Reconstitution of immune cell populations in multiple sclerosis patients after autologous stem cell transplantation.

Multiple sclerosis is an inflammatory T cell-mediated autoimmune disease. In a Phase II clinical trial, high-dose immunosuppressive therapy combined with autologous CD34+ haematopoietic stem cell transplant resulted in 69·2% of subjects remaining disease-free without evidence of relapse, loss of neurological function or new magnetic resonance imaging (MRI) lesions to year 5 post-treatment. A combination of CyTOF mass cytometry and multi-parameter flow cytometry was used to explore the reconstitution kinetics of immune cell subsets in the periphery post-haematopoietic cell transplant (HSCT) and the impact of treatment on the phenotype of circulating T cells in this study population. Repopulation of immune cell subsets progressed similarly for all patients studied 2 years post-therapy, regardless of clinical outcome. At month 2, monocytes and natural killer (NK) cells were proportionally more abundant, while CD4 T cells and B cells were reduced, relative to baseline. In contrast to the changes observed at earlier time-points in the T cell compartment, B cells were proportionally more abundant and expansion in the proportion of naive B cells was observed 1 and 2 years post-therapy. Within the T cell compartment, the proportion of effector memory and late effector subsets of CD4 and CD8 T cells was increased, together with transient increases in proportions of CD45RA-regulatory T cells (Tregs ) and T helper type 1 (Th1 cells) and a decrease in Th17·1 cells. While none of the treatment effects studied correlated with clinical outcome, patients who remained healthy throughout the 5-year study had significantly higher absolute numbers of memory CD4 and CD8 T cells in the periphery prior to stem cell transplantation.

Minimal information about T cell assays: the process of reaching the community of T cell immunologists in cancer and beyond.

Many assays to evaluate the nature, breadth, and quality of antigen-specific T cell responses are currently applied in human medicine. In most cases, assay-related protocols are developed on an individual laboratory basis, resulting in a large number of different protocols being applied worldwide. Together with the inherent complexity of cellular assays, this leads to unnecessary limitations in the ability to compare results generated across institutions. Over the past few years a number of critical assay parameters have been identified which influence test performance irrespective of protocol, material, and reagents used. Describing these critical factors as an integral part of any published report will both facilitate the comparison of data generated across institutions and lead to improvements in the assays themselves. To this end, the Minimal Information About T Cell Assays (MIATA) project was initiated. The objective of MIATA is to achieve a broad consensus on which T cell assay parameters should be reported in scientific publications and to propose a mechanism for reporting these in a systematic manner. To add maximum value for the scientific community, a step-wise, open, and field-spanning approach has been taken to achieve technical precision, user-friendliness, adequate incorporation of concerns, and high acceptance among peers. Here, we describe the past, present, and future perspectives of the MIATA project. We suggest that the approach taken can be generically applied to projects in which a broad consensus has to be reached among scientists working in fragmented fields, such as immunology. An additional objective of this undertaking is to engage the broader scientific community to comment on MIATA and to become an active participant in the project.

Normal lymphocyte development but delayed humoral immune response in CD81-null mice.

CD81 is a cell surface molecule expressed on many cell types and associated with the CD19/CD21/Leu13 signal-transducing complex on B cells. A recent report implies that CD81 expression on thymic stromal cells is important in the maturation of thymocytes from CD4-CD8- to CD4+CD8+. However, we have produced CD81-null mice by gene targeting, and find that they undergo normal development of thymocytes and express normal numbers of T cells. B cells are also found in normal numbers in the spleen, blood, and peritoneal cavity of CD81-null mice, but they express a lower level of CD19 compared to heterozygous littermates. Finally, early antibody responses to the protein antigen ovalbumin are weaker in CD81-null mice compared to their heterozygous littermates. This is consistent with the proposed role of the CD19/CD21/CD81-signaling complex in lowering the threshold for B cell responses. These results show that CD81 is not required for maturation of T cells, but is important for optimal expression of CD19 on B cells and optimal stimulation of antibody production.

Detection of CD4 T-cell responses to a tumor vaccine by cytokine flow cytometry.

Cytokine flow cytometry (CFC) is a simple and powerful method for measuring antigen-specific T-cell responses by detection of intracellular cytokine staining. We applied this method to the detection of CD4 T-cell responses to tumor vaccines. Patients with multiple myeloma were immunized against their autologous tumor immunoglobulin idiotype, using antigen-pulsed dendritic cell vaccination. Blood samples were drawn before and after vaccination, and CFC and proliferation assays were performed. For CFC, whole blood was incubated overnight with antigen in the presence of costimulatory antibodies to CD28 and CD49d. The blood was then treated with EDTA, erythrocytes were lysed, and leukocytes were fixed, permeabilized, and stained for intracellular cytokines [tumor necrosis factor-alpha (TNF-alpha) or IFN-gamma], CD4, and CD69. Cells were analyzed by flow cytometry and cytokine-producing CD69+ cells enumerated as a percentage of CD4 cells. Of nine patients analyzed, three demonstrated detectable CFC responses to tumor immunoglobulin and/or keyhole limpet hemocyanin (KLH) after vaccination. One of these patients responded only to KLH, whereas the other two responded to both tumor immunoglobulin and KLH. Most responses were detected with both TNF-alpha and IFN-gamma, but one patient's KLH response was detected only with TNF-alpha. There was a positive, but not strong, correlation of cytokine responses with proliferative responses to KLH. Although further follow-up and correlation with clinical outcome is needed, CFC may represent a simple yet detailed assessment of T-cell frequencies and subsets responding to cancer vaccines.

Isolation of anti-idiotypic antibodies to T cells using an anti-framework determinant antibody.

A panel of anti-idiotypic antibodies to the T cell line HPB-ALL was produced by screening with a novel enzyme-linked immunoadsorption assay (ELISA). Using the beta framework I (beta F1) monoclonal antibody directed at a common determinant on the human T cell receptor beta subunit, we were able to specifically capture the receptor molecule from a cell lysate preparation and use this as the basis of an ELISA assay. Hybridoma supernatants were tested for their ability to bind to the receptor thus captured. A total of four antibodies were isolated by this method, and they were shown to immunoprecipitate a disulfide-linked heterodimer composed of alpha (49 kDa) and beta (40 kDa) subunits from HPB-ALL cells, similar to the subunits recognized by the beta F1 antibody. Furthermore, all four antibodies blocked the binding of T40/25, an anti-idiotype to HPB-ALL. Three of these antibodies blocked the binding of anti-Leu 4 to a similar degree as did T40/25, while one did not. This suggests that these new anti-idiotypic antibodies recognize distinct but associated idiotypic determinants. The isolation of such antibodies for any particular T cell line or tumor promises to be useful for biological studies of T cell malignancy in humans.

Use of overlapping peptide mixtures as antigens for cytokine flow cytometry.

Intracellular cytokine staining and flow cytometry can be used to measure T-cell responses to defined antigens. Although CD8+ T-cell responses to soluble proteins are inefficiently detected by this approach, peptides can be used as antigens. Using overlapping peptides spanning an entire protein sequence, CD8+ T-cell responses can be detected to multiple epitopes, regardless of HLA type. In this study, overlapping peptide mixes of various lengths were compared and 15 amino acid peptides with 11 amino acid overlaps were found to stimulate both CD4+ and CD8+ T-cell responses. Such peptide mixes stimulated CD4+ T-cell responses equivalent to those observed with whole recombinant protein, while simultaneously stimulating CD8+ T-cell responses much higher than those observed with whole protein. Although 8-12 amino acid peptides produced the highest level of CD8+ T-cell responses, 15 amino acid peptides were still very effective. Peptides that were 20 amino acids in length, however, did not stimulate strong CD8+ T-cell responses at the same peptide dose. The cytokine responses to individual epitopes added up approximately to the response to the entire mix, demonstrating that large mixes can detect responses in a quantitative fashion. Unlike whole protein antigens, peptide mixes were effective at stimulating responses in both cryopreserved PBMC and blood stored for 24 h at room temperature. Thus, overlapping 15 amino acid peptide mixes may facilitate the analysis of antigen-specific CD4+ and CD8+ T-cell responses by cytokine flow cytometry, using clinical specimens that include shipped blood or cryopreserved PBMC.

Direct functional analysis of epitope-specific CD8+ T cells in peripheral blood.

The functional status of virus-specific CD8+ T cells is important for the outcome and the immunopathogenesis of viral infections. We have developed an assay for the direct functional analysis of antigen-specific CD8+ T cells, which does not require prolonged in vitro cultivation and amplification of T cells. Whole blood samples were incubated with peptide antigens for <5 h, followed by staining with peptide-MHC tetramers to identify epitope-specific T cells. The cells were also stained for the activation marker CD69 or for the production of cytokines such as interferon-gamma (IFNgamma) or tumor necrosis factor-alpha (TNFalpha). With the combined staining with tetramer and antibodies to CD69 or cytokines the number of antigen-specific CD8+ T cells as well as the functional response of each individual cell to the cognate antigen can be determined in a single experiment. Virus-specific CD8+ T cells that are nonfunctional, as well as those that are functional under the same stimulating conditions can be simultaneously detected with this assay, which is not possible by using other T-cell functional assays including cytotoxicity assay, intracellular cytokine staining, and enzyme-linked immunospot (ELISPOT) assay.

Cytokine fusion constructs as DNA vaccines against tumors.

Various studies have used DNA vaccination as a method of immunizing against tumors (1-12). As with any tumor vaccine, one challenge is to find a truly tumor-specific antigen (13,14). The majority of immunologically targeted tumor antigens are also expressed on a subset of normal host cells. Examples of such antigens include prostate-specific antigen, and CD20, a B cell marker. Some tumor antigens are specific for activated cells of certain types, such as carcinoembryonic antigen (CEA) or the IL-2 receptor. These are often found on embryonic or fetal cells as well as tumor cells. The carbohydrate antigens of melanomas and the immunoglobulin (Ig) idiotype of B cell lymphomas represent tumor-specific antigens (TSA). Unfortunately, TSA have not been identified in more common malignancies. Furthermore, the antigenic determinants of known TSA may differ between patients; for example, the tumor idiotype (Id) of B cell lymphoma is highly patient-specific and must be determined for each case.

Differential expression of murine CD81 highlighted by new anti-mouse CD81 monoclonal antibodies.

We describe the use of a soluble CD81-Fc fusion protein to screen for novel monoclonal antibody (MAb) reactive with the extracellular loops of murine CD81 (TAPA-1). Two such MAbs, Eat1 and Eat2 (for Extracellular Anti-TAPA1), were used to assess the expression and function of CD81 on murine lymphocytes. Although CD81 is expressed uniformly on all human lymphocytes, murine CD81 was found to be expressed at much higher levels on resting B cells than on resting T cells. This was particularly evident when staining with the new MAbs, Eat1 and Eat2. The molecule is also functionally active on B cells, as Eat1 and Eat2 induce homotypic adhesion of B lymphocytes. Stimulated B cells undergo early apoptotic events in the presence of Eat2, as shown by binding of Annexin V-fluorescein isothiocyanate (FITC). Polyclonal activation of murine T cells also induces higher level CD81 expression, and many immortalized murine T-cell lines express high levels of the protein. In contrast to human CD81, which is expressed equally on all thymocytes, murine CD81 is induced during thymic development, being expressed at high levels on CD4+CD8+ thymocytes, in contrast to other subsets of thymocytes. Finally, murine dendritic cells, splenic macrophages, and non-killer (NK) cells all express high levels of CD81. We conclude that CD81 is differentially expressed in the murine immune system, and is involved in regulating the adhesion and activation of murine B cells.

NK cells are dysfunctional in human chronic myelogenous leukemia before and on imatinib treatment and in BCR-ABL-positive mice.

Although BCR-ABL+ stem cells in chronic myeloid leukemia (CML) resist elimination by targeted pharmacotherapy in most patients, immunological graft-versus-leukemia effects can cure the disease. Besides cytotoxic T cells, natural killer (NK) cells may have a role in immune control of CML. Here, we explored the functionality of NK cells in CML patients and in a transgenic inducible BCR-ABL mouse model. Compared with controls, NK-cell proportions among lymphocytes were decreased at diagnosis of CML and did not recover during imatinib-induced remission for 10-34 months. Functional experiments revealed limited in vitro expansion of NK cells from CML patients and a reduced degranulation response to K562 target cells both at diagnosis and during imatinib therapy. Consistent with the results in human CML, relative numbers of NK1.1+ NK cells were reduced following induction of BCR-ABL expression in mice, and the defects persisted after BCR-ABL reversion. Moreover, target-induced degranulation by expanded BCR-ABL+ NK cells was compromised. We conclude that CML is associated with quantitative and functional defects within the NK-cell compartment, which is reproduced by induced BCR-ABL expression in mice. Further work will aim at identifying the mechanisms of NK-cell deficiency in CML and at developing strategies to exploit NK cells for immunotherapy.

Cytokine flow cytometry: multiparametric approach to immune function analysis.

More precise quantitation of cellular immune responses has become possible with the advent of single-cell assays of immune function, such as cytokine flow cytometry, enzyme-linked immunospot (ELISPOT), and MHC-peptide multimers. Cytokine flow cytometry is an attractive technique because it allows the detection of responses to whole antigens without regard to MHC restriction, while also collecting additional information on responding cells via multiparameter flow cytometry. In this review, we compare cytokine flow cytometry with other assays of immune function, summarize some of that data that have been collected in various disease states using cytokine flow cytometry, and describe some methodological improvements designed to increase the robustness, throughput, and information content of this technique. We hypothesize that a new generation of automated cytokine flow cytometry assays will allow elucidation of the correlates of protection for diseases involving cellular immunity, through application of these assays in more and large clinical trials.

Spontaneous T cell antigen receptor variants of a human T leukemia cell line.

We have sought to address the question of clonal variation of TCR within a human T leukemia cell line, HPB-ALL. To do so, a panel of anti-idiotypic antibodies was produced and the cell line examined for variants. We isolated both spontaneous idiotype and receptor-negative variants without applying mutagens or any selective pressure other than sorting the cells. These sorted and cloned populations are all clonally related to each other as shown by their beta-TCR locus gene rearrangements. The idiotype variants have alpha-chains which are differentially glycosylated, but they have the same size core protein after treatment with peptide N-glycosidase F to remove their carbohydrate side chains. This probably accounts for their idiotypic difference, since the antibody that distinguishes them appears dependent upon glycosylation for its binding, as shown by immunoprecipitation in the presence versus the absence of tunicamycin, which inhibits glycosylation from occurring. The idiotype variants differed from one another in variable region sequences by only a single amino acid substitution in the beta-chain, which is likely not important for the idiotypic difference. The receptor-negative variant produces both alpha- and beta-mRNA and cytoplasmic protein for TCR, but fails to transport this protein to the cell surface. We conclude that idiotype and receptor-negative variants of a T cell clone can occur in the absence of appreciable somatic mutation.

Prevalence of antigen receptor variants in human T cell lines and tumors.

Previously, we have shown that a human T acute lymphoblastic leukemia cell line, HPB-ALL, exhibits clonal heterogeneity within its Ag receptor, as revealed by varying reactivity patterns with a panel of anti-idiotype mAb. We now extend these findings to another human T acute lymphoblastic leukemia cell line, SUP-T13, and to two fresh human chronic lymphocytic leukemias, JE and EF. In the two cell lines, two types of Ag receptor variants could be found: those that retained a receptor molecule but lost reactivity with an anti-idiotype mAb (idiotype variants), and those which had lost surface receptor expression completely (receptor-negative variants). The idiotype variants, at least in HPB-ALL, have differentially glycosylated receptor alpha-chains from the parent. The receptor-negative cells, in HPB-ALL as well as in SUP-T13, produce cytoplasmic receptor and CD3 proteins but do not transport them to the surface. Neither idiotype nor receptor-negative variants could be detected in either of the fresh tumors of chronic lymphocytic leukemias. The limit of sensitivity in these analyses was about 0.05%. We conclude that antigen receptor variants can spontaneously occur in cell lines derived from acute lymphoblastic leukemias, but are infrequent in chronic lymphocytic leukemias in vivo, and that therapy with anti-idiotype mAb may be a viable strategy for these malignancies.

Optimization of whole blood antigen-specific cytokine assays for CD4(+) T cells.

BACKGROUND: The analysis of cytokine production is a valuable component of studies of immune response to stimulation such as pathogens, vaccines, and other immunological challenges. One highly sensitive method of cytokine evaluation involves three-color flow cytometric analysis of cytokine production in individual CD4(+) T cells. METHODS: We present four methods to enhance the acquisition and analysis of cells secreting the cytokines interferon gamma (IFNgamma), tumor necrosis factor alpha (TNFalpha), interleukin-2 (IL-2), and interleukin-4 (IL-4). Using cytomegalovirus (CMV) as the antigenic model, titration and kinetic experiments were carried out in whole blood from CMV-seropositive donors. RESULTS: CMV is most effective as a stimulating antigen when used at a dose of 5 microg/ml and for a period of at least 6 h, the first 2 h in the absence of 10 microg/ml Brefeldin A. This period of incubation can be made more convenient by the use of a "timed cooling" device, whereby the samples are automatically cooled and held at 4 degrees C at the end of incubation. Such timed cooling does not affect backgrounds or the proportion of responding cells. For certain samples, a high background can be reduced by adding fourth-color reagents. They identify and allow for elimination of monocytes and activated platelets, which contribute to false positive staining. CONCLUSIONS: These optimizations make the assay both convenient for use in whole blood samples and highly reproducible (intra-assay variability is less than 10%; interassay variability is less than 25%).

The tetraspanin superfamily: molecular facilitators.

A legacy of molecular evolution is the formation of gene families encoding proteins that often serve related functions. One such family gaining recent attention is the tetraspanin superfamily, whose membership has grown to nearly 20 known genes since its discovery in 1990. All encode cell-surface proteins that span the membrane four times, forming two extracellular loops. Some of these genes are found in organisms as primitive as schistosomes and nematodes. Alternately known as the transmembrane 4 (TM4) superfamily or the TM4SF, 4TM, or tetraspan family, we propose here that the name tetraspanins be used for the purpose of standardization. What do the tetraspanins do? Awaiting definitive functional studies, we can only put together pieces of a puzzle that has been built by raising antibodies against these proteins and looking at their distribution, associations, and functions. A brief overview indicates that some tetraspanins are found in virtually all tissues (CD81, CD82, CD9, CD63), whereas others are highly restricted, such as CD37 (B cells) or CD53 (lymphoid and myeloid cells). Many of these proteins have a flair for promiscuous associations with other molecules, including lineage-specific proteins, integrins, and other tetraspanins. In terms of function, they are involved in diverse processes such as cell activation and proliferation, adhesion and motility, differentiation, and cancer. We propose that these functions may all relate to their ability to act as "molecular facilitators," grouping specific cell-surface proteins and thus increasing the formation and stability of functional signaling complexes.

In situ detection and characterization of apoptotic thymocytes in human thymus. Expression of bcl-2 in vivo does not prevent apoptosis.

Apoptosis plays a crucial role in shaping the T cell repertoire during T cell development in the thymus. The observed disappearance in the thymus of CD4+ CD8+ thymocytes with a specific TCR, and the lack of CD4+ or CD8+ single positive mature cells expressing the same TCR specificity in the periphery have led to the conclusion that deletion occurs at the CD4+ CD8+ double positive stage; however, there is no direct evidence demonstrating apoptotic CD4+ CD8+ cells in situ. Apoptosis of thymocytes in situ at other stages of T cell development has also not been reported. Using three-color immunofluorescence and flow cytometric assays on frozen human thymic tissue and freshly isolated human thymocytes respectively, we directly identify CD4+ CD8+ and CD4- CD8- thymocytes in newborn human thymus that contain intracellular fragmented DNA and are therefore apoptotic. We determine that 75% of the apoptotic thymocytes are CD4+ CD8+ double positive apoptotic thymocytes, and interestingly, that 13% are CD4- CD8- double negative thymocytes. The majority of apoptotic thymocytes in situ are detected at the cortical-medullary junction; however, apoptotic thymocytes are also found scattered throughout the cortex. Furthermore, we determine that within the apoptotic thymocyte population, 54% express the apoptotic regulatory protein bcl-2 in vivo, whereas 32% are bcl-2 negative. Thus, our in vivo data directly demonstrate that both CD4+ CD8+ and CD4- CD8- human thymocytes die in situ via an apoptotic process, and that expression of the bcl-2 protein in situ does not prevent immature thymocytes from apoptosis.

Immunofluorescence analysis of T-cell responses in health and disease.

The use of flow cytometry to study the functional responses of T cells by immunofluorescent staining for intracellular cytokines and other markers is a growing field of clinical interest. In this article, we describe methods for the rapid evaluation of T-cell responses to mitogens and specific antigens and explore how these assays might be valuable in various clinical settings.

CD81 on B cells promotes interleukin 4 secretion and antibody production during T helper type 2 immune responses.

Mice lacking CD81 (TAPA-1), a widely expressed tetraspanin molecule, have impaired antibody responses to protein antigens. This defect is specific to antigens that preferentially stimulate a T helper 2 response (ovalbumin or keyhole limpet hemocyanin in alum) and is only seen with T cell-dependent antigens. Absence of CD81 on B cells is sufficient to cause the defect. Also, antigen-specific interleukin (IL) 4 production is greatly reduced in the spleen and lymph nodes of CD81-null mice compared with heterozygous littermates. Thus, expression of CD81 on B cells is critical for inducing optimal IL-4 and antibody production during T helper 2 responses. These findings suggest that CD81 may interact with a ligand on T cells to signal IL-4 production. By using a soluble form of CD81 as a probe, a putative ligand for CD81 was identified on a subset of B and T cells. Two possible models for the interaction of CD81 on B cells with a potential ligand on either B or T cells are proposed.

CD81 (TAPA-1): a molecule involved in signal transduction and cell adhesion in the immune system.

CD81 (TAPA-1) is a widely expressed cell-surface protein involved in an astonishing variety of biologic responses. It has been cloned independently several times for different functional effects and is reported to influence adhesion, morphology, activation, proliferation, and differentiation of B, T, and other cells. On B cells CD81 is part of a complex with CD21, CD19, and Leu13. This complex reduces the threshold for B cell activation via the B cell receptor by bridging Ag specific recognition and CD21-mediated complement recognition. Similarly on T cells CD81 associates with CD4 and CD8 and provides a costimulatory signal with CD3. In fetal thymic organ culture, mAb to CD81 block maturation of CD4-CD8- thymocytes, and expression of CD81 on CHO cells endows those cells with the ability to support T cell maturation. However, CD81-deficient mice express normal numbers and subsets of T cells. These mice do exhibit diminished antibody responses to protein antigens. CD81 is also physically and functionally associated with several integrins. Anti-CD81 can activate integrin alpha 4 beta 1 (VLA-4) on B cells, facilitating their adhesion to tonsilar interfollicular stroma. Similarly, anti-CD81 can activate alpha L beta 2 (LFA-1) on human thymocytes. CD81 can also affect cognate B-T cell interactions because anti-CD81 increases IL-4 synthesis by T cells responding to antigen presented by B cells but not by monocytes. The tetraspanin superfamily (or TM4SF) includes CD81, CD9, CD37, CD53, CD63, CD82, CD151, and an increasing number of additional proteins. Like CD81, several tetraspanins are involved in cell adhesion, motility, and metastasis, as well as cell activation and signal transduction.