P-selectin and P-selectin glycoprotein ligand 1 are major determinants for Th1 cell recruitment to nonlymphoid effector sites in the intestinal lamina propria.

The recruitment of activated T cell subsets to sites of effector immune responses is mediated by homing receptors induced upon activation in secondary lymphoid tissue. Using an adoptive transfer model, the intestinal recruitment of CD4+ T cells activated with intraperitoneal antigen in complete Freund's adjuvant was examined. The data demonstrate that activated CD4+ T cells recruited to intestinal Peyer's patches (PP) and lamina propria (LP) up-regulate functional P-selectin glycoprotein ligand 1 (PSGL-1). Blockade of IL-12 inhibited functional PSGL-1 expression and reduced PP and LP CD4+ T cell recruitment by >40%. P-selectin blockade reduced LP recruitment of activated cells by 56% without affecting PP recruitment. Studies of mice examined 3 d after adoptive transfer of differentiated T cell subsets revealed that Th1 but not Th2 cells were recruited to small intestine PP and LP. Mucosal addressin cell adhesion molecule blockade reduced Th1 recruitment to PP by 90% and to LP by >72%, whereas P-selectin blockade reduced Th1 recruitment to PP by 18% and Th1 recruitment to LP by 84%. These data suggest that IL-12-induced functional PSGL-1 expression is a major determinant for the recruitment of Th1 effector cells to noninflamed as well as inflamed intestine.

Vaccine-induced HIV seropositivity/reactivity in noninfected HIV vaccine recipients.

CONTEXT: Induction of protective anti-human immunodeficiency virus (HIV) immune responses is the goal of an HIV vaccine. However, this may cause a reactive result in routine HIV testing in the absence of HIV infection. OBJECTIVE: To evaluate the frequency of vaccine-induced seropositivity/reactivity (VISP) in HIV vaccine trial participants. DESIGN, SETTING, AND PARTICIPANTS: Three common US Food and Drug Administration-approved enzyme immunoassay (EIA) HIV antibody kits were used to determine VISP, and a routine diagnostic HIV algorithm was used to evaluate VISP frequency in healthy, HIV-seronegative adults who completed phase 1 (n = 25) and phase 2a (n = 2) vaccine trials conducted from 2000-2010 in the United States, South America, Thailand, and Africa. MAIN OUTCOME MEASURE: Vaccine-induced seropositivity/reactivity, defined as reactive on 1 or more EIA tests and either Western blot-negative or Western blot-indeterminate/atypical positive (profile consistent with vaccine product) and HIV-1-negative by nucleic acid testing. RESULTS: Among 2176 participants free of HIV infection who received a vaccine product, 908 (41.7%; 95% confidence interval [CI], 39.6%-43.8%) had VISP, but the occurrence of VISP varied substantially across different HIV vaccine product types: 399 of 460 (86.7%; 95% CI, 83.3%-89.7%) adenovirus 5 product recipients, 295 of 552 (53.4%; 95% CI, 49.2%-57.7%) recipients of poxvirus alone or as a boost, and 35 of 555 (6.3%; 95% CI, 4.4%-8.7%) of DNA-alone product recipients developed VISP. Overall, the highest proportion of VISP (891/2176 tested [40.9%]) occurred with the HIV 1/2 (rDNA) EIA kit compared with the rLAV EIA (150/700 tested [21.4%]), HIV-1 Plus O Microelisa System (193/1309 tested [14.7%]), and HIV 1/2 Peptide and HIV 1/2 Plus O (189/2150 tested [8.8%]) kits. Only 17 of the 908 participants (1.9%) with VISP tested nonreactive using the HIV 1/2 (rDNA) kit. All recipients of a glycoprotein 140 vaccine (n = 70) had VISP, with 94.3% testing reactive with all 3 EIA kits tested. Among 901 participants with VISP and a Western blot result, 92 (10.2%) had a positive Western blot result (displaying an atypical pattern consistent with vaccine product), and 592 (65.7%) had an indeterminate result. Only 8 participants with VISP received a vaccine not containing an envelope insert. CONCLUSIONS: The induction of VISP in HIV vaccine recipients is common, especially with vaccines containing both the HIV-1 envelope and group-specific core antigen gene proteins. Development and detection of VISP appear to be associated with the immunogenicity of the vaccine and the EIA assay used.

Benefits of a comprehensive quality program for cryopreserved PBMC covering 28 clinical trials sites utilizing an integrated, analytical web-based portal.

The HIV Vaccine Trials Network (HVTN) is a global network of 28 clinical trial sites dedicated to identifying an effective HIV vaccine. Cryopreservation of high-quality peripheral blood mononuclear cells (PBMC) is critical for the assessment of vaccine-induced cellular immune functions. The HVTN PBMC Quality Management Program is designed to ensure that viable PBMC are processed, stored and shipped for clinical trial assays from all HVTN clinical trial sites. The program has evolved by developing and incorporating best practices for laboratory and specimen quality and implementing automated, web-based tools. These tools allow the site-affiliated processing laboratories and the central Laboratory Operations Unit to rapidly collect, analyze and report PBMC quality data. The HVTN PBMC Quality Management Program includes five key components: 1) Laboratory Assessment, 2) PBMC Training and Certification, 3) Internal Quality Control, 4) External Quality Control (EQC), and 5) Assay Specimen Quality Control. Fresh PBMC processing data is uploaded from each clinical site processing laboratory to a central HVTN Statistical and Data Management Center database for access and analysis on a web portal. Samples are thawed at a central laboratory for assay or specimen quality control and sample quality data is uploaded directly to the database by the central laboratory. Four year cumulative data covering 23,477 blood draws reveals an average fresh PBMC yield of 1.45×10(6)±0.48 cells per milliliter of useable whole blood. 95% of samples were within the acceptable range for fresh cell yield of 0.8-3.2×10(6) cells/ml of usable blood. Prior to full implementation of the HVTN PBMC Quality Management Program, the 2007 EQC evaluations from 10 international sites showed a mean day 2 thawed viability of 83.1% and a recovery of 67.5%. Since then, four year cumulative data covering 3338 specimens used in immunologic assays shows that 99.88% had acceptable viabilities (>66%) for use in cellular assays (mean, 91.46% ±4.5%), and 96.2% had acceptable recoveries (50%-130%) with a mean of recovery of 85.8% ±19.12% of the originally cryopreserved cells. EQC testing revealed that since August 2009, failed recoveries dropped from 4.1% to 1.6% and failed viabilities dropped from 1.0% to 0.3%. The HVTN PBMC quality program provides for laboratory assessment, training and tools for identifying problems, implementing corrective action and monitoring for improvements. These data support the benefits of implementing a comprehensive, web-based PBMC quality program for large clinical trials networks.

A novel HIV T helper epitope-based vaccine elicits cytokine-secreting HIV-specific CD4+ T cells in a Phase I clinical trial in HIV-uninfected adults.

A Phase I human vaccine trial of a novel polypeptide vaccine of HIV T helper epitopes (EP-1043) and a DNA vaccine of HIV CTL epitopes was conducted in 84 healthy adult volunteers. The vaccine immunogenicity was assessed by an intracellular cytokine staining assay for IL-2, IL-4, TNF-alpha and IFN-gamma. Sixty eight percent (32/47) of subjects had a positive CD4+ T response after receiving two vaccinations of the polypeptide vaccine. The responding CD4+ T cells made various combinations of IL-2, IL-4, IFN-gamma, and TNF-alpha. The study demonstrated that the EP-1043 vaccine is safe, well-tolerated, and immunogenic.

T cell receptor revision does not solely target recent thymic emigrants.

CD4(+)Vbeta5(+) T cells enter one of two tolerance pathways after recognizing a peripherally expressed superantigen encoded by an endogenous retrovirus. One pathway leads to deletion, while the other, termed TCR revision, results in cellular rescue upon expression of an alternate TCR that no longer recognizes the tolerogen. TCR revision requires the rearrangement of novel TCR beta-chain genes and depends on recombinase-activating gene (RAG) expression in peripheral T cells. In line with recent findings that RAG(+) splenic B cells are immature cells that have maintained RAG expression, it has been hypothesized that TCR revision is limited to recent thymic emigrants that have maintained RAG expression and TCR loci in a recombination-permissive configuration. Using mice in which the expression of green fluorescent protein is driven by the RAG2 promoter, we now show that in vitro stimulation can drive reporter expression in noncycling, mature, peripheral CD4(+) T cells. In addition, thymectomized Vbeta5 transgenic RAG reporter mice are used to demonstrate that TCR revision can target peripheral T cells up to 2 mo after thymectomy. Both sets of experiments strongly suggest that reinduction of RAG genes triggers TCR revision. Approximately 3% of CD4(+)Vbeta5(+) T cells in thymectomized Vbeta5 transgenic reporter mice have undergone TCR revision within the previous 4-5 days. TCR revision can also occur in Vbeta5(+) T cells from nontransgenic mice, illustrating the relevance of this novel tolerance mechanism in unmanipulated animals.

Differential regulation of peripheral CD4+ T cell tolerance induced by deletion and TCR revision.

In Vbeta5 transgenic mice, mature Vbeta5(+)CD4(+) T cells are tolerized upon recognition of a self Ag, encoded by a defective endogenous retrovirus, whose expression is confined to the lymphoid periphery. Cells are driven by the tolerogen to enter one of two tolerance pathways, deletion or TCR revision. CD4(+) T cells entering the former pathway are rendered anergic and then eliminated. In contrast, TCR revision drives gene rearrangement at the endogenous TCR beta locus and results in the appearance of Vbeta5(-), endogenous Vbeta(+), CD4(+) T cells that are both self-tolerant and functional. An analysis of the molecules that influence each of these pathways was conducted to understand better the nature of the interactions that control tolerance induction in the lymphoid periphery. These studies reveal that deletion is efficient in reconstituted radiation chimeras and is B cell, CD28, inducible costimulatory molecule, Fas, CD4, and CD8 independent. In contrast, TCR revision is radiosensitive, B cell, CD28, and inducible costimulatory molecule dependent, Fas and CD4 influenced, and CD8 independent. Our data demonstrate the differential regulation of these two divergent tolerance pathways, despite the fact that they are both driven by the same tolerogen and restricted to mature CD4(+) T cells.

Continued maturation of thymic emigrants in the periphery.

Developing thymocytes are selected for recognition of molecules encoded by the major histocompatibility complex, purged of self-reactive cells and committed to either the CD4 or CD8 lineage. The 1% of thymocytes that complete these tasks emigrate and join the population of peripheral lymphocytes. Whether T cell maturation is complete at the time of thymic exit has been a subject of debate. Using mice transgenic for green fluorescent protein driven by the recombination activating gene 2 promoter to identify recent thymic emigrants, we now show that T cell differentiation continues post-thymically, with progressive maturation of both surface phenotype and immune function. In addition, the relative contribution of CD4 and CD8 recent thymic emigrants was modulated as they entered the peripheral T cell pool. Thus, T cell maturation and subset contribution are both finalized in the lymphoid periphery.

The state of CD4+ T cell activation is a major factor for determining the kinetics and location of T cell responses to oral antigen.

Current models suggest that inductive immune responses to enteric Ag are initiated in Peyer's patches (PP) and mesenteric lymph nodes (MLN) followed by migration of activated, memory-like CD4(+) T cells to extralymphoid sites in the intestinal lamina propria (LP). The resultant immune system contains both naive and activated T cells. To examine the differential responses of naive and memory-like T cells to oral Ag, bone marrow chimeras (BMC) were generated. Irradiated BALB/c hosts were reconstituted with a mix of DO11.10 x RAG-1(-/-) and BALB/c bone marrow. In unprimed DO11.10 and BMC models, LP and PP DO11.10 T cells responded to oral Ag with similar kinetics. Responses of activated, memory-like T cells to oral Ag were examined in thymectomized BMC 60 days after i.p. immunization with OVA peptide in Freund's adjuvant (OVA(323-339)/CFA). Results indicate that i.p. OVA(323-339)/CFA generated a high proportion of memory-like CD45RB(low) DO11.10 T cells in peripheral lymphoid (40%) and intestinal LP (70%) tissue. Previously activated DO11.10 T cells in the LP responded to oral Ag earlier and at 50% higher levels compared with memory CD4(+) T cells localized to PP tissue. These data indicate that responses to oral Ag in antigenically naive animals are initiated in PP whereas in Ag-experienced animals LP T cells respond earlier and more vigorously than cells in PP. Taken together, these data suggest that previous activation alters the hierarchy of T cell responses to oral Ag by enhancing the efficiency of LP T cell activation.

Cutting edge: TCR revision occurs in germinal centers.

Mouse CD4(+)Vbeta5(+) T cells recognize a peripherally expressed superantigen encoded by an endogenous retrovirus. Ag encounter tolerizes the mature CD4 T cell compartment, either by deletion of autoreactive cells or by TCR revision. This latter process is driven by TCRbeta rearrangement through RAG activity and results in the rescue of cells expressing novel TCRs that no longer recognize the tolerogen. Consistent with the notion that revising T cells represent a distinct peripheral T cell population, we now show that these lymphocyte blasts express a hybrid effector/memory phenotype and are not undergoing cell division. A population of revising T cells is CD40(+), expresses the germinal center (GC) marker CXCR5, and is Vbeta5(low)Thy-1(low). Histology reveals that, consistent with their surface Ag phenotype, T cells undergoing TCR revision are enriched in splenic GCs. These data demonstrate that TCR revision is a multistep tolerance pathway supported by the unique microenvironment provided by GCs.