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Abstract Background The detection of insulin autoantibodies (IAA) aids in the prediction of autoimmune diabetes development. However, the long-standing, gold standard 125I-insulin radiobinding assay (RBA) has low reproducibility between laboratories, long sample processing times and requires the use of newly synthesized radiolabeled insulin for each set of assays. Therefore, a rapid, non-radioactive, and reproducible assay is highly desirable. Methods We have developed electrochemiluminescence (ECL)-based assays that fulfill these criteria in the measurement of IAA and anti-insulin antibodies (IA) in non-obese diabetic (NOD) mice and in type 1 diabetic individuals, respectively. Using the murine IAA ECL assay, we examined the correlation between IAA, histopathological insulitis, and blood glucose in a cohort of female NOD mice from 4 up to 36 weeks of age. We developed a human IA ECL assay that we compared to conventional RBA and validated using samples from 34 diabetic and 59 non-diabetic individuals in three independent laboratories. Results Our ECL assays were rapid and sensitive with a broad dynamic range and low background. In the NOD mouse model, IAA levels measured by ECL were positively correlated with insulitis severity, and the values measured at 8-10 weeks of age were predictive of diabetes onset. Using human serum and plasma samples, our IA ECL assay yielded reproducible and accurate results with an average sensitivity of 84% at 95% specificity with no statistically significant difference between laboratories. Conclusions These novel, non-radioactive ECL-based assays should facilitate reliable and fast detection of antibodies to insulin and its precursors sera and plasma in a standardized manner between laboratories in both research and clinical settings. Our next step is to evaluate the human IA assay in the detection of IAA in prediabetic subjects or those at risk of type 1 diabetes and to develop similar assays for other autoantibodies that together are predictive for the diagnosis of this common disorder, in order to improve prediction and facilitate future therapeutic trials.

Abstract Background This study assessed the Granzyme B (GrB) ELISPOT as a viable alternative to the 51Cr-release assay for measuring cytotoxic activity of innate immune effector cells. We strategically selected the GrB ELISPOT assay because GrB is a hallmark effector molecule of cell-mediated destruction of target cells. Methods We optimized the GrB ELISPOT assay using the human-derived TALL-104 cytotoxic cell line as effectors against K562 target cells. Titration studies were performed to assess whether the ELISPOT assay could accurately enumerate the number of GrB-secreting effector cells. TALL-104 were treated with various secretion inhibitors and utilized in the GrB ELISPOT to determine if GrB measured in the ELISPOT was due to degranulation of effector cells. Additionally, CD107a expression on effector cells after effector-target interaction was utilized to further confirm the mechanism of GrB release by TALL-104 and lymphokine-activated killer (LAK) cells. Direct comparisons between the GrB ELISPOT, the IFN-γ ELISPOT and the standard 51Cr-release assays were made using human LAK cells. Results Titration studies demonstrated a strong correlation between the number of TALL-104 and LAK effector cells and the number of GrB spots per well. GrB secretion was detectable within 10 min of effector-target contact with optimal secretion observed at 3–4 h; in contrast, optimal IFN-γ secretion was not observed until 24 h. The protein secretion inhibitor, brefeldin A, did not inhibit the release of GrB but did abrogate IFN-γ production by TALL-104 cells. GrB secretion was abrogated by BAPTA-AM (1,2-bis-(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid tetra(acetoxymethyl) ester), which sequesters intracellular Ca2+, thereby preventing degranulation. The number of effector cells expressing the degranulation associated glycoprotein CD107a increased after interaction with target cells and correlated with the stimulated release of GrB measured in the ELISPOT assay. Conclusions Because of its high sensitivity and ability to estimate cytotoxic effector cell frequency, the GrB ELISPOT assay is a viable alternative to the 51Cr-release assay to measure MHC non-restricted cytotoxic activity of innate immune cells. Compared to the IFN-γ ELISPOT assay, the GrB ELISPOT may be a more direct measure of cytotoxic cell activity. Because GrB is one of the primary effector molecules in natural killer (NK) cell-mediated killing, detection and enumeration of GrB secreting effector cells can provide valuable insight with regards to innate immunological responses.

Abstract Background The desired outcome of cancer vaccination is to induce a potent T cell response which can specifically recognize and eliminate autologous tumor cells in vivo. Accordingly, immunological assays that demonstrate recognition of native tumor cells (tumor-specific) may be more clinically relevant than assays that demonstrate recognition of tumor protein or peptide (antigen-specific). Methods Towards this goal, we adapted the IFN-γ ELISPOT assay to measure immune responses against autologous primary tumor cells in vaccinated cancer patients. As a model system to develop the assay, we utilized peripheral blood mononuclear cells (PBMC) directly isolated from follicular lymphoma patients vaccinated with tumor-derived idiotype protein. Results After optimizing several variables, we demonstrated that the modified IFN-γ ELISPOT assay could be used to reliably and reproducibly determine the tumor-reactive T cell frequency in the PBMC of these patients. The precursor frequency of tumor-reactive T cells was significantly higher in the postvaccine PBMC, compared with prevaccine samples in all patients tested. Furthermore, the specificity of these T cells was established by the lack of reactivity against autologous normal B cells. Conclusions These results demonstrate the feasibility of quantitating tumor-specific T cell responses when autologous, primary tumor cells are available as targets.

Abstract Background The interferon-γ (IFN-γ) ELISPOT assay is one of the most useful techniques for immunological monitoring of cancer vaccine trials and has gained increased application as a measure of specific T cell activation. However, it does not assess cell-mediated cytotoxicity directly as IFN-γ secretion is not limited to only cytolytic cells. Granzyme B (GrB) is a key mediator of target cell death via the granule-mediated pathway. Therefore, the release of GrB by cytolytic lymphocytes upon effector-target interaction may be a more specific indicator of CTL and NK cytotoxic ability than IFN-γ secretion. Methods We assessed whether the GrB ELISPOT assay is a viable alternative to the 51Cr-release and IFN-γ ELISPOT assays for measuring antigen-specific CTL cytotoxicity. Direct comparisons between the three assays were made using human CTL cell lines (αEN-EBV and αJY) and an in vitro stimulated anti-Flu matrix peptide (FMP)-specific CTL. Results When the GrB ELISPOT was directly compared to the IFN-γ ELISPOT and 51Cr-release assays, excellent cross-correlation between all three assays was shown. However, measurable IFN-γ secretion in the ELISPOT assay was observed only after 1 hour of incubation and cytotoxicity assessed via the 51Cr-release assay after 4 hours, whereas GrB secretion was detectable within 10 min of effector-target contact with significant secretion observed after 1 h. Titration studies demonstrated a strong correlation between the number of effector cells and GrB spots per well. Irrelevant targets or antigens did not induce significant GrB secretion. Additionally, GrB secretion was abrogated when CTL cultures were depleted of CD8+ cells. Conclusion Our findings demonstrate that the GrB ELISPOT assay is a superior alternative to the 51Cr-release assay since it is significantly more sensitive and provides an estimation of cytotoxic effector cell frequency. Additionally, unlike the IFN-γ ELISPOT assay, the GrB ELISPOT directly measures the release of a cytotolytic protein. Detection of low frequency tumor-specific CTL and their specific effector functions can provide valuable insight with regards to immunological responses.

Purpose: Cancers overexpressing the HER2/neu gene are usually more aggressive and are associated with poor prognosis. Although trastuzumab has significantly improved the outcome, many tumors do not respond or acquire resistance to current therapies. To provide an alternative HER2-targeted therapy, we have developed and characterized a novel recombinant protein combining an HER2-specific Affibody and modified Pseudomonas aeruginosa exotoxin A (PE 38), which, after binding to HER2, is internalized and delivered to the cytosol of the tumor cell, where it blocks protein synthesis by ADP ribosylation of eEF-2., Experimental Design: The effect of the Affitoxin on cell viability was assessed using CellTiter-Glo (Promega). To assess HER2-specific efficacy, athymic nude mice bearing BT-474 breast cancer, SK-OV-3 ovarian cancer, and NCI-N87 gastric carcinoma xenografts were treated with the Affitoxin (HER2- or Tag-specific), which was injected every third day. Affitoxin immunogenicity in female BALB/c mice was investigated using standard antibody production and splenocyte proliferation assays., Results: In vitro experiments proved that HER2-Affitoxin is a potent agent that eliminates HER2-overexpressing cells at low picomolar concentrations. Therapeutic efficacy studies showed complete eradication of relatively large BT-474 tumors and significant effects on SK-OV-3 and NCI-N87 tumors. HER2-Affitoxin cleared quickly from circulation (T(1/2) < 10 minutes) and was well tolerated by mice at doses of 0.5 mg/kg and below. Immunogenicity studies indicated that HER2-Affitoxin induced antibody development after the third injected dose., Conclusions: Our findings showed that HER2-Affitoxin is an effective anticancer agent and a potential candidate for clinical studies., (©2011 AACR.)

In this report, we studied T cell responses to a prostate cancer Ag by adoptively transferring tumor Ag-specific T cells into prostate tumor-bearing mice. Our findings demonstrate that CD8(+) T cells initially encountered tumor Ag in the lymph node and underwent an abortive proliferative response. Upon isolation from the tumor, the residual tumor-specific T cells were functionally tolerant of tumor Ag as measured by their inability to degranulate and secrete IFN-gamma and granzyme B. We next sought to determine whether providing an ex vivo-matured, peptide-pulsed dendritic cell (DC) vaccine could overcome the tolerizing mechanisms of tumor-bearing transgenic adenocarcinoma of the mouse prostate model mice. We demonstrate that tumor Ag-specific T cells were protected from tolerance following provision of the DC vaccine. Concurrently, there was a reduction in prostate tumor size. However, even when activated DCs initially present tumor Ag, T cells persisting within the tolerogenic tumor environment gradually lost Ag reactivity. These results suggest that even though a productive antitumor response can be initiated by a DC vaccine, the tolerizing environment created by the tumor still exerts suppressive effects on the T cells. Furthermore, our results demonstrate that when trying to elicit an effective antitumor immune response, two obstacles must be considered: to maintain tumor Ag responsiveness, T cells must be efficiently primed to overcome tumor Ag presented in a tolerizing manner and protected from the suppressive mechanisms of the tumor microenvironment.

It is widely recognized that the immune system plays a role in cancer progression and that some tumors are inherently immunogenic. The identification of tumor-associated antigens (TAAs) has stimulated research focused on immunotherapies to mediate the regression of established tumors. Cancer-specific immunity has traditionally been aimed at activating CD8+ cytotoxic T lymphocytes (CTLs) directed against major histocompatibility complex (MHC) class I-binding peptide epitopes. Other approaches utilize T cell adoptive therapy where autologous, tumor-specific T cells propagated in vitro are transferred back into recipients. However, these strategies have met with limited success in part due to the regulatory mechanisms of T cell tolerance, which poses a considerable challenge to cancer immunotherapy. Our laboratory utilizes the TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) model, a murine model of prostate cancer, to study mechanisms of T cell tolerization to tumor antigens. We previously demonstrated that upon encounter with their cognate antigen in the tumor microenvironment, naive T cell become tolerized. Our ongoing studies are testing whether provision of CD4+ T cells can enhance tumor immunity by preventing CD8+ T cell tolerance. A greater understanding of the interaction between various tumor-specific T cell subsets will facilitate the design of novel approaches to stimulate a more potent antitumor immune response.

We have developed a modification of the ELISPOT assay that measures Granzyme B (GrB) release from cytotoxic T lymphocytes (CTLs). The GrB ELISPOT assay is a superior alternative to the 51Cr-release assay since it is significantly more sensitive and provides an estimation of cytotoxic effector cell frequency. Additionally, unlike the IFN-gamma ELISPOT assay, the GrB ELISPOT directly measures the release of a cytolytic protein. We report that the GrB ELISPOT can be utilized to measure ex vivo antigen-specific cytotoxicity of peripheral blood mononuclear cells (PBMCs) from cancer patients vaccinated with a peptide-based cancer vaccine. We compare the reactivity of patients' PBMCs in the GrB ELISPOT, with reactivity in the tetramer, IFN-gamma ELISPOT and chromium (51Cr)-release assays. Differences in immune response over all assays tested were found between patients, and four response patterns were observed. Reactivity in the GrB ELISPOT was more closely associated with cytotoxicity in the 51Cr-release assay than the tetramer or IFN-gamma ELISPOT assays. We also optimized the GrB ELISPOT assay to directly measure immune responses against autologous primary tumor cells in vaccinated cancer patients. A perforin ELISPOT assay was also adapted to evaluate peptide-stimulated reactivity of PMBCs from vaccinated melanoma patients. Modifications of the ELISPOT assay described in this chapter allow a more comprehensive evaluation of low-frequency tumor-specific CTLs and their specific effector functions and can provide a valuable insight into immune responses in cancer vaccine trials.

Granzyme B (GrB) is present in the granules of cytolytic lymphocytes and is a key mediator of cell-mediated target cell death via the granule-mediated pathway. The release of GrB can be used as an indicator of a cytotoxic T lymphocyte response. Herein, we report that the GrB enzyme-linked immunospot assay (ELISPOT) can be used to measure ex vivo antigen-specific cytotoxicity of peripheral blood mononuclear cells from cancer patients vaccinated with a peptide-based cancer vaccine. We compare the reactivity of patients' peripheral blood mononuclear cells in the GrB ELISPOT with reactivity in the tetramer, interferon (IFN)-gamma ELISPOT, and Cr-release assays. Differences in immune response over all assays tested were found between patients and 4 response patterns were observed. Reactivity in the GrB ELISPOT was more closely associated with cytotoxicity in the Cr-release assay than the tetramer or IFN-gamma ELISPOT assays. Moreover, the higher affinity g209-2M peptide (used for vaccination) elicited greater GrB secretion than the native g209 peptide, although this difference was not observed with IFN-gamma secretion. Taken together with the fact that GrB is a specific mediator released by cytotoxic T lymphocytes, these results show that simultaneous use of the GrB ELISPOT assay with other immunologic assays may provide important additional immunologic insight into patient responses to cancer vaccines.

We have shown previously that IFN-gamma-inducing cytokines such as IL-12 can mediate potent antitumor effects against murine solid tumors. IL-27 is a newly described IL-12-related cytokine that potentiates various aspects of T and/or NK cell function. We hypothesized that IL-27 might also mediate potent antitumor activity in vivo. TBJ neuroblastoma cells engineered to overexpress IL-27 demonstrated markedly delayed growth compared with control mice, and complete durable tumor regression was observed in >90% of mice bearing either s.c. or orthotopic intra-adrenal tumors, and 40% of mice bearing induced metastatic disease. The majority of mice cured of their original TBJ-IL-27 tumors were resistant to tumor rechallenge. Furthermore, TBJ-IL-27 tumors were heavily infiltrated by CD8(+) T cells, and draining lymph node-derived lymphocytes from mice bearing s.c. TBJ-IL-27 tumors are primed to proliferate more readily when cultured ex vivo with anti-CD3/anti-CD28 compared with lymphocytes from mice bearing control tumors, and to secrete higher levels of IFN-gamma. In addition, marked enhancement of local IFN-gamma gene expression and potent up-regulation of cell surface MHC class I expression are noted within TBJ-IL-27 tumors compared with control tumors. Functionally, these alterations occur in conjunction with the generation of tumor-specific CTL reactivity in mice bearing TBJ-IL-27 tumors, and the induction of tumor regression via mechanisms that are critically dependent on CD8(+), but not CD4(+) T cells or NK cells. Collectively, these studies suggest that IL-27 could be used therapeutically to potentiate the host antitumor immune response in patients with malignancy.

The leukocytes in milk consist of lymphocytes, neutrophil polymorphonuclear leukocytes (PMN) and macrophages. Lymphocytes together with antigen-presenting cells function in the generation of an effective immune response. Lymphocytes can be divided into two distinct subsets, T- and B-lymphocytes, that differ in function and protein products. The professional phagocytic cells of the bovine mammary gland are PMN and macrophages. In the normal mammary gland macrophages are the predominate cells which act as sentinels to invading mastitis causing pathogens. Once the invaders are detected, macrophages release chemical messengers called chemoattractants that cause the directed migration of PMN into the infection. Migration of neutrophils into mammary tissue provides the first immunological line of defense against bacteria that penetrate the physical barrier of the teat canal. However, their presence is like a double-edged sword. While the PMN are phagocytosing and destroying the invading pathogens, they inadvertently release chemicals which induces swelling of secretory epithelium cytoplasm, sloughing of secretory cells, and decreased secretory activity. Permanent scarring will result in a loss of milk production. Resident and newly migrated macrophages help reduce the damage to the epithelium by phagocytosing PMN that undergo programmed cell death through a process called apoptosis. Specific ligands on the neutrophil surface are required for directed migration and phagocytosis. In response to infection, freshly migrated leukocytes express greater numbers of cell surface receptors for immunoglobulins and complement and are more phagocytic than their counterparts in blood. However, phagocytic activity rapidly decreases with continued exposure to inhibitory factors such as milk fat globules and casein in mammary secretions. Compensatory hypertrophy in non-mastitic quarters partially compensates for lost milk production in diseased quarters. Advances in molecular biology are making available the tools, techniques, and products to study and modulate host-parasite interactions. For example the cloning and expression of proteins that bind endotoxin may provide ways of reducing damaging effects of endotoxin during acute coliform mastitis. The successful formation of bifunctional monoclonal antibodies for the targeted lysis of mastitis causing bacteria represents a new line of therapeutics for the control of mastitis in dairy cows.

This study determined the cytokine profile of CD4+ T-helper cells to elucidate the specific CD4+ T-helper phenotype during the postpartum period. Peripheral blood mononuclear cells were isolated from cows during periods of increased susceptibility (3 d postpartum, n = 7) and decreased susceptibility (mid- to late lactation, n = 6) to mastitis. Isolated mononuclear cells were magnetically separated into CD4(+)-enriched or CD4(+)-depleted populations using specific bovine monoclonal antibodies and were confirmed to be enriched or depleted by flow cytometric analysis. T-helper-1 and T-helper-2 subpopulations were distinguished by cytokine profiles, at both the molecular and protein level, by competitive quantitative reverse transcriptase-polymerase chain reaction and specific bioassays, respectively. The CD4(+)-enriched cultures isolated postpartum had enhanced interleukin-4 and interleukin-10 mRNA transcript expression; cultures isolated during the mid- to late lactating period had enhanced interleukin-2 mRNA transcripts. Depletion of CD4+ lymphocytes decreased, and enrichment of CD4+ lymphocytes increased interferon-gamma transcripts in cultures isolated from mid- to late lactation cows. Interferon-gamma and interleukin-2 bioassays revealed that cytokine secretion paralleled mRNA transcript levels. These data suggest that CD4+ lymphocytes act predominantly as T-helper-2 compared with T-helper-1 within 3 d after calving. Alterations in the T-helper-1 and T-helper-2 responses, and therefore the repertoire of cytokines produced, may be an underlying reason for diminished host immune response during the postpartum period.

This study examined the immunoregulatory role of CD8+ lymphocytes during the postpartum period. Peripheral blood cells were isolated from postpartum and mid to late lactating animals. Flow cytometric analysis was performed to determine the frequencies of relevant cell populations. Depletion of CD8+ lymphocytes from whole cultures significantly decreased proliferation and cytotoxic ability of cells isolated from mid to late lactating animals. Enrichment of whole cultures with CD8+ lymphocytes further decreased their proliferative ability but pure CD8+ lymphocyte had increased cytotoxic activity. In contrast, neither depletion nor enrichment of whole cultures with CD8+ lymphocytes altered the already diminished proliferative responses of cells isolated from postpartum cows. No cytotoxic activity was observed by cells isolated from postpartum animals. Cultures from mid to late lactating cows mainly expressed IFN-gamma mRNA where as IL-4 mRNA was mainly expressed by cultures isolated from postpartum animals. Flow cytometric analysis revealed that CD8+ lymphocytes have a high level of activation and expression of the beta-chain during the postpartum compared with the mid to late lactating period. These data indicate that CD8+ lymphocytes are of the suppressor compared to the cytotoxic nature immediately following parturition.

The antibacterial activity of bovine lymphocytes was evaluated following in vitro stimulation with interleukin-2. Mononuclear cells were isolated from the blood, lymph node, and mammary parenchymal tissue of four lactating and four periparturient dairy cows. These cells were evaluated for antibacterial activity against Staphylococcus aureus following incubation for 48 h with or without interleukin-2. Cultures stimulated with interleukin-2 had higher bactericidal activity of all three isolated cell populations than did unstimulated cultures, regardless of lactational stage. This observation suggests that this effector function may possibly be activated in vivo and may potentially increase mammary gland resistance to bacterial infections during periods of increased susceptibility. Flow cytometric analysis of the cultured cells revealed that antibacterial effector cells were mainly CD2+ and were depleted of macrophages. Despite shifts in CD4+, CD8+, and gamma delta T lymphocytes during the periparturient period, bactericidal activity was similar among the three cell sources. This similarity suggests that these lymphocyte phenotypes might not be directly responsible for this effector function. Identification of the antibacterial effector phenotype and its mechanism of action may lead to immunoregulatory strategies aimed at enhancing this novel bactericidal function.

Once activated, lymphocytes can regulate both specific and nonspecific immune responses. Alterations in lymphocyte function may increase the host's vulnerability to bacterial infections such as mastitis. Susceptibility to mastitis as well as diminished leukocyte functional capabilities have been shown to be influenced by lactational stage. Therefore, the present study characterized the phenotypes and functions of several bovine lymphoid populations at two points in the lactational cycle. Mononuclear cells were isolated from peripheral blood, supramammary lymph nodes, and mammary parenchyma of mid-lactating and postpartum dairy cows. The phenotypic composition, proliferative ability, cytokine secretion, and cytotoxic activity of isolated leukocytes were assessed with respect to lactational stage and tissue source. Lower percentages of T lymphocytes were consistent with diminished mitogen-stimulated proliferation and spontaneous cytotoxic activity by lymphocytes isolated from postpartum compared with mid-lactating animals. Stimulation with interleukin-2 did not enhance the cytotoxic activity or proliferative ability of lymphocytes isolated postpartum to similar levels observed for those isolated from mid-lactating animals. These data indicate that certain diminished lymphocyte functions observed during the postpartum period may result from shifts in leukocyte trafficking patterns.