Johnson T. Wong

Optimization of methods to assess human mucosal T-cell responses to HIV infection

The majority of HIV-1 infections occur via sexual transmission at mucosal epithelia lining the vagina, cervix or rectum. Mucosal tissues also serve as viral reservoirs. However, our knowledge of human mucosal T-cell responses is limited. There is a need for reliable, sensitive, and reproducible methods for assessing mucosal immunity. Here we report on the collaborative efforts of two laboratories to optimize methods for processing, culturing, and analyzing mucosal lymphocytes. Rectal biopsy tissue was obtained by flexible sigmoidoscopy, which is rapid, minimally invasive, and well tolerated. Of the four methods compared for isolating mucosal mononuclear cells (MMC), collagenase digestion reproducibly yielded the most lymphocytes (4-7 x 10(6)). Furthermore, 0.5-1 x 10(6) MMC could be polyclonally expanded to yield 17 x 10(6) CD8+ T cells allowing mapping of responses to overlapping peptides spanning the HIV-1 genome using IFN-gamma enzyme-linked immunospot (ELISpot). Expansion also reduced the spontaneous IFN-gamma production normally detected in fresh MMC. Piperacillin-tazobactam and amphotericin B reduced contamination of MMC cultures to 4%. Taken together, these methods will be useful for studies of mucosal immunity to HIV-1 and other pathogens during natural infection and following vaccination.

Risk stratification for desensitization of patients with carboplatin hypersensitivity: Clinical presentation and management

BACKGROUND Women with ovarian cancer treated with chemotherapeutic platinum agents frequently develop hypersensitivity reactions (HSRs). How best to risk-stratify patients for desensitization is uncertain. OBJECTIVES To evaluate skin test (ST) reactivity to carboplatin in patients with recent and remote histories of carboplatin HSR and to review the relationship between skin test reactivity and tolerance of subsequent carboplatin desensitization. METHODS Thirty-eight women with carboplatin HSR were evaluated by ST to carboplatin. Thirty women subsequently underwent 106 desensitizations to carboplatin. RESULTS Carboplatin ST was positive in 25 of 38 patients (66%). Of patients with recent HSR (<3 months), 20 of 24 (83%) tested positive, whereas 5 of 14 (36%) with remote HSR (>9 months) tested positive (P < .01). Nineteen carboplatin ST+ and 11 ST- patients underwent desensitization to carboplatin. Seven ST+ patients (37%) had mild HSR during desensitization but completed the desensitization with additional treatment or protocol modification. ST- patients with a recent history of HSR (n = 3) tolerated a rapid protocol without HSR and remained ST- with repeated testing. Six of 8 ST- patients (75%) with remote HSR reacted during desensitization. The HSRs were more severe and often associated with an elevated tryptase level. Five of 7 patients retested became ST+ before the second desensitization. Carboplatin desensitization was successfully completed in 105 of 106 (99%) treatment courses. CONCLUSIONS The timing of carboplatin ST in relation to initial HSR is vital for risk stratification and subsequent desensitization. Initial ST- patients with a remote history of HSR are at high risk for conversion to ST+ and can develop more severe HSR.

Viral Inhibition Assay: A CD8 T Cell Neutralization Assay for Use in Clinical Trials of HIV-1 Vaccine Candidates

We have characterized an assay measuring CD8 T cell-mediated inhibition of human immunodeficiency virus (HIV) type 1 replication, demonstrating specificity and reproducibility and employing a panel of primary HIV-1 isolates. The assay uses relatively simple autologous cell culture and enzyme-linked immunosorbent assay, avoids generation of T cell clones, and can be performed with <2 million peripheral blood mononuclear cells. Efficient CD8 T cell-mediated cross-clade inhibition of HIV-1 replication in vitro was demonstrated in antiretroviral therapy-naive HIV-1-infected subjects with controlled viral replication in vivo but not in viremic subjects. An HIV-1 vaccine candidate, consisting of DNA and recombinant adenovirus 5 vectors tested in a phase I clinical trial, induced CD8 T cells that efficiently inhibited HIV-1 in a HLA-I-dependent manner. Assessment of direct antiviral T cell function by this assay provides additional information to guide vaccine design and the prioritizing of candidates for further clinical trials.

Parallel human immunodeficiency virus type 1-specific CD8(+) T-lymphocyte responses in blood and mucosa during chronic infection

ABSTRACT Gut-associated lymphoid tissue is the major reservoir of lymphocytes and human immunodeficiency virus type 1 (HIV-1) replication in vivo, yet little is known about HIV-1-specific CD8+ T-lymphocyte (CTL) responses in this compartment. Here we assessed the breadth and magnitude of HIV-1-specific CTL in the peripheral blood and sigmoid colon mucosa of infected subjects not on antiretroviral therapy by enzyme-linked immunospot analysis with 53 peptide pools spanning all viral proteins. Comparisons of blood and mucosal CTL revealed that the magnitude of pool-specific responses is correlated within each individual (mean r2 = 0.82 ± 0.04) and across all individuals (r2 = 0.75; P < 0.001). Overall, 85.1% of screened peptide pools yielded concordant negative or positive results between compartments. CTL targeting was also closely related between blood and mucosa, with Nef being the most highly targeted (mean of 2.4 spot-forming cells [SFC[/106 CD8+ T lymphocytes/amino acid [SFC/CD8/aa]), followed by Gag (1.5 SFC/CD8/aa). Finally, comparisons of peptide pool responses seen in both blood and mucosa (concordant positives) versus those seen only in one but not the other (discordant positives) showed that most discordant results were likely an artifact of responses being near the limit of detection. Overall, these results indicate that HIV-1-specific CTL responses in the blood mirror those seen in the mucosal compartment in natural chronic infection. For protective or immunotherapeutic vaccination, it will be important to determine whether immunity is elicited in the mucosa, which is a key site of initial infection and subsequent HIV-1 replication in vivo.

Simultaneous Assessment of Cytotoxic T Lymphocyte Responses Against Multiple Viral Infections by Combined Usage of Optimal Epitope Matrices, Anti- CD3 mAb T-cell Expansion and "RecycleSpot"

The assessment of cellular anti-viral immunity is often hampered by the limited availability of adequate samples, especially when attempting simultaneous, high-resolution determination of T cell responses against multiple viral infections. Thus, the development of assay systems, which optimize cell usage, while still allowing for the detailed determination of breadth and magnitude of virus-specific cytotoxic T lymphocyte (CTL) responses, is urgently needed. This study provides an up-to-date listing of currently known, well-defined viral CTL epitopes for HIV, EBV, CMV, HCV and HBV and describes an approach that overcomes some of the above limitations through the use of peptide matrices of optimally defined viral CTL epitopes in combination with anti-CD3 in vitro T cell expansion and re-use of cells from negative ELISpot wells. The data show that, when compared to direct ex vivo cell preparations, antigen-unspecific in vitro T cell expansion maintains the breadth of detectable T cell responses and demonstrates that harvesting cells from negative ELISpot wells for re-use in subsequent ELISpot assays (RecycleSpot), further maximized the use of available cells. Furthermore when combining T cell expansion and RecycleSpot with the use of rationally designed peptide matrices, antiviral immunity against more than 400 different CTL epitopes from five different viruses can be reproducibly assessed from samples of less than 10 milliliters of blood without compromising information on the breadth and magnitude of these responses. Together, these data support an approach that facilitates the assessment of cellular immunity against multiple viral co-infections in settings where sample availability is severely limited.

Selective reduction and proliferation of the CD4+ and CD8+ T cell subsets with bispecific monoclonal antibodies: Evidence for inter-T cell-mediated cytolysis

CD3,4 (anti-CD3:anti-CD4) bispecific monoclonal antibodies (BSMAB) cause a profound decrease in CD4+ T cells and a marked proliferation of CD8+ T cells in peripheral blood mononuclear cells in vitro. CD3,8 (anti-CD3:anti-CD8) BSMAB causes a reciprocal decrease in CD8+ T cells and a proliferation of CD4+ T cells. The major effector of CD4+ T cell cytolysis in the presence of CD3,4 resides in the CD8+ T cell population. In contrast, both the CD4+ and CD8+ T cells are effective mediators of cytolysis of the CD8+ T cells in the presence of the CD3,8. The likely underlying mechanism in each case is bridging of the CD4 and CD8 of the target cells to the CD3 complexes of the effector cells by antibodies, mimicking the natural encounter between a cytolytic T cell and its target. Proliferation studies indicated that CD3,4 and CD3,8 each can induce proliferation of both CD4+ and CD8+ T cells in the presence of accessory cells. These results suggest that the major selection of the BSMABs occurs via selective destruction of one T cell subset with concurrent stimulation of the remaining CD3+ population. Potential applications of the selective destruction and proliferation include study and manipulation of the T cell subsets in HIV infections, tumor infiltrating lymphocytes, autoimmune diseases, and graft rejection.

Ten-year study of causes of moderate to severe angioedema seen by an inpatient allergy/immunology consult service

The causes of angioedema are not well described, especially in the inpatient setting. The purpose of this study was to examine the causes of moderate to severe angioedema in patients requiring inpatient treatment. We performed a retrospective review in patients requiring inpatient consultation by the Division of Allergy and Immunology at our institution between 1995 and 2004. We focused on potential interactions among medications that elicited life-threatening angioedema requiring intubation. The allergy/immunology service was consulted on 69 patients with moderate to severe angioedema. Medications were the most common cause of angioedema (n = 64, 93%). In most cases (n = 46, 67%), the angioedema was attributed to two or more medications. Patients previously stable on ACE inhibitors (ACEI), aspirin (ASA), or non-steroidal anti-inflammatory drugs (NSAIDs) appeared more likely to develop angioedema soon after the addition of another drug (i.e., ACEI, ASA/NSAIDs, direct mast cell degranulators, and antibiotics). ACEI, ASA/NSAID, and direct mast cell degranulators were contributing causes in 36 patients (56%), 45 patients (70%), and 23 patients (36%), respectively. Twenty patients required intubation, 14 (70%) patients were on ACEI, 12 (60%) patients were on ASA/NSAID, and 7 (35%) patients were on direct mast cell degranulators. ACEI, ASA/NSAID, or direct mast cell degranulators were a cause in 95% (n = 19) of patients requiring intubation. The combination of ACEI and ASA/NSAID was the most frequent cause of angioedema among all patients (n = 17, 25%) and those requiring intubation (n = 8, 40%). Moderate to severe angioedema often is a result of interactions between two or more medications involved in different pathways causing angioedema. In particular, combinations of ACEI, ASA/NSAID, or direct mast cell degranulators may lead to life-threatening angioedema requiring intubation.

Differential immunogenicity of vaccinia and HIV-1 components of a human recombinant vaccine in mucosal and blood compartments

Mucosal immune responses induced by HIV-1 vaccines are likely critical for prevention. We report a Phase 1 safety and immunogenicity trial in eight participants using the vaccinia-based TBC-3B vaccine given subcutaneously to determine the relationship between HIV-1 specific systemic and gastrointestinal mucosal responses. Across all subjects, detectable levels of blood vaccinia- and HIV-1-specific antibodies were elicited but none were seen mucosally. While the vaccinia component was immunogenic for CD8(+) T lymphocyte (CTL) responses in both blood and mucosa, it was greater in blood. The HIV-1 component of the vaccine was poorly immunogenic in both blood and mucosa. Although only eight volunteers were studied intensively, the discordance between mucosal and blood responses may highlight mechanisms contributing to recent vaccine failures.

Improved CD4 Lymphocyte Outgrowth in Response to Effective Antiretroviral Therapy

CD4 lymphocyte regenerative capacity was evaluated by use of an ex vivo outgrowth assay in human immunodeficiency virus (HIV)-1-infected subjects enrolled in a clinical trial (Merck 039). CD4 lymphocytes were selectively expanded in vitro by T cell receptor triggering, which also induces HIV production from latently infected cells. CD4 cell expansion and lack of virus production in cultures correlated well with clinical responses and were best in those receiving an aggressive antiretroviral three-drug regimen. Twelve clinical responders receiving triple-drug therapy monitored for 60 weeks had both excellent ex vivo CD4 cell expansion and lack of HIV replication, often in the absence of added drug in culture. Breakthrough viruses recovered from drug-containing arms of the cultures showed phenotypic resistance to the drugs used in vivo. This CD4 lymphocyte outgrowth assay correlates well with clinical outcome in subjects receiving potent antiretroviral regimens and may predict the emergence of early drug resistance.

Immunogenic epitopes of the p55 chain of the IL-2 receptor. Relationships to high-affinity IL-2 binding and modulation of the p55 chain.

Monoclonal antibodies were used to determine the relationships between epitopes on the p55 chain of the IL-2 receptor and high-affinity IL-2 binding. Five monoclonal antibodies to the human P55 chain of the IL-2 receptor were induced by immunizing mice with murine L cells that were transfected with human p55 cDNA. Since the p55 chain is the only human antigen expressed on these cells, all antihuman MABs thus generated were directed against this molecule. These antibodies were used to map epitopes on the p55 chain and determine their relationship to high-affinity IL-2 binding. Extensive flow cytometric studies with these MABs and a large panel of other anti-p55 MABs revealed three major patterns of competition. Type I MABs compete with anti-Tac extensively but not with antibodies of other groups. Type II MABs do not block anti-Tac but do block 7E11. Type III MABs do not block either type I or type II antibodies. 125I-IL2 competition studies under high-affinity conditions revealed that types I and II MABs inhibit IL-2 binding. Type III MABs can be resolved into two subgroups, one that inhibits IL-2 binding and one that does not. Together these data suggest that there are at least four distinct immunogenic epitopes on the human p55 chain, with three epitopes related to IL-2 binding. The competitive component evident by a change in Kd on the Scatchard plots suggests that all three epitopes are close to or part of the IL-2-binding site of the p55 chain. The noncompetitive component, as evidenced by the lower number of high-affinity IL-2 receptors induced by these antibodies, suggests that the same epitopes are also close to the site(s) of interaction between the p55 and p70 chains to form the high-affinity receptor. These studies indicated that the IL-2-binding site and site of interaction between the p55 and p70 chains are close together or identical. Modulation studies revealed that one type II antibody (7E11) modulates the p55 chain in the absence of IL2 and the p70 chain, thus revealing that modulation of the p55 chain can occur by an active process, and not merely passively comodulate by the p70 chain upon IL-2 binding. Modulation of the p55 chain alone has no proliferative effect on IL-2-responsive T lymphoblasts. Potentially this antibody-dependent modulation may be used to deliver toxin to activated lymphocytes.