and Immuplex^®to determine the effects of these products on cytokine production in activated T lymphocytes.</p> <p>Results</p> <p>Congaplex^®enhanced phytohemagglutinin/phorbol 12-myristate 13-acetate (PHA/PMA) stimulation of both CEM and Jurkat cells as measured by the production of cytokines, while Immuplex^®suppressed PHA/PMA-induced production of cytokines, with the exception of interleukin (IL)-8 which was enhanced by Immuplex^®. <it>In vitro </it>treatment of PBMCs from 10 healthy donors with Congaplex^®or Immuplex^®decreased PHA-stimulated production of interferon (IFN)-γ but increased the production of IL-13.</p> <p>Conclusions</p> <p>While the effects of Congaplex^®and Immuplex^®differed in these two models, these data demonstrate that the aqueous extracts from these two dietary supplements can affect the inflammatory response of T lymphocytes.</p

Interactions between lymphocytes and myeloid cells regulate pro- versus anti-tumor immunity

Tumor-associated myeloid cells have been implicated in regulating many of the “hallmarks of cancer” and thus fostering solid tumor development and metastasis. However, the same innate leukocytes also participate in anti-tumor immunity and restraint of malignant disease. While many factors regulate the propensity of myeloid cells to promote or repress cancerous growths, polarized adaptive immune responses by B and T lymphocytes have been identified as regulators of many aspects of myeloid cell biology by specifically regulating their functional capabilities. Here, we detail the diversity of heterogeneous B and T lymphocyte populations and their impacts on solid tumor development through their abilities to regulate myeloid cell function in solid tumors

NK Cells Promote Th-17 Mediated Corneal Barrier Disruption in Dry Eye

The conjunctiva contains a specialized population of lymphocytes that reside in the epithelium, named intraepithelial lymphocytes (IEL).Here we characterized the IEL population prior to and after experimental desiccating stress (DS) for 5 or 10 days (DS5, DS10) and evaluated the effect of NK depletion on DS. The frequency of IELs in normal murine conjunctiva was CD3(+)CD103(+) (~22%), CD3(+)γδ(+) (~9.6%), CD3(+)NK(+) (2%), CD3(-)NK(+) (~4.4%), CD3(+)CD8α (~0.9%), and CD4 (~0.6%). Systemic depletion of NK cells prior and during DS led to a decrease in the frequency of total and activated DCs, a decrease in T helper-17(+) cells in the cervical lymph nodes and generation of less pathogenic CD4(+)T cells. B6.nude recipient mice of adoptively transferred CD4(+)T cells isolated from NK-depleted DS5 donor mice showed significantly less corneal barrier disruption, lower levels of IL-17A, CCL20 and MMP-3 in the cornea epithelia compared to recipients of control CD4(+)T cells.Taken together, these results show that the NK IELs are involved in the acute immune response to desiccation-induced dry eye by activating DC, which in turn coordinate generation of the pathogenic Th-17 response

T-cell regulation in Erythema Nodosum Leprosum.

Leprosy is a disease caused by Mycobacterium leprae where the clinical spectrum correlates with the patient immune response. Erythema Nodosum Leprosum (ENL) is an immune-mediated inflammatory complication, which causes significant morbidity in affected leprosy patients. The underlying cause of ENL is not conclusively known. However, immune-complexes and cell-mediated immunity have been suggested in the pathogenesis of ENL. The aim of this study was to investigate the regulatory T-cells in patients with ENL. Forty-six untreated patients with ENL and 31 non-reactional lepromatous leprosy (LL) patient controls visiting ALERT Hospital, Ethiopia were enrolled to the study. Blood samples were obtained before, during and after prednisolone treatment of ENL cases. Peripheral blood mononuclear cells (PBMCs) were isolated and used for immunophenotyping of regulatory T-cells by flow cytometry. Five markers: CD3, CD4 or CD8, CD25, CD27 and FoxP3 were used to define CD4+ and CD8+ regulatory T-cells. Clinical and histopathological data were obtained as supplementary information. All patients had been followed for 28 weeks. Patients with ENL reactions had a lower percentage of CD4+ regulatory T-cells (1.7%) than LL patient controls (3.8%) at diagnosis of ENL before treatment. After treatment, the percentage of CD4+regulatory T-cells was not significantly different between the two groups. The percentage of CD8+ regulatory T-cells was not significantly different in ENL and LL controls before and after treatment. Furthermore, patients with ENL had higher percentage of CD4+ T-ells and CD4+/CD8+ T-cells ratio than LL patient controls before treatment. The expression of CD25 on CD4+ and CD8+ T-cells was not significantly different in ENL and LL controls suggesting that CD25 expression is not associated with ENL reactions while FoxP3 expression on CD4+ T-cells was significantly lower in patients with ENL than in LL controls. We also found that prednisolone treatment of patients with ENL reactions suppresses CD4+ T-cell but not CD8+ T-cell frequencies. Hence, ENL is associated with lower levels of T regulatory cells and higher CD4+/CD8+ T-cell ratio. We suggest that this loss of regulation is one of the causes of ENL

Immune Cell Composition in Human Non-small Cell Lung Cancer

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death in the world. Immunological analysis of the tumor microenvironment (immunoscore) shows great promise for improved prognosis and prediction of response to immunotherapy. However, the exact immune cell composition in NSCLC remains unclear. Here, we used flow cytometry to characterize the immune infiltrate in NSCLC tumors, non-cancerous lung tissue, regional lymph node, and blood. The cellular identity of &gt;95% of all CD45+ immune cells was determined. Thirteen distinct immune cell types were identified in NSCLC tumors. T cells dominated the lung cancer landscape (on average 47% of all CD45+ immune cells). CD4+ T cells were the most abundant T cell population (26%), closely followed by CD8+ T cells (22%). Double negative CD4−CD8− T cells represented a small fraction (1.4%). CD19+ B cells were the second most common immune cell type in NSCLC tumors (16%), and four different B cell sub-populations were identified. Macrophages and natural killer (NK) cells composed 4.7 and 4.5% of the immune cell infiltrate, respectively. Three types of dendritic cells (DCs) were identified (plasmacytoid DCs, CD1c+ DCs, and CD141+ DCs) which together represented 2.1% of all immune cells. Among granulocytes, neutrophils were frequent (8.6%) with a high patient-to-patient variability, while mast cells (1.4%), basophils (0.4%), and eosinophils (0.3%) were less common. Across the cohort of patients, only B cells showed a significantly higher representation in NSCLC tumors compared to the distal lung. In contrast, the percentages of macrophages and NK cells were lower in tumors than in non-cancerous lung tissue. Furthermore, the fraction of macrophages with high HLA-DR expression levels was higher in NSCLC tumors relative to distal lung tissue. To make the method readily accessible, antibody panels and flow cytometry gating strategy used to identify the various immune cells are described in detail. This work should represent a useful resource for the immunomonitoring of patients with NSCLC

Anchor Side Chains of Short Peptide Fragments Trigger Ligand-Exchange of Class II MHC Molecules

Class II MHC molecules display peptides on the cell surface for the surveillance by CD4+ T cells. To ensure that these ligands accurately reflect the content of the intracellular MHC loading compartment, a complex processing pathway has evolved that delivers only stable peptide/MHC complexes to the surface. As additional safeguard, MHC molecules quickly acquire a ‘non-receptive’ state once they have lost their ligand. Here we show now that amino acid side chains of short peptides can bypass these safety mechanisms by triggering the reversible ligand-exchange. The catalytic activity of dipeptides such as Tyr-Arg was stereo-specific and could be enhanced by modifications addressing the conserved H-bond network near the P1 pocket of the MHC molecule. It affected both antigen-loading and ligand-release and strictly correlated with reported anchor preferences of P1, the specific target site for the catalytic side chain of the dipeptide. The effect was evident also in CD4+ T cell assays, where the allele-selective influence of the dipeptides translated into increased sensitivities of the antigen-specific immune response. Molecular dynamic calculations support the hypothesis that occupation of P1 prevents the ‘closure’ of the empty peptide binding site into the non-receptive state. During antigen-processing and -presentation P1 may therefore function as important “sensor” for peptide-load. While it regulates maturation and trafficking of the complex, on the cell surface, short protein fragments present in blood or lymph could utilize this mechanism to alter the ligand composition on antigen presenting cells in a catalytic way