Treatment of non-small-cell lung cancer after progression on nivolumab or pembrolizumab.

Background: Although PD-1 antibodies (PD1 Ab) are the standard of care for advanced non-small-cell lung cancer (ansclc), most patients will progress. We compared survival outcomes for patients with ansclc who received systemic therapy (st) after progression and for those who did not. Additionally, clinical characteristics that predicted receipt of st after PD1 Ab failure were evaluated. Methods: All patients with ansclc in British Columbia initiated on nivolumab or pembrolizumab between June 2015 and November 2017, with subsequent progression, were identified. Eligibility criteria for additional st included an Eastern Cooperative Oncology Group (ecog) performance status (ps) of 3 or less and survival for more than 30 days from the last PD1 Ab treatment. Post-progression survival (pps) was assessed by landmark analysis. Baseline characteristics associated with pps were identified by multivariable analysis. Results: Of 94 patients meeting the eligibility criteria, 33 received st after progression. In 75.6%, a PD1 Ab was received as first- or second-line treatment. The most common sts were erlotinib (36.4%) and docetaxel (27.3%). No statistically significant difference in median pps was observed between patients who did and did not receive st within 30 days of their last PD1 Ab treatment (6.9 months vs. 3.6 months, log-rank p = 0.15.) In multivariable analysis, factors associated with increased pps included an ecog ps of 0 or 1 compared with 2 or 3 [hazard ratio (hr): 0.42; 95% confidence interval (ci): 0.24 to 0.73; p = 0.002] and any response compared with no response to PD1 Ab (hr: 0.54; 95% ci: 0.33 to 0.90; p = 0.02). Conclusions: In this cohort, only 35.1% of patients eligible for post-PD1 Ab therapy received st. Post-progression survival was not significantly affected by receipt of post-progression therapy. Prospective trials are needed to clarify the benefit of post-PD1 Ab treatments.

Characterization of synovial fluid metabolomic phenotypes of cartilage morphological changes associated with osteoarthritis.

OBJECTIVE: Osteoarthritis (OA) is a multifactorial disease with etiological heterogeneity. The objective of this study was to classify OA subgroups by generating metabolomic phenotypes from human synovial fluid. DESIGN: Post mortem synovial fluids (n = 75) were analyzed by high performance-liquid chromatography mass spectrometry (LC-MS) to measure changes in the global metabolome. Comparisons of healthy (grade 0), early OA (grades I-II), and late OA (grades III-IV) donor populations were considered to reveal phenotypes throughout disease progression. RESULTS: Global metabolomic profiles in synovial fluid were distinct between healthy, early OA, and late OA donors. Pathways differentially activated among these groups included structural deterioration, glycerophospholipid metabolism, inflammation, central energy metabolism, oxidative stress, and vitamin metabolism. Within disease states (early and late OA), subgroups of donors revealed distinct phenotypes. Synovial fluid metabolomic phenotypes exhibited increased inflammation (early and late OA), oxidative stress (late OA), or structural deterioration (early and late OA) in the synovial fluid. CONCLUSION: These results revealed distinct metabolic phenotypes in human synovial fluid, provide insight into pathogenesis, represent novel biomarkers, and can move toward developing personalized interventions for subgroups of OA patients.

Pathogenesis-related proteins of plants as allergens.

OBJECTIVE: Many pathogenesis-related (PR) proteins from plants are allergenic. We review the evidence that PR proteins represent an increasingly important group of plant-derived allergens. DATA SOURCES: A detailed literature search was conducted through PubMed and GenBank databases. STUDY SELECTION: All reports in PubMed and GenBank related to PR protein allergens for which at least partial amino acid sequence is known were included. RESULTS: Production of PR proteins by plants is induced in plants by stress. Members of PR-protein groups 2, 3, 4, 5, 8, 10, and 14 have demonstrated allergenicity. PR2-, 3-, 4-, and 8-homologous allergens are represented by the latex allergens. Cross-reactivity of PR3 latex allergen, Hev b 6.02, with some fruit allergens may be a reflection of the representation of homologous PR proteins among varied plants. The expression of one of the representative PR5-homologous cedar pollen allergens, Jun a 3, is highly variable across years and geographic areas, possibly because of variable induction of this PR protein by environmental factors. PR10-homologous birch pollen allergen, Bet v 1, is structurally similar to and cross-reacts with PR10 proteins from fruits (eg, Mal d 1) which cause oral allergy syndrome. PR14 allergens (eg, Zea m 14) consist of lipid transfer proteins found in grains and fruits and are inducers of anaphylaxis. CONCLUSIONS: PR-homologous allergens are pervasive in nature. Similarity in the amino acid sequences among members of PR proteins may be responsible for cross-reactivity among allergens from diverse plants. Induced expression of PR-homologous allergens by environmental factors may explain varying degrees of allergenicity. Man-made environmental pollutants may also alter the expression of some PR protein allergens.

Negative selection of T cells by Helicobacter pylori as a model for bacterial strain selection by immune evasion.

The majority of humans infected with Helicobacter pylori maintain a lifelong infection with strains bearing the cag pathogenicity island (PAI). H. pylori inhibits T cell responses and evades immunity so the mechanism by which infection impairs responsiveness was investigated. H. pylori caused apoptotic T cell death, whereas Campylobacter jejuni did not. The induction of apoptosis by H. pylori was blocked by an anti-Fas Ab (ZB4) or a caspase 8 inhibitor. In addition, a T cell line with the Fas rendered nonfunctional by a frame shift mutation was resistant to H. pylori-induced death. H. pylori strains bearing the cag PAI preferentially induced the expression of Fas ligand (FasL) on T cells and T cell death, whereas isogenic mutants lacking these genes did not. Inhibiting protein synthesis blocked FasL expression and apoptosis of T cells. Preventing the cleavage of FasL with a metalloproteinase inhibitor increased H. pylori-mediated killing. Thus, H. pylori induced apoptosis in Fas-bearing T cells through the induction of FasL expression. Moreover, this effect was linked to bacterial products encoded by the cag PAI, suggesting that persistent infection with this strain may be favored through the negative selection of T cells encountering specific H. pylori Ags.

Identification of mutations in the genes for the pollen allergens of eastern red cedar (Juniperus virginiana).

BACKGROUND: Cedar pollens are important causes of seasonal allergic disease in diverse geographical areas. However, pollens from different families and species vary in their propensity to induce allergic responses. OBJECTIVE: To compare the structure of potential allergens from eastern red cedar (Juniperus virginiana) pollen with those of the highly allergenic cedar (mountain cedar, J. ashei) pollens. MATERIALS AND METHODS: The cDNAs for potential pollen allergens, Jun v 1 and Jun v 3, were amplified by reverse transcriptase-polymerase chain reaction, cloned and sequenced. Expression of the native proteins in pollen was characterized by SDS-PAGE and immunoblotting. RESULTS: The cDNA sequence for one potential major allergen, Jun v 1, was highly homologous to those of the other cedar pollens. The second potential allergen, Jun v 3, was also highly homologous to its counterpart in mountain cedar, but a stop codon in the mRNA would result in a protein of only 91 amino acids, which would lack potential N-glycosylation sites and the IgE binding epitopes of the 199 amino acid homologue from mountain cedar pollen, Jun a 3. IgE from the sera of patients with hypersensitivity to cedar pollen bound to eastern red cedar proteins of four different sizes. N-terminal amino acid sequence analysis indicated that two of these proteins (43 and 30 kDa) were either isoforms or processed Jun v 1. No Jun v 3 protein was detected. The N-terminal sequence of an additional 145-kDa allergen, termed Jun v 4, was not homologous to any previously described allergens. CONCLUSION: These findings suggest that mutations in the genes or post-translational modifications of two potentially allergenic proteins might help to explain why the pollen of eastern red cedar is reported to be less allergenic than those of other members of Cupressaceae and Taxodiaceae families.

V(D)J recombination defects in lymphocytes due to RAG mutations: severe immunodeficiency with a spectrum of clinical presentations.

Severe combined immunodeficiency (SCID) comprises a heterogeneous group of primary immunodeficiencies, a proportion of which are due to mutations in either of the 2 recombination activating genes (RAG)-1 and -2, which mediate the process of V(D)J recombination leading to the assembly of antigen receptor genes. It is reported here that the clinical and immunologic phenotypes of patients bearing mutations in RAGs are more diverse than previously thought and that this variability is related, in part, to the specific type of RAG mutation. By analyzing 44 such patients from 41 families, the following conclusions were reached: (1) null mutations on both alleles lead to the T-B-SCID phenotype; (2) patients manifesting classic Omenn syndrome (OS) have missense mutations on at least one allele and maintain partial V(D)J recombination activity, which accounts for the generation of residual, oligoclonal T-lymphocytes; (3) in a third group of patients, findings were only partially compatible with OS, and these patients, who also carried at least one missense mutation, may be considered to have atypical SCID/OS; (4) patients with engraftment of maternal T cells as a complication of a transplacental transfusion represented a fourth group, and these patients, who often presented with a clinical phenotype mimicking OS, may be observed regardless of the type of RAG gene mutation. Analysis of the RAG genes by direct sequencing is an effective way to provide accurate diagnosis of RAG-deficient as opposed to RAG-independent V(D)J recombination defects, a distinction that cannot be made based on clinical and immunologic phenotype alone.

Homology modeling and characterization of IgE binding epitopes of mountain cedar allergen Jun a 3.

The Jun a 3 protein from mountain cedar (Juniperus ashei) pollen, a member of group 5 of the family of plant pathogenesis-related proteins (PR-proteins), reacts with serum IgE from patients with cedar hypersensitivity. We used the crystal structures of two other proteins of this group, thaumatin and an antifungal protein from tobacco, both approximately 50% identical in sequence to Jun a 3, as templates to build homology models for the allergen. The in-house programs EXDIS and FANTOM were used to extract distance and dihedral angle constraints from the Protein Data Bank files and determine energy-minimized structures. The mean backbone deviations for the energy-refined model structures from either of the templates is <1 A, their conformational energies are low, and their stereochemical properties (determined with PROCHECK) are acceptable. The circular dichroism spectrum of Jun a 3 is consistent with the postulated beta-sheet core. Tryptic fragments of Jun a 3 that reacted with IgE from allergic patients all mapped to one helical/loop surface of the models. The Jun a 3 models have features common to aerosol allergens from completely different protein families, suggesting that tertiary structural elements may mediate the triggering of an allergic response.

Mutations in conserved regions of the predicted RAG2 kelch repeats block initiation of V(D)J recombination and result in primary immunodeficiencies.

The V(D)J recombination reaction is composed of multiple nucleolytic processing steps mediated by the recombination-activating proteins RAG1 and RAG2. Sequence analysis has suggested that RAG2 contains six kelch repeat motifs that are predicted to form a six-bladed beta-propeller structure, with the second beta-strand of each repeat demonstrating marked conservation both within and between kelch repeat-containing proteins. Here we demonstrate that mutations G95R and DeltaI273 within the predicted second beta-strand of repeats 2 and 5 of RAG2 lead to immunodeficiency in patients P1 and P2. Green fluorescent protein fusions with the mutant proteins reveal appropriate localization to the nucleus. However, both mutations reduce the capacity of RAG2 to interact with RAG1 and block recombination signal cleavage, therefore implicating a defect in the early steps of the recombination reaction as the basis of the clinical phenotype. The present experiments, performed with an extensive panel of site-directed mutations within each of the six kelch motifs, further support the critical role of both hydrophobic and glycine-rich regions within the second beta-strand for RAG1-RAG2 interaction and recombination signal recognition and cleavage. In contrast, multiple mutations within the variable-loop regions of the kelch repeats had either mild or no effects on RAG1-RAG2 interaction and hence on the ability to mediate recombination. In all, the data demonstrate a critical role of the RAG2 kelch repeats for V(D)J recombination and highlight the importance of the conserved elements of the kelch motif.

Helicobacter pylori modulates lymphoepithelial cell interactions leading to epithelial cell damage through Fas/Fas ligand interactions.

Helicobacter pylori causes a common chronic infection of humans that leads to epithelial cell damage. Studies have shown that apoptosis of the gastric epithelium is increased during infection and this response is associated with an expansion of gastric T-helper type 1 (Th1) cells. We report that gastric T cells contribute to apoptosis of the epithelium by a Fas/Fas ligand (FasL) interaction. Fas receptor expression was detected on freshly isolated gastric epithelial cells by flow cytometry and immunohistochemistry, and this level of expression was increased during infection with H. pylori. The expression of Fas receptor on three gastric epithelial cell lines was increased by H. pylori, either alone or in combination with gamma interferon or tumor necrosis factor alpha. The role of Fas in apoptosis of gastric epithelial cell lines was evidenced by DNA fragmentation after cross-linking of Fas with specific antibodies. FasL expression was detected by immunohistochemistry on mononuclear cells in gastric biopsy specimens of infected but not uninfected subjects. Gastric T-cell lines were also shown to express FasL, as evidenced by reverse transcription-PCR and killing of target cells expressing Fas receptor. Moreover, these T-cell lines were capable of killing cultured gastric epithelial target cells and antibodies that block the interaction between Fas receptor and FasL inhibited this cytotoxic activity. These observations demonstrate that local Th1 cells may contribute to the pathogenesis of gastric disease during H. pylori infection by increasing the expression of Fas on gastric epithelial cells and inducing apoptosis through Fas/FasL interactions.

Variable expression of pathogenesis-related protein allergen in mountain cedar (Juniperus ashei) pollen.

Allergic diseases have been increasing in industrialized countries. The environment is thought to have both direct and indirect modulatory effects on disease pathogenesis, including alterating on the allergenicity of pollens. Certain plant proteins known as pathogenesis-related proteins appear to be up-regulated by certain environmental conditions, including pollutants, and some have emerged as important allergens. Thus, the prospect of environmentally regulated expression of plant-derived allergens becomes yet another potential environmental influence on allergic disease. We have identified a novel pathogenesis-related protein allergen, Jun a 3, from mountain cedar (Juniperus ashei) pollen. The serum IgE from patients with hypersensitivity to either mountain cedar or Japanese cedar were shown to bind to native and recombinant Jun a 3 in Western blot analysis and ELISA. Jun a 3 is homologous to members of the thaumatin-like pathogenesis-related (PR-5) plant protein family. The amounts of Jun a 3 extracted from mountain cedar pollen varied up to 5-fold in lots of pollen collected from the same region in different years and between different regions during the same year. Thus, Jun a 3 may contribute not only to the overall allergenicity of mountain cedar pollen, but variable levels of Jun a 3 may alter the allergenic potency of pollens produced under different environmental conditions.

Isolation and characterization of the mountain cedar (Juniperus ashei) pollen major allergen, Jun a 1.

BACKGROUND: Cedar pollens are important causes of seasonal allergic disease in diverse geographic areas. OBJECTIVE: A major allergen from mountain cedar (Juniperus ashei) pollen, termed Jun a 1, was isolated and characterized. METHODS: Water-soluble pollen glycoproteins were extracted, salt precipitated, and purified with use of concanavalin A affinity chromatography or HPLC. The purified fractions were characterized by SDS-PAGE, immunoblotting, and N-terminal amino acid sequence analysis. Binding of allergen-specific IgE from the sera of cedar-hypersensitive patients was detected by ELISA and antigen-specific responses of peripheral blood T cells by tritiated thymidine incorporation. RESULTS: The major extractable cedar pollen glycoprotein had a molecular weight and N-terminal amino acid sequence that was similar to that of the major allergen Cha o 1, from Japanese cypress (Chamaecyparis obtusa), and Cry j 1, from Japanese cedar (Cryptomeria japonica). IgE from cedar-hypersensitive patients' sera bound to the isolated glycoprotein. CONCLUSION: The predominance of Jun a 1 in the soluble proteins of mountain cedar pollen and its high degree of homology with Cha o 1 and Cry j 1 make it likely to be the major allergen of this pollen. Amino acid sequence conservation also makes Jun a 1 a potential target for cross-reactivity between these pollen allergens. The observed reactivity of IgE from the sera of Japanese cedar-sensitive patients with Jun a 1 is consistent with this proposition.

Molecular cloning of the mountain cedar (Juniperus ashei) pollen major allergen, Jun a 1.

BACKGROUND: Cedar pollens cause allergic disease in diverse geographic areas. We have recently purified and characterized the major mountain cedar (Juniperus ashei) pollen allergen, Jun a 1. OBJECTIVE: A full-length complementary DNA for Jun a 1 was cloned and sequenced, and the recombinant protein was expressed. METHODS: Messenger RNA from mountain cedar pollen was purified and Jun a 1 sequences were established with use of reverse transcriptase-PCR and primers based on the N-terminal amino acid sequence of Jun a 1 and the homologous protein Cry j 1. Portions of the nucleotide sequence were confirmed by comparison with N-terminal amino acid sequencing of the intact tryptic fragments of the purified native protein. Recombinant Jun a 1 was cloned into pET 30, expressed in BL21, and purified by HPLC, and its allergenicity was analyzed by Western blotting with patient sera. RESULTS: Jun a 1 possesses a high level of amino acid sequence homology with Cha o 1 and Cry j 1, the major allergens of Japanese cypress and Japanese cedar. The amino acid sequence of a region with putative pectate lyase activity was identical to that of Cry j 1 and Cha o 1. Jun a 1 contained 2 potential N-glycosylation sites that were distinct from those found in Cry j 1. The IgE from patient sera bound recombinant Jun a 1 in Western blot analysis. CONCLUSION: The high degree of homology of Jun a 1 with Cha o 1 and Cry j 1 may explain the cross-reactivity of conifer pollens. Differences in N-glycosylation suggest little overlap of glycopeptide epitopes.

A mathematical model predicting the frequency of aberrant rearrangements in the T-cell receptor gene.

The T-cell receptor (TCR) genetic loci undergo an orderly process of recombination in ontogeny in order to generate a diverse array of antigen receptors. Normally occurring, out-of-frame and incomplete rearrangements produce non-productive TCR transcripts. Abnormalities in the rearrangement process occur at very low frequencies but may predominate in inborn errors of recombination. Detecting these abnormalities in surviving pools of lymphocytes is difficult and typically focuses on identification of abnormally rearranged alleles or on detecting abnormalities in recombinase proteins. Thus, there currently exists no rapid screening method to identify aberrant V(D)J recombination. To address this issue, a mathematical model was developed to predict the error rate from the measured proportions of different non-productive TCR alleles. Since the proportions of different non-productive rearrangements vary in a characteristic fashion in response to abnormalities in the recombination process, the mathematical model presented here provides a tool to indirectly assess the error rate of TCR recombination. The model was applied to a group of patients with Omenn's syndrome, most of whom had an unknown primary defect. The results indicate that these patients had a > 90% rate of aberrant TCR recombination.

T-cell receptor analysis in Omenn's syndrome: evidence for defects in gene rearrangement and assembly.

Patients with Omenn's syndrome have a form of severe immune deficiency that is associated with pathological features of graft-versus-host disease, except for the lack of foreign engraftment. It has been hypothesized that the disease's unique clinical features are mediated by an expanded population of autologous self-reactive T cells of limited clonality. In the current study, an investigation of the T-cell receptor (TCR) repertoire was undertaken to identify defects in T-cell rearrangement and development. The TCR repertoire in this group of patients was exquisitely restricted in the number of different TCR clonotypes, and some of these clonotypes seemed to have similar recognition motifs in the antigen-binding region, indicating antigen-driven proliferation of T lymphocytes. The TCRs from some patients lacked N- or P-nucleotide insertions and used proximal variable and joining gene segments, suggesting abnormal intrathymic T-cell development. Finally, abnormal assembly of gene segments and truncated rearrangements within nonproductive alleles suggested abnormalities in TCR rearrangement mechanisms. Overall, the findings suggest that inefficient and/or abnormal generation of TCRs may be a consistent feature of this disease.

Lessons from an evaluation of the effectiveness of an asthma day camp.

PURPOSE: To evaluate the effectiveness of applied teaching methodologies in a camp setting on asthma self-management skills in school-age children. DESIGN: This was a descriptive pilot study using a one-group pretest-posttest design. SAMPLE: Thirty-four subjects, ages 6 to 12 years, representing a typical clinical population of children with asthma. METHODS: Children's asthma knowledge, symptoms, behavior, and mastery, as well as peak-flow technique, were measured 2 to 3 weeks before camp and then again on the last day of camp. Baseline measures of parents' asthma knowledge and family stress related to asthma were obtained. Outcomes specific to asthma management, such as missed school days, emergency room visits, and hospitalizations, were evaluated by parent report the year before and after the intervention. RESULTS: Significant improvement in peak-flow technique and a reported reduction in emergency room visits and missed school days after camp were found. CLINICAL IMPLICATIONS: In this pilot study, the use of an applied teaching format for school-age children in an asthma day camp resulted in some learning. More rigorous design and instrumentation are important for better evaluation of programs such as this. Nurses working with these populations should plan structured evaluations of the programs so they can best meet the children's needs.

Lymphocytes in the human gastric mucosa during Helicobacter pylori have a T helper cell 1 phenotype.

BACKGROUND & AIMS: Studies have shown that gastric T cells are increased during Helicobacter pylori infection. The purpose of this study was to characterize the human gastric T-cell responses in the presence or absence of H. pylori. METHODS: T-cell surface antigens were examined by immunohistochemistry or after isolation for evaluation of surface antigens and cytoplasmic cytokines using flow cytometry. RESULTS: CD4+ and CD8+ T cells were increased in situ during infection with H. pylori. Freshly isolated gastric T cells expressed cytoplasmic interferon gamma (IFN-gamma) and interleukin (IL)-2 after a brief stimulation. Simultaneous four-color flow cytometry demonstrated that both CD8+ and CD4+ T cells expressed IFN-gamma. Because stimulation through CD30 favors the induction of IL-5 and Th2 cells, gastric and colonic T cells were examined for CD30 expression. Consistent with the notion that Th2 cells are found in the intestine, CD30 was evident throughout the lamina propria of the colon but was virtually absent in the stomach. Furthermore, freshly isolated gastric T cells produced little IL-4 and virtually no IL-5 or tumor necrosis factor beta. CONCLUSIONS: These observations show that gastric T cells resemble the Th1 type, which may explain their failure to induce immunity to H. pylori and their ability to contribute to the pathogenesis of gastric disease.

Missense mutation in exon 7 of the common gamma chain gene causes a moderate form of X-linked combined immunodeficiency.

Clinical and immunologic features of a recently recognized X-linked combined immunodeficiency disease (XCID) suggested that XCID and X-linked severe combined immunodeficiency (XSCID) might arise from different genetic defects. The recent discovery of mutations in the common gamma chain (gamma c) gene, a constituent of several cytokine receptors, in XSCID provided an opportunity to test directly whether a previously unrecognized mutation in this same gene was responsible for XCID. The status of X chromosome inactivation in blood leukocytes from obligate carriers of XCID was determined from the polymorphic, short tandem repeats (CAG), in the androgen receptor gene, which also contains a methylation-sensitive HpaII site. As in XSCID, X-chromosome inactivation in obligate carriers of XCID was nonrandom in T and B lymphocytes. In addition, X chromosome inactivation in PMNs was variable. Findings from this analysis prompted sequencing of the gamma c gene in this pedigree. A missense mutation in the region coding for the cytoplasmic portion of the gamma c gene was found in three affected males but not in a normal brother. Therefore, this point mutation in the gamma c gene leads to a less severe degree of deficiency in cellular and humoral immunity than that seen in XSCID.

Thymic hypoplasia and T-cell deficiency in ectodermal dysplasia: case report and review of the literature.

Ectodermal dysplasia is a heterogeneous disorder that includes a constellation of congenital malformations occasionally associated with mild to moderate immune dysfunction. In this report, we describe a female infant with ectodermal dysplasia who was found to have thymic hypoplasia but no other phenotypic features of the DiGeorge anomalad. She experienced Candida parapsilosis sepsis at 1 week of age and a skin infection with Mycobacterium chelonii at 6 months. The numbers of blood B cells were normal and serum immunoglobulins normal to slightly reduced, but serum antibody responses of all immunoglobulin isotypes to protein immunogens were absent. Blood T cells were profoundly reduced and proliferative responses of T cells to mitogens were blunted. In contrast, there was an increased number of natural killer (NK) cells and increased NK activity in the blood. Over the first year of life, some of the immunodeficiencies resolved. Although the numbers of blood T cells (17% of total lymphocytes) remained low, proliferative responses to mitogens normalized and specific antibody responses improved. It seems likely that the thymic hypoplasia in this case was due to a paucity of ectodermal elements in the developing thymus, and that the immune defects were largely secondary to that event. In that respect, this human model of ectodermal dysplasia and thymic hypoplasia resembled the ectodermal/thymic defects found in the nude mouse.

Human T-cell receptor (TCR) alpha/beta + CD4-CD8- T cells express oligoclonal TCRs, share junctional motifs across TCR V beta-gene families, and phenotypically resemble memory T cells.

Most human T cells express the TCR alpha/beta and either CD4 or CD8 molecules (single positive, SP); however, small numbers lack CD4 and CD8. In inbred mice, alpha/beta CD4-CD8- (double negative, DN) T cells preferentially express certain beta variable region (V beta) families and may arise via unique developmental pathways. Increased percentages of alpha/beta DN T cells have been identified in some human and murine autoimmune and immunodeficiency diseases. However, their contribution to disease pathology or normal immunity is unknown. To study the cell surface phenotype and TCR diversity of human alpha/beta DN T cells, these cells were isolated from the peripheral blood of healthy adults. The proportion of alpha/beta DN T cells expressing molecules associated with activation (HLA-DR), previous exposure to antigen (CD45RO), and cytotoxic function (CD56, CD57, and CD11b) was increased relative to SP T cells. The TCR V beta repertoire of alpha/beta DN T cells was different from that of alpha/beta SP T cells, although most major gene families were present. For example, higher proportions of V beta 11, a minor gene family in peripheral blood leukocytes, were found in most alpha/beta DN T-cell samples. In contrast to mice, no dominant V beta family was used consistently in different human individuals. Within an individual alpha/beta DN T cells possessed an oligoclonal TCR beta repertoire with conservation of several distinct junctional amino acid motifs with one joined to three different V beta genes in two individuals, suggesting that these cells have undergone a selection process driven by a limited set of ligands. The possibility that they may represent, at least in part, originally SP T cells anergized by down-modulation of CD4 or CD8 must also be entertained. Overall, this study demonstrates that human peripheral blood alpha/beta DN T cells possess unique phenotypic and TCR beta repertoire characteristics when compared with the major alpha/beta SP T cell populations and thus may serve specialized immunologic functions and/or have an unusual origin.

In vivo and in vitro suppression of T-cell receptor alpha/beta CD4- CD8- T lymphocytes by cyclosporine A.

We treated a patient with a combined immunodeficiency and disease pathology resembling GvHD with cyclosporine. This disorder was characterized by exfoliative dermatitis, lymphadenopathy, and lymphocytosis of a novel T-cell phenotype (CD3+ TCR alpha/beta+ CD4- CD8-). The patient's peripheral blood T cells had elevated cytolytic activity and expressed increased levels of IL2R, HLA-DR, and CD45RO. Treatment with CsA resulted in marked clinical improvement, resolution of the lymphocytosis, and reduced cytolytic activity of peripheral blood T cells. T-cell HLA-DR and IL2R expression was reduced by cyclosporine, but CD45RO remained intact on virtually all circulating T cells. CsA also inhibited the cytolytic activity and cytokine production of in vitro cultured TCR alpha/beta+ CD4- CD8- cell lines. Our data suggest that alleviation of the patient's clinical symptoms resulted from cyclosporine-mediated suppression of proliferation, cytotoxicity, and inflammatory cytokine production of TCR alpha/beta+ CD4- CD8- T lymphocytes in vivo. The response of this patient to cyclosporine, which was similar to that seen in true GvHD, provides further evidence that these conditions share common pathogenetic pathways.