Cellular Immune Responses In Patients Research Articles

Identification of new NY‐ESO‐1 epitopes recognized by CD4+ T cells and presented by HLA‐DQ B1 03011

NY-ESO-1 is one of the most immunogenic cancer antigens eliciting strong humoral and cellular immune responses in patients with NY-ESO-1-expressing malignancies. Since CD4+ T cells play a critical role in generating and maintaining antigen-specific cellular and humoral immune responses, we searched for new NY-ESO-1 epitopes presented by MHC class II molecules. CD4+ T cells of patients with NY-ESO-1-expressing cancer were presensitized with 18-mer overlapping synthetic peptides spanning the entire sequence of NY-ESO-1. Two partly overlapping NY-ESO-1 epitopes p49-66 and p55-72 were identified as targets for NY-ESO-1-specific CD4+ T cells. Peptide-specific CD4+ T-cell clones were generated by repetitive stimulation with NY-ESO-1 p49-66 and p55-72. Further experiments confirmed distinct specificities for the CD4+ T-cell clones indicating that at least 2 different CD4+ T-cell epitopes are located in the region p49-72 of the NY-ESO-1 sequence. Using a set of partially histocompatible EBV-B cell lines and MHC class II-specific antibodies, we found that both CD4+ T-cell epitopes were presented in the context of HLA-DQ B1 03011(DQ7). Natural processing and presentation of these epitopes was demonstrated by recognition of an HLA-DQ B1 03011- and NY-ESO-1-expressing lymphoma cell line and by recognition of dendritic cells (DC) exogenously loaded with NY-ESO-1 protein or infected with recombinant NY-ESO-1 adenoviral constructs. The specific production of IFN-gamma and TNF-alpha suggests that the NY-ESO-1-specific CD4+ T-cell clones belong to the Th1 subtype. The characterization of the new HLA-DQ B1 03011-restricted NY-ESO-1 peptides broadens the repertoire of epitopes that can be used to monitor NY-ESO-1-specific spontaneous and vaccine-induced T-cell responses in cancer patients.

RHAMM/CD168-R3 Peptide Vaccination of HLA-A2+ Patients with Acute Myeloid Leukemia (AML), Myelodysplastic Syndrome (MDS) and Multiple Myeloma (MM).

The receptor for hyaluronic acid mediated motility (RHAMM/CD168) has been described as a leukemia-associated antigen (LAA) eliciting both humoral and cellular immune responses in patients with hematological malignancies. RHAMM/CD168 is expressed in more than 80% of patients with acute myeloid leukemia (AML) or multiple myeloma (MM). Recently, we characterized the RHAMM/CD168-derived peptide R3 (ILSLELMKL) as a CD8+ T cell epitope. R3-primed CD8+ T lymphocytes were able to lyse autologous RHAMM/CD168+ AML blasts in a MHC class I-restricted and epitope-specific manner. Therefore, we initiated a phase I/II R3 peptide vaccination trial for patients with AML, MDS or MM overexpressing RHAMM/CD168. Patients were included with positive RHAMM/CD168 expression but with a limited tumor load. 300 mcg RHAMM R3 peptide emulsified with the incomplete Freund's adjuvant (ISA-51, Montanide; day 3) and GM-CSF (Leukine, days 1–5) was administrated four times subcutaneously at a biweekly interval. The primary aim of the study is safety and feasibility of this peptide vaccination, secondary aims the evaluation of a specific T cell immune response to RHAMM/CD168 R3 peptide and the assessment of the influence of the R3 peptide vaccination on the remission status. Since January 2005, ten patients were enrolled in this study. The first eight patients (2 AML, 3 MDS, 3 MM) have completed the course of four vaccinations and four patients have been evaluated. The only side effects observed under R3-peptide vaccination were erythema and induration of the skin at the site of injection (CTC I°). In 2/4 patients, we found in the peripheral blood a significant increase of specific CD8+ T cells (from 0.01% to 0.8%) recognizing the R3 peptide in ELISPOT analysis and tetramer staining, one patient showed already initially a high number of HLA-A2/R3 tetramer+CCR7-CD27-CD45RA+ effector T cells and maintained this level of T cell response. Clinical responses have been assessed by the examination of peripheral blood and bone-marrow samples before and after vaccination. Patients showed a reduction of the tumor-specific expressed antigen RHAMM/CD168 in real-time RT-PCR analysis after vaccination. 2/4 patients with myeloid disorders (1 AML, 1 MDS RAEB1) showed a reduction of CD33+ cells in FACS analysis of the bone-marrow after 4 vaccinations from 10 and 7 % to 1–2 and <1%, respectively. One patient with MM showed a reduction of plasma cells in bone-marrow and a stable quantity of light chains in peripheral blood, one patient with AML showed a progressive disease. In summary, RHAMM/CD168 is a promising target antigen for immunotherapies in patients with hematological malignancies.

Immune Monitoring in a Phase 1 Trial of a PSA DNA Vaccine in Patients with Hormone-Refractory Prostate Cancer

Prostate cancer remains a leading cause of cancer illness and death among men in Europe. No curative treatment exists when the disease has spread beyond the prostate. Immunotherapy with DNA vaccines has emerged as a potential therapeutic approach for the induction of antitumor specific cytotoxic T lymphocytes. In this study six patients with hormone-refractory prostate cancer were monitored for their ability to mount PSA-specific cellular responses after receiving a pVAX/PSA DNA vaccine (patients 1-3, 100 microg; patients 7-9, 900 microg) with recombinant GM-CSF and IL-2 as adjuvants. IFNgamma ELISPOT showed that naturally processed PSA protein and PSA peptides are recognized by T cells in the blood of some prostate cancer patients after a PSA DNA vaccine. Analysis of other cytokines showed the production of IL-4 and IL-6 but importantly did not show an increase in the number of IL-10-producing cells after vaccination in any of the patients. The authors conclude that a pVAX/PSA DNA vaccine can induce PSA-specific cellular immune responses in patients with hormone-refractory prostate cancer, thus emphasizing the potential for PSA as a target molecule for the immunotherapy of prostate cancer.

Immunomodulation of responses to active immunization with a polyvalent immunotherapeutic: cyclophosphamide, H2 blockade, IL-2/IFNα and GM-CSF

2564 Background: Strong humoral and cellular immune responses in patients with melanoma receiving a specific active immunotherapeutic (Canvaxin; CancerVax Corp, Carlsbad, CA) have been correlated with improved survival. We examined the impact of several immunomodulators on the strength of immune response. Methods: Analysis of sequential phase II clinical trials using Canvaxin alone (n=446) or in combination with cyclophosphamide (n=92), cimetidine (n= 357), indomethacin (n=54) or IL-2/INFα (n=71) was performed with regard to maximal immune response within 16 weeks of treatment. A randomized clinical trial (n= 97) of Canvaxin with or without the addition of granulocyte-macrophage colony-stimulating factor (GM-CSF) was also analyzed. Delayed-type hypersensitivity (DTH) to vaccine was measured in all trials. IgM and IgG responses to the TA90 tumor-associated antigen were measured prospectively in the GM-CSF trial. Results: DTH responses were greater in patients with stage IV melanoma with concurrent cyclophosphamide (mean induration 20.6 mm with cyclophosphamide vs. 14.2 without, p=0.011). This was not seen in patients with stage III disease or in any stage with cimetidine, indomethacin or IL-2/IFNα treatment. The randomized GM-CSF trial did not show a significant difference (by logistic regression) in maximal DTH response (15.9± 9.7 mm with GM-CSF vs. 16.7 ± 9.9 mm without). There was a significant increase in maximal anti-TA90 IgM titers in the GM-CSF arm (797 ± 622 vs. 541 ± 533; p=0.0123), but no difference in anti-TA90 IgG (680 ± 557 with GM-CSF vs. 612 ± 510 without GM-CSF.) Maximal absolute lymphocyte counts during treatment were also elevated in the GM-CSF arm. Adverse event profiles were not markedly different, but GM-CSF patients reported more grade 1–2 fatigue. Conclusions: These data suggest that the addition of cyclophosphamide to adjuvant active specific immunotherapy in stage IV melanoma impacts DTH responses, and GM-CSF increases IgM response. Since this represents an impact on both the cellular (DTH) and humoral (IgM) arms of immunity, further study of modulator combinations is warranted. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration CancerVax CancerVax

Comparative Analysis of Immunophenotypic Abnormalities in Cellular Immunity of Uremic Patients Undergoing Either Hemodialysis or Continuous Ambulatory Peritoneal Dialysis

Aim. To investigate the abnormalities of cellular immune responses in patients on hemodialysis (HD) and in those on continuous ambulatory peritoneal dialysis (CAPD). Patients and Methods. Forty-five (45) healthy volunteers, 34 patients on HD therapy, and 37 patients on CAPD were recruited for the present study. Lymphocyte subpopulations (CD2 + , CD3 + , CD3 + /CD4 + , CD3 + /CD8 + , CD3 − /16 + 56 + , CD19, and CD4/CD8) were determined by flow cytometry. Results. Lymphopenia, decreased absolute counts, and altered percentage values of CD3 + , CD3 + /4 + , and CD19 + subpopulations were found in both patient groups. The HD and CAPD patients showed increased percentages of natural killer cells (CD3 − /16 + 56 + ) compared to controls but CD4 + /CD8 + ratio showed no significant changes among uremic patients and controls. Conclusions. Replacement therapy may contribute to the quantitative alterations of immune subsets found in HD and CAPD patients compared to normal subjects. We speculate that these changes account, at least in part, for the immune dysregulation observed in patients with chronic renal failure. Analysis of lymphocyte subsets will help the research and the evaluation of the possible causes of immunodeficiency in uremic patients undergoing replacement therapy and will probably contribute to more efficient and preventive strategies.

Dendritic Cell Vaccination: Potential Immunotherapy for Adrenocortical Carcinoma

The ineffectiveness of conventional therapies in adrenocortical carcinoma (ACC) emphasize the need to search for novel treatment protocols. Diverse studies suggest that immunization with autologous dendritic cells (DCs) pulsed with tumor antigen result in protective immunity and rejection of established tumors. The DC vaccination outcomes mainly depend on 1) the generation and activation of DCs, 2) the number of DCs applied and 3) the (tumor) antigens used for immunization. Several protocols have been generated to obtain large numbers of activated human DCs either from CD34+ bone-marrow cells or from peripheral blood monocytes by culturing in media supplemented with GM-CSF and IL-4, followed by stimulation with TNF-α or monocyte-conditioned medium. Using the first protocol, we were able to induce a Th1 immune response (shown by an increased interferon-γ secretion) towards the polypeptide hormone calcitonin in 5 of 7 patients with metastasized medullary thyroid carcinoma. Immunohistochemical analyses of delayed-type hypersensitivity skin reactions revealed an antigen-specific perivascular and epidermal infiltration with CD4+ T-helper (Th) cells and CD8+ cytotoxic T cells. One of these patients showed a temporary clinical effect with a decrease in liver and lung metastases. A more recent study with an improved DC generation protocol with GM-CSF (1000 U/ml) and IFNalpha (1000 U/ml) following leukapheresis resulted in the induction of a Th1 immunity (IFN-γ intracytoplasmic cytokine staining) and cytotoxic CTLs (granzyme ELISpot assay) in 2 of 5 patients each (follow-up: 2–5 months). Our data indicate that vaccination with polypeptide hormone-pulsed DC result in the induction of a cellular immune response in patients with endocrine carcinomas, leading to clinical response in some. This approach may represent the basis for the development of a new therapeutic strategy in ACC as well. We have now started a DC-based vaccination trial in two patients with ACC (follow-up: ½ and 2 months each). Further follow-up of these and other patients enrolled into the study will answer the question of whether this strategy may represent a new therapy approach in ACC.

Quantitation of immune responses against the cancer testis antigen NY-ESO-1 following vaccination with protein with and without ISCOMATRIX adjuvant

2518 Background: One major challenge for cancer immunology is the measurement of the immune response so that any relationship to clinical outcomes can be evaluated using stringent criteria. Methods: We have applied a novel scoring system in order to better quantitate integrated antibody and cellular immune responses in patients vaccinated with a full-length protein cancer testis antigen, NY-ESO-1. The score accounts for rate of onset, duration and intensity of an immune response (the immune equivalent of AUC). Forty-six patients (pts), 42 with melanoma, all with fully resected disease, were vaccinated IM on three occasions at monthly intervals. NY-ESO-1 protein was administered with or without the ISCOMATRIX adjuvant, which has been shown to induce both antibody and T cell responses. NY-ESO-1 ISCOMATRIX vaccine doses were:10μg (n=3), 30μg (n=3), and 100μg (n=16). 16 pts received 100μg NY-ESO-1 protein alone and 8 pts received placebo. NY-ESO-1 immunity was assessed by Delayed Type Hypersensitivity (DTH) to NY-ESO-1 protein, by ELISA for antibody and by tetramer and ICS for the number of reactive NY-ESO-1 CD8+ & CD4+ T cells. Results: Although low level immune responses were seen against protein alone, the addition of adjuvant greatly increased immune responses in a dose-dependent fashion. In an unplanned analysis, the relapse free survival (RFS) of melanoma pts was assessed. Despite small numbers, patients in cohorts with higher immune response scores appeared to have longer RFS than those from cohorts with low scores. Conclusions: A prospective evaluation of this is required determine whether immune responses to NY-ESO-1 is correlated clinical outcome of pts with tumours which express this antigen. Using a system to quantitate immune responses will also facilitate strategies aimed at optimising vaccine potency. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration CSL Ltd. CSL Ltd.

Quantitation of immune responses against the cancer testis antigen NY-ESO-1 following vaccination with protein with and without ISCOMATRIX adjuvant

2518 Background: One major challenge for cancer immunology is the measurement of the immune response so that any relationship to clinical outcomes can be evaluated using stringent criteria. Methods: We have applied a novel scoring system in order to better quantitate integrated antibody and cellular immune responses in patients vaccinated with a full-length protein cancer testis antigen, NY-ESO-1. The score accounts for rate of onset, duration and intensity of an immune response (the immune equivalent of AUC). Forty-six patients (pts), 42 with melanoma, all with fully resected disease, were vaccinated IM on three occasions at monthly intervals. NY-ESO-1 protein was administered with or without the ISCOMATRIX adjuvant, which has been shown to induce both antibody and T cell responses. NY-ESO-1 ISCOMATRIX vaccine doses were:10μg (n=3), 30μg (n=3), and 100μg (n=16). 16 pts received 100μg NY-ESO-1 protein alone and 8 pts received placebo. NY-ESO-1 immunity was assessed by Delayed Type Hypersensitivity (DTH) to NY-ESO-1 protein, by ELISA for antibody and by tetramer and ICS for the number of reactive NY-ESO-1 CD8+ & CD4+ T cells. Results: Although low level immune responses were seen against protein alone, the addition of adjuvant greatly increased immune responses in a dose-dependent fashion. In an unplanned analysis, the relapse free survival (RFS) of melanoma pts was assessed. Despite small numbers, patients in cohorts with higher immune response scores appeared to have longer RFS than those from cohorts with low scores. Conclusions: A prospective evaluation of this is required determine whether immune responses to NY-ESO-1 is correlated clinical outcome of pts with tumours which express this antigen. Using a system to quantitate immune responses will also facilitate strategies aimed at optimising vaccine potency. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration CSL Ltd. CSL Ltd.

Cellular immune response of patients with neurocysticercosis (inflammatory and non-inflammatory phases)

The cellular immune response in neurocysticercosis (NC) was studied in 22 patients, 11 (50%) of them in the inflammatory phase of the disease, by means of immunophenotyping of cells in cerebrospinal fluid (CSF) and peripheral blood (PB), lymphoproliferation assay with Taenia solium total saline extract (Tso) and Taenia crassiceps vesicular fluid (Tcra) as stimuli, and by determining the cytokine production profile in the cell culture supernatant. A higher mean percentage of CD19+ and CD56+ cells was observed in the CSF samples from inflammatory (16.8 and 11.3%) and non-inflammatory NC-patients (14.1 and 8.4%) when compared with the control group (CG, 7.6 and 5.4%). The CSF samples from inflammatory NC-patients also showed a higher percentage of HCAM (19.1%) and ICAM (44.9%) adhesion molecules when compared to CG (3.1 and 4.8%). The inflammatory phase showed predominance of CD8+ cells (CSF 26.6% and PB 36.2%) when compared with non-inflammatory phase (CSF 21.5% and PB 29.0%). All cell populations identified in the CSF from NC-patients showed cell activation (CD69+). The cell populations identified in PB showed higher expression of CD69 during the inflammatory phase, while only CD4+ cells presented no cell activation during the non-inflammatory phase. The antigen-specific lymphoproliferation assay showed mean positive results (stimulation index, SI ≥ 2.5) only for cells from inflammatory NC-patients (Tcra 3.2 and Tso 5.4), but less intense than the CG (Tcra 5.7 and Tso 8.9). The cytokine production profile when using Tso antigen as stimuli showed differences between NC-patients with inflammatory (production of IL-4/IL-12/TNF-α/ICAM/VCAM) and non-inflammatory phase (production of IL-6/IL-10/IL-12/TNF-α/ICAM/VCAM). A prevalence of Th2 profile was observed in nine (69%) of the 13 (62% of total) NC-patients presenting positive SI. Cells from inflammatory NC-patients showed a predominance of a Th1 response upon in vitro stimulation, while those from non-inflammatory NC-patients showed a mixed Th1/Th2 pattern.

NY-ESO-1 expression and its serum immunoreactivity in esophageal cancer.

NY-ESO-1, a member of the cancer/testis antigen (CTA) family, elicits humoral and cellular immune responses in patients with advanced cancer. Unresectable or metastatic esophageal carcinoma patients do not benefit from the present multimodality treatment regimens in terms of survival. The objectives of this study were to analyze the antibody response to NY-ESO-1 antigen in patients with esophageal cancer and to determine the potential of NY-ESO-1 for use in tumor-specific immunotherapy. Serum from 69 patients with esophageal cancer was investigated for antibody production against NY-ESO-1 by Western blot analysis. Also analyzed by immunohistochemistry were 56 tissue samples from these patients for NY-ESO-1 protein expression. NY-ESO-1 protein expression was found in 18 of 56 (32%) esophageal carcinomas. Serum immunoreactivity specific for NY-ESO-1 was found in 9 patients (13%) of whom 8 were in the advanced stage (stages III and IV). There was no relationship between clinicopathologic features and serum immunoreactivity for NY-ESO-1. NY-ESO-1 protein expression was detected in three of five antibody-positive patients whose tissue was available for analysis. Survival analysis showed no significant difference between antibody-positive and antibody-negative patient groups. A humoral immune response to NY-ESO-1 antigen was established in patients with advanced esophageal cancer. NY-ESO-1 is a good candidate for vaccine-based immunotherapy for advanced esophageal carcinoma.

The receptor for hyaluronic acid mediated motility (RHAMM/CD168) is a leukemia associated antigen eliciting both humoral and cellular immune responses in patients with acute myeloid leukemia (AML)

RHAMM/CD168 was defined as a LAA using the approach of serological screening of expression libraries (SEREX), eliciting IgG responses in 42% of patients with AML, but not in healthy volunteers (HV) or patients with autoimmune diseases. mRNA for RHAMM was demonstrated by qRT-PCR to be expressed in leukemic blasts of more than 80% of the AML patients, but not in PBMN or CD34+ stem cells of healthy volunteers. Among normal tissues, only testis, placenta and thymus showed significant mRNA expression for the antigen, therefore sharing the expression profile of some other SEREX antigens.

Oxidative stress as a trigger for cellular immune responses in patients with alcoholic liver disease.

Serum antibodies reactive with neo-antigens generated during ethanol metabolism have been identified in patients with alcoholic liver disease (ALD), although their role in the pathogenesis of disease remains unclear. In this study, we characterized peripheral blood mononuclear cell (PBMC) T-cell and antibody responses to human serum albumin (HAS) adducted with acetaldehyde under reducing conditions (AcA-HSA) or with malondialdehyde (MDA-HSA) in patients with advanced ALD (AALD, n = 28), heavy drinkers with no liver disease (NALD, n = 14), and mild/moderate drinking controls (n = 22). Peak proliferative responses of PBMC were assessed in vitro by tritiated thymidine incorporation after the addition of optimized concentrations of antigen or OKT3. Antibody titers were determined by enzyme-linked immunosorbent assay (ELISA). MDA-HSA induced PBMC T-cell proliferation was significantly higher in ALD than in NALD or control patients. Moreover, 10 of 28 (36%) of ALD patients had significant T-cell proliferative responses to MDA-HSA compared to 0 of 14 (0%, P =.02) of the NALD group and 2 of 22 (9%, P <.05) of controls. No significant difference in PBMC T-cell response to Aca-HSA was seen between subject groups. Patients with positive cellular responses to MDA had higher serum anti-MDA antibody titers than those not exhibiting a positive cellular response (P <.005). In conclusion, the pattern of cellular and humoral responses to MDA adducts suggests that the development of these responses may be a susceptibility factor for the development of advanced alcoholic liver disease. The apparent importance of T-cell responses to MDA adducts suggests that oxidative stress may represent an important stimulus for the development of cellular immune responses associated with advanced ALD.

Evaluation of cellular immune response in patients with recurrent candidiasis

Recurrent cutaneous or mucosal candidiasis is characterized by the occurrence of at least four candidiasis episodes within a one-year period. The factors involved in recurrence of infection are still unknown. In the present study the lymphoproliferative response and the IFN-gamma production by candidiasis patients were evaluated. The stimulation index of mononuclear cell cultures of candidiasis patients stimulated with Candida albicans antigen, PPD and TT were 6 +/- 8, 17 +/- 20 and 65 +/- 30, respectively. The addition of monoclonal antibody anti-IL-10 to Candida albicans antigen stimulated cultures raised the lymphoproliferative response from 735 +/- 415 to 4143 +/- 1746 cpm. The IFN-gamma production by cells of candidiasis patients stimulated with Candida albicans antigen was 162 +/- 345 pg/ml. Candidiasis patients have an impairment in the lymphoproliferative response specific to C. albicans antigen and on IFN-gamma production and the lymphoproliferative response can be partially restored, in vitro, by IL-10 neutralization.

Infection with Schistosoma mansoni correlates with altered immune responses to Ascaris lumbricoides and hookworm

Studies were performed on humoral and cellular immune responses of patients from areas in Brazil endemic for hookworm and Ascaris lumbricoides, and either endemic or non-endemic for Schistosoma mansoni. Humoral and cellular responses were evaluated by enzyme-linked immunosorbant assay (ELISA) and peripheral blood mononuclear cell (PBMC) proliferation assays against larval hookworm antigens, A. lumbricoides egg antigens, and soluble egg antigens (SEA) or soluble whole adult antigenic preparation (SWAP) from S. mansoni. Patients from S. mansoni-endemic areas, who currently had only hookworm or Ascaris infections, expressed lower humoral and cellular responses to hookworm or Ascaris antigens, respectively, than did their counterparts from areas not endemic for S. mansoni. Individuals from S. mansoni endemic area, although without detectable S. mansoni infection, do mount humoral and cellular responses to SEA and SWAP. This group of individuals has been probably in contact with S. mansoni antigens, since the groups harboring A. lumbricoides or hookworm infections from non- S. mansoni endemic areas do not have detectable anti- S. mansoni responses. PBMC proliferative responses discriminated well between patients with active hookworm infections versus ascariasis, if they were from areas not endemic for S. mansoni.

Immune and hormonal activity in adults suffering from depression.

An association between depression and altered immune and hormonal systems has been suggested by the results of many studies. In the present study we carried out immune and hormonal measurements in 40 non-medicated, ambulatory adult patients with depression determined by CID-10 criteria and compared with 34 healthy nondepressed subjects. The severity of the condition was determined with the Hamilton Depression Rating Scale. Of 40 depressed patients, 31 had very severe and 9 severe or moderate depression, 29 (72.5%) were females and 11 (27.5%) were males (2.6:1 ratio). The results revealed a significant reduction of albumin and elevation of alpha-1, alpha-2 and beta-globulins, and soluble IL-2 receptor in patients with depression compared to the values obtained for nondepressed subjects (P<0.05). The decrease lymphocyte proliferation in response to a mitogen was significantly lower in severely or moderately depressed patients when compared to control (P<0.05). These data confirm the immunological disturbance of acute phase proteins and cellular immune response in patients with depression. Other results may be explained by a variety of interacting factors such as number of patients, age, sex, and the nature, severity and/or duration of depression. Thus, the data obtained should be interpreted with caution and the precise clinical relevance of these findings requires further investigation.

The T-cell response in patients with cancer

Publisher Summary The chapter examines several methods to measure human T-cell responses, including ELISPOT analysis, intracellular cytokine staining of immune cells after antigenic stimulation, limiting dilution analysis, conventional cloning, and molecular definition of the T-cell response either in the peripheral circulation or in situ in patients with cancer The chapter presents the cellular immune response in patients with cancer. The chapter explores the recent studies that suggest humoral immunity and T-cell-mediated immunity are closely linked. In addition, most of the data concerning antitumor immune responses have been generated using MHC class I tetramer reagents. The ultimate goal of immunomonitoring in patients with cancer is the association of the biomarkers with the clinical outcome or with response to therapy. The enumeration of MHC-restricted and antigen-specific T cells in patients with cancer allows to address the question of whether the number of antigen-experienced T cells are associated with the disease, or alternatively, with antigen-specific vaccination. The ultimate goal would be that either the number of antigen-specific T cells or the quality of T cells that is, defined by cytokine release, or by homing markers be associated with a good or bad prognosis in the course of the natural disease or predict response to vaccination.

Evidence for prostaglandin-producing supressor cells in HCV patients with normal ALT.

Our objective was to verify the presence of prostaglandin-producing suppressor cells in response to hepatitis C virus antigens in peripheral blood mononuclear cells proliferation. Standard proliferation tests were performed in 31 patients: 20 with chronic hepatitis C after antiviral treatment [7 long-term responders (LTR), 7 relapsers (RR), 6 nonresponders (NR)], 7 with HCV infection with persistently normal aminotransferase levels (PNAL), and 4 with hepatocellular carcinoma. Six antigens were used from the core and NS3 regions. A modified proliferation assay consisting of the addition of indomethacin was also done. Lymphoproliferative responses to the HCV antigens were detectable in 27% (11/41) of test points of LTR, 10% (3/31) of RR, 26% (9/35) of NR, and 18% (7/39) of patients with PNAL. Indomethacin only had effect in PNAL patients, by increasing the frequency of reactivity from 18% (7/39) to 36% (14/39) tests points (P = 0.037); also, in three of these patients (43%) indomethacin strongly modified proliferation to core 31-50 and NS3 1248-1261 antigens, increasing both the frequency and stimulation index from 33% (6/18) to 72% (13/18) (chi2 = 5.43, P = 0.019) and 1.89 +/- 0.43 to 6.18 +/- 4.74 (P = 0.028), respectively. These results suggest that prostaglandin-producing suppressor may play a role in chronic HCV infection by inhibiting cellular immune responses in patients with persistently normal ALT.

JC virus-specific cytotoxic T lymphocytes in individuals with progressive multifocal leukoencephalopathy.

Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease of the central nervous system caused by a reactivation of the polyomavirus JC (JCV) within a setting of immunosuppression. The nature of the immune response that contains replication of this virus is unknown. We have explored JCV-specific cellular immune responses in patients with PML and control subjects. JCV antigen-stimulated peripheral blood mononuclear cells (PBMC) of four human immunodeficiency virus (HIV)-infected patients who were survivors of PML and one HIV-uninfected patient recently diagnosed with PML lysed autologous B-lymphoblastoid cell lines expressing either the JCV T regulatory protein or the VP1 major capsid protein. This lysis was mediated by CD8(+) T lymphocytes and was major histocompatibility complex class I restricted. These cells were therefore cytotoxic T lymphocytes (CTL). JCV-specific CTL could not be detected in PBMC of three HIV-infected PML patients who had progressive neurologic disease and an eventual fatal outcome. These data suggest that the JCV-specific cellular immune response may play a crucial role in the containment of PML. This finding may also prove useful as a favorable prognostic marker in the clinical management of these patients.

Bovine beta-casein antibodies in breast- and bottle-fed infants: their relevance in Type 1 diabetes.

Bovine beta-casein is a cow's milk protein that targets both humoral and cellular immune responses in patients with Type 1 diabetes and, to a lesser degree, also in normal subjects. In this study we aimed to determine whether the avoidance of cow's milk consumption early in life could prevent the development of antibody response to bovine beta-casein despite the mother being exposed on a daily basis to cow's milk consumption. We measured the antibody response to bovine beta-casein using an ELISA method in 28 healthy infants under 4 months of age, of whom 16 were exclusively breast-fed and 12 were bottle-fed with cow's milk. In addition, beta-casein antibodies were measured in 37 prepubertal children with Type 1 diabetes and in 31 healthy children who were exposed to cow's milk or dairy products to see whether differences in antibody titers exist in this young age group. Antibodies binding to beta-casein were also evaluated by immunoblotting analysis. Elevated levels of beta-casein antibodies were found in bottle-fed infants compared to breast-fed infants (p<0.0001). Antibody levels to bovine beta-casein were also significantly higher in children with Type 1 diabetes compared to age-matched controls (p=0.03). By western blot analysis we confirmed specific binding to bovine beta-casein in bottle-fed infants, in children with Type 1 diabetes and in controls exposed to cow's milk, but not in infants who were exclusively breast-fed. The results of this study indicate that breastfeeding within the first 4 months of life prevents the generation of antibody response to bovine beta-casein despite the mothers' consumption of cow's milk during the breastfeeding period. These findings may have relevance for disease prevention.

Effect of IL‐12 on T‐cell immune responses in patients with chronic HCV infection

As the host's immune responses may determine the outcome of hepatitis C virus (HCV) infection, and interleukin (IL)‐12 plays an essential role in host defense against infectious diseases, we studied the antigen‐specific and non‐specific cellular immune responses in patients with chronic HCV infection. A proliferative response to phytohemagglutinin (PHA) was found in all 20 patients. Of the 20, 8 (40%) displayed a lymphocyte proliferation in response to HCV antigen c22, 2 (10%) to c33, 6 (30%) to c100–3, and 1 (5%) to NS5. The addition of rhIL‐12 to cultures of peripheral blood mononuclear cells (PBMC) stimulated with PHA significantly enhanced the proliferative responses in normal controls as well as in HCV‐infected subjects. The increased proliferation was also observed in HCV‐infected patients when PBMC were co‐cultured with HCV antigens c22 and c 100–3 in the presence of rhIL‐12. The production of interferon γ (IFNγ), IL‐2, IL‐4 and IL‐10 was observed in 7 (58.3%), 5 (41.7%,), 3 (25.0%) and 5 (41.7%) HCV‐infected individuals stimulated with c22, and in 4 (33.3%), 2 (16.7%), 2 (16.7%) and 2 (16.7%) with c100–3, respectively. All HCV‐infected individuals had increased production of cytokines IFNγ, IL‐2, IL‐4 and IL‐10 in supernatants of PBMC after stimulation with PHA. IL‐12 significantly augmented Th1 cytokine production in HCV‐infected individuals stimulated with PHA and with HCV antigens. In conclusion, deficient cellular immune responses are present in HCV‐infected patients and IL‐12 can enhance the immune responses in these patients.