The crossroads: divergent roles of virus-specific CD4+ T lymphocytes in determining the outcome for human papillomavirus infection.

Despite the widespread availability of effective prophylactic vaccines to prevent human papillomavirus (HPV) infection, HPV remains a major health burden. For health care systems in countries with the capacity for vaccine roll out, incomplete strategies result in citizens with naturally occurring infection, who are at an a posteriori risk of HPV-driven disease. Genital HPV infection is the most common sexually transmitted virus globally. Those classified as high-risk HPV strains are more likely to generate persistent disease. Within this group, HPV16 and 18 are the most prevalent and likely to induce persistent high-grade squamous intraepithelial neoplasia; neoplasia is a large step toward cancerous growth known as a squamous cell carcinoma which contribute to all cervical, 70% of oropharyngeal, 78% of vaginal and 88% of anal cancers. This review will illuminate the relevance of CD4+ T lymphocytes in determining the outcome of papillomavirus infection from the perspective of oropharyngeal and anogenital HPV-driven disease in the immune competent and immunocompromised. The focus is on recent investigations for this "silent" pandemic among current global health crises that should not be forgotten. Informing effective strategies that control viral infection through naturally acquired or induced immunity will identify aspects of scientific and clinical practice that may improve outcome.

Parallel analysis of multiple human memory CD4+ T-cell subsets within antigen-specific responses using cell proliferation dyes.

Activation induced marker (AIM) assays are being used increasingly to measure antigen-specific T-cell responses, but this activation can alter cell lineage defining phenotypic markers. We aimed to extend the utility of AIM assays to enable pre-activation defined cell populations to be tracked and quantified within T-cell memory responses. We sorted three ex vivo CD4+ T-cell populations prior to any activation using well defined ex vivo lineage surface marker combinations. These populations were memory non-Tregs, CD39+ Tregs and CD39neg Tregs, although any three memory CD4+ T-cell populations able to be isolated by cell surface markers could potentially be tracked. These cells were labeled with three distinct fluorescent cell proliferation dyes (CFSE, CellTrace Violet and Cell Proliferation Dye eF670) and then all autologous PBMCs were reconstituted maintaining ex vivo cell ratios and CD25/OX40 AIM assays performed with CMV and HSV antigens. This approach enabled tracking of pre-defined cell populations within antigen stimulated responses using both activation marker and cell proliferation readouts. We confirmed that although CD39+ Tregs comprise a substantial proportion of AIM assay responses, they do not make substantial contributions to the proliferative response. This extends the utility of AIM assays to enable parallel analysis of the relative contribution of several CD4+ memory T-cell subsets to recall responses.

Navigating the complexity of chronic HIV-1 associated immune dysregulation.

Despite successful viral suppression with antiretroviral therapy, chronic HIV-1 infection is associated with ongoing immune dysfunction. Investigation of the complex immune response in treated and untreated individuals with chronic HIV-1 infection is warranted. Immune alterations such as monocyte phenotype and Th-17/Treg ratios often persist years after the reduction in viraemia and predispose many individuals to long-term comorbidities such as cardiovascular disease or cancer. Furthermore, while there has been extensive research on the latent reservoir of treated patients with chronic HIV-1, which prevents the discontinuation of treatment, the mechanism behind this remains elusive and needs further investigation. In this review, we assist in navigating the recent research on these groups of individuals and provide a basis for further investigation.

Evaluation of Commercially Available Viral Transport Medium (VTM) for SARS-CoV-2 Inactivation and Use in Point-of-Care (POC) Testing.

Critical to facilitating SARS-CoV-2 point-of-care (POC) testing is assurance that viruses present in specimens are inactivated onsite prior to processing. Here, we conducted experiments to determine the virucidal activity of commercially available Viral Transport Mediums (VTMs) to inactivate SARS-CoV-2. Independent testing methods for viral inactivation testing were applied, including a previously described World Health Organization (WHO) protocol, in addition to a buffer exchange method where the virus is physically separated from the VTM post exposure. The latter method enables sensitive detection of viral viability at higher viral titre when incubated with VTM. We demonstrate that VTM formulations, Primestore® Molecular Transport Medium (MTM) and COPAN eNAT™ completely inactivate high-titre SARS-CoV-2 virus (>1 × 107 copies/mL) and are compatible with POC processing. Furthermore, full viral inactivation was rapidly achieved in as little as 2 min of VTM exposure. We conclude that adding certain VTM formulations as a first step post specimen collection will render SARS-CoV-2 non-infectious for transport, or for further in-field POC molecular testing using rapid turnaround GeneXpert platforms or equivalent.

Circulating gluten-specific, but not CMV-specific, CD39+ regulatory T cells have an oligoclonal TCR repertoire.

OBJECTIVES: Understanding the T cell receptor (TCR) repertoire of regulatory CD4+ T-cell (Treg) populations is important for strategies aiming to re-establish tolerance in autoimmune diseases. We studied circulating deamidated gluten-epitope-specific CD39+ Tregs in patients with coeliac disease following an oral gluten challenge, and we used cytomegalovirus (CMV)-specific CD39+ Tregs from healthy controls as a comparator population. METHODS: We used the OX40 assay to isolate antigen-specific Tregs by induced surface co-expression of CD25, OX40 and CD39. RACE PCR amplification and Sanger sequencing of the TCR ß chain were used to analyse repertoire diversity. RESULTS: We found that, following oral gluten challenge, circulating gluten-specific CD39+ Tregs had an oligoclonal TCR repertoire that contained public clonotypes. Conversely, the TCR repertoire of CMV-epitope-specific CD39+ Tregs from healthy controls was polyclonal. DISCUSSION: These data indicate that a biased TCR repertoire is not inherent to CD39+ Tregs, and, in this case, is apparently driven by the HLA-DQ2.5-restricted deamidated gluten peptide in coeliac disease patients. CONCLUSION: This is the first assessment of the TCR repertoire within circulating human Tregs specific for foreign antigen. These data enhance our understanding of antigen-specific CD4+ responses in the settings of chronic inflammation and infection and may help guide immunomonitoring strategies for CD4+ T cell-based therapies, particularly for coeliac disease.

HIV-1 DNA Is Maintained in Antigen-Specific CD4+ T Cell Subsets in Patients on Long-Term Antiretroviral Therapy Regardless of Recurrent Antigen Exposure.

Memory CD4+ T cells (mCD4s) containing integrated HIV DNA are considered the main barrier to a cure for HIV infection. Here, we analyzed HIV DNA reservoirs in antigen-specific subsets of mCDs to delineate the mechanisms by which HIV reservoirs persist during antiretroviral therapy (ART). HIV Gag, cytomegalovirus (CMV), and tetanus toxoid (TT)-specific mCD4s were isolated from peripheral blood samples obtained from 11 individual subjects, 2-11 years after commencing ART. Antigen-specific mCD4s were identified by the sensitive OX40 assay and purified by cell sorting. Total HIV DNA levels were quantified by real-time PCR, and clonal viral sequences generated from mCD4 subsets and pre-ART plasma samples. Quantitative results and sequence analysis were restricted to five and three study participants, respectively, which was likely due to the low frequency of the antigen-specific mCD4s and relatively low HIV DNA proviral loads. Median HIV Gag-, CMV-, and TT-specific mCD4s were 0.61%, 2.46%, and 0.78% of total mCD4s, and they contained a median of 2.50, 2.38, and 2.55 log10 copies of HIV DNA per 106 cells, respectively. HIV DNA sequences were derived from antigen-specific mCD4s clustered with sequences derived from pre-ART plasma samples. There was a trend toward increased viral diversity in clonal viral sequences derived from CMV-specific mCD4s relative to TT-specific mCD4s. Despite limitations, this study provides direct evidence that HIV reservoirs persist in memory CD4+ T cell subsets maintained by homeostatic proliferation (TT) and adds to growing evidence against viral evolution during ART. Similar future studies require techniques that sample diverse HIV reservoirs and with improved sensitivity.

Circulating gluten-specific FOXP3+CD39+ regulatory T cells have impaired suppressive function in patients with celiac disease.

BACKGROUND: Celiac disease is a chronic immune-mediated inflammatory disorder of the gut triggered by dietary gluten. Although the effector T-cell response in patients with celiac disease has been well characterized, the role of regulatory T (Treg) cells in the loss of tolerance to gluten remains poorly understood. OBJECTIVE: We sought to define whether patients with celiac disease have a dysfunction or lack of gluten-specific forkhead box protein 3 (FOXP3)+ Treg cells. METHODS: Treated patients with celiac disease underwent oral wheat challenge to stimulate recirculation of gluten-specific T cells. Peripheral blood was collected before and after challenge. To comprehensively measure the gluten-specific CD4+ T-cell response, we paired traditional IFN-γ ELISpot with an assay to detect antigen-specific CD4+ T cells that does not rely on tetramers, antigen-stimulated cytokine production, or proliferation but rather on antigen-induced coexpression of CD25 and OX40 (CD134). RESULTS: Numbers of circulating gluten-specific Treg cells and effector T cells both increased significantly after oral wheat challenge, peaking at day 6. Surprisingly, we found that approximately 80% of the ex vivo circulating gluten-specific CD4+ T cells were FOXP3+CD39+ Treg cells, which reside within the pool of memory CD4+CD25+CD127lowCD45RO+ Treg cells. Although we observed normal suppressive function in peripheral polyclonal Treg cells from patients with celiac disease, after a short in vitro expansion, the gluten-specific FOXP3+CD39+ Treg cells exhibited significantly reduced suppressive function compared with polyclonal Treg cells. CONCLUSION: This study provides the first estimation of FOXP3+CD39+ Treg cell frequency within circulating gluten-specific CD4+ T cells after oral gluten challenge of patients with celiac disease. FOXP3+CD39+ Treg cells comprised a major proportion of all circulating gluten-specific CD4+ T cells but had impaired suppressive function, indicating that Treg cell dysfunction might be a key contributor to disease pathogenesis.

Cytotoxic CD4 T Cells-Friend or Foe during Viral Infection?

CD4 T cells with cytotoxic function were once thought to be an artifact due to long-term in vitro cultures but have in more recent years become accepted and reported in the literature in response to a number of viral infections. In this review, we focus on cytotoxic CD4 T cells in the context of human viral infections and in some infections that affect mice and non-human primates. We examine the effector mechanisms used by cytotoxic CD4 cells, the phenotypes that describe this population, and the transcription factors and pathways that lead to their induction following infection. We further consider the cells that are the predominant targets of this effector subset and describe the viral infections in which CD4 cytotoxic T lymphocytes have been shown to play a protective or pathologic role. Cytotoxic CD4 T cells are detected in the circulation at much higher levels than previously realized and are now recognized to have an important role in the immune response to viral infections.

The primary immune response to Vaccinia virus vaccination includes cells with a distinct cytotoxic effector CD4 T-cell phenotype.

BACKGROUND: Smallpox was eradicated by a global program of inoculation with Vaccinia virus (VV). Robust VV-specific CD4 T-cell responses during primary infection are likely essential to controlling VV replication. Although there is increasing interest in cytolytic CD4 T-cells across many viral infections, the importance of these cells during acute VV infection is unclear. METHODS: We undertook a detailed functional and genetic characterization of CD4 T-cells during acute VV-infection of humans. VV-specific T-cells were identified by up-regulation of activation markers directly ex vivo and through cytokine and co-stimulatory molecule expression. At day-13-post primary inoculation with VV, CD38highCD45RO+ CD4 T-cells were purified by cell sorting, RNA isolated and analysed by microarray. Differential expression of up-regulated genes in activated CD4 T-cells was confirmed at the mRNA and protein levels. We compared analyses of VV-specific CD4 T-cells to studies on 12 subjects with primary HIV infection (PHI). VV-specific T-cells lines were established from PBMCs collected post vaccination and checked for cytotoxicity potential. RESULTS: A median 11.9% CD4 T-cells were CD38highCD45RO+ at day-13 post-VV inoculation, compared to 3.0% prior and 10.4% during PHI. Activated CD4 T-cells had an up-regulation of genes related to cytolytic function, including granzymes K and A, perforin, granulysin, TIA-1, and Rab27a. No difference was seen between CD4 T-cell expression of perforin or TIA-1 to VV and PHI, however granzyme k was more dominant in the VV response. At 25:1 effector to target ratio, two VV-specific T-cell lines exhibited 62% and 30% cytotoxicity respectively and CD107a degranulation. CONCLUSIONS: We show for the first time that CD4 CTL are prominent in the early response to VV. Understanding the role of CD4 CTL in the generation of an effective anti-viral memory may help develop more effective vaccines for diseases such as HIV.

Innate and Adaptive Immunity in Long-Term Non-Progression in HIV Disease.

Long-term non-progressors (LTNP) were identified after 10-15 years of the epidemic, and have been the subject of intense investigation ever since. In a small minority of cases, infection with nef/3'LTR deleted attenuated viral strains allowed control over viral replication. A common feature of LTNP is the readily detected proliferation of CD4 T-cells in vitro, in response to p24. In some cases, the responding CD4 T-cells have cytotoxic effector function and may target conserved p24 epitopes, similar to the CD8 T-cells described below. LTNP may also carry much lower HIV DNA burden in key CD4 subsets, presumably resulting from lower viral replication during primary infection. Some studies, but not others, suggest that LTNP have CD4 T-cells that are relatively resistant to HIV infection in vitro. One possible mechanism may involve up-regulation of the cell cycle regulator p21/waf in CD4 T-cells from LTNP. Delayed progression in Caucasian LTNP is also partly associated with heterozygosity of the Δ32 CCR5 allele, probably through decreased expression of CCR5 co-receptor on CD4 T-cells. However, in approximately half of Caucasian LTNP, two host genotypes, namely HLA-B57 and HLA-B27, are associated with viral control. Immunodominant CD8 T-cells from these individuals target epitopes in p24 that are highly conserved, and escape mutations have significant fitness costs to the virus. Furthermore, recent studies have suggested that these CD8 T-cells from LTNP, but not from HLA-B27 or HLA-B57 progressors, can cross-react with intermediate escape mutations, preventing full escape via compensatory mutations. Humoral immunity appears to play little part in LTNP subjects, since broadly neutralizing antibodies are rare, even amongst slow progressors. Recent genome-wide comparisons between LTNP and progressors have confirmed the HLA-B57, HLA-B27, and delta32 CCR5 allelic associations, plus indicated a role for HLA-C/KIR interactions, but have not revealed any new genotypes so far. Nevertheless, it is hoped that studying the mechanisms of intracellular restriction factors, such as the recently identified SAMHD1, will lead to a better understanding of non-progression.

A molecular basis for the control of preimmune escape variants by HIV-specific CD8+ T cells.

The capacity of the immune system to adapt to rapidly evolving viruses is a primary feature of effective immunity, yet its molecular basis is unclear. Here, we investigated protective HIV-1-specific CD8+ T cell responses directed against the immunodominant p24 Gag-derived epitope KK10 (KRWIILGLNK263-272) presented by human leukocyte antigen (HLA)-B∗2705. We found that cross-reactive CD8+ T cell clonotypes were mobilized to counter the rapid emergence of HIV-1 variants that can directly affect T cell receptor (TCR) recognition. These newly recruited clonotypes expressed TCRs that engaged wild-type and mutant KK10 antigens with similar affinities and almost identical docking modes, thereby accounting for their antiviral efficacy in HLA-B∗2705+ individuals. A protective CD8+ T cell repertoire therefore encompasses the capacity to control TCR-accessible mutations, ultimately driving the development of more complex viral escape variants that disrupt antigen presentation.

Escape from highly effective public CD8+ T-cell clonotypes by HIV.

Mapping the precise determinants of T-cell efficacy against viruses in humans is a public health priority with crucial implications for vaccine design. To inform this effort, we performed a comprehensive analysis of the effective CD8(+) T-cell clonotypes that constitute responses specific for the HIV p24 Gag-derived KK10 epitope (KRWIILGLNK; residues 263-272) restricted by HLA-B*2705, which are known to confer superior control of viral replication in HIV-infected individuals. Particular KK10-specific CD8(+) T-cell clonotypes, characterized by TRBV4-3/TRBJ1-3 gene rearrangements, were found to be preferentially selected in vivo and shared between individuals. These "public" clonotypes exhibit high levels of TCR avidity and Ag sensitivity, which impart functional advantages and enable effective suppression of HIV replication. The early L(268)M mutation at position 6 of the KK10 epitope enables the virus to avoid recognition by these highly effective CD8(+) T-cell clonotypes. However, alternative clonotypes with variant reactivity provide flexibility within the overall KK10-specific response. These findings provide refined mechanistic insights into the workings of an effective CD8(+) T-cell response against HIV.

Persistent survival of prevalent clonotypes within an immunodominant HIV gag-specific CD8+ T cell response.

CD8(+) T cells play a significant role in the control of HIV replication, yet the associated qualitative and quantitative factors that determine the outcome of infection remain obscure. In this study, we examined Ag-specific CD8(+) TCR repertoires longitudinally in a cohort of HLA-B*2705(+) long-term nonprogressors with chronic HIV-1 infection using a combination of molecular clonotype analysis and polychromatic flow cytometry. In each case, CD8(+) T cell populations specific for the immunodominant p24 Gag epitope KRWIILGLNK (KK10; residues 263-272) and naturally occurring variants thereof, restricted by HLA-B*2705, were studied at multiple time points; in addition, comparative data were collected for CD8(+) T cell populations specific for the CMV pp65 epitope NLVPMVATV (NV9; residues 495-503), restricted by HLA-A*0201. Dominant KK10-specific clonotypes persisted for several years and exhibited greater stability than their contemporaneous NV9-specific counterparts. Furthermore, these dominant KK10-specific clonotypes exhibited cross-reactivity with antigenic variants and expressed significantly higher levels of CD127 (IL-7Rα) and Bcl-2. Of note, we also found evidence that promiscuous TCR α-chain pairing associated with alterations in fine specificity for KK10 variants could contribute to TCR ß-chain prevalence. Taken together, these data suggest that an antiapoptotic phenotype and the ability to cross-recognize variant epitopes contribute to clonotype longevity and selection within the peripheral memory T cell pool in the presence of persistent infection with a genetically unstable virus.

HIV immune escape at an immunodominant epitope in HLA-B*27-positive individuals predicts viral load outcome.

The CTL response in HLA-B*27(+) HIV-infected individuals is characterized by an immunodominant response to a conserved epitope in gag p24 (aa 263-272, KRWIILGLNK; KK10). Mutations resulting in substitution of the arginine (R264) at position 2 of this epitope have been identified as escape mutations. Nineteen HLA-B*27(+) long-term nonprogressors were identified from an Australian cohort with an average follow-up of 16 y following infection. Viral and host genetic factors impacting on disease progression were determined at multiple time points. Twelve of 19 had wild-type sequences at codon 264 at all time points; 7 of 19 carried CTL escape variants. Median viral load and CD4(+) T cell counts were not significantly different between these groups at enrollment. Viral load, as judged by levels at their last visit (1,700 and 21,000 RNA copies/ml, respectively; p = 0.01) or by time-weighted area under the curve was higher in the escape group (p = 0.02). Escape mutants at other HLA-B*27-restricted epitopes were uncommon. Moreover, host polymorphisms, such as CCR5Δ32, CCR2-64I, and SDF1-3'A, or breadth of TCR repertoire responding to KK10 did not segregate to wild-type or escape groups. Host and viral factors were examined for a relationship to viral load. The only factor to affect viral load was the presence of the R264 escape mutations at the immunodominant epitope. CTL escape at R264 in the KK10 epitope is a major determinant of subsequent viral load in these HLA-B*27(+) individuals.

Development of non-antibiotic-resistant, chromosomally based, constitutive and inducible expression systems for aroA-attenuated Salmonella enterica Serovar Typhimurium.

Live-vaccine delivery systems expressing two model antigens from Mycoplasma hyopneumoniae, F2(P97) (Adh) and NrdF, were constructed using Salmonella enterica serovar Typhimurium aroA (STM-1), and immunogenicity in mice was evaluated. Recombinant plasmid-based expression (PBE) and chromosomally based expression (CBE) systems were constructed. The PBE system was formed by cloning both antigen genes into pJLA507 to create an operon downstream of temperature-inducible promoters. Constitutive CBE was achieved using a promoter-trapping technique whereby the promoterless operon was stably integrated into the chromosome of STM-1, and the expression of antigens was assessed. The chromosomal position of the operon was mapped in four clones. Inducible CBE was obtained by using the in vivo-induced sspA promoter and recombining the expression construct into aroD. Dual expression of the antigens was detected in all systems, with PBE producing much larger quantities of both antigens. The stability of antigen expression after in vivo passage was 100% for all CBE strains recovered. PBE and CBE strains were selected for comparison in a vaccination trial. The vaccine strains were delivered orally into mice, and significant systemic immunoglobulin M (IgM) and IgG responses against both antigens were detected among all CBE groups. No significant immune response was detected using PBE strains. Expression of recombinant antigens in S. enterica serovar Typhimurium aroA from chromosomally located strong promoters without the use of antibiotic resistance markers is a reliable and effective method of inducing a significant immune response.

Proliferation of weakly suppressive regulatory CD4+ T cells is associated with over-active CD4+ T-cell responses in HIV-positive patients with mycobacterial immune restoration disease.

The role of Treg in patients with late-stage HIV disease, who commence combination antiretroviral therapy (cART) and develop pathogen-specific immunopathology manifesting as immune restoration disease (IRD) remains unclear. We hypothesised that Treg could be defective in either numbers and/or function and therefore unable to ensure the physiological equilibrium of the immune system in patients with IRD. Phenotypic and functional CD4(+) T-cell subsets of eight late-stage HIV patients with nadir CD4 count <50 cells/microL, who developed mycobacterial IRD upon commencing cART were compared with six therapy naive HIV(+) patients (nadir CD4 count <50 cells/microL), who did not develop an IRD after cART. Mycobacterium-avium-specific CD4(+) T cells from IRD patients produced high levels of IFN-gamma and IL-2 compared with controls (p<0.001). Surprisingly, we found a significant expansion of CD127(lo)Foxp3(+)CD25(+) Treg in IRD patients and a higher ratio of Treg to effector/memory subsets (p<0.001). In vitro suppression assays demonstrated reduced functional capacity of suppressor cells and diminished IL-10 secretion in IRD patients. Plasma levels of IL-7 were increased in patients and, interestingly, exogenous IL-7 and other cytokines strongly inhibited Treg suppression. These data suggest that despite substantial Treg expansion in IRD, their ability to induce suppression, and thereby downregulate aberrant immune responses, is compromised.

TCR beta-chain sharing in human CD8+ T cell responses to cytomegalovirus and EBV.

The CD8(+) TCR repertoires specific for many immunogenic epitopes of CMV and EBV are dominated by a few TCR clonotypes and involve public TCRs that are shared between many MHC-matched individuals. In previous studies, we demonstrated that the observed sharing of epitope-specific TCRbeta chains between individuals is strongly associated with TCRbeta production frequency, and that a process of convergent recombination facilitates the more efficient production of some TCRbeta sequences. In this study, we analyzed a total of 2836 TCRbeta sequences from 23 CMV-infected and 10 EBV-infected individuals to investigate the factors that influence the sharing of TCRbeta sequences in the CD8(+) T cell responses to two immunodominant HLA-A*0201-restricted epitopes from these viruses. The most shared TCRbeta amino acid sequences were found to have two features that indicate efficient TCRbeta production, as follows: 1) they required fewer nucleotide additions, and 2) they were encoded by a greater variety of nucleotide sequences. We used simulations of random V(D)J recombination to demonstrate that the in silico TCRbeta production frequency was predictive of the extent to which both TCRbeta nucleotide and amino acid sequences were shared in vivo. These results suggest that TCRbeta production frequency plays an important role in the interindividual sharing of TCRbeta sequences within CD8(+) T cell responses specific for CMV and EBV.

T-cell responses in primary HIV-1 infection.

PURPOSE OF REVIEW: Important immunological events, especially involving T cells, occur during primary HIV-1 infection. The qualitative nature of the primary immune response to the virus may determine long-term outcome. Whereas CD4 T cells are being rapidly depleted, CD8 T cells play an important role in the initial control of viral replication. There is significant individual variability in the extent of viral control. Understanding the mechanisms underlying these differences and the causes of the development of dysfunctional T-cell responses will allow the identification of opportunities for therapeutic intervention that might change the long-term outcome. RECENT FINDINGS: Recent studies have revealed early dysfunction of T cells demonstrating increased expression of PD-1, CTLA-4 and reduced expression of CD127. Those studies suggest disruption of the interaction between CD4 and CD8 T cells. In addition, a few regions, mainly within the Gag protein, have been highlighted as potentially important targets for effective immune responses inducing viral control. SUMMARY: Despite recent studies emphasizing the critical nature of acute HIV-1 infection, current intervention strategies have failed to influence disease progression. Recent findings have indicated potential new strategies to re-enable functional properties of T cells and direct these responses towards critical regions of the virus.

Validation of RNA-based molecular clonotype analysis for virus-specific CD8+ T-cells in formaldehyde-fixed specimens isolated from peripheral blood.

Recent advances in the field of molecular clonotype analysis have enabled detailed repertoire characterization of viably isolated antigen-specific T cell populations directly ex vivo. However, in the absence of a biologically contained FACS facility, peripheral blood mononuclear cell (PBMC) preparations derived from patients infected with agents such as HIV must be formaldehyde fixed to inactivate the pathogen; this procedure adversely affects nucleic acid template quality. Here, we developed and validated a method to amplify and sequence mRNA species derived from formaldehyde fixed PBMC specimens. Antigen-specific CD8+ cytotoxic T-lymphocyte populations were identified with standard fluorochrome-conjugated peptide-major histocompatibility complex class I tetramers refolded around synthetic peptides representing immunodominant epitopes from HIV p24 Gag (KRWII[M/L]GLNK/HLA B*2705) and CMV pp65 (NLVPMVATV/HLA A*0201 and TPRVTGGGAM/HLA B*0702), and acquired in separate laboratories with or without fixation. In the presence of proteinase K pre-treatment, the observed antigen-specific CD8+ T-cell repertoire determined by molecular clonotype analysis was statistically no different whether derived from fixed or unfixed PBMC. However, oligo-dT recovery methods were not suitable for use with fixed tissue as significant skewing of clonotypic representation was observed. Thus, we have developed a reliable RNA-based method for molecular clonotype analysis that is compatible with formaldehyde fixation and therefore suitable for use with primary human samples isolated by FACS outside the context of a biological safety level 3 containment facility.

A new variant cytotoxic T lymphocyte escape mutation in HLA-B27-positive individuals infected with HIV type 1.

The immune response in HIV-infected individuals who carry HLA-B27 is characterized by an immunodominant cytotoxic T lymphocyte (CTL) response to a conserved epitope corresponding to amino acids 263-272 of HIV-1 p24 gag. The arginine at position 264 is a crucial anchor residue. Amino acid substitution at 264 from arginine (R) to glycine (G), lysine (K), or threonine (T) results in a low affinity peptide that binds to HLA-B27 inefficiently and is poorly recognized by T cells that respond to the wild-type peptide. These mutants have been characterized as CTL escape mutations. We studied the plasma virus of 20 HLA-B27 longterm nonprogressors: 14 were wild type and 6 were found to be mutant. Five of these carried known escape mutations coding for K or G at position 264. One patient demonstrated a previously undescribed R264Q mutation in 30/31 clones. This altered epitope failed to elicit an IFN-gamma response from PBMC isolated from any of four HLA-B27-positive individuals with strong responses to wild-type peptide. A peptide binding assay confirmed that the R264Q mutant peptide had 30-fold lower binding affinity to HLA-B27 compared to wild type. Therefore, the R264Q variant is a likely novel escape mutation in HLA-B27-positive individuals.