Precise CD4 T-cell counting using red diode laser excitation: for richer,for poorer

BACKGROUND: Measuring CD4 T-cell counts at low cost is relevant in dealing with the human immunodeficiency virus (HIV) epidemic throughout the developing world. The recently introduced novel concepts in gating strategies and sample stabilization facilitate affordable immunophenotyping by flow cytometry. However, the impact of these developments is still limited by the high cost of currently available flow cytometers. METHODS: Diode lasers emitting 10-15 mW at 635 nm are one-tenth the size and cost and require one thousandth the power of an equivalent 488-nm argon ion laser. We used the available 635-nm diode-based flow cytometers, including PA-II, Luminex 100, SuperMot, and FACSCalibur, to investigate whether these instruments can generate reliable CD4 counts when used with allophycocyanin (APC) and cyanin-5 (Cy5)-labeled CD4 antibodies. RESULTS: We document the feasibility of obtaining leucocyte differential counts using orthogonal side scatter (SSC) without the need for forward scatter (FSC). Accurate CD4% values among lymphocytes and leucocytes can be obtained by primary CD4 gating using a single CD4 monoclonal antibody conjugated to APC or Cy5. Double immunofluorescence (IF) staining with CD4-APC (FL1) and CD45-APC-Cy7 (FL2) introduces pan-leucogating for a convenient assessment of absolute CD4 counts on double platforms. We demonstrate that small flow cytometers with laser diodes are capable of delivering absolute CD4 T-cell counts with a precision similar to the performance of the current state-of-the-art single-platform instruments (e.g., the CytoronAbsolute; R(2) = 0.961). In this respect, they appear to be superior to the nonflow CD4 counting techniques. CONCLUSIONS: Accurate CD4 counts can be generated at minimal cost on red diode laser-operated flow cytometers, retaining the potential for high throughput capacity without compromising precision. With further improvements in volumetric technology and clinical software, these cytometers may develop into a new generation of inexpensive battery-operated laboratory hardware that combines cellular phenotyping with bead-based multiplexing immunoassays for (HIV) serology.     

Evaluation of two human plasma pools as candidate international standard preparations for syphilitic antibodies

A collaborative study was designed to asses two freeze-dried human plasma preparations containing anti-Treponema pallidum antibodies, 05/132 and 05/122, for their suitability as international reference reagents for syphilis serology. Both preparations are intended as replacements of the first international standard (IS) for syphilitic serum antibodies (HS). Samples were tested by eight laboratories using the T. pallidum passive particle agglutination assay (TPPA), the venereal disease research laboratory test (VDRL) and the rapid plasma reagin test (RPR). In addition a range of immunoassays was also used. The outcome of the collaborative study revealed that candidate standard 05/132 contains T. pallidum-specific IgG and IgM and is reactive in VDRL or RPR, and that 05/122 contains T. pallidum-specific IgG but is not reactive in either the VDRL or RPR test. Both 05/132 and 05/122 are reactive in the TPPA. On the basis of these results the Expert Committee on Biological Standardization of the World Health Organization designated 05/132 as the 1st IS for human syphilitic plasma IgG and IgM with a unitage of 3 IU per ampoule relative to HS and 05/122 as the 1st IS for human syphilitic plasma IgG with a unitage of 300 mIU per ampoule relative to 05/132.     

Metadherin peptides containing CD4+ and CD8+ T cell epitopes as a therapeutic vaccine candidate against cancer

The concept of peptide‐based vaccines against cancer has made noteworthy progress. Metadherin (MTDH) overexpression and its role in the development of diverse cancers make it an attractive target for cancer immunotherapy. In the current study, six different T cell epitope prediction tools were run to identify MTDH peptides with multiple immunogenic regions. Further, molecular docking was performed to assess HLA‐peptide binding interactions. Nine and eleven peptides fragments containing multiple CD8 ⁺ and CD4 ⁺ T‐cell epitopes, ranging from 9 to 20 amino acids, respectively, were obtained using a consensus immunoinformatics approach. The three peptides that were finally identified as having overlapping CD4 ⁺ and CD8 ⁺ T‐ cell epitopes are ARLREMLSVGLGFLRTELG, FLLGYGWAAACAGAR, YIDDEWSGLNGLSSADP. These peptides were found to not only have multiple T cell epitopes but also to have binding affinity with wide HLA molecules. A molecular docking study revealed that the predicted immunogenic peptides (with single or multiple T cell epitopes) of MTDH have comparable binding energies with naturally bound peptides for both HLA classes I and II. Thus, these peptides have the potential to induce immune responses that could be considered for developing synthetic peptide vaccines against multiple cancers.     

Analyses of quality assessment studies using CD45 for gating lymphocytes for CD3(+)4(+)%

BACKGROUND: The purpose of this study was to assess whether laboratories which do not use CD45 for gating lymphocytes with three- (or four-) color flow cytometry (non-CD45 laboratories) for CD3(+)4(+)% and CD3(+)8(+)% do worse on quality assessment (QA) studies than laboratories which do use CD45 (CD45 laboratories). METHODS: Data came from blood specimens donated by 62 donors (50 HIV-positive) assayed over 2 years (November, 1996-October, 1998) by 35 laboratories in the NIAID DAIDS Flow Cytometry QA Program. RESULTS: Non-CD45 laboratories were significantly more likely to be classified as having unacceptable inter-laboratory results (far from the group median) than CD45 laboratories (5.6% vs 1.5%, P = 0.005 for CD3(+)4(+)%; 10.4% vs 5.0%, P = 0.007 for CD3(+)8(+)%). The intra-laboratory range of results on blinded replicates was significantly more likely to be deemed unacceptable (range >4%) in non-CD45 laboratories than in CD45 laboratories for CD3(+)8(+)% (14. 5% vs 3.5%, P = 0.002) but not for CD3(+)4(+)% (2.6% vs 1.5%, P = 0. 62). These differences in favor of CD45 gating were observed even though the non-CD45 laboratories had been doing three-color flow cytometry in the QA program significantly longer (P = 0.05) than the CD45 laboratories, and so would be expected to have fewer problems with the assay. CONCLUSIONS: Laboratories which choose to use a single CD3/CD4/CD8 tube for immunophenotyping may be sacrificing both accuracy and reproducibility.     

Identification of two T-cell epitopes on the candidate Epstein-Barr virus vaccine glycoprotein gp340 recognized by CD4+ T-cell clones.

Current efforts to develop an Epstein-Barr virus subunit vaccine are based on the major envelope glycoprotein gp340. Given the central role of CD4+ T cells in regulating immune responses to subunit vaccine antigens, the present study has begun the work of identifying linear epitopes which are recognized by human CD4+ T cells within the 907-amino-acid sequence of gp340. A panel of gp340-specific CD4+ T-cell clones from an Epstein-Barr virus-immune donor were first assayed for their proliferative responses to a series of truncated gp340 molecules expressed from recombinant DNA vectors in rat GH3 cells, by using an autologous B lymphoblastoid cell line as a source of antigen-presenting cells. The first four T-cell clones analyzed all responded to a truncated form of gp340 which contained only the first 260 N-terminal amino acids. These clones were subsequently screened for responses to each of a panel of overlapping synthetic peptides (15-mers) corresponding to the primary amino acid sequence of the first 260 N-terminal amino acids of gp340. One clone (CG2.7) responded specifically to peptides from the region spanning amino acids 61 to 81, while three other clones (CG5.15, CG5.24, and CG5.36) responded specifically to peptides from the region spanning amino acids 163 to 183. Work with individual peptides from these regions allowed finer mapping of the T-cell epitopes and also revealed the highly dose-dependent nature of peptide-induced responses, with inhibitory effects apparent when the most antigenic peptides were present at supraoptimal concentrations. Experiments using homozygous typing B lymphoblastoid cell lines as antigen-presenting cells showed that the T-cell clones with different epitope specificities were restricted through different HLA class II antigens; clone CG2.7 recognized epitope 61-81 in the context of HLA DRw15, whereas clones CG5.15, CG5.24, and CG5.36 recognized epitope 163-183 in the context of HLA DRw11. The present protocol therefore makes a systematic analysis of CD4+ T-cell epitopes within gp340 possible; it will be necessary to screen gp340-specific T-cell clones from a variety of donors to assess the wider influence of HLA class II polymorphism upon epitope choice.     

Evaluation of stabilized whole blood control materials for lymphocyte immunophenotyping

We evaluated stabilized whole blood control materials for their performance as a routine control material for immunophenotyping using flow cytometry. These materials serve as the method control, controlling for both the binding of monoclonal antibodies to the cells and the efficiency of the red-blood-cell lysing reagent. Three products (FluoroTrol, StatusFlow, and CD-Chex): were tested. The controls performed very well in evaluating both CD4 and CD8 percentages and absolute cell counts using flow cytometry. Light scattering patterns and fluorescence intensity of antibody binding were slightly different from those of normal whole blood. These products are not appropriate for quantitative fluorescence standards. Cytometry (Comm. Clin. Cytometry) 38:268-273, 1999. Published 1999 Wiley-Liss, Inc.     

Prediction of CD34(+) cell yield in hematopoietic cell products from children by peripheral blood CD34(+) cell counts

Background aimsPeripheral blood stem cells (PBSC) are increasingly used as an alternative to bone marrow in autologous transplantations. In adult patients, the peripheral blood CD34 ⁺ cell count is a good predictor of CD34 ⁺ cell yield in apheresis. However, the determinants of stem cell yield in the pediatric population have not been well established.MethodsWe retrospectively studied 396 apheresis procedures in 301 pediatric patients. Receiver operating characteristic (ROC) curves based on pre-apheresis peripheral blood CD34 ⁺ cell counts were generated to facilitate prediction of the optimal timing of PBSC collection. The associations between CD34 ⁺ cell yield and age and mobilization regimen were analyzed.ResultsSignificant differences in CD34 ⁺ cell yield among different age groups were observed. Furthermore, higher CD34 ⁺ cell yields were obtained in patients receiving chemotherapy as part of the mobilization regimen than those without chemotherapy. A correlation was noted between the CD34 ⁺ cell yield and blood surrogate markers, including white blood cell count, absolute neutrophil count and pre-apheresis peripheral blood CD34 ⁺ cell count. Cut-off values of > 35 CD34 ⁺ cells/μL in patients < 15 years old and > 45 CD34 ⁺ cells/μL in patients ≥ 15 years old were strong predictors of an adequate PBSC collection in one apheresis session. For clinical use, ROC curves and tables were generated to assist advance planning for PBSC collection.ConclusionsThe pre-apheresis peripheral blood CD34 ⁺ cell count is most useful in predicting PBSC yield. Our new cut-off values have better operating characteristics for children than the conventional value of 20 CD34 ⁺ cells/μL used for adults.     

Association study and expression analysis of CD9 as candidate gene for boar sperm quality and fertility traits

Cluster-of-differentiation antigen 9 (CD9) gene expressed in the male germ line stem cells is crucial for sperm-egg fusion, and was therefore selected as candidate gene for boar semen quality. The association of CD9 with boar sperm quality and fertility trait was analyzed using a total of 340 boars both from purebred Pietrain and Pietrain×Hampshire crosses. A single nucleotide polymorphism (g.358A>T) in intron 6 was significantly associated with sperm motility (MOT) (P<0.001), plasma droplet rate (PDR) (P<0.001) and abnormal spermatozoa rate (ASR) (P<0.01). Boars were divided into two groups with group 1 (G-I) boars having a higher SCON and SMOT, lower SVOL (sperm volume) and group 2 (G-II) having a lower SCON and SMOT, higher SVOL. The mRNA and protein expression levels were evaluated in reproductive, non-reproductive tissues and spermatozoa from G-I and G-II animals by using quantitative real-time PCR and western blotting. When both reproductive and non-reproductive tissues were examined, highest mRNA was expressed in prostate gland, then in the body of the epididymis, vas deferens and tail of the epididymis. In case of reproductive tissues, CD9 expression was higher in tissues and spermatozoa collected from G-I boars than those collected from G-II boars. The mRNA expression was significantly different (P<0.05) in body of epididymis from G-I and G-II boars. The CD9 protein expression results from western blot were coincided with the results of qRT-PCR. Moreover, CD9 protein localization in Leydig cells, Sertoli cells, epithelial cells and spermatozoa was remarkable which indicated the important role of CD9 in spermatogenesis process. By using mRNA and protein expression profiles, it could be shown that CD9 plays a crucial role during sperm development, especially within the epididymis where the maturation of the sperm, a key process for the sperm quality and motility takes place. These results will improve the understanding of the functions of the CD9 in spermatogenesis within the reproductive tracts and will shed light on CD9 as a candidate gene in the selection of good sperm quality boars.     

Granulysin-expressing CD4+ T cells as candidate immune marker for tuberculosis during childhood and adolescence

Background

Granulysin produced by cytolytic T cells directly contributes to immune defense against tuberculosis (TB). We investigated granulysin as a candidate immune marker for childhood and adolescent TB.

Methods

Peripheral blood mononuclear cells (PBMC) from children and adolescents (1–17 years) with active TB, latent TB infection (LTBI), nontuberculous mycobacteria (NTM) infection and from uninfected controls were isolated and restimulated in a 7-day restimulation assay. Intracellular staining was then performed to analyze antigen-specific induction of activation markers and cytotoxic proteins, notably, granulysin in CD4⁺ CD45RO⁺ memory T cells.

Results

CD4⁺ CD45RO⁺ T cells co-expressing granulysin with specificity for Mycobacterium tuberculosis (Mtb) were present in high frequency in TB-experienced children and adolescents. Proliferating memory T cells (CFSE_lowCD4⁺CD45RO⁺) were identified as main source of granulysin and these cells expressed both central and effector memory phenotype. PBMC from study participants after TB drug therapy revealed that granulysin-expressing CD4⁺ T cells are long-lived, and express several activation and cytotoxicity markers with a proportion of cells being interferon-gamma-positive. In addition, granulysin-expressing T cell lines showed cytolytic activity against Mtb-infected target cells.

Conclusions

Our data suggest granulysin expression by CD4⁺ memory T cells as candidate immune marker for TB infection, notably, in childhood and adolescence.     

Identification of hexon-specific CD4 and CD8 T-cell epitopes for vaccine and immunotherapy

Adenoviral infections in the immunocompromised host are associated with significant morbidity and mortality. Although the adoptive transfer of adenovirus-specific T cells may prevent and treat such infections, the T-cell immune response to the multiplicity of adenovirus serotypes and subspecies that infect humans has not been well characterized, impeding the development of such approaches. We have, therefore, analyzed the specificities of T-cell responses to the viral capsid hexon antigen, since this structure is highly conserved in human pathogens. We screened 25 human cytotoxic T-cell lines with adenovirus specificity to extensively characterize their responses to adenoviral hexon and to identify a panel of novel CD4⁺ and CD8⁺ T-cell epitopes. Using a peptide library spanning the entire sequence of the hexon protein, we confirmed the responsiveness of these cytotoxic T-cell lines to seven peptides described previously and also identified 33 new CD4- or CD8-restricted hexon epitopes. Importantly, the majority of these epitopes were shared among different adenovirus subspecies, suggesting that T cells with such specificities could recognize and be protective against multiple serotypes, simplifying the task of effective adoptive transfer or vaccine-based immunotherapy for treating infection by this virus.     

Characterization of a high-affinity monoclonal antibody specific for CD44v6 as candidate for immunotherapy of squamous cell carcinomas

 Variant isoforms of CD44, a family of cell-surface glycoproteins generated by alternative splicing and post-translational modifications, are expressed in a variety of human tumors and play important roles in tumor progression and metastasis formation. The murine monoclonal IgG1 antibody VFF18, specific for an epitope encoded by human CD44 variant exon 6, binds with high affinity to the recombinant protein (K _d = 1.7×10^–10 M) as well as to tumor cell lines in vitro, and is suitable for immunohistochemical analysis of human tumors. Screening of more than 500 tumor samples of different histogenesis showed that VFF18 most strongly and uniformly reacts with squamous cell carcinomas (SCC). Detailed analysis of 185 SCC (head and neck, lung, skin) confirmed reactivity of the antibody with 99% of the samples, with intense and homogeneous staining of the tumor cells in the majority of cases, whereas reactivity of VFF18 with normal tissues is limited to certain epithelia and activated lymphocytes. When radiolabelled VFF18 was administered to nude mice bearing human epidermoid carcinoma (A-431) xenograft, fast and selective tumor uptake of the radioimmunoconjugate with a maximum of 18% of the injected dose per gram of tissue was observed. Taken together, these data suggest that mAb VFF18 is a promising targeting vehicle for radioimmunotherapy of squamous cell carcinomas in humans. Received: 9 September 1996 / Accepted: 25 September 1996     

Electroinsertion of full length recombinant CD4 into red blood cell membrane

Electroinsertion is a novel technique of protein implantation in cell membranes using electrical pulses, of field strength between 1.3 kV/cm and 2.1 kV/cm and up to 1 ms duration. The full length recombinant CD4 receptor could thus be inserted in human and murine red blood cell (RBC) membranes. 100% of the RBC subjected to this procedure were shown to expose different CD4 epitopes after electroinsertion. An average of 5000 epitopes per cell has been detected by immunofluorescence assay using flow cytometry and whole cell ELISA. CD4 electroinserted in red blood cell membranes showed upon reaction with monoclonal antibody significant patching similar to that observed in T4 cells expressing CD4. Furthermore, the fluorescent enhancement coming from accumulation of immune complex phycoerythrin-antiphycoerythrin was similar for both native CD4 on T4 cells or CD4 electroinserted into erythrocyte membrane. Attempts to electroinsert proteins without a membrane spanning sequence have consistently failed, suggesting that adsorption is not responsible for the observed phenomena.     

Chimeric yellow fever/dengue virus as a candidate dengue vaccine: quantitation of the dengue virus-specific CD8 T-cell response

We have constructed a chimeric yellow fever/dengue (YF/DEN) virus, which expresses the premembrane (prM) and envelope (E) genes from DEN type 2 (DEN-2) virus in a YF virus (YFV-17D) genetic background. Immunization of BALB/c mice with this chimeric virus induced a CD8 T-cell response specific for the DEN-2 virus prM and E proteins. This response protected YF/DEN virus-immunized mice against lethal dengue encephalitis. Control mice immunized with the parental YFV-17D were not protected against DEN-2 virus challenge, indicating that protection was mediated by the DEN-2 virus prM- and E-specific immune responses. YF/DEN vaccine-primed CD8 T cells expanded and were efficiently recruited into the central nervous systems of DEN-2 virus challenged mice. At 5 days after challenge, 3 to 4% of CD8 T cells in the spleen were specific for the prM and E proteins, and 34% of CD8 T cells in the central nervous system recognized these proteins. Depletion of either CD4 or CD8 T cells, or both, strongly reduced the protective efficacy of the YF/DEN virus, stressing the key role of the antiviral T-cell response.     

Epstein-Barr Virus (EBV)-derived BARF1 encodes CD4- and CD8-restricted epitopes as targets for T-cell immunotherapy

Background aims

EBV type II latency tumors, such as Hodgkin lymphoma (HL), Non-Hodgkin lymphoma (NHL) and nasopharyngeal carcinoma, express a limited array of EBV antigens including Epstein-Barr nuclear antigen (EBNA)1, latent membrane protein (LMP)1, LMP2, and BamH1-A right frame 1 (BARF1). Adoptive immunotherapy for these malignancies have focused on EBNA1, LMP1 and LMP2 because little is known about the cellular immune response to BARF1.

Evaluation of tafazzin as candidate for dilated cardiomyopathy in Irish wolfhounds

Dilated cardiomyopathy (DCM) is a common disease in humans and dogs. Large-breed dogs and especially Irish wolfhounds belong to the frequently affected breeds. Male Irish wolfhounds show a significantly higher prevalence of DCM than females. Therefore, we evaluated X chromosome markers for linkage with DCM as well as a human candidate gene on the X chromosome. A set of X chromosomal microsatellites was genotyped in Irish wolfhound families segregating for DCM. In addition, exon and intron sequences of the tafazzin (TAZ) gene were assayed for polymorphisms segregating in these families. Statistical analysis of the microsatellite markers did not reveal linkage to DCM. Furthermore, all Irish wolfhounds included in this study were monomorphic for TAZ, and only 8 sequence differences to the Dog Genome Assembly 2.1 could be found. The results indicate that due to the lack of mutations, TAZ is unlikely to cause DCM in Irish wolfhounds.     

The use of OsO4 as fixative for Feulgen-stained preparations

OsO(4) has many advantages over Carnoy's fixative mixture for the Feulgen nuclear staining in the protozoan Tokophrya infusionum. While Carnoy's fluid used prior to the Feulgen reaction produces shrinkage of the macronucleus and coarse clumping of its chromatin bodies, OsO(4) preserves faithfully the size and shape of the macronucleus and its chromatin material. This finding seems to be of special importance in view of the fact that electron microscopy relies on OsO(4) fixation. The satisfactory preservation of structured detail in Feulgen-stained preparations is of importance for the correlation of histochemical and morphological information.     

Importance of the CD3 marker for evaluating changes in rhesus macaque CD4/CD8 T-cell ratios

BACKGROUND: Until recently, there were no CD3 antibodies that crossreacted with rhesus macaque T cells. Consequently, studies relying on CD8 counts or CD4/CD8 ratios enumerated this subpopulation on the basis of CD8+ or CD8bright+ staining. We used a rhesus-specific, anti-CD3 antibody to better define the CD8+ T-cell population, and to show the effects of better measurements on CD4/CD8 ratios and changes in T cells as macaques age. METHODS: We used three-color flow cytometry to measure CD4 and CD8 populations with and without CD3 costaining. Venous blood samples were obtained from 52 colony-bred macaques between 2 months and 9 years of age. RESULTS: The CD8+ T cells defined by CD3 and CD8 double staining were approximately 60% of all cells that were stained by CD8 alone. Improved detection of this lymphocyte subset showed that CD4/CD8 ratios were close to the range of 1.5-2.0. Declining CD4/CD8 ratios during aging are predominantly due to decreasing CD4+ T-cell counts. CONCLUSIONS: Better quantitation of the CD8+ T-cell population showed that the CD4/CD8 ratio was not inverted as had been reported, but is actually very similar to the values observed in human beings. Although the two species differ in the pattern of CD8 expression, the general immune system characteristics are very similar.