Enzyme-linked Immunosorbent Spot Assay Research Articles

Specific CD8+ T cell responses correlate with control of simian immunodeficiency virus replication in Mauritian cynomolgus macaques.

Specific major histocompatibility complex (MHC) class I alleles are associated with an increased frequency of spontaneous control of human and simian immunodeficiency viruses (HIV and SIV). The mechanism of control is thought to involve MHC class I-restricted CD8(+) T cells, but it is not clear whether particular CD8(+) T cell responses or a broad repertoire of epitope-specific CD8(+) T cell populations (termed T cell breadth) are principally responsible for mediating immunologic control. To test the hypothesis that heterozygous macaques control SIV replication as a function of superior T cell breadth, we infected MHC-homozygous and MHC-heterozygous cynomolgus macaques with the pathogenic virus SIVmac239. As measured by a gamma interferon enzyme-linked immunosorbent spot assay (IFN-γ ELISPOT) using blood, T cell breadth did not differ significantly between homozygotes and heterozygotes. Surprisingly, macaques that controlled SIV replication, regardless of their MHC zygosity, shared durable T cell responses against similar regions of Nef. While the limited genetic variability in these animals prevents us from making generalizations about the importance of Nef-specific T cell responses in controlling HIV, these results suggest that the T cell-mediated control of virus replication that we observed is more likely the consequence of targeting specificity rather than T cell breadth.

Elite Controllers with Low to Absent Effector CD8 + T Cell Responses Maintain Highly Functional, Broadly Directed Central Memory Responses

Analyses of the breadth and specificity of virus-specific CD8(+) T cell responses associated with control of HIV have largely relied on measurement of cytokine secretion by effector T cells. These have resulted in the identification of HIV elite controllers with low or absent responses in which non-T-cell mechanisms of control have been suggested. However, successful control of HIV infection may be associated with central memory T cells, which have not been consistently examined in these individuals. Gag-specific T cells were characterized using a peptide-based cultured enzyme-linked immunosorbent spot assay (ELISpot). Peripheral blood mononuclear cells from HIV elite controllers (n = 10), progressors (n = 12), and antiretroviral-treated individuals (n = 9) were cultured with overlapping peptides for 12 days. Specificity was assessed by tetramer staining, functional features of expanded cells were assessed by cytokine secretion, and virus inhibition and phenotypic characteristics were assessed by cell sorting and coculture assays. After peptide stimulation, elite controllers showed a greater number of previously undetectable (new) responses compared to progressors (P = 0.0008). These responses were highly polyfunctional, with 64.5% of responses having 3 to 5 functions. Expandable epitope-specific CD8(+) T cells from elite controllers had strong virus inhibitory capacity and predominantly displayed a central memory phenotype. These data indicate that elite controllers with minimal T cell responses harbor a highly functional, broadly directed central memory T cell population that is capable of suppressing HIV in vitro. Comprehensive examination of this cell population could provide insight into the immune responses associated with successful containment of viremia.

Human Cytomegalovirus-Specific T-Cell Immune Reconstitution in Preemptively Treated Heart Transplant Recipients Identifies Subjects at Critical Risk for Infection

Human cytomegalovirus (CMV) infection represents a major threat for heart transplant recipients (HTXs). CMV-specific T cells effectively control virus infection, and thus, assessment of antiviral immune recovery may have clinical utility in identifying HTXs at risk of infection. In this study, 10 CMV-seropositive (R(+)) pretransplant patients and 48 preemptively treated R(+) HTXs were examined before and after 100 days posttransplant. Preemptive treatment is supposed to favor the immune recovery. CMV DNAemia and gamma interferon enzyme-linked immunosorbent spot (ELISPOT) assay were employed to assess the viremia and immune reconstitution. HTXs could be categorized into three groups characterized by high (>100), medium (50 to 100), and low (<50) spot levels. Early-identified high responders efficiently controlled the infection and also maintained high immunity levels after 100 days after transplant. No episodes of grade ≥2R rejection occurred in the high responders. Midresponders were identified as a group with heterogeneous trends of immune reconstitution. Low responders were 41% and 21% of HTXs before and after 100 days posttransplant, respectively. Low responders were associated with a higher incidence of infection. The effect of viremia on immune recovery was investigated: a statistically significant inverse correlation between magnitude of viremia and immune recovery emerged; in particular, each 10-fold increase in viremia (>4 log(10) DNAemia/ml) was associated with a 36% decrease of the ELISPOT assay spot levels. All episodes of high viremia (>4 log(10) DNAemia/ml) occurred from 1 to 60 days after transplant. Thus, the concomitant evaluation of viremia and CMV immune reconstitution has clinical utility in identifying HTXs at risk of infection and may represent a helpful guide in making therapeutic choices.

Added Value of Use of a Purified Protein Derivative-Based Enzyme-Linked Immunosorbent Spot Assay for Patients with Mycobacterium bovis BCG Infection after Intravesical BCG Instillations

In this case series, we describe four cases in which the use of gamma interferon release assays with purified protein derivative (PPD) as a stimulating antigen was able to demonstrate PPD-specific immune activation. This may help to improve the adequate diagnosis of (systemic) Mycobacterium bovis BCG infections after intravesical BCG instillations for bladder carcinoma.

Independence of measles-specific humoral and cellular immune responses to vaccination

With a larger, independent cohort and more sophisticated measures, we sought to confirm our work that indicated independence of humoral and cellular immunity following measles vaccination. We recruited an age-stratified random cohort of 764 healthy subjects from all socioeconomic strata, all with medical-record documentation of 2 age-appropriate doses of measles-containing vaccine. We quantified measles-specific neutralizing antibody levels and assayed the interferon-γ (IFN-γ) enzyme-linked immunosorbent spot assay (ELISPOT) response to measles virus. We also measured secreted cytokines from the peripheral blood mononuclear cells (PBMCs) in response to measles virus by performing enzyme-linked immunosorbent assays as secondary measures of cellular immune status. The median antibody level and median IFN-γ ELISPOT response were 844 mIU/mL (interquartile range [IQR]: 418-1,752) and 36 (IQR: 13.00-69.00) spot-forming cells (per 2 × 10(5) PBMCs), respectively. We observed only a very weak and negative correlation (Spearman's r(s) or ρ of -0.090 [95% confidence interval: -0.162--0.018]). We observed a similar lack of quantitatively important correlations between the neutralizing antibody level and any of the secondary measures. Our data confirm the independence of humoral and cellular immune responses after the second dose of measles vaccination. As researchers pursue novel measles vaccine and measles vaccine delivery systems, they must not infer that humoral responses predict cellular responses.

Pretransplant IFN-γ ELISPOT assay as a potential screening test to select immunosuppression protocols for patients receiving basiliximab induction therapy

The use of basiliximab induction therapy has increased in standard immunological risk patients. The objective of this study was to identify whether pretransplant donor-reactive interferon-γ enzyme-linked immunosorbent spot (ELISPOT) assay results were associated with post-transplant clinical outcomes in patients receiving basiliximab induction therapy and whether this could be helpful for choosing an efficacious immunosuppressive regimen. In 154 living donor renal transplant recipients who received basiliximab induction therapy without desensitization, we determined pretransplant ELISPOT frequencies and correlated the results with clinical outcomes based on the use of calcineurin inhibitors (tacrolimus [TAC] or cyclosporine [CSA]). The ELISPOT (+) patients had higher rate of post-transplant biopsy-proven acute rejection (AR) than ELISPOT (-) patients (P = 0.001) regardless of immunosuppressive regimen. In the logistic and multivariate regression analysis, ELISPOT was the only significant correlate of AR (P = 0.002), and the patients with increased ELISPOT results and CSA therapy were associated with AR. Our results suggest that the pretransplant ELISPOT (+) may assess the risk of poor post-transplant outcomes in patients with basiliximab induction.

Innate stimulation of B1a cells enhances the autoreactive IgM repertoire in the NOD mouse: implications for type 1 diabetes

We sought to determine whether the presence of natural autoreactive antibodies of B1a cell origin would play a role in the initiation of type 1 diabetes. We compared IgM repertoires and B1a cell compartments in NOD and C57BL/6 mice. Serum IgM autoreactivity profiles were determined by ELISA and the secretory properties and activation status of B1a cells were characterised by enzyme-linked immunosorbent spot (ELISPOT) assay and flow cytometry. B1a cell response to innate activation was analysed by gene expression assays, ELISA and [(3)H]thymidine incorporation. The effect of NOD IgM produced by B1a cells on NOD.severe combined immunodeficient (SCID) beta cells was examined in co-cultures: IgM binding was measured by flow cytometry and real-time PCR was used to study oxidative stress responses. NOD mice displayed increased levels of serum anti-insulin IgM that were independent of the H2 locus, that were maintained up to prediabetic stages and that correlated with the NOD B1a cell secretion profile. NOD B1a cells had a naturally increased pattern of activation, expressed higher levels of toll-like-receptors (Tlrs) and responded to TLR stimulation in vitro with higher proliferation and increased capacity to secrete anti-type-1-diabetes-related IgM, but produced lower amounts of IL10. IgM of NOD B1a cell origin was able to bind to pancreatic beta cells in vitro and induce expression of inducible nitric oxide synthase (Nos2). NOD B1a cells had a lower innate activation threshold for secretion of autoreactive IgM capable of triggering oxidative stress responses on binding to pancreatic beta cells; this provides an early mechanism that contributes to diabetes in a mouse model of type 1 diabetes.

Diagnostic value of an IFN-γ ELISPOT assay to detect latent tuberculosis infection in hepatitis C patients

The rapid diagnosis of tuberculosis (TB) and latent tuberculosis infections (LTBI) is a significant problem in clinical practice. The aim of this study was to evaluate the diagnostic value of an enzyme-linked immunosorbent spot (ELISPOT) assay measuring interferon-γ in hepatitis C patients with LTBI. A total of 160 hepatitis C patients at the Jilin University Hospital, Changchun, China, were prospectively enrolled from January 2009 to December 2010; 43 had been positively diagnosed with TB, 38 with non-TB diseases, and 79 with a history of TB. All patients were evaluated by the tuberculin skin test (TST) and ELISPOT assays. Among the 43 diagnosed TB patients, the ELISPOT assay had a sensitivity of 92.1%, compared to a sensitivity of 60.5% for the TST. Among the 79 TB exposure patients, the ELISPOT assay was more sensitive (90%) than the TST (61.5%), the specificity of the ELISPOT assay was 90%, and the specificity of the TST was 61.5% in LTBI. Among the 38 subjects with non-TB diseases, the specificity of the ELISPOT was better than the TST’s. In conclusion, this ELISPOT assay could provide useful support in diagnosing LTBI in hepatitis C patients and may provide guidance regarding the treatment of LTBI and hepatitis C co-infection. Key words: Latent tuberculosis infections (LTBI), Hepatitis C virus (HCV), enzyme-linked immunosorbent spot (ELISPOT) assay, CFP-10/ESAT-6

Widespread impact of HLA restriction on immune control and escape pathways of HIV-1.

The promiscuous presentation of epitopes by similar HLA class I alleles holds promise for a universal T-cell-based HIV-1 vaccine. However, in some instances, cytotoxic T lymphocytes (CTL) restricted by HLA alleles with similar or identical binding motifs are known to target epitopes at different frequencies, with different functional avidities and with different apparent clinical outcomes. Such differences may be illuminated by the association of similar HLA alleles with distinctive escape pathways. Using a novel computational method featuring phylogenetically corrected odds ratios, we systematically analyzed differential patterns of immune escape across all optimally defined epitopes in Gag, Pol, and Nef in 2,126 HIV-1 clade C-infected adults. Overall, we identified 301 polymorphisms in 90 epitopes associated with HLA alleles belonging to shared supertypes. We detected differential escape in 37 of 38 epitopes restricted by more than one allele, which included 278 instances of differential escape at the polymorphism level. The majority (66 to 97%) of these resulted from the selection of unique HLA-specific polymorphisms rather than differential epitope targeting rates, as confirmed by gamma interferon (IFN-γ) enzyme-linked immunosorbent spot assay (ELISPOT) data. Discordant associations between HLA alleles and viral load were frequently observed between allele pairs that selected for differential escape. Furthermore, the total number of associated polymorphisms strongly correlated with average viral load. These studies confirm that differential escape is a widespread phenomenon and may be the norm when two alleles present the same epitope. Given the clinical correlates of immune escape, such heterogeneity suggests that certain epitopes will lead to discordant outcomes if applied universally in a vaccine.

The clinical relevance of persistent recombinant immunoblot assay–indeterminate reactions: insights into the natural history of hepatitis C virus infection and implications for donor counseling

Recombinant immunoblot assay (RIBA) is used to determine the specificity of antibody to hepatitis C virus (anti-HCV). The RIBA result is recorded as positive, negative, or indeterminate. The interpretation and significance of RIBA-indeterminate reactions are unclear. We addressed the clinical relevance of these reactions in the context of the natural history of HCV infection in a prospectively followed cohort of anti-HCV-positive blood donors. Donor demographics, exposure history, and humoral and cell-mediated immunity (CMI) were compared in 15 RIBA-indeterminate subjects, nine chronic HCV carriers, and eight spontaneously recovered subjects. Serum samples were tested for anti-HCV by a quantitative, liquid luciferase immunoprecipitation system (LIPS). CMI was assessed by interferon-γ enzyme-linked immunosorbent spot assay. In the LIPS assay, the sum of antibody responses to six HCV antigens showed significant (p < 0.001) stepwise diminution progressing from chronic carriers to spontaneously recovered to RIBA-indeterminate subjects. CMI responses in RIBA-indeterminate subjects were similar to spontaneously recovered subjects and greater than chronic carriers and controls (p < 0.008). A parenteral risk factor was identified in only 13% of RIBA-indeterminate subjects compared to 89% of chronic carriers and 87% of spontaneously recovered subjects. RIBA-indeterminate donors were older than the other groups. The CMI and LIPS results suggest that persistent RIBA-indeterminate reactions represent waning anti-HCV responses in persons who have recovered from a remote HCV infection. In such cases, detectable antibody may ultimately disappear leaving no residual serologic evidence of prior HCV infection, as reported in a minority of long-term HCV-recovered subjects.

Arthritis development in germ free mice deficient for reactive oxygen species

BackgroundThe Ncf1 protein is a subunit of the nicotinamide adenine dinucleotide phosphate oxidase (NADPH) oxidase complex, which is responsible for producing the precursors of reactive oxygen species (ROS). ROS is...

CD8+T Cells Provide an Immunologic Signature of Tuberculosis in Young Children

The immunologic events surrounding primary Mycobacterium tuberculosis infection and development of tuberculosis remain controversial. Young children who develop tuberculosis do so quickly after first exposure, thus permitting study of immune response to primary infection and disease. We hypothesized that M. tuberculosis-specific CD8(+) T cells are generated in response to high bacillary loads occurring during tuberculosis. To determine if M. tuberculosis-specific T cells are generated among healthy children exposed to M. tuberculosis and children with tuberculosis. Enzyme-linked immunosorbent spot assays were used to measure IFN-γ production in response to M. tuberculosis-specific proteins ESAT-6/CFP-10 by peripheral blood mononuclear cells and CD8(+) T cells isolated from Ugandan children hospitalized with tuberculosis (n = 96) or healthy tuberculosis contacts (n = 62). The proportion of positive CD8(+) T-cell assays and magnitude of CD8(+) T-cell responses were significantly greater among young (<5 yr) tuberculosis cases compared with young contacts (P = 0.02, Fisher exact test, P = 0.01, Wilcoxon rank-sum, respectively). M. tuberculosis-specific T-cell responses measured in peripheral blood mononuclear cells were equivalent between groups. Among young children, M. tuberculosis-specific CD8(+) T cells develop in response to high bacillary loads, as occurs during tuberculosis, and are unlikely to be found after M. tuberculosis exposure. T-cell responses measured in peripheral blood mononuclear cells are generated after M. tuberculosis exposure alone, and thus cannot distinguish exposure from disease. In young children, IFN-γ-producing M. tuberculosis-specific CD8(+) T cells provide an immunologic signature of primary M. tuberculosis infection resulting in disease.

Monitoring of peptide-specific and gamma interferon-productive T cells in patients with active and convalescent tuberculosis using an enzyme-linked immunosorbent spot assay.

To establish a high-efficiency gamma interferon-specific enzyme-linked immunosorbent spot assay (IFN-γ ELISPOT assay) for detection of tuberculosis (TB), peptides (E6, E7, and C14) and peptide mixtures (E6 plus E7 and E6 plus E7 plus C14) were used to monitor peripheral blood (PBL) samples from patients with pulmonary TB (PTB), as well as control samples. The positive detection rates of the five IFN-γ ELISPOT assays were 78.38%, 74.86%, 55.83%, 90.43%, and 91.51%, respectively, and there were similar detection rates between the two combined peptide mixture IFN-γ ELISPOT assays and the tuberculin skin test (TST) (90.62% versus 95.59%). No significant difference was found between the detection rates of the two combined peptide mixture IFN-γ ELISPOT assays and the T-SPOT.TB assay for 86 patients with PTB (P > 0.05), but the median number of spot-forming cells/10(6) cells (SFP value) for positive results was higher by the former than by the latter assay (P < 0.05). In contrast, the 29.93% positive detection rate and median SFP value of 482 by the two combined peptide mixture IFN-γ ELISPOT assays were significantly higher than the corresponding values of 14.29% and 152 by T-SPOT.TB assay for the same 147 community donors (P < 0.05). For nine PTB patients tracked, the SFP value of 7 for the two peptide mixture IFN-γ ELISPOT assays began to decrease from the second month after regular treatment. A relatively low, almost normal, SFP level was reached and maintained after the third or fourth month. Two in-house IFN-γ ELISPOT assays and the T-SPOT.TB assay could reduce the false-positive and false-negative detection rates of TST and sputum acid-fast staining. Therefore, these two combined peptide mixture IFN-γ ELISPOT assays have a potential advantage, beyond their greater specificity and sensitivity, for use in screening and detection of active TB infection (TBI) and latent TB infection (LTBI) in China.

Involvement of intron A into eukaryotic expression vector to improve immunogenicity of mycobacterial heat-shock protein 65 DNA vaccine in mice

To explore the involvement of intron A into eukaryotic expression vector to improve antigen expression efficiency and enhance immunogenicity of DNA vaccine in mice. As model antigen, the coding gene of mycobacterial Hsp65 was cloned into eukaryotic expression vector pCMV4.0 with intron A involved and pVAX1 without intron A involved, respectively. The resulted recombinant expression vectors were transfected into 293T cells and were then injected into BALB/c mice as DNA vaccines. Anti-Hsp65 specific IgG and isotype were detected by ELISA and T cell immune response was analyzed by enzyme-linked immunosorbent spot (ELISPOT) assay and intracellular cytokine staining. Compared with non-intron A pVAX1hsp65, the recombinant plasmid pCMV4.0hsp65 involved with intron A pVAX1hsp65 caused higher expression level of Hsp65 in 293T cells, and enhanced Th1 type immune response, which was defined as higher level of anti-Hsp65 specific total IgG level (3.76 ± 0.23 vs 3.15 ± 0.22, P < 0.01) and IgG2a/IgG1 ratio (4.08 ± 0.04 vs 2.23 ± 0.12, P < 0.01) and more IFN-γ-secreting CD4(+) ((2.0 ± 0.058)% vs (1.5 ± 0.087)%, t = 4.804, P < 0.01) and CD8(+) ((0.6 ± 0.058)% vs (1.0 ± 0.115)%, t = 3.098, P < 0.05) T lymphocytes. The difference showed statistical significance. Intron A can improve the expression efficiency of mycobacterial Hsp65 antigen and enhance immunogenicity of DNA vaccine in mice when involved into eukaryotic expression vector.

Humoral and Cellular Immune Responses to Yersinia pestis Infection in Long-Term Recovered Plague Patients

Plague is one of the most dangerous diseases and is caused by Yersinia pestis. Effective vaccine development requires understanding of immune protective mechanisms against the bacterium in humans. In this study, the humoral and memory cellular immune responses in plague patients (n = 65) recovered from Y. pestis infection during the past 16 years were investigated using a protein microarray and an enzyme-linked immunosorbent spot assay (ELISpot). The seroprevalence to the F1 antigen in all recovered patients is 78.5%. In patients infected more than a decade ago, the antibody-positive rate still remains 69.5%. There is no difference in the antibody presence between gender, age, and infected years, but it seems to be associated with the F1 antibody titers during infection (r = 0.821; P < 0.05). Except F1 antibody, the antibodies against LcrV and YopD were detected in most of the patients, suggesting they could be the potential diagnostic markers for detecting the infection of F1-negative strains. Regarding cellular immunity, the cell number producing gamma interferon (IFN-γ), stimulated by F1 and LcrV, respectively, in vitro to the peripheral blood mononuclear cells of 7 plague patients and 4 negative controls, showed no significant difference, indicating F1 and LcrV are not dominant T cell antigens against plague for a longer time in humans. Our findings have direct implications for the future design and development of effective vaccines against Y. pestis infection and the development of new target-based diagnostics.

Toxoplasma gondii HLA-B*0702-restricted GRA720-28 peptide with adjuvants and a universal helper T cell epitope elicits CD8+ T cells producing interferon-γ and reduces parasite burden in HLA-B*0702 mice

The ability of CD8(+) T cells to act as cytolytic effectors and produce interferon-γ (IFN-γ) was demonstrated to mediate resistance to Toxoplasma gondii in murine models because of the recognition of peptides restricted by murine major histocompatibility complex (MHC) class I molecules. However, no T gondii-specific HLA-B07-restricted peptides were proven protective against T gondii. Recently, 2 T gondii-specific HLA-B*0702-restricted T cell epitopes, GRA7(20-28) (LPQFATAAT) and GRA3(27-35) (VPFVVFLVA), displayed high-affinity binding to HLA-B*0702 and elicited IFN-γ from peripheral blood mononuclear cells of seropositive HLA-B*07 persons. Herein, these peptides were evaluated to determine whether they could elicit IFN-γ in splenocytes of HLA-B*0702 transgenic mice when administered with adjuvants and protect against subsequent challenge. Peptide-specific IFN-γ-producing T cells were identified by enzyme-linked immunosorbent spot and proliferation assays utilizing splenic T lymphocytes from human lymphocyte antigen (HLA) transgenic mice. When HLA-B*0702 mice were immunized with one of the identified epitopes, GRA7(20-28) in conjunction with a universal CD4(+) T cell epitope (PADRE) and adjuvants (CD4(+) T cell adjuvant, GLA-SE, and TLR2 stimulatory Pam(2)Cys for CD8(+) T cells), this immunization induced CD8(+) T cells to produce IFN-γ and protected mice against high parasite burden when challenged with T gondii. This work demonstrates the feasibility of bioinformatics followed by an empiric approach based on HLA binding to test this biologic activity for identifying protective HLA-B*0702-restricted T gondii peptides and adjuvants that elicit protective immune responses in HLA-B*0702 mice.

Spontaneous autoimmune gastritis and hypochlorhydria are manifest in the ileitis-prone SAMP1/YitFcs mice

SAMP1/YitFcs mice serve as a model of Crohn's disease, and we have used them to assess gastritis. Gastritis was compared in SAMP1/YitFcs, AKR, and C57BL/6 mice by histology, immunohistochemistry, and flow cytometry. Gastric acid secretion was measured in ligated stomachs, while anti-parietal cell antibodies were assayed by immunofluorescence and enzyme-linked immunosorbent spot assay. SAMP1/YitFcs mice display a corpus-dominant, chronic gastritis with multifocal aggregates of mononuclear cells consisting of T and B lymphocytes. Relatively few aggregates were observed elsewhere in the stomach. The infiltrates in the oxyntic mucosa were associated with the loss of parietal cell mass. AKR mice, the founder strain of the SAMP1/YitFcs, also have gastritis, although they do not develop ileitis. Genetic studies using SAMP1/YitFcs-C57BL/6 congenic mice showed that the genetic regions regulating ileitis had comparable effects on gastritis. The majority of the cells in the aggregates expressed the T cell marker CD3 or the B cell marker B220. Adoptive transfer of SAMP1/YitFcs CD4(+) T helper cells, with or without B cells, into immunodeficient recipients induced a pangastritis and duodenitis. SAMP1/YitFcs and AKR mice manifest hypochlorhydria and anti-parietal cell antibodies. These data suggest that common genetic factors controlling gastroenteric disease in SAMP1/YitFcs mice regulate distinct pathogenic mechanisms causing inflammation in separate sites within the digestive tract.

Coating Human Pancreatic Islets With CD4+CD25highCD127− Regulatory T Cells as a Novel Approach for the Local Immunoprotection

To develop a novel approach for local immunoprotection using CD4(+)CD25(high)CD127(-) T regulatory cells (Tregs) attached to the surface of the islets before transplantation. Tregs expanded ex vivo can control allo and autoreactivity, therefore, Treg-based therapy may offer more effective protection for transplanted islets from immunologic attack than currently used immunosuppression. Local application of Tregs can make such therapy more clinically feasible and efficient. Human islets were isolated and coated with allogeneic ex vivo expanded Tregs using biotin-poly(ethylene glycol)-N-hydroxysuccinimide ester (biotin-PEG-NHS) and streptavidin as binding molecules. Coating pancreatic islets with Tregs did not affect islet viability (>90% fluorescein diacetate/propidium iodide) or the insulin secretion profile in dynamic islet perifusion assays. After in vitro incubation with allogeneic T effector cells, Treg-coated islets revealed preserved function with higher insulin secretion compared with controls-native islets, coated islets with T effector cells or when Tregs were added to the culture, but not attached to islets (P < 0.05). In addition, the Enzyme-linked immunosorbent spot (ELISPOT) assay revealed suppression of interferon (IFN)-γ secretion, when T effector cells were challenged with Treg-coated islets comparing to controls (99 ± 7 vs 151 ± 8 dots, respectively; P < 0.01). We demonstrated, for the first time, the ability to bind immune regulatory cells to target cells with preservation of their viability and function and protective activity against immune attack. If successfully tested in an animal model, local delivery of immunoprotective Tregs on the surface of transplanted pancreatic islets may be an alternative or improvement to the currently used immunosuppression.

Limited immunogenicity of HIV CD8+ T-cell epitopes in acute Clade C virus infection.

Human immunodeficiency virus type 1 (HIV-1)-specific CD8(+) responses contribute to the decline in acute peak viremia following infection. However, data on the relative immunogenicity of CD8(+) T-cell epitopes during and after acute viremia are lacking. We characterized CD8(+) T-cell responses in 20 acutely infected, antiretroviral-naive individuals with HIV-1 subtype C infection using the interferon-γ enzyme-linked immunosorbent spot assay. Eleven of these had not fully seroconverted at the time of analysis. Viruses from plasma were sequenced within defined cytotoxic T-lymphocyte (CTL) cell epitopes for selected subjects. At approximately 28 days after estimated initial infection, CD8(+) T-cell responses were directed against an average of 3 of the 410 peptides tested (range, 0-6); 2 individuals had no detectable responses at this time. At 18 weeks, the average number of peptides targeted had increased to 5 (range 0-11). Of the 56 optimal Gag CTL epitopes sequenced, 31 were wild-type in the infecting viruses, but only 11 of 31 elicited measurable CD8(+) T-cell responses. These data demonstrate that the majority of CD8(+) responses are not elicited during acute HIV infection despite the presence of the cognate epitope in the infecting strain. There is a need to define factors that influence lack of induction of effective immune responses and the parameters that dictate immunodominance in acute infection.

Dynamics of cell-mediated immune responses to cytomegalovirus in pediatric transplantation recipients

CMI responses, combined with quantification of CMV DNA (DNAemia), may identify transplantation recipients at risk for invasive disease. PBMC were collected in pediatric transplantation candidates at one, three, and six months post-transplant in 10 subjects (six renal, three cardiac, one stem cell) and at single time points in eight HC and 14 children greater than one yr post-transplant (LTTx). Cells were stimulated with anti-CD3mAb or CMV pp65 peptide pools and responses assessed by IFNG enzyme-linked immunosorbent spot assay and cytokine secretion. IFNG responses to anti-CD3mAb were significantly lower pretransplant relative to HC and were further decreased at one and three months post-transplant, but recovered to levels comparable to HC by six months. Responses to pp65 among CMV-seropositive recipients followed a similar pattern but recovered by three months. CMV-seropositive LTTx and HC showed a Th1 cytokine response to pp65 stimulation. Three LTTx subjects developed CMV DNAemia; two demonstrated decreased responses to anti-CD3mAB (and pp65 in the CMV seropositive subject) at the onset of DNAemia, which recovered as DNAemia resolved. Monitoring CMI in children is feasible and may provide an adjunct biomarker to predict CMV progression and recovery.