Sensitive and selective detection of mycoplasma in cell culture samples using cantilever sensors

In this article we report a new biosensor‐based method that is more sensitive and rapid than the current approach for detecting mycoplasma in cell culture samples. Piezoelectric‐excited millimeter‐sized cantilever (PEMC) sensors respond to mass change via resonant frequency change. They are sensitive at femtogram level and can be used directly in liquid for label‐free detection. Common cell culture contaminant, Acholeplasma laidlawii was detected in both buffer and cell culture medium. Two different sources (positive control from a commercial kit and ATCC 23206) were analyzed using antibody‐immobilized PEMC sensor. Resonant frequency decrease caused by binding of A. laidlawii was monitored in real‐time using an impedance analyzer. Positive detection was confirmed by a second antibody binding. The limit of detection (LOD) was lower than 10³ CFU/mL in cell culture medium using PEMC sensor while parallel ELISA assays showed LOD as 10⁷ CFU/mL. This study shows that PEMC sensor can be used for sensitive and rapid mycoplasma detection in cell culture samples. Biotechnol. Bioeng. 2010;105: 1069–1077. © 2009 Wiley Periodicals, Inc.     

Preparation of reference strains for validation and comparison of mycoplasma testing methods

Aims: To optimize growth conditions for preparation of stocks of mycoplasma reference strains to obtain highly viable and disperse samples with low ratios of genomic copy (GC) number to that of colony forming units (CFU). These stocks are required for assessment of relative limits of detection (LOD) of alternative nucleic acid testing (NAT)‐based methods in comparison to the conventional microbiological methods. Methods and Results: A kinetics study was used to assess the changes in ratios between the numbers of GC and CFU at different growth phases of six different mycoplasma cultures Acholeplasma laidlawii, Mycoplasma gallisepticum, Mycoplasma arginini, Mycoplasma fermentans, Mycoplasma orale and Mycoplasma pneumoniae. All tested mycoplasmas demonstrated low GC/CFU ratios (≤10) within the log and early stationary growth phases. A significant increase in GC/CFU ratios was observed at the very late stationary and death phases, when the titre of cultures has declined. Similar patterns of GC/CFU profiles were observed for A. laidlawii and Myc. gallisepticum co‐cultured with suspension of Chinese hamster ovary (CHO) cells. Conclusions: Tested mycoplasma strains harvested at the exponential‐early stationary phases of growth demonstrated the lowest GC/CFU ratios and low propensity to form filamentous structures or aggregates under proposed conditions and can be used for the preparation of a mycoplasma reference panel for methods comparability study. Significance and Impact of the Study: This study shows that the preparation and use of viable mycoplasma reference strains with low CG/CFU ratios is the most reliable way to adequately evaluate the LOD of alternative NAT‐based mycoplasma testing methods.     

Construction of recA mutants of Acholeplasma laidlawii by insertional inactivation with a homologous DNA fragment

Mycoplasmas (class Mollicutes) are wall-less prokaryotes phylogenetically related to gram-positive bacteria. This study describes the construction of recA mutants of the mycoplasma Acholeplasma laidlawii. An internal fragment of the recA gene from A. laidlawii was cloned into a plasmid that does not replicate in this organism. When this plasmid construct was used to transform A. laidlawii, it inserted into the chromosome, disrupting the recA gene. The pheno-type of the resulting recA mutant was compared to that of wild-type cells and to that of a strain that has a naturally occurring ochre mutation in its recA gene. As found in other bacterial systems, loss of RecA activity resulted in cells deficient in DNA repair.     

Activity of Lectins of Winter Wheat Seedlings Infected with Mycoplasma and Treated with Salicylic Acid

Changes in activity of soluble and cell wall lectins have been revealed in seedlings of winter wheat Triticum aestivum L. cultivar Mironovskaya 808 after infection with mycoplasma Acholeplasma laidlawii 118. The protective effect of salicylic acid was manifested as negating the bursts of lectin activity induced by mycoplasma infection.__________Translated from Izvestiya Akademii Nauk, Seriya Biologicheskaya, No. 4, 2005, pp. 423–427.Original Russian Text Copyright © 2005 by Trifonova, Maksyutova, Timofeeva, Chernov.     

PCR-based detection and eradication of mycoplasmal infections from various mammalian cell lines: a local experience

A total of 200 cell lines including different human, monkey, mice, hamster and rat cell types were examined for mycoplasma infection status. PCR assay using generic-specific universal primers showed that 40 (20%) of the cell lines are contaminated with mycoplasma. Employment of species-specific primers within these infected cell lines revealed infection with M. hyorhinis (42.5%), M. fermentas (37.5%), M. arginini (37.5%), M. orale (12.5%) and A. laidlawii (7.5%). A number of the cultures were coinfected with 2 or 3 different species. Contaminated samples were treated with BM-Cyclin, Ciprofloxacin and mycoplasma removal agent (MRA). Mycoplasma eradication was subsequently checked by PCR following 2 weeks continuous culture of treated cells in antibiotic free culture medium. Mycoplasmal infections were eradicated in 100, 70 and 42% of infected cell lines when the samples were treated with BM-Cyclin, MRA and Ciprofloxacin, respectively. However, 12% (BM-Cyclin), 62.5% (MRA) and 82.5% (Ciprofloxacin) of mycoplasma regrowth was observed 4 months after the treatment. Notably, the risk of spontaneous culture death was 17.5, 12.5 and 0% for BM-Cyclin, MRA and Ciprofloxacin, respectively.     

Immune Responses in Acute and Convalescent Patients with Mild,Moderate and Severe Disease during the 2009 Influenza Pandemic in Norway

Increased understanding of immune responses influencing clinical severity during pandemic influenza infection is important for improved treatment and vaccine development. In this study we recruited 46 adult patients during the 2009 influenza pandemic and characterized humoral and cellular immune responses. Those included were either acute hospitalized or convalescent patients with different disease severities (mild, moderate or severe). In general, protective antibody responses increased with enhanced disease severity. In the acute patients, we found higher levels of TNF-α single-producing CD4⁺T-cells in the severely ill as compared to patients with moderate disease. Stimulation of peripheral blood mononuclear cells (PBMC) from a subset of acute patients with peptide T-cell epitopes showed significantly lower frequencies of influenza specific CD8⁺ compared with CD4⁺ IFN-γ T-cells in acute patients. Both T-cell subsets were predominantly directed against the envelope antigens (HA and NA). However, in the convalescent patients we found high levels of both CD4⁺ and CD8⁺ T-cells directed against conserved core antigens (NP, PA, PB, and M). The results indicate that the antigen targets recognized by the T-cell subsets may vary according to the phase of infection. The apparent low levels of cross-reactive CD8⁺ T-cells recognizing internal antigens in acute hospitalized patients suggest an important role for this T-cell subset in protective immunity against influenza.     

Mycoplasma orale infection affects K+ and Cl− currents in the HSG salivary gland cell line

Summary The relations between K⁺ channel and Cl⁻ channel currents and mycoplasma infection status were studied longitudinally in HSG cells, a human submandibular gland cell line. The K⁺ channel currents were disrupted by the occurrence of mycoplasma infection: muscarinic activation of K⁺ channels and K⁺ channel expression as estimated by ionomycin- or hypotonically induced K⁺ current responses were all decreased. Similar decreases in ionomycin- and hypotonically induced responses were observed for Cl⁻ channels, but only the latter decrease was statistically significant. Also, Cl⁻ currents could be elicited more frequently than K⁺ currents (63% of cases versus 0%) in infected cells when tested by exposure to hypotonic media, indicating that mycoplasma infection affects K⁺ channels relatively more than Cl⁻ channels. These changes occurred in the originally infected cells, were ameliorated when the infection was cleared with sparfloxacin, and recurred when the cells were reinfected. Such changes would be expected to result in hyposecretion of salivary fluid if they occurredin vivo.     

PC61 (Anti-CD25) Treatment Inhibits Influenza A Virus-Expanded Regulatory T Cells and Severe Lung Pathology during a Subsequent Heterologous Lymphocytic Choriomeningitis Virus Infection

Prior immunity to influenza A virus (IAV) in mice changes the outcome to a subsequent lymphocytic choriomeningitis virus (LCMV) infection and can result in severe lung pathology, similar to that observed in patients that died of the 1918 H1N1 pandemic. This pathology is induced by IAV-specific memory CD8⁺ T cells cross-reactive with LCMV. Here, we discovered that IAV-immune mice have enhanced CD4⁺ Foxp3⁺ T-regulatory (Treg) cells in their lungs, leading us to question whether a modulation in the normal balance of Treg and effector T-cell responses also contributes to enhancing lung pathology upon LCMV infection of IAV-immune mice. Treg cell and interleukin-10 (IL-10) levels remained elevated in the lungs and mediastinal lymph nodes (mLNs) throughout the acute LCMV response of IAV-immune mice. PC61 treatment, used to decrease Treg cell levels, did not change LCMV titers but resulted in a surprising decrease in lung pathology upon LCMV infection in IAV-immune but not in naive mice. Associated with this decrease in pathology was a retention of Treg in the mLN and an unexpected partial clonal exhaustion of LCMV-specific CD8⁺ T-cell responses only in IAV-immune mice. PC61 treatment did not affect cross-reactive memory CD8⁺ T-cell proliferation. These results suggest that in the absence of IAV-expanded Treg cells and in the presence of cross-reactive memory, the LCMV-specific response was overstimulated and became partially exhausted, resulting in a decreased effector response. These studies suggest that Treg cells generated during past infections can influence the characteristics of effector T-cell responses and immunopathology during subsequent heterologous infections. Thus, in humans with complex infection histories, PC61 treatment may lead to unexpected results.     

Early Gag Immunodominance of the HIV-Specific T-Cell Response during Acute/Early Infection Is Associated with Higher CD8+ T-Cell Antiviral Activity and Correlates with Preservation of the CD4+ T-Cell Compartment

The important role of the CD8⁺ T-cell response on HIV control is well established. Moreover, the acute phase of infection represents a proper scenario to delineate the antiviral cellular functions that best correlate with control. Here, multiple functional aspects (specificity, ex vivo viral inhibitory activity [VIA] and polyfunctionality) of the HIV-specific CD8⁺ T-cell subset arising early after infection, and their association with disease progression markers, were examined. Blood samples from 44 subjects recruited within 6 months from infection (primary HIV infection [PHI] group), 16 chronically infected subjects, 11 elite controllers (EC), and 10 healthy donors were obtained. Results indicated that, although Nef dominated the anti-HIV response during acute/early infection, a higher proportion of early anti-Gag T cells correlated with delayed progression. Polyfunctional HIV-specific CD8⁺ T cells were detected at early time points but did not associate with virus control. Conversely, higher CD4⁺ T-cell set points were observed in PHI subjects with higher HIV-specific CD8⁺ T-cell VIA at baseline. Importantly, VIA levels correlated with the magnitude of the anti-Gag cellular response. The advantage of Gag-specific cells may result from their enhanced ability to mediate lysis of infected cells (evidenced by a higher capacity to degranulate and to mediate VIA) and to simultaneously produce IFN-γ. Finally, Gag immunodominance was associated with elevated plasma levels of interleukin 2 (IL-2) and macrophage inflammatory protein 1β (MIP-1β). All together, this study underscores the importance of CD8⁺ T-cell specificity in the improved control of disease progression, which was related to the capacity of Gag-specific cells to mediate both lytic and nonlytic antiviral mechanisms at early time points postinfection.     

Detection of mycoplasmas infecting cell cultures by DNA hybridization

Summary Infection of cell cultures by mycoplasmas can be detected and the mycoplasma identified by Southern blot hybridization of theEco RI-digested DNA of the suspected cell cultures with a nick-translated probe consisting of cloned ribosomal RNA genes ofMycoplasma capricolum. The probe does not hybridize with eukaryotic DNA. The hybridization pattern with mycoplasmal DNA is species specific, enabling the identification of the four most prevalent mycoplasma contaminants,Mycoplasma orale, Mycoplasma hyorhinis, Mycoplasma arginini, andAcholeplasma laidlawii. The test is also very sensitive and can detect as little as 1 ng of mycoplasmal DNA, roughly equivalent to the DNA content of 10⁵ mycoplasmas. The study was supported by the U.S. Public Health Service Grant GM25286 awarded to G. G. and by a grant from the United States-Israel Binational Agricultural Research Development Fund (BARD) awarded to S. R.     

T-cell Receptor-optimized Peptide Skewing of the T-cell Repertoire Can Enhance Antigen Targeting

Altered peptide antigens that enhance T-cell immunogenicity have been used to improve peptide-based vaccination for a range of diseases. Although this strategy can prime T-cell responses of greater magnitude, the efficacy of constituent T-cell clonotypes within the primed population can be poor. To overcome this limitation, we isolated a CD8⁺ T-cell clone (MEL5) with an enhanced ability to recognize the HLA A*0201-Melan A_27–35 (HLA A*0201-AAGIGILTV) antigen expressed on the surface of malignant melanoma cells. We used combinatorial peptide library screening to design an optimal peptide sequence that enhanced functional activation of the MEL5 clone, but not other CD8⁺ T-cell clones that recognized HLA A*0201-AAGIGILTV poorly. Structural analysis revealed the potential for new contacts between the MEL5 T-cell receptor and the optimized peptide. Furthermore, the optimized peptide was able to prime CD8⁺ T-cell populations in peripheral blood mononuclear cell isolates from multiple HLA A*0201⁺ individuals that were capable of efficient HLA A*0201⁺ melanoma cell destruction. This proof-of-concept study demonstrates that it is possible to design altered peptide antigens for the selection of superior T-cell clonotypes with enhanced antigen recognition properties.     

Nutritional effects of culture media on mycoplasma cell size and removal by filtration

Careful media filtration prior to use is an important part of a mycoplasma contamination prevention program. This study was conducted to increase our knowledge of factors that influence efficient filtration of mycoplasma. The cell size of Acholeplasma laidlawii was measured after culture in various nutritional conditions using scanning electron microscopy. The maximum cell size changed, but the minimum cell size remained virtually unchanged and all tested nutritional conditions resulted in a population of cells smaller than 0.2 μm. Culture in Tryptic Soy Broth (TSB) resulted in an apparent increase in the percentage of very small cells which was not reflected in increased penetration of non-retentive 0.2 μm rated filters. A. laidlawii cultured in selected media formulations was used to challenge 0.2 μm rated filters using mycoplasma broth base as the carrier fluid. We used 0.2 μm rated filters as an analytical tool because A. laidlawii is known to penetrate 0.2 μm filters and the degrees of penetration can be compared. Culture of A. laidlawii in TSB resulted in cells that did not penetrate 0.2 μm rated filters to the same degree as cells cultured in other media such as mycoplasma broth or in TSB supplemented with 10% horse serum.     

Characterization of variant and parental-cross-protective immunity to immunogenic variants of a murine fibrosarcoma using the local adoptive transfer assay

Summary The purpose of this study was to characterize the lymphocyte populations responsible for rejection of immunogenic (Imm⁺) tumor variants, and the crossprotective immunity engendered by Imm⁺ variants against the weakly immunogenic parental tumor. Immunogenic clones of the weakly immunogenic methylcholanthreneinduced fibrosarcoma MCA-F have been generated using 1-methyl-3-nitro-1-nitrosoguanidine, 5-aza-2-deoxycytidine, or ultraviolet radiation (UV-B; 280–320 nm). These clones grow progressively in immunosuppressed adult-thymectomized irradiated mice, but are rejected by immunocompetent syngeneic hosts. The parental MCA-F tumor grows progressively in both groups. Mice that have rejected a challenge of 1 × 10⁵ Imm⁺ cells show an anamnestic immune response against both the Imm⁺ clone and the parental MCA-F tumor. Using the local adoptive transfer assay and depletion of T-cell subsets with antibody plus complement, we show that immunity induced by the Imm⁺ variants against the parent MCA-F was mediated by the Thy1.2⁺, L3T4a⁺ population without an apparent contribution by Lyt2.1⁺ cells. Although antivariant immunity was also dependent upon Thy1.2⁺ cells, depletion of either the L3T4a⁺ or the Lyt2.1⁺ cells failed to abolish immunity against the variant. A role for Lyt2.1⁺ T lymphocytes in antivariant immunity, but not antiparent immunity, was supported by the results of cytotoxic T lymphocyte (CTL) assays. Following immunization with high numbers (1 × 10⁵ to 5 × 10⁵) of viable Imm⁺ cells, antivariant, but not antiparent CTL activity was detected in mixed lymphocyte tumor cell cultures. Immunization with lower numbers (3 × 10⁴) of viable Imm⁺ or with high numbers of mitomycin-C-treated Imm⁺ engenders only antivariant immunity without parental cross-protection. Under these conditions lymphocytes mediating immunity against the variant in the local adoptive transfer assay were exclusively of the Thyl1.2⁺, L3T4a⁺ phenotype, with no contribution from the Lyt2.1⁺ cells. Identical results were obtained for Imm⁺ clones of MCA-F induced by methylnitronitrosoguanidine, 5-azadeoxycytidine, and UV-B, suggesting that the nature of the antitumor immunity engendered by Imm⁺ is not significantly affected by the agent used. Furthermore, these results demonstrate that the cross-reactivity and cellular effectors of antitumor immunity in this system are influenced by the immunizing dose of Imm⁺ cells: the predominant effectors of both antivariant and parental-cross-reactive immunity were of the CD4⁺ T cell subclass, with a CD8⁺ cytotoxic population contributing to antivariant immunity only after highdose immunization.Supported by Biomedical Research Support Grant RR5511-23 awarded by the National Cancer Institute, DHHSRecipient of an R. E. Bob Smith Predoctoral Fellowship     

Hantaan Virus Infection Induces Both Th1 and ThGranzyme B+ Cell Immune Responses That Associated with Viral Control and Clinical Outcome in Humans

Hantaviruses infection causing severe emerging diseases with high mortality rates in humans has become public health concern globally. The potential roles of CD4⁺T cells in viral control have been extensively studied. However, the contribution of CD4⁺T cells to the host response against Hantaan virus (HTNV) infection remains unclear. Here, based on the T-cell epitopes mapped on HTNV glycoprotein, we studied the effects and characteristics of CD4⁺T-cell responses in determining the outcome of hemorrhagic fever with renal syndrome. A total of 79 novel 15-mer T-cell epitopes on the HTNV glycoprotein were identified, among which 20 peptides were dominant target epitopes. Importantly, we showed the presence of both effective Th1 responses with polyfunctional cytokine secretion and ThGranzyme B⁺ cell responses with cytotoxic mediators production against HTNV infection. The HTNV glycoprotein-specific CD4⁺T-cell responses inversely correlated with the plasma HTNV RNA load in patients. Individuals with milder disease outcomes showed broader epitopes targeted and stronger CD4⁺T-cell responses against HTNV glycoproteins compared with more severe patients. The CD4⁺T cells characterized by broader antigenic repertoire, stronger polyfunctional responses, better expansion capacity and highly differentiated effector memory phenotype(CD27^-CD28^-CCR7^-CD45RA-CD127^hi) would elicit greater defense against HTNV infection and lead to much milder outcome of the disease. The host defense mediated by CD4⁺T cells may through the inducing antiviral condition of the host cells and cytotoxic effect of ThGranzyme B⁺ cells. Thus, these findings highlight the efforts of CD4⁺T-cell immunity to HTNV control and provide crucial information to better understand the immune defense against HTNV infection.     

Role of CD4+ and CD8α+ T cells in protective immunity against Edwardsiella tarda infection of ginbuna crucian carp,Carassius auratus langsdorfii

Edwardsiella tarda is an intracellular pathogen that causes edwardsiellosis in fish. Our previous study suggests that cell-mediated immunity (CMI) plays an essential role in protection against E. tarda infection. In the present study, we adoptively transferred T-cell subsets sensitized with E. tarda to isogenic naïve ginbuna crucian carp to determination the T-cell subsets involved in protecting fish from E. tarda infection. Recipients of CD4⁺ and CD8α⁺ cells acquired significant resistance to infection with E. tarda 8 days after sensitization, indicating that helper T cells and cytotoxic T lymphocytes plays crucial roles in protective immunity to E. tarda. Moreover, transfer of sensitized CD8α⁺ cells up-regulated the expression of genes encoding interferon-γ (IFN-γ) and perforin, suggesting that protective immunity to E. tarda involves cell-mediated cytotoxicity and interferon-γ-mediated induction of CMI. The results establish that CMI plays a crucial role in immunity against E. tarda. These findings provide novel insights into understanding the role of CMI to intracellular pathogens of fish.     

Dual roles of CagA protein in Helicobacterpylori-induced chronic gastritis in mice

Cytotoxin-associated gene A (CagA) acts directly on gastric epithelial cells. However, the roles of CagA in host adaptive immunity against Helicobacter pylori (H. pylori) infection are not fully understood. In this study, to investigate the roles of CagA in the development of H. pylori-induced chronic gastritis, we used an adoptive-transfer model in which spleen cells from C57BL/6 mice with or without H. pylori infection were transferred into RAG2^−/− mice, with gastric colonization of either CagA⁺H. pylori or CagA⁻H. pylori. Colonization of CagA⁺H. pylori but not CagA⁻H. pylori in the host gastric mucosa induced severe chronic gastritis in RAG2^−/− mice transferred with spleen cells from H. pylori-uninfected mice. In addition, when CagA⁺H. pylori-primed spleen cells were transferred into RAG2^−/− mice, CD4⁺ T cell infiltration in the host gastric mucosa were observed only in RAG2^−/− mice infected with CagA⁺H. pylori but not CagA⁻H. pylori, suggesting that colonization of CagA⁺H. pylori in the host gastric mucosa is essential for the migration of H. pylori-primed CD4⁺ T cells. On the other hand, transfer of CagA⁻H. pylori-primed spleen cells into CagA⁺H. pylori-infected RAG2^−/− mice induced more severe chronic gastritis with less Foxp3⁺ regulatory T-cell infiltration as compared to transfer of CagA⁺H. pylori-primed spleen cells. In conclusion, CagA in the stomach plays an important role in the migration of H. pylori-primed CD4⁺ T cells in the gastric mucosa, whereas CagA-dependent T-cell priming induces regulatory T-cell differentiation, suggesting dual roles for CagA in the pathophysiology of H. pylori-induced chronic gastritis.     

Change in Lectin Specificity of Winter Wheat Seedlings in the Course of Infection with Mycoplasms

The activity of soluble lectins in leaves and roots of seedlings of winter wheat (Triticum aestivum L.) cultivar Mironovskaya 808 increased 1 day and 2 days, respectively, after infection with the mycoplasma Acholeplasma laidlawii 118. Analysis of acid-soluble proteins of wheat leaves by PAGE revealed the appearance of 22- and 20-kDa polypeptides, the disappearance of a 14-kDa polypeptide, and an increase in the content of polypeptides with molecular weights of 76, 48, 25, and 18 kDa. The 18-kDa polypeptide is a subunit of wheat germ agglutinin. A change in the activity of lectins may be a nonspecific response of plants to infection with the pathogen.     

Quantitation of Anaplasma marginale major surface protein (MSP)1a and MSP2 epitope-specific CD4+ T lymphocytes using bovine DRB3*1101 and DRB3*1201 tetramers

Antigen-specific CD4⁺ T cells play a critical role in protective immunity to many infectious pathogens. Although the antigen-specific CD4⁺ T cells can be measured by functional assays such as proliferation or cytokine enzyme-linked immunospot, such assays are limited to a specific function and cannot quantify anergic or suppressed T cells. In contrast, major histocompatiblity complex (MHC) class II tetramers can enumerate epitope-specific CD4⁺ T cells independent of function. In this paper, we report the construction of bovine leukocyte antigen MHC class II tetramers using a novel mammalian cell system to express soluble class II DRA/DRB3 molecules and defined immunodominant peptide epitopes of Anaplasma marginale major surface proteins (MSPs). Phycoerythrin-labeled tetramers were either loaded with exogenous peptide or constructed with the peptide epitope linked to the N terminus of the DRB3 chain. A DRB3*1101 tetramer loaded with MSP1a peptide F2-5B (ARSVLETLAGHVDALG) and DRB3*1201 tetramers loaded with MSP1a peptide F2-1-1b (GEGYATYLAQAFA) or MSP2 peptide P16-7 (NFAYFGGELGVRFAF) specifically stained antigen-specific CD4⁺ T cell lines and clones. Tetramers constructed with the T-cell epitope linked to the DRB3 chain were slightly better at labeling CD4⁺ T cells. In one cell line, the number of tetramer-positive T cells increased to approximately 94% of the CD4⁺ T cells after culture for 21 weeks with specific antigen. This novel technology should be useful to track the fate of antigen-specific CD4⁺ T-cell responses in cattle after immunization or infection with persistent pathogens, such as A. marginale, that modulate the host immune response.     

Fine specificity of a proliferating T-cell clone activated by a conformational determinant of the I-Ek molecule

We have examined the fine specificity of a stable Thy-1.2⁺, Lyt-1.2⁺, Lyt-2^–, and I-A^s– anti-I-E^k proliferating T-cell clone isolated from an A.TH anti-A.TL secondary mixed lymphocyte culture. Spleen cells from various I-A^k, E^k strains induced either a strong (A.TL, OH, and CBA) or a weak (AKR and B10.BR) proliferative response, although such cells expressed at their surface similar amounts of I-E^k antigens. Analysis of H-2 recombinant strains indicated that this clone recognized a conformational determinant carried by the E ^k E ^k dimer, but not on the Ea chain per se. Among the Fl hybrid strains in which the combinatorial E ^k E ^k product was detected by cellular binding with monoclonal E ^k -specific antibodies (mAb), some [(BIO.S(8R) × BlO.HTT) but not others (for example, B10.A(4R) × B10.A(5R)] were stimulatory. Seventeen anti-E^k mAb, regardless of the three spatially separated domains that they defined by antibody binding competition, completely inhibited the restimulation of this clone, whereas 15 other anti-A^k mAb failed to do so. This clone was not reactivated by stimulating cells from strains with the H-2 haplotypes p, j, v, b, r, and s but it proliferated strongly against cells from several H-2 ^d or H-2 ^q strains. Genetic evidence or blocking studies with selected mAb assigned these cross-reactive mixed lymphocyte reaction determinants to the A^d or A^q molecules, respectively. The data support the conclusion that alloreactive T cells may define a polymorphism of I-region coded products not detected by serological analyses and extend at the T-cell level the observations of serological cross-reactions between A and E molecules.     

Determination of a T cell receptor of potent CD8+ T cells against simian immunodeficiency virus infection in Burmese rhesus macaques

Cumulative studies on human immunodeficiency virus (HIV)-infected individuals have shown association of major histocompatibility complex class I (MHC-I) polymorphisms with lower viral load and delayed AIDS progression, suggesting that HIV replication can be controlled by potent CD8⁺ T-cell responses. We have previously established an AIDS model of simian immunodeficiency virus (SIV) infection in Burmese rhesus macaques and found a potent CD8⁺ T cell targeting the Mamu-A1*065:01-restricted Gag_241-249 epitope, which is located in a region corresponding to the HIV Gag_240-249 TW10 epitope restricted by a protective MHC-I allele, HLA-B*57. In the present study, we determined a T cell receptor (TCR) of this Gag_241-249 epitope-specific CD8⁺ T cell. cDNA clones encoding TCR-α and TCR-β chains were obtained from a Gag_241-249-specific CD8⁺ T-cell clone. Coexpression of these TCR-α and TCR-β cDNAs resulted in reconstitution of a functional TCR specifically detected by Gag_241-249 epitope-Mamu-A1*065:01 tetramer. Two of three previously-reported CD8⁺ T-cell escape mutations reduced binding affinity of Gag_241-249 peptide to Mamu-A1*065:01 but the remaining one not. This is consistent with the data obtained by molecular modeling of the epitope-MHC-I complex and TCR. These results would contribute to understanding how viral CD8⁺ T-cell escape mutations are selected under structural constraint of viral proteins.