Functionally restricted, allospecific, human helper T cell lines that amplify either B cell or cytolytic T cell responses.

Using a panel of partially cloned, OKT4+, DRw-1-specific, alloproliferative human T cell lines, we have identified two functionally restricted and reciprocating types of helper T cells. One provides major histocompatibility complex-restricted help for plaque-forming cell responses by DRw 1+ allogeneic B cells; the other preferentially amplifies the generation of allospecific cytotoxic T lymphocytes (CTL) from CTL precursors that have been suboptimally triggered by alloantigen.

Dendritic cells are potent antigen-presenting cells for microbial superantigens.

Dendritic cells are a small subset of human blood mononuclear cells that are potent stimulators of several T cell functions. Here we show they are 10-50-fold more potent than monocytes or B cells in inducing T cell responses to a panel of superantigens. Furthermore, dendritic cells can present femtomolar concentrations of superantigen to T cells even at numbers where other antigen-presenting cells (APCs) are inactive. Although dendritic cells express very high levels of the major histocompatibility complex products that are required to present superantigens, it is only necessary to pulse these APCs for 1 hour with picomolar levels of one superantigen, staphylococcal enterotoxin B, to maximally activate T cells. Our results suggest that very small amounts of superantigen will be immunogenic in vivo if presented on dendritic cells.

A cloned major histocompatibility complex-restricted trinitrophenyl-reactive human helper T cell line that activates B cell subsets via two distinct pathways.

A cloned, trinitrophenyl (TNP)-specific helper T cell line (TCL), termed E-11, has been established in long-term, interleukin 2-dependent culture and used to study human T helper (Th)-B cell collaboration. Co-culture of E-11 with TNP-modified, but not unmodified or FITC-modified, autologous B cells results in a vigorous, polyclonally plaque-forming cell (PFC) response. E-11 helper activity is not constitutive, but requires antigen-specific, major histocompatibility complex-restricted activation of the TCL cells by interaction with TNP-modified autologous or DR 5+ allogeneic macrophages. Using B cell subsets isolated by discontinuous density gradient cengrifugation as responder populations, we determined that E-11 activates B cell subsets via two distinct mechanisms: (a) E-11 polyclonally activates large B cells in an unrestricted and nonspecific manner; and (b) E-11 preferentially induces a PFC response by TNP-modified small B cells. These results suggest that the large B cell subset is activated by helper signals generated during the Th-antigen-presenting cell interaction, while small B cells require an additional stimulus that is provided by antigen-specific Th-B cell contact.

Direct T helper-B cell interactions induce an early B cell activation antigen.

We have explored the consequences for the B cell of cognate interaction with T cells. Early expression of the B cell-restricted cell surface activation antigen, BLAST-2, has been used as an assay system to measure direct T-B cell collaboration. BLAST-2 is preferentially expressed by allogenic B cells cultured with MHC class II antigen-restricted Th clone cells matched to the DR specificity of the target B cells. B cells cultured with DR-mismatched allospecific Th cells express minimal BLAST-2. Th cell-induced BLAST-2 expression appears to be accessory cell independent and occurs as early as 8 h after initiation of culture, with peak expression at 18 h. Direct T-B cell contact, rather than Th-derived lymphokines, provides the most efficient stimulus for BLAST-2 expression. Crosslinking of sIg on B cells is a poor stimulus for BLAST-2 expression. The BLAST-2 assay permits the evaluation of early events associated with B cell activation through cognate interactions, and may facilitate subsequent studies of the mechanism of B cell differentiation.

Interleukin 1 production during accessory cell-dependent mitogenesis of T lymphocytes.

We have studied the control and significance of IL-1 production in human leukocyte cultures during accessory cell-dependent, T lymphocyte mitogenesis using sensitive bioassays and immunolabeling techniques. In primary antigen-dependent systems like the MLR, IL-1 production was not detected in accessory cells (monocytes, dendritic cells) or T cells, suggesting that it is not an early product in these responses. However, monocytes could be induced to make IL-1 after interacting with sensitized antigen-specific T cells. Both alloreactive T cell clones or freshly prepared lymphoblasts induced IL-1 provided the monocytes carried the HLA-DR antigens to which the T cells were initially sensitized. Even in these circumstances, dendritic cells and B cells failed to make IL-1. The mechanism whereby activated T cells induce IL-1 in monocytes was explored. Supernatants from cocultures of monocytes and T cells or several recombinant cytokines induced little or no IL-1. A more potent antigen independent pathway of IL-1 induction was identified. IL-1 could be induced in third-party HLA-DR nonspecific monocytes in cocultures of alloreactive T cell clones or blasts and HLA-DR-specific dendritic cells. The induction was factor independent since dendritic cells and T blasts placed in a chamber separate from third-party monocytes by a semipermeable membrane did not induce monocyte IL-1. These results suggest that a cell contact mechanism rather than an IL-1-inducing factor leads to IL-1 production. The role of IL-1 in T cell proliferation was tested with a polyclonal anti-IL-1 antibody. The antibody failed to block the proliferation of primary T cells, or alloreactive T cell clones and blasts stimulated with HLA-specific monocytes or dendritic cells, even though IL-1 in the medium was neutralized.

Helper T cell-dependent human B cell differentiation mediated by a mycoplasmal superantigen bridge.

Experimentally induced murine graft-vs.-host disease may be characterized by hypergammaglobulinemia, autoantibody formation, and immune complex-mediated organ system damage that mimics SLE. These autoimmune phenomena are mediated by abnormal Th-B cell cooperation, across MHC disparities, in which donor-derived allospecific Th cells recognize and interact with MHC class II antigens on the surface of recipient B cells. Microbial toxins, termed superantigens, which bind to MHC class II molecules and activate selected T cells based on TCR variable gene usage, may induce a similar form of Th-B cell interaction. In the present study, we generated and characterized human Th cell lines reactive with the Mycoplasma arthritidis superantigen (MAM). The essential observation is that resting human B cells bind MAM and present it to superantigen-reactive autologous or allogeneic Th cells, resulting in both Th cell activation and a consequent polyclonal Ig response by the superantigen-bearing B cells.

Antigen-specific, MHC nonrestricted T helper cell-induced B cell activation.

We used a cloned, TNP-specific, MHC-restricted, human Th cell line, E-11, and an assay of cognate Th-B cell interaction, BLAST-2 antigen expression on the B cell surface, to investigate the functional nature of the Th cell antigen receptor. We observed that E-11 induces BLAST-2 expression by resting B cells in a hapten-dependent, hapten-specific, but MHC nonrestricted manner. The implication of these results for the Th cell receptor are discussed.

CD40 ligation induces Apo-1/Fas expression on human B lymphocytes and facilitates apoptosis through the Apo-1/Fas pathway.

The Apo-1/Fas antigen (CD95) mediates programmed cell death of lymphocytes when bound by Fas ligand or anti-Apo-1/Fas antibody. In contrast, the CD40 antigen provides a potent activation and survival signal to B lymphocytes when it is engaged by its T cell ligand (CD40L, gp39) or cross-linked by anti-CD40 antibody. In this study, we use human tonsillar B cells and the Ramos Burkitt's lymphoma B cell line, which serves as a model for human germinal center B lymphocytes, to study the effectors of Apo-1/Fas expression and apoptosis of human B cells. We found that Apo-1/Fas expression was upregulated on both malignant and normal human B lymphocytes after CD40 ligation induced by (a) cognate T helper-B cell interaction mediated by microbial superantigen (SAg); (b) contact-dependent interaction with CD40L+, but not CD40L- Jurkat mutant T cell clones; and (c) monoclonal anti-CD40, but not any of a panel of control antibodies. Enhanced B cell Fas/Apo-1 expression is functionally significant. Coculture of Ramos Burkitt's lymphoma line cells with irradiated SAg-reactive CD4+ T cells with SAg or CD40L+ Jurkat T cells results in B cell apoptosis, evidenced by reduced cell viability and DNA laddering. This process is augmented by the addition of anti-Apo-1/Fas monoclonal antibody, consistent with an acquired susceptibility to Apo-1/Fas-mediated apoptosis. These data support an immunoregulatory pathway in which seemingly contradictory signals involving the B cell proliferation/survival antigen CD40, as well as the Apo-1/Fas molecule, which mediates programmed cell death of lymphocytes, are linked in the process of human B cell activation.

Functional analysis of human T cell subsets defined by monoclonal antibodies. IV. Induction of suppressor cells within the OKT4+ population.

In this report, we explored the functional heterogeneity within the OKT4+ subset of human T cells. Evidence was obtained that although in vitro pokeweed mitogen-activated OKT4+ cells can function as radioresistant helper cells, these activated OKT4+ cells could also exert potent feedback suppression. Despite the induction of suppressor cells after pokeweed mitogen activation, the OKT4+ population maintains its original OKT3+, OKT4+, nd OKT8- surface phenotype. The suppressor cells contained within the activated OKT4+ population were found to be radiosensitive. Importantly, the suppression mediated by activated OKT4+ cells required the presence of radiosensitive cells contained within the resting OKT4+ population. Taken together, these results suggest that the OKT4+ subset of human T cells contains cells that can be activated to differentiate into suppressor cells independent of OKT8+ cells.

Human HLA-A0201-restricted cytotoxic T lymphocyte recognition of influenza A is dominated by T cells bearing the V beta 17 gene segment.

The major histocompatibility complex class I-restricted cytotoxic T lymphocyte (CTL) response is important in the clearance of viral infections in humans. After influenza A infection, a peptide from the matrix protein, M58-66, is presented in the context of the MHC allele HLA-A0201 and the resulting CTL response is detectable in most HLA-A0201 subjects. An initial study suggested that M58-66-specific CTL clones show conserved T cell receptor (TCR) alpha and beta gene segments. We have addressed the significance of this observation by determining the expression of V beta 17 during the development of M58-66-specific CTL lines in 21 unrelated HLA-A0201 subjects, and analyzing TCR usage by M58-66-specific CTL clones. TCR V beta 17 was the dominant V beta segment used and CD8 V beta 17 expansion correlated with M58-66-specific lysis. Limiting dilution analysis from five subjects showed the M58-66 CTL precursor frequency to vary between 1/54,000 and less than 1/250,000, and that up to 85% of the matrix peptide (M58-66)-specific CTL used the V beta 17 gene segment. The M58-66 specific CTL response was dependent on previous viral exposure and specific V beta 17 expansion, as it was not found in cord blood, despite a readily expandable V beta 17+ CD8+ T cell subpopulation. Sequence analysis of 38 M58-66-specific V beta 17 transcripts from 13 subjects revealed extensive conservation in the CDR3 region including conservation of an arginine-serine motif. To test the dependence of this CTL response on the V beta 17 gene segment, peripheral blood lymphocytes were depleted of CD8+ TCR V beta 17+ cells, before the generation of M58-66-specific CTL. In most cases such depletion blocked or severely reduced the generation of the M58-66-specific response, and under limiting dilution conditions could abolish M58-66-specific CTL precursors. These studies reveal the dependence of this natural human immune response on a particular TCR gene segment.

Characterization of human T cells reactive with the Mycoplasma arthritidis-derived superantigen (MAM): generation of a monoclonal antibody against V beta 17, the T cell receptor gene product expressed by a large fraction of MAM-reactive human T cells.

While all known microbial superantigens are mitogenic for human peripheral blood lymphocytes (PBL), the functional response induced by Mycoplasma arthritidis-derived superantigen (MAM) is unique in that MAM stimulation of PBL consistently results in T cell-dependent B cell activation characterized by polyclonal IgM and IgG production. These immunostimulatory effects of MAM on the humoral arm of the human immune system warranted a more precise characterization of MAM-reactive human T cells. Using an uncloned MAM reactive human T cell line as immunogen, we have generated a monoclonal antibody (mAb) (termed C1) specific for the T cell receptor V beta gene expressed by the major fraction of MAM-reactive human T cells, V beta 17. In addition, a V beta 17- MAM-reactive T cell population exists, assessed by MAM, induced T cell proliferation and cytotoxic T cell activity. mAb C1 will be useful in characterizing the functional properties of V beta 17+ T cells and their potential role in autoimmune disease.

Co-managed care for fragility hip fractures (Rochester model).

Hip fractures in older adults are a common event with a high risk of morbidity and mortality. Patients who sustain a hip fracture often present with multiple co-morbid conditions that can benefit from co-management by orthopedic surgeons and geriatricians. This manuscript describes a co-managed model of care for patients with hip fractures. This model of care will be explained, and the benefits and results will be described. Retrospective review of the care of all native non-pathological hip fracture patients aged 60 years and older admitted between April 2005 and March 2009 to a 261-bed community teaching hospital. The outcome measures include patient characteristics, length of stay, mortality, 30-day readmission, re-operation, and costs of care. Seven hundred fifty-eight patients were identified with an average age of 84.8 (SD 8.4); 77.8% of the patients were female, 94.7% Caucasian, and 37.3% from nursing homes, and the mean Charlson score is 2.9 (SD 2.1). The length of stay was 4.3 days, 30-day readmission rate was 10.4%, 17-month re-operation rate was 1.9%, and costs of care to the system were $15,188. The 1-year mortality rate was 21.2%. This model of care resulted in improvements in all measures studied. Previous studies have shown reduction in in-hospital complications. Additional studies are needed to show if this model of care can be translated to other systems or to other surgical conditions. Wide application of this model care could substantially improve the quality of care and cost of caring for frail elders with hip fractures.

Ortho-geriatric service--a literature review comparing different models.

In the fast-growing geriatric population, we are confronted with both osteoporosis, which makes fixation of fractures more and more challenging, and several comorbidities, which are most likely to cause postoperative complications. Several models of shared care for these patients are described, and the goal of our systematic literature research was to point out the differences of the individual models. A systematic electronic database search was performed, identifying articles that evaluate in a multidisciplinary approach the elderly hip fracture patients, including at least a geriatrician and an orthopedic surgeon focused on in-hospital treatment. The different investigations were categorized into four groups defined by the type of intervention. The main outcome parameters were pooled across the studies and weighted by sample size. Out of 656 potentially relevant citations, 21 could be extracted and categorized into four groups. Regarding the main outcome parameters, the group with integrated care could show the lowest in-hospital mortality rate (1.14%), the lowest length of stay (7.39 days), and the lowest mean time to surgery (1.43 days). No clear statement could be found for the medical complication rates and the activities of daily living due to their inhomogeneity when comparing the models. The review of these investigations cannot tell us the best model, but there is a trend toward more recent models using an integrated approach. Integrated care summarizes all the positive features reported in the various investigations like integration of a Geriatrician in the trauma unit, having a multidisciplinary team, prioritizing the geriatric fracture patients, and developing guidelines for the patients' treatment. Each hospital implementing a special model for geriatric hip fracture patients should collect detailed data about the patients, process of care, and outcomes to be able to participate in audit processes and avoid peerlessness.

Pulmonary Artery Catheter Seemingly Entrapped in the Skull.

A pulmonary artery catheter is an important tool for the monitoring of hemodynamics in patients. Unfortunately, misplacement of a catheter tip may occur in the vasculature local to the intended placement. Misplacement of the catheter can be further complicated by entrapment at the unintended destination. We present a case of a misplaced and entrapped pulmonary artery catheter in a patient with worsening pulmonary disease. After multiple unsuccessful attempts to float the catheter, it was partially retracted and found to be stuck. Imaging showed the tip terminating in the right internal jugular vein at the level of the jugular foramen. It was initially suspected that the catheter had become looped, knotted, or otherwise entangled within the vasculature of the skull and surgical removal would be necessary. Before surgical removal was performed, it was instead determined that the catheter had become kinked and entrapped at the end of the introducer sheath, and noninvasive removal was accomplished by first removing the introducer sheath.

Biochemical basis of synergy between antigen and T-helper (Th) cell-mediated activation of resting human B cells.

We have utilized CD23 expression as a marker for B cell activation in order to investigate the biochemical basis for synergy between antigen and T helper (Th) cells in the activation of resting human B cells. Our results confirm that while ligation of surface immunoglobulin (sIg) receptors by antigen analogues (e.g., F(ab')2 goat anti-human IgM) does not lead to CD23 expression, this stimulus markedly enhances CD23 expression induced during antigen specific Th-B cell interaction or by rIL-4. Utilizing a panel of monoclonal anti-human IgM antibodies, we observed a positive correlation between the capacity of a particular antibody to synergize with rIL-4 in CD23 expression and with B cell growth factor in B cell proliferation; suggesting that synergy in CD23 expression reflects the transduction of a functionally important signal via the sIg receptor. We next assayed analogues of the "second messenger" molecules, released during inositol lipid hydrolysis, for their capacity to amplify CD23 expression. These studies showed that protein kinase C (PKC) activating phorbol esters and the synthetic diacylgylcerol analogue, DiC8, synergize with either Th cells or rIL-4 in CD23 expression, while under no experimental condition does increasing B cell [Ca2+]i with ionomycin enhance CD23 expression. Taken together, these data suggest that activation of B cell PKC is the crucial biochemical event that primes antigen-activated B cells to respond more vigorously to interaction with Th cells and/or their soluble products.

CDR3 sequence motifs shared by oligoclonal rheumatoid arthritis synovial T cells. Evidence for an antigen-driven response.

T lymphocytes reactive with as yet undefined joint-localized foreign or autoantigens may be important in the pathogenesis of RA. Molecular studies demonstrating skewed T cell antigen receptor (TCR) variable gene usage and selective expansion of particular T cell clones within the synovial compartment support this view. Based on our recent study documenting selective expansion of V beta 17+ T cells in RA, we have pursued the identification of T cells relevant to the disease process, in an informative patient, by combining molecular analysis of freshly explanted RA synovial tissue V beta 17 TCR transcripts with in vitro expansion of V beta 17+ synovial tissue T cell clones. Peripheral blood V beta 17 cDNA transcripts proved heterogeneous. In contrast, two closely related sequences, not found in the peripheral blood, dominated synovial tissue V beta 17 transcripts, suggesting selective localization and oligoclonal expansion at the site of pathology. CD4+, V beta 17+ synovial tissue-derived T cell clones, isolated and grown in vitro, were found to express TCR beta chain transcripts homologous to the dominant V beta 17 synovial tissue sequences. One clone shares with a dominant synovial tissue sequence a conserved cluster of 4/5 amino acids (IGQ-N) in the highly diverse antigen binding CDR3 region, suggesting that the T cells from which these transcripts derive may recognize the same antigen. These findings have permitted a complete characterization of the alpha/beta TCR expressed by putatively pathogenic T cell clones in RA. Functional analysis suggests that the conserved CDR3 sequence may confer specificity for, or restriction by, the MHC class II antigen, DR4.

Amplification of altered self-reactive cytolytic T lymphocyte responses by cloned, allospecific human Th cells.

The effect of a cloned allospecific human Th cell, termed 86, on the in vitro generation of altered self-reactive cytolytic T lymphocytes (CTL) was investigated. Utilizing the induction of hapten altered self-reactive CTL as a model for virus or tumor-specific cell-mediated immunity, we determined that the presence of small numbers of clone 86 cells markedly amplified the generation of hapten altered self-reactive CTL. The killer cells induced belong to the CD4-, CD8+ subset, are specific for the hapten-modified autologous stimulator cells present in culture, and are MHC class I restricted. The CTL induced under these culture conditions are readily expanded in the presence of IL-2 with maintenance of efficient and specific altered self-killing. Of interest, clone 86 cells preferentially enhance the growth of CD8+ T cells and selectively amplify altered self-cytolysis but not NK cell activity. Although in vitro clone 86 cells mediate help for CTL generation via the production of lymphokines (IL-4 but little IL-2), one can envision immunotherapeutic strategies for human disease that involve the adoptive transfer of Th cells functionally analogous to clone 86.

Human immunodeficiency virus infection of helper T cell clones. Early proliferative defects despite intact antigen-specific recognition and interleukin 4 secretion.

HIV selectively inhibited the proliferative response of clonal CD4+ T lymphocytes to alloantigen while other alloantigen-dependent responses were unperturbed. Specifically, impaired blastogenesis could be dissociated from alloantigen-specific induction of the B cell activation molecule CD23, IL-4 release, and inositol lipid hydrolysis. In addition, membrane expression of pertinent T cell receptor molecules, including CD2, CD3, and T cell antigen receptor (Ti), remained intact. Using two MHC class II-specific human CD4+ helper T cell clones, the proliferative defect was shown to be an early consequence of HIV infection, occurring within 4 d of viral inoculation and preceding increases in mature virion production. It was generalizable to three distinct methods of T cell activation, all independent of antigen-presenting cells: anti-CD3 mediated cross-linking of the CD3/Ti complex; anti-CD2 and phorbol 12-myristic 13-acetate (PMA); and anti-CD28 plus PMA. These abnormalities were not mitigated by addition of exogenous IL-2, even though expression of the IL-2 receptor (CD25) was unaltered. These studies define a selective blockade in T cell function early after HIV exposure that could serve as a model for certain in vivo manifestations of AIDS.

PolyI.polyC12U-mediated inhibition of loss of alloantigen responsiveness viral replication in human CD4+ T cell clones exposed to human immunodeficiency virus in vitro.

Two alloreactive human CD4+ T cell clones, recognizing HLA-DR2 and HLA-DR1 determinants, lost their specific proliferative capacity after infection with HIV. This system was used to explore the effect of polyI.polyC12U on HIV replication and immune suppression. The mismatched double-stranded RNA blocked HIV-associated particulate reverse transcriptase activity and viral-mediated cytopathic effects. Also, polyI.polyC12U preserved the alloreactivity of T cell clones after exposure to HIV.PolyI.polyC12U appeared to act at a level subsequent to host cell infection and reverse transcription. It had no effect on the enhancement of gene expression by the HIV transcription unit tatIII. These findings indicate that early in the course of infection of CD4+ T lymphocytes, HIV can directly abrogate proliferation to specific allodeterminants, and that this function is preserved in the presence of polyI.polyC12U. They also provide insight into the mechanism of antiviral action of a class of agent with potential clinical utility in AIDS.

The apoptosis-1/Fas protein in human systemic lupus erythematosus.

Three independent mutations involving the apoptosis-1 (APO-1)/Fas receptor or its putative ligand have led to lupuslike diseases associated with lymphadenopathy in different strains of mice. To determine whether humans with SLE also have a defect in this apotosis pathway, we analyzed the expression of APO-1 on freshly isolated blood mononuclear cells and on lymphocytes activated in vitro using flow cytometry and the monoclonal antibody anti-APO-1. Significantly higher level of APO-1 expression were detected on freshly isolated peripheral B cells and both CD4+ and CD8+ T lymphocyte populations obtained from lupus patients when compared with normal controls (P < 0.001). Almost 90% of the cells that stained positive for APO-1 also expressed the CD29 antigen, suggesting that APO-1 was upregulated after lymphocyte activation in vivo. No defect in APO-1 regulation was detected after activation of SLE T (with anti-CD3) or B (with Staphylococcus aureus Cowan 1) lymphocytes in the presence of IL-2 in vitro. Similarly, the anti-APO-1 antibody induced apoptosis in 74 +/- 5% of activated SLE T cells in vitro compared with 79 +/- 6% of the normal controls (P > 0.05). These results reveal that, while APO-1/Fas may play an important role in the regulation of lymphocyte survival in SLE, no consistent defect in the expression or function of the receptor could be detected in these studies.