BW5147 and Derivatives for the Study of T Cells and their Antigen Receptors.

Like B cells, T cells can be immortalized through hybridization with lymphoma cells, a technique that has been particularly useful in the study of the T cell receptors (TCR) for antigen. In T cell hybridizations, the AKR mouse strain-derived thymus lymphoma BW5147 is by far the most popular fusion line. However, the full potential of this technology had to await inactivation of the productively rearranged TCR-α and -ß genes in the lymphoma. BWα-ß-, the TCR-gene deficient variant of the original lymphoma, which has become the fusion line of choice for αß T cells, is now available with numerous modifications, enabling the investigation of many aspects of TCR-mediated responses and TCR-structure. Unexpectedly, inactivating BW's functional TCR-α gene also rendered the lymphoma more permissive for the expression of TCR-γδ, facilitating the study of γδ T cells, their TCRs, and their TCR-mediated reactivity.

How C-terminal additions to insulin B-chain fragments create superagonists for T cells in mouse and human type 1 diabetes.

In type 1 diabetes (T1D), proinsulin is a major autoantigen and the insulin B:9-23 peptide contains epitopes for CD4+ T cells in both mice and humans. This peptide requires carboxyl-terminal mutations for uniform binding in the proper position within the mouse IAg7 or human DQ8 major histocompatibility complex (MHC) class II (MHCII) peptide grooves and for strong CD4+ T cell stimulation. Here, we present crystal structures showing how these mutations control CD4+ T cell receptor (TCR) binding to these MHCII-peptide complexes. Our data reveal stricking similarities between mouse and human CD4+ TCRs in their interactions with these ligands. We also show how fusions between fragments of B:9-23 and of proinsulin C-peptide create chimeric peptides with activities as strong or stronger than the mutated insulin peptides. We propose transpeptidation in the lysosome as a mechanism that could accomplish these fusions in vivo, similar to the creation of fused peptide epitopes for MHCI presentation shown to occur by transpeptidation in the proteasome. Were this mechanism limited to the pancreas and absent in the thymus, it could provide an explanation for how diabetogenic T cells escape negative selection during development but find their modified target antigens in the pancreas to cause T1D.

A Large Randomized Trial: Effects of Mindfulness-Based Stress Reduction (MBSR) for Breast Cancer (BC) Survivors on Salivary Cortisol and IL-6.

Breast cancer survivors (BCS) often experience psychological and physiological symptoms after cancer treatment. Mindfulness-based stress reduction (MBSR), a complementary and alternative therapy, has reduced subjective measures of stress, anxiety, and fatigue among BCS. Little is known, however, about how MBSR affects objective markers of stress, specifically the stress hormone cortisol and the pro-inflammatory cytokine interleukin-6 (IL-6). In the present study, BCS ( N = 322) were randomly assigned to a 6-week MBSR program for BC or usual-care control. Measurements of cortisol, IL-6, symptoms, and quality of life were obtained at orientation and 6 weeks. Cortisol and IL-6 were also measured prior to and after the MBSR(BC) class Weeks 1 and 6. The mean age of participants was 56.6 years and 69.4% were White non-Hispanic. Most had Stage I (33.8%) or II (35.7%) BC, and 35.7% had received chemotherapy and radiation. Cortisol levels were reduced immediately following MBSR(BC) class compared to before the class Weeks 1 and 6 (Wilcoxon-signed rank test; p < .01, d = .52-.56). IL-6 was significantly reduced from pre- to postclass at Week 6 (Wilcoxon-signed rank test; p < .01, d = .21). No differences were observed between the MBSR(BC) and control groups from baseline to Week 6 using linear mixed models. Significant relationships with small effect sizes were observed between IL-6 and both symptoms and quality of life in both groups. Results support the use of MBSR(BC) to reduce salivary cortisol and IL-6 levels in the short term in BCS.

Class II major histocompatibility complex mutant mice to study the germ-line bias of T-cell antigen receptors.

The interaction of αß T-cell antigen receptors (TCRs) with peptides bound to MHC molecules lies at the center of adaptive immunity. Whether TCRs have evolved to react with MHC or, instead, processes in the thymus involving coreceptors and other molecules select MHC-specific TCRs de novo from a random repertoire is a longstanding immunological question. Here, using nuclease-targeted mutagenesis, we address this question in vivo by generating three independent lines of knockin mice with single-amino acid mutations of conserved class II MHC amino acids that often are involved in interactions with the germ-line-encoded portions of TCRs. Although the TCR repertoire generated in these mutants is similar in size and diversity to that in WT mice, the evolutionary bias of TCRs for MHC is suggested by a shift and preferential use of some TCR subfamilies over others in mice expressing the mutant class II MHCs. Furthermore, T cells educated on these mutant MHC molecules are alloreactive to each other and to WT cells, and vice versa, suggesting strong functional differences among these repertoires. Taken together, these results highlight both the flexibility of thymic selection and the evolutionary bias of TCRs for MHC.

The Enhancing Connections Program: a six-state randomized clinical trial of a cancer parenting program.

OBJECTIVE: The purpose of this study was to test the efficacy of a cancer parenting program for child rearing mothers with breast cancer, the Enhancing Connections Program. Primary goals were to decrease maternal depressed mood and anxiety, improve parenting quality, parenting skills and confidence, and enhance the child's behavioral-emotional adjustment to maternal breast cancer. METHOD: A total of 176 mothers diagnosed within 6 months with Stage 0 to Stage III breast cancer and their 8- to 12-year-old child were recruited from medical providers in 6 states: Washington, California, Pennsylvania, Minnesota, Arizona, and Indiana. After consenting and obtaining baseline measures, study participants were randomized into experimental or control groups. Experimental mothers received 5, 1-hr educational counseling sessions at 2-week intervals; controls received a booklet and phone call on communicating and supporting their child about the mother's cancer. Outcomes were assessed at 2 and 12 months. RESULTS: Compared to controls, at 2 months experimental mothers significantly improved on depressed mood and parenting skills; experimental children improved on behavioral-emotional adjustment: total behavior problems, externalizing problems, and anxiety/depressed mood significantly declined. At 1 year, experimental children remained significantly less depressed than controls on both mother- and child-reported measures. The intervention failed to significantly affect parenting self-efficacy or maternal anxiety. CONCLUSIONS: The Enhancing Connections Program benefitted mothers and children in specific areas and warrants refinement and further testing.

Influence of mindfulness-based stress reduction (MBSR) on telomerase activity in women with breast cancer (BC).

Mindfulness-based stress reduction (MBSR) reduces symptoms of depression, anxiety, and fear of recurrence among breast cancer (BC) survivors. However, the effects of MBSR (BC) on telomere length (TL) and telomerase activity (TA), known markers of cellular aging, psychological stress, and disease risk, are not known. This randomized, wait-listed, controlled study, nested within a larger trial, investigated the effects of MBSR (BC) on TL and TA. BC patients (142) with Stages 0-III cancer who had completed adjuvant treatment with radiation and/or chemotherapy at least 2 weeks prior to enrollment and within 2 years of completion of treatment with lumpectomy and/or mastectomy were randomly assigned to either a 6-week MBSR for BC program or a usual care. Assessments of TA and TL were obtained along with psychological measurements at baseline, 6 weeks, and 12 weeks after completing the MBSR(BC) program. The mean age of 142 participants was 55.3 years; 72% were non-Hispanic White; 78% had Stage I or II cancer; and 36% received both chemotherapy and radiation. In analyses adjusted for baseline TA and psychological status, TA increased steadily over 12 weeks in the MBSR(BC) group (approximately 17%) compared to essentially no increase in the control group (approximately 3%, p < .01). In contrast, no between-group difference was observed for TL (p = .92). These results provide preliminary evidence that MBSR(BC) increases TA in peripheral blood mononuclear cells from BC patients and have implications for understanding how MBSR(BC) may extend cell longevity at the cellular level.

Lymphocyte recovery after breast cancer treatment and mindfulness-based stress reduction (MBSR) therapy.

OBJECTIVES: This randomized controlled trial was conducted to examine immune recovery following breast cancer (BC) therapy and evaluate the effect of mindfulness-based stress reduction therapy (MBSR) on immune recovery with emphasis on lymphocyte subsets, T cell activation, and production of T-helper 1 (Th1; interferon [IFN]-γ) and T-helper 2 (Th2; interleukin-4 [IL-4]) cytokines. METHOD: Participants who completed the study consisted of 82 patients diagnosed with Stage 0-III BC, who received lumpectomy and adjuvant radiation ± chemotherapy. Patients were randomized into an MBSR(BC) intervention program or a control (usual care) group. Immune cell measures were assessed at baseline and within 2 weeks after the 6-week intervention. The numbers and percentages of lymphocyte subsets, activated T cells, and Th1 and Th2 cells in peripheral blood samples were determined by immunostaining and flow cytometry. RESULTS: Immune subset recovery after cancer treatment showed positive associations with time since treatment completion. The B and natural killer (NK) cells were more susceptible than T cells in being suppressed by cancer treatment. Women who received MBSR(BC) had T cells more readily activated by the mitogen phytohemagglutinin (PHA) and an increase in the Th1/Th2 ratio. Activation was also higher for the MBSR(BC) group if <12 weeks from the end of treatment and women in MBSR(BC) <12 weeks had higher T cell count for CD4(+). CONCLUSION: MBSR(BC) promotes a more rapid recovery of functional T cells capable of being activated by a mitogen with the Th1 phenotype, whereas substantial recovery of B and NK cells after completion of cancer treatment appears to occur independent of stress-reducing interventions.

Ikaros promotes rearrangement of TCR α genes in an Ikaros null thymoma cell line.

Ikaros is important in the development and maintenance of the lymphoid system, functioning in part by associating with chromatin-remodeling complexes. We have studied the functions of Ikaros in the transition from pre-T cell to the CD4(+) CD8(+) thymocyte using an Ikaros null CD4(-) CD8(-) mouse thymoma cell line (JE131). We demonstrate that this cell line carries a single functional TCR ß gene rearrangement and expresses a surface pre-TCR. JE131 cells also carry nonfunctional rearrangements on both alleles of their TCR α loci. Retroviral reintroduction of Ikaros dramatically increased the rate of transcription in the α locus and TCR Vα/Jα recombination resulting in the appearance of many new αßTCR(+) cells. The process is RAG dependent, requires switch/sucrose nonfermentable chromatin-remodeling complexes and is coincident with the binding of Ikaros to the TCR α enhancer. Furthermore, knockdown of Mi2/nucleosome remodeling and deacetylase complexes increased the frequency of TCR α rearrangement. Our data suggest that Ikaros controls Vα/Jα recombination in T cells by controlling access of the transcription and recombination machinery to the TCR α loci. The JE131 cell line should prove to be a very useful tool for studying the molecular details of this and other processes involved in the pre-T cell to αßTCR(+) CD4(+) CD8(+) thymocyte transition.

A pilot study evaluating the effect of mindfulness-based stress reduction on psychological status, physical status, salivary cortisol, and interleukin-6 among advanced-stage cancer patients and their caregivers.

PURPOSE: To investigate whether a mindfulness-based stress reduction program for cancer (MBSR-C) improved psychological and physical symptoms, quality of life (QOL), and stress markers among advanced-stage cancer patients and caregivers. DESIGN: A pilot within-subject design was used. METHOD: Patients previously diagnosed with advanced-stage breast, colon, lung, or prostate cancer and on treatment were recruited from the Moffitt Cancer Center and Research Institute. Twenty-six patient-caregiver dyads completed a modified 6-week, self-study MBSR-C program based on the Kabat-Zinn model. Psychological and physical symptoms and QOL were compared pre- and post-MBSR-C sessions. Salivary cortisol and interleukin-6 were assessed pre- and post-MBSR-C session at 1, 3, and 6 weeks. FINDINGS: Following the 6-week MBSR program, patients showed improvements in stress and anxiety (p < .05); caregivers' psychological and QOL also improved but were not statistically significant. Both patients and caregivers had decreases in cortisol at Weeks 1 and 3 (p < .05) but not at Week 6. Similar to cortisol levels at Week 6, salivary interleukin-6 levels were lower overall (before/after an MBSR-C session), compared with Week 1 for patients and caregivers. CONCLUSIONS: MBSR-C may be a beneficial intervention for reducing stress, anxiety, cortisol levels, and symptoms in advanced-stage cancer patients and may also benefit caregivers.

Mindfulness based stress reduction in post-treatment breast cancer patients: an examination of symptoms and symptom clusters.

To investigate prevalence and severity of symptoms and symptom clustering in breast cancer survivors who attended MBSR(BC). Women were randomly assigned into MBSR(BC) or Usual Care (UC). Eligible women were ≥ 21 years, had been diagnosed with breast cancer and completed treatment within 18 months of enrollment. Symptoms and interference with daily living were measured pre- and post-MBSR(BC) using the M.D. Anderson Symptom Inventory. Symptoms were reported as highly prevalent but severity was low. Fatigue was the most frequently reported and severe symptom among groups. Symptoms clustered into 3 groups and improved in both groups. At baseline, both MBSR(BC) and the control groups showed similar mean symptom severity and interference; however, after the 6-week post-intervention, the MBSR(BC) group showed statistically-significant reduction for fatigue and disturbed sleep (P < 0.01) and improved symptom interference items, compared to the control group. For the between-group comparisons, 11 of 13 symptoms and 5 of 6 interference items had lower means in the MBSR(BC) condition than the control condition. These results suggest that MBSR(BC) modestly decreases fatigue and sleep disturbances, but has a greater effect on the degree to which symptoms interfere with many facets of life. Although these results are preliminary, MBSR intervention post-treatment may effectively reduce fatigue and related interference in QOL of breast cancer survivors.

Feasibility of a mindfulness-based stress reduction program for early-stage breast cancer survivors.

PURPOSE: To assess the feasibility of whether mindfulness-based stress reduction (MBSR) has a positive effect on breast cancer survivors' psychological status, psychosocial characteristics, symptoms, and quality of life (QOL) during the critical transition period from end of treatment to resumption of daily activities. DESIGN: Single-group, quasi-experimental, pretest-posttest design. METHOD: A sample of 19 women who completed breast cancer treatment with lumpectomy, radiation, and/or chemotherapy was recruited from the Moffitt Cancer Center and Research Institute, a National Cancer Institute- designated cancer center, and the University of South Florida. The authors assessed the feasibility, compliance, and whether an 8-week MBSR program positively influenced changes in psychological status (fear of recurrence, perceived stress, anxiety, depression), psychosocial characteristics (optimism, social support, spirituality), physical symptoms, and QOL. FINDINGS: Seventeen women (89.5%) completed the study. The mean age was 57 years; the majority of participants (94%) were White. The estimated compliance rate for the program was 67%. Paired t tests indicated significant improvements fear of recurrence, perceived stress, anxiety, depression, and QOL through MBSR participation. CONCLUSIONS: Participants enrolled in the MBSR classes generally were compliant. Significant improvement in psychological status, symptoms, and QOL can be achieved with MBSR use in this population.

Comparison of blood glucose management strategies to achieve control following cardiac surgery (computerized versus paper).

INTRODUCTION: Blood glucose control can be time-consuming and difficult to achieve. We hypothesized that a computerized system to obtain glucose control would enable faster "time to target" and produce less variability in blood glucose levels. METHODS: Patients who underwent cardiac surgery at a community hospital between January and December 2007 (n = 1131) with glucose control obtained under a paper protocol were compared with similar patients operated on between January and December 2008 (n = 769) whose glucose control was obtained with a computer-driven protocol. RESULTS: Glucose control was achieved in both groups. The computer group had less variability in glucose levels than the paper group. The mean time to target for the computer group was 3.5 (+/-1.3) hours. The time to target for the paper group was quite skewed; therefore, the median time to target was 6 hours. CONCLUSIONS: The computer-driven protocol achieved excellent glycemic control.

A conserved CXXC motif in CD3epsilon is critical for T cell development and TCR signaling.

Virtually all T cell development and functions depend on its antigen receptor. The T cell receptor (TCR) is a multi-protein complex, comprised of a ligand binding module and a signal transmission module. The signal transmission module includes proteins from CD3 family (CD3epsilon, CD3delta, CD3gamma) as well as the zeta chain protein. The CD3 proteins have a short extracellular stalk connecting their Ig-like domains to their transmembrane regions. These stalks contain a highly evolutionarily conserved CXXC motif, whose function is unknown. To understand the function of these two conserved cysteines, we generated mice that lacked endogenous CD3epsilon but expressed a transgenic CD3epsilon molecule in which these cysteines were mutated to serines. Our results show that the mutated CD3epsilon could incorporate into the TCR complex and rescue surface TCR expression in CD3epsilon null mice. In the CD3epsilon mutant mice, all stages of T cell development and activation that are TCR-dependent were impaired, but not eliminated, including activation of mature naïve T cells with the MHCII presented superantigen, staphylococcal enterotoxin B, or with a strong TCR cross-linking antibody specific for either TCR-Cbeta or CD3epsilon. These results argue against a simple aggregation model for TCR signaling and suggest that the stalks of the CD3 proteins may be critical in transmitting part of the activation signal directly through the membrane.

Weight patterns in children with higher risk ALL: A report from the Children's Oncology Group (COG) for CCG 1961.

BACKGROUND: This retrospective analysis defined and described patterns and predictors of weight change during treatment in children with acute lymphocytic leukemia (ALL) with high-risk features who received treatment on Children's Cancer Group protocol CCG 1961. PROCEDURE: Patients (1,638) were enrolled in CCG 1961 from November 1996 to May 2002. Weight was measured as BMI percent (%), specific for age and gender, and defined as 100 x ln(BMI/median BMI). RESULTS: By the end of treatment, 23% of children were obese (BMI >or=95%), compared with 14% at diagnosis. Children who received post-induction intensified therapy (arms C, D, SER with Doxorubicin or Idarubicin) had higher gastrointestinal toxicities and lower BMI% from consolidation through interim maintenance 1. BMI% then increased for all arms between delayed intensification and maintenance 1 or 2. Children who were of Black or Hispanic race, obese at diagnosis, or who had grade 3 or 4 pancreatitis/glucose toxicities during induction had higher BMI% throughout treatment. Children were more likely to be obese at the end of the study if they were aged 5-9 years at diagnosis or female gender. Cranial radiation was not a predictor of obesity. CONCLUSIONS: Successful treatment of higher risk childhood ALL was associated with obesity, independent of cranial irradiation. The beginning of maintenance therapy may be the best time to intervene with nutritional and behavioral interventions, particularly for children who are obese or aged 5-9 years at diagnosis, female, Black or Hispanic, or those with metabolic toxicities during induction.

Germline-encoded amino acids in the alphabeta T-cell receptor control thymic selection.

An alphabeta T-cell response depends on the recognition of antigen plus major histocompatibility complex (MHC) proteins by its antigen receptor (TCR). The ability of peripheral alphabeta T cells to recognize MHC is at least partly determined by MHC-dependent thymic selection, by which an immature T cell survives only if its TCR can recognize self MHC. This process may allow MHC-reactive TCRs to be selected from a repertoire with completely random and unbiased specificities. However, analysis of thymocytes before positive selection indicated that TCR proteins might have a predetermined ability to bind MHC. Here we show that specific germline-encoded amino acids in the TCR promote 'generic' MHC recognition and control thymic selection. In mice expressing single, rearranged TCR beta-chains, individual mutation of amino acids in the complementarity-determining region (CDR) 2beta to Ala reduced development of the entire TCR repertoire. Altogether, these results show that thymic selection is controlled by germline-encoded MHC contact points in the alphabeta TCR and indicate that the diversity of the peripheral T-cell repertoire is enhanced by this 'built-in' specificity.

Case-control study to evaluate predictors of lymphedema after breast cancer surgery.

PURPOSE/OBJECTIVES: To identify risk factors for lymphedema after breast cancer surgery. DESIGN: Multisite case-control study. SETTING: Lymphedema clinics in the upper midwestern region of the United States. SAMPLE: 94 patients with lymphedema and 94 controls without lymphedema, matched on type of axillary surgery and surgery date. METHODS: The Measure of Arm Symptom Survey, a patient-completed tool, assessed potential risk factors for lymphedema. Severity of lymphedema was measured by arm circumference, and disease and treatment factors were collected via chart review. MAIN RESEARCH VARIABLES: Risk factors for lymphedema after breast cancer surgery. FINDINGS: On univariate analysis, patients with lymphedema were more likely than controls to be overweight (body mass index >or= 25) (p = 0.009). They also were more likely to have had axillary radiation (p = 0.011), mastectomy (p = 0.008), chemotherapy (p = 0.033), more positive nodes (p = 0.009), fluid aspirations after surgery (p = 0.005), and active cancer status (p = 0.008). Strength training (p = 0.014) and air travel (p = 0.0005) were associated with less lymphedema occurrence. On multivariate analysis, the only factor significantly associated with lymphedema was being overweight (p = 0.022). CONCLUSIONS: Being overweight is an important modifiable risk factor for lymphedema. Axillary radiation, more extensive surgery, chemotherapy, and active cancer status also were predictive of lymphedema. IMPLICATIONS FOR NURSING: This study provides evidence that excess weight contributes to lymphedema; strength training and airline travel did not contribute to lymphedema.

Transgenic Bcl-3 slows T cell proliferation.

Immunological adjuvants, such as bacterial LPS, increase the mRNA levels of the IkB-related NF-kappaB transcriptional transactivator, Bcl-3, in activated T cells. Adjuvants also increase the life expectancy of activated T cells, as does over-expression of Bcl-3, suggesting that Bcl-3 is part of the pathway whereby adjuvants affect T cell lifespans. However, previous reports, confirmed here, show that adjuvants also increase the life expectancies of Bcl-3-deficient T cells, making Bcl-3's role and effects in adjuvant-induced survival uncertain. To investigate the functions of Bcl-3 further, here we confirm the adjuvant-induced expression of Bcl-3 mRNA and show Bcl-3 induction at the protein level. Bcl-3 was expressed in mice via a transgene driven by the human CD2 promoter. Like other protective events, over-expression of Bcl-3 slows T cell activation very early in T cell responses to antigen, both in vitro and in vivo. This property was intrinsic to the T cells over-expressing the Bcl-3 and did not require Bcl-3 expression by other cells such as antigen-presenting cells.

Randomized controlled trial of mindfulness-based stress reduction (MBSR) for survivors of breast cancer.

OBJECTIVES: Considerable morbidity persists among survivors of breast cancer (BC) including high levels of psychological stress, anxiety, depression, fear of recurrence, and physical symptoms including pain, fatigue, and sleep disturbances, and impaired quality of life. Effective interventions are needed during this difficult transitional period. METHODS: We conducted a randomized controlled trial of 84 female BC survivors (Stages 0-III) recruited from the H. Lee Moffitt Cancer and Research Institute. All subjects were within 18 months of treatment completion with surgery and adjuvant radiation and/or chemotherapy. Subjects were randomly assigned to a 6-week Mindfulness-Based Stress Reduction (MBSR) program designed to self-regulate arousal to stressful circumstances or symptoms (n=41) or to usual care (n=43). Outcome measures compared at 6 weeks by random assignment included validated measures of psychological status (depression, anxiety, perceived stress, fear of recurrence, optimism, social support) and psychological and physical subscales of quality of life (SF-36). RESULTS: Compared with usual care, subjects assigned to MBSR(BC) had significantly lower (two-sided p<0.05) adjusted mean levels of depression (6.3 vs 9.6), anxiety (28.3 vs 33.0), and fear of recurrence (9.3 vs 11.6) at 6 weeks, along with higher energy (53.5 vs 49.2), physical functioning (50.1 vs 47.0), and physical role functioning (49.1 vs 42.8). In stratified analyses, subjects more compliant with MBSR tended to experience greater improvements in measures of energy and physical functioning. CONCLUSIONS: Among BC survivors within 18 months of treatment completion, a 6-week MBSR(BC) program resulted in significant improvements in psychological status and quality of life compared with usual care.