A T cell clone's avidity is a function of its activation state.

At present it is unclear how Ag dose-dependent T cell functions, such as cytokine production, reflect TCR affinity and how the signal strength afforded by the Ag dose affects the kinetics of cytokine production by the individual T cell. We used a computer-assisted ELISPOT approach to address these issues. IFN-gamma release by a clonal population of CD4 T cells was monitored on a clonal population of APC while titrating the nominal peptide. The frequency of cytokine-producing cells, the net per-cell output of cytokine, and the onset of cytokine production were each found to be functions of the signal strength. Sigmoidal dose-response curves were seen at the clonal population level, but the activation thresholds for the individual T cells followed a Gaussian distribution. Moreover, the overall dose-response curve of the T cell clone revealed cyclic changes, becoming increasingly shifted toward lower Ag concentrations with the duration of time that elapsed since the last restimulation with Ag. Therefore, responsiveness to Ag ("functional avidity") is not a constant parameter of a T cell clone but a function of the T cell's history of last Ag encounter. The implications of such shifting activation thresholds are discussed for autoimmune disease.

Protective anti-Helicobacter immunity is induced with aluminum hydroxide or complete Freund's adjuvant by systemic immunization.

To determine whether systemic immunization against Helicobacter pylori could be achieved with an adjuvant approved for human use, the efficacy of vaccination with Helicobacter antigen in combination with aluminum hydroxide (AlOH) was evaluated in a murine model of Helicobacter infection. Immunization with antigen and AlOH induced interleukin-5-secreting, antigen-specific T cells, and immunization with antigen and complete Freund's adjuvant induced interferon-gamma-secreting, antigen-specific T cells, as determined by ELISPOT assay. Both immune responses conferred protection after challenge with either H. pylori or H. felis, as confirmed by the complete absence of any bacteria, as assessed by both histology and culture of gastric biopsy samples. Protection was antibody independent, as demonstrated with antibody-deficient muMT mice (immunoglobulin-gene knockout mice), and CD4(+) spleen T cells from immunized mice were sufficient to transfer protective immunity to otherwise immunodeficient rag1(-/-) recipients. These results suggest an alternative and potentially more expeditious strategy for development of a human-use H. pylori vaccine.

Immunomodulatory effect of extracorporeal photopheresis after successful treatment of resistant renal allograft rejection.

BACKGROUND: Acute renal allograft rejection contributes to patient morbidity. Standard immunosuppressives are only partially effective and have significant side effects. Extracorporeal photopheresis (ECP) has been effective in reversing the acute rejection process. T cell cytokine expression is implicated in rejection and tolerance but actual changes in the cytokine profile of ECP-treated individuals have not been documented. METHODS: ECP was administered to a patient with acute renal allograft rejection resistant to other immunosuppressives. Enzyme-linked immunosorbent spot (ELISPOT) assay was performed to determine the frequency of mitogen-induced cytokine-producing cells before and after ECP. RESULTS: ECP resulted in resolution of rejection; serum creatinine concentration fell from 7.1 to 2.2 mg/dl; ELISPOT revealed a three-fold increase in the frequency of IL-5 producing cells; IFN-gamma:IL-5 ratio shifted from 2.73 pre-treatment to 1.01 post-treatment. CONCLUSION: Effective therapy of acute allograft rejection with ECP alters the peripheral blood cytokine profile towards "type 2" cytokines, suggesting that alteration of T cell cytokine profiles may contribute to the resolution of the process.

Staphylococcal enterotoxin-B-induced lethal shock in mice is T-cell-dependent, but disease susceptibility is defined by the non-T-cell compartment.

Here we introduce a murine model for SEB-induced lethal shock that relies on the administration of SEB alone and does not involve hepatotoxicity by avoiding pretreatment with the hepatotoxin D-galactosamine. In the absence of D-gal, we first identified SEB-susceptible and -resistant H-2(k)-congenic mouse strains. In contrast with what is well established for the classic D-gal-dependent model and what therefore is anticipated for the human disease, the levels of TNF produced did not define susceptibility in our model. The SEB-induced TNF response in vitro and in vivo was stronger in resistant B10.BR mice than in susceptible C3H/HeJ mice. Neither the magnitude nor the quality of the T cell response induced by SEB defined susceptibility. Adoptive transfer experiments in C3H-SCID recipient mice demonstrated that induction of the disease is T-cell-dependent. T cells from resistant and susceptible mice both transferred disease susceptibility to H-2(k)-congenic C3H-SCID mice, indicating that disease susceptibility is downstream from T cell activation, at the level of the target organ itself, which responds differently to T-cell-induced inflammation.

Pulmonary mononuclear cell responses to antigens of Mycobacterium tuberculosis in healthy household contacts of patients with active tuberculosis and healthy controls from the community.

Protective immunity against Mycobacterium tuberculosis requires CD4+ lymphocyte-mediated immune responses and IFN-gamma activity. As the primary portal of entry of M. tuberculosis is the lung, pulmonary immune responses against multiple M. tuberculosis Ags were compared between both M. tuberculosis-exposed tuberculin skin test-positive healthy household contacts (HHC) of patients with active sputum smear and culture-positive tuberculosis and tuberculin skin test-positive healthy control individuals from the community (CC). Frequencies of M. tuberculosis Ag-specific IFN-gamma-producing cells, IFN-gamma concentrations in culture supernatants, and DNA synthesis in bronchoalveolar cells (BAC) and PBMC were studied in HHC (n = 10) and CC (n = 15). Using enzyme-linked immunospot assay we found higher frequencies of IFN-gamma-producing cells with specificity to M. tuberculosis-secreted Ag 85 (Ag 85) in BAC from HHC than in BAC from CC (p < 0.022) and relative to autologous PBMC, indicating compartmentalization of Ag 85-specific cells to the lungs. Further, IFN-gamma-producing cells with specificity to components A and B of Ag 85 were specifically compartmentalized to the lungs in HHC (p < 0. 05). IFN-gamma concentrations in culture supernatants of BAC and Ag-specific DNA synthesis were low and comparable in the two subject groups. Increased immune responses to Ag 85 at the site of repeated exposure to M. tuberculosis (the lung) may represent an important component of protective immunity against M. tuberculosis. Correlates of protective immunity against M. tuberculosis are required for assessment of the efficiency of anti-tuberculous vaccines.

Quantification of self-recognition in multiple sclerosis by single-cell analysis of cytokine production.

Identifying and quantifying autoaggressive responses in multiple sclerosis (MS) has been difficult in the past due to the low frequency of autoantigen-specific T cells, the high number of putative determinants on the autoantigens, and the different cytokine signatures of the autoreactive T cells. We used single-cell resolution enzyme-linked immunospot (ELISPOT) assays to study, directly ex vivo, proteolipid protein (PLP)-specific memory cell reactivity from MS patients and controls. Overlapping 9-aa-long peptides, spanning the entire PLP molecule in single amino acid steps, were used to determine the frequency and fine specificity of PLP-specific lymphocytes as measured by their IFN-gamma and IL-5 production. MS patients (n = 22) responded to 4 times as many PLP peptides as did healthy controls (n = 22). The epitopes recognized in individual patients, up to 22 peptides, were scattered throughout the PLP molecule, showing considerable heterogeneity among MS patients. Frequency measurements showed that the number of PLP peptide-specific IFN-gamma-producing cells averaged 11 times higher in MS patients than in controls. PLP peptide-induced IL-5-producing T cells occurred in very low frequencies in both MS patients and controls. This first comprehensive assessment of the anti-PLP-Th1/Th2 response in MS shows a greatly increased Th1 effector cell mass in MS patients. Moreover, the highly IFN-gamma-polarized, IL-5-negative cytokine profile of the PLP-reactive T cells suggests that these cells are committed Th1 cells. The essential absence of uncommitted Th0 cells producing both cytokines may explain why therapeutic strategies that aim at the induction of immune deviation show little efficacy in the established disease.

Granzyme B ELISPOT assay for ex vivo measurements of T cell immunity.

A major goal in immunodiagnostics has been the development of assay systems that can measure CD8(+) T cell immunity in humans, directly ex vivo, at high resolution, and with high throughput. We established granzyme B (grB) enzyme-linked immunospot assay (ELISPOT) in conjunction with image analysis to this end. Using grB transfected and untransfected Chinese hamster ovary (CHO) cells and T cell lines, we show that the antibody pair utilized was grB-specific and that only activated T cells secrete grB. GrB release began within 4 h after antigen stimulation and stopped within 40 h. Side-by-side comparison showed grB ELISPOT assays to have a higher resolution than classic chromium-release assays in terms of signal-to-noise ratio. The linearity of the relation of the number of CD8(+) effector T cells plated to grB spots detected suggests that grB ELISPOT assays measure the frequencies of grB-secreting cells directly. Reactivity to HIV peptides was seen in grB ELISPOT assays of freshly isolated PBMC from HIV patients, consistent with the detection of peptide-specific memory cells. The higher resolution and lower labor and material investment should make grB ELISPOT assays an attractive alternative to chromium-release assays in monitoring the clonal sizes of specific CD8 memory cells in vivo.

Direct visualization of cytokine-producing recall antigen-specific CD4 memory T cells in healthy individuals and HIV patients.

We have used computer-assisted cytokine ELISA spot analysis to measure the frequencies, the type of cytokine, and the amount of cytokine produced by individual recall Ag-specific CD4 memory cells in freshly isolated blood. We studied the memory cells specific for tetanus toxoid and purified protein derivative in 18 healthy individuals and in 22 HIV-infected patients on highly active antiretroviral therapy (HAART). In healthy individuals, the frequency, cytokine signature, and cytokine production per cell of these memory cells were stable over time. Although it is presently unclear whether the maintenance of the memory T cell pool depends upon Ag persistence, cross-reactive Ag stimulation, or cytokine-driven bystander stimulations and expansions, our data strongly argue for a stable memory cell pool in healthy individuals. In HIV patients, however, the frequency of these memory cells was a function of the viral load. The decreased numbers of functional memory cells in patients with high viral loads might provide one mechanism behind the immunodeficient state. Although the cytokine output per cell was unaffected in most patients (20 of 24), in some patients (4 of 24) it was >100-fold reduced, which might provide an additional mechanism to account for the reduced immunocompetence of these patients. The ability to visualize directly and quantify the cytokine produced by the low frequency memory cells in freshly isolated blood that have been physiologically stimulated by Ag should aid comprehensive studies of the Ag-specific memory cell pool in vivo, in health and disease.

Single-cytokine-producing CD4 memory cells predominate in type 1 and type 2 immunity.

The patterns of Ag-induced cytokine coexpression in normal, in vivo-primed CD4 memory T cells has remained controversial because the low frequency at which these cells occur has effectively prevented direct ex vivo measurements. We have overcome this limitation by using two-color cytokine enzyme-linked immunospot assays and computer-assisted image analysis. We found CD4 memory cells that simultaneously expressed IL-2, IL-3, IL-4, IL-5, and IFN-gamma to be rare (0-10%). This cytokine segregation was seen in adjuvant-induced type 1, type 2, and mixed immunity to OVA, in Leishmania infection regardless of the Ag dose used or how long after immunization the assay was performed. The data suggest that type 1 and type 2 immunity in vivo is not mediated by classic Th1 or Th2 cells but by single-cytokine-producing memory cells.

Pretransplant frequency of donor-specific, IFN-gamma-producing lymphocytes is a manifestation of immunologic memory and correlates with the risk of posttransplant rejection episodes.

While matching for MHC Ags improves renal allograft survival, closely matched grafts sometimes fail due to rejection, and poorly matched allografts are often well tolerated by the recipient. The severity of the rejection process may partially depend on the presence of environmentally primed T cells in the recipient that cross-react with donor Ags. To test for the presence of primed, donor-specific T cells in humans before transplantation, we used an enzyme-linked immunospot assay for detection of allospecific cytokines produced by individual human PBLs. We demonstrate that this approach detects cytokine production at single cell resolution and detects production of IFN-gamma only when there is defined immunologic priming, thus representing a measure of primed donor-specific immunity. Because the environmental Ag exposure of the recipient is not a function of the HLA mismatch between donor and potential recipient, the number of HLA mismatches may not correlate with the frequency of pretransplant, donor-specific IFN-gamma-producing PBLs. Studies of donor-specific IFN-gamma-producing lymphocytes in a cohort of patients being evaluated for renal transplantation corroborated this hypothesis. Moreover, for recipients of both living and cadaver renal allografts, the pretransplant frequency of donor-specific memory cells correlated with the posttransplant risk of developing acute rejection episodes. This improved ability to define the strength of the allospecific immune response by enzyme-linked immunospot assay may allow improved pairing of recipients with donors and identification of kidney allograft donor-recipient pairs at high risk for acute rejection, thus permitting targeted interventions aimed at prolonging graft survival.

Prevention of murine EAE by oral hydrolytic enzyme treatment.

Clinical trials that test the efficacy of Phlogenzym (consisting of the hydrolytic enzymes bromelain and trypsin and the anti-oxidant rutosid) as a treatment for T cell-mediated autoimmune diseases including multiple sclerosis (MS), type 1 diabetes and rheumatoid arthritis are presently ongoing. We tested the effects of Phlogenzym treatment in the murine model for MS, experimental allergic encephalomyelitis (EAE), a disease induced in SJL mice by immunization with proteolipid protein (PLP) peptide 139-151. Oral administration of Phlogenzym resulted in complete protection from EAE. In Phlogenzym-treated mice, the dose response curve of the PLP:139-151-specific T cell response was shifted to the right, that is, the primed T cells required higher peptide concentrations to become activated. Additionally, the T cell response to this peptide was shifted towards the T helper 2 cytokine profile. Both effects are consistent with an increased T cell activation threshold. In support of this interpretation, we found that the accessory molecules CD4, CD44, and B7-1 (all of which are involved in T cell co-stimulation) were cleaved by Phlogenzym, while CD3 and MHC class II molecules (which are involved in the recognition of antigens by T cells) and LFA-1 were unaffected. These data show the efficacy of oral Phlogenzym treatment in an animal model of T cell-mediated autoimmune disease and suggest that the protective effect might be the result of an increase in the activation threshold of the autoreactive T lymphocytes brought about by the cleavage of accessory molecules involved in the interaction of T cells and antigen presenting cells.

Adjuvant-guided type-1 and type-2 immunity: infectious/noninfectious dichotomy defines the class of response.

Traditionally, protein Ags have been injected in CFA (oil with inactivated mycobacteria) to induce immunity and with IFA (oil alone) to induce tolerance. We report here that injection of hen eggwhite lysozyme, a prototypic Ag, in CFA-induced and IFA-induced pools of hen eggwhite lysozyme-specific memory T cells of comparable fine specificity, clonal size, and avidity spectrum, but with type-1 and type-2 cytokine signatures, respectively. This adjuvant-guided induction of virtually unipolar type-1 and type-2 immunity was observed with seven protein Ags and in a total of six mouse strains. Highly polarized type-1 and type-2 immunity are thus readily achievable through the choice of adjuvant, irrespective of the genetic bias of the host and of the nature of the protein Ag. This finding should have far-reaching implications for the development of vaccines against infectious and autoimmune diseases. Furthermore, our demonstration that Ag injected with IFA is as strongly immunogenic for T cells as it is with CFA shows that the presence of the mycobacteria determines not the priming of naive T cells through the second-signal link but the path of downstream differentiation toward CD4 memory cells that express either type-1 or type-2 cytokines.

Enhanced responses to Mycobacterium tuberculosis antigens by human alveolar lymphocytes during active pulmonary tuberculosis.

Responses to mycobacterial and nonmycobacterial antigens were examined in bronchoalveolar cells (BAC) and peripheral blood mononuclear cells (PBMC) from patients with active pulmonary tuberculosis (n=16) and healthy subjects (n=23). DNA synthesis in BAC (but not PBMC) from tuberculosis patients was significantly increased in response to the mycobacterial antigens purified protein derivative (PPD), antigen 85, and mannose-capped lipoarabinomannan but not to nonmycobacterial antigens. The response to PPD was also increased in enriched alveolar lymphocytes from tuberculosis patients (P<.05). The frequency of interferon-gamma but not interleukin-4- or -10-producing cells by ELISAspot was increased in PPD-stimulated BAC from patients with tuberculosis (P<.05). Accessory function of alveolar macrophages for T lymphocyte responses was similar and suppressive activity was variably decreased in tuberculosis patients. Thus, there is compartmentalization of mycobacterial antigen-specific lymphocytes to the lungs during active tuberculosis that on challenge produce a Th1-type cytokine host response.

Enzyme-linked immunosorbent assay spot detection of interferon-gamma and interleukin 5-producing cells as a predictive marker for renal allograft failure.

BACKGROUND: Despite improvements in the short-term survival of renal transplants, many allografts fail over the 5-10 years after transplantation. We sought to identify an immunologic assay that could identify those patients at high risk for future allograft failure. METHODS: Blood samples were obtained from 23 renal allograft recipients with acute and/or chronic graft dysfunction and from 22 controls. Isolated peripheral blood lymphocytes (PBLs) were tested for interferon (IFN)-gamma and interleukin (IL)-5 production using an enzyme-linked immunosorbent spot assay. IFN-gamma:IL-5 ratios were calculated and compared between groups. Among the 23 patients with graft dysfunction, the ratios were also compared with graft function at 6 months. RESULTS: IFN-gamma:IL-5 ratios of > or = 15 were associated with allograft rejection episodes in 8 of 12 cases, whereas 10 of 11 episodes of graft dysfunction from other causes (infection, drug toxicity, obstruction) were associated with values <15. All normal controls had values <15 (22/22). Among the graft recipients with acute renal failure, all patients with IFN-gamma:IL-5 ratios <15 exhibited improved renal function at 6-month follow-up (14/14), whereas 8 of 9 patients with IFN-gamma:IL-5 ratios > or = 15 developed allograft failure at 6 months (sensitivity 100%, specificity 93.3%). CONCLUSION: In renal transplant recipients with acute allograft dysfunction, mitogen-induced peripheral blood lymphocyte IFN-gamma:IL-5 ratios > or = 15 were highly predictive of allograft failure within 6 months of the assay. This test may be a useful prognostic marker for identification of transplant recipients with acute graft dysfunction who are at high risk for future graft loss and thus allow targeted therapeutic interventions to prolong graft survival.

Identification of LFA-1 as a candidate autoantigen in treatment-resistant Lyme arthritis.

Treatment-resistant Lyme arthritis is associated with immune reactivity to outer surface protein A (OspA) of Borrelia burgdorferi, the agent of Lyme disease, and the major histocompatibility complex class II allele DRB1*0401. The immunodominant epitope of OspA for T helper cells was identified. A homology search revealed a peptide from human leukocyte function-associated antigen-1 (hLFA-1) as a candidate autoantigen. Individuals with treatment-resistant Lyme arthritis, but not other forms of arthritis, generated responses to OspA, hLFA-1, and their highly related peptide epitopes. Identification of the initiating bacterial antigen and a cross-reactive autoantigen may provide a model for development of autoimmune disease.

The human immune system in hu-PBL-SCID mice.

Severe combined immunodeficiency (SCID) mice can be stably grafted with human peripheral blood lymphocytes, creating hu-PBL-SCID chimeras; essentially, these are mice with a human immune system. Here, Magdalena Tary-Lehmann, Andrew Saxon and Paul Lehmann discuss the immunobiology of these chimeras. The authors propose that hu-PBL-SCID chimerism evolves in two phases. During the first three weeks after grafting, many of the injected cells survive and the human immune system is functional. Subsequently, anti-mouse-reactive clones are selected and the immune system becomes nonfunctional. The implications of this scenario for the utilization of the hu-PBL-SCID model are discussed.

Anti-SCID mouse reactivity shapes the human CD4+ T cell repertoire in hu-PBL-SCID chimeras.

Injecting human peripheral blood mononuclear cells into severe combined immunodeficient (SCID) mice results in long-term engraftment of human lymphocytes, of which > 98% are phenotypically mature, activated T cells. Here we have characterized the human T cells that populate such hu-PBL-SCID chimeras. We report that these human T cells do not mobilize Ca2+ after CD3 stimulation, i.e., their T cell receptor (TCR)-mediated signal transduction is deficient. Chimera-derived human T cells do not secrete lymphokines or undergo blastogenesis after CD3 stimulation, but proliferate in response to interleukin 2 (IL-2), defining the chimera derived human T cells as anergic. Anergy was seen in both the CD4+ and the CD8+ subpopulations. We established human T cell lines from chimeras. These T cells retained their anergic state for 1-2 mo in culture, after which they simultaneously regained the ability to mobilize Ca2+, secrete lymphokines, and to undergo blastogenesis following stimulation via the TCR. Once regaining proliferative responsiveness to CD3 stimulation, these CD4+ T cell lines displayed anti-SCID mouse reactivity and showed no specificity for recall antigens. All CD3-responsive CD4+ T cell clones obtained from such lines were SCID mouse specific, recognizing native major histocompatibility complex class II products on the murine cells. In contrast, chimera-derived human CD8+ cell lines and clones did not display detectable anti-mouse reactivity. The data show that the human T cell system in long term hu-PBL-SCID chimeras is nonfunctional due to both anergy and the limitation of the CD4+ repertoire to xenoreactive clones. The data suggest that long-term hu-PBL-SCID chimerism represents an atypical graft-versus-host reaction in which the human effector T cells become anergic in the murine environment.

The SCID mouse environment causes immunophenotypic changes in human immature T-cell lines.

In order to evaluate whether the SCID mouse can provide the microenvironment for the growth of human immature T-cell leukemias and if in vivo growth alters their phenotype, we examined the behavior of 3 well-characterized T-cell acute lymphoblastic leukemia (T-ALL)-derived T-cell lines which are at different stages of maturation (CEM, SUP-T3 and MOLT-4f) after transfer to non-irradiated SCID mice. All 3 T-cell lines engrafted and proliferated to form tumors in the mice and showed dissemination patterns in the SCID mouse comparable to those of T-ALL in man: i.e., human cells were detectable by flow cytometry or were cultured from mouse bone marrow, spleen, liver or thymus. CEM, which is the most immature T-cell line, readily formed tumors after injection of cells. The more mature T-cell lines, SUP-T3 and MOLT-4f, required a longer time period, even after injection of higher cell numbers. Whereas no changes in the configuration of the rearranged T-cell receptor genes were detected, striking phenotypic changes were observed in all 3 leukemias growing in the SCID mice after injection. SCID-CEM cells showed an increase in the surface expression of CD3 and CD8 and a decrease in the expression of CD1 and CD71 (transferrin-receptor). SCID-MOLT-4f cells showed an increase in CD5 and CD8 expression and a decrease in CD45RA expression. SCID-SUP-T3 cells showed increased expression of CD8 and CD45RA. Apparently, the mouse environment caused changes in cell-surface antigen expression on the T-ALL-derived T-cell lines. Some immunophenotypic changes remained stable during subsequent growth in culture of SUP-T3 cells, suggesting that maturation of the cell line occurred in vivo. The other cell lines, CEM and MOLT-4f after undergoing in vivo-induced changes, reverted to the original immunophenotype, suggesting transitory activation in vivo. These data point out the importance of stromal factors in defining growth and maturation of human leukemic cells in vivo, in SCID mice.

Human mature T cells that are anergic in vivo prevail in SCID mice reconstituted with human peripheral blood.

In these studies we have characterized the human cells that repopulate severe combined immunodeficient (SCID) mice after injection of adult peripheral blood or cord blood (hu-PBL-SCID mice). In all organs of the chimeras, and at any time point tested, single-positive (CD4+ or CD8+) T cells that expressed the alpha/beta T cell receptor (TCR) prevailed. All T cells were CD45RO+ and the majority were also HLA-DR+. Thus, the human T cells in the chimeras exhibited the phenotype of mature, memory cells that showed signs of recent activation. Cell cycle studies revealed a mitotically active human T cell population in the murine host. However, when freshly isolated from the chimeras, the human T cells were refractory to stimulation by anti-CD3 antibody but proliferated in response to exogenous interleukin 2. Chimera-derived human T cell lines retained this state of unresponsiveness to TCR-triggered proliferation for 4-6 wk in vitro. Subsequently, the T cell lines developed responses to anti-CD3 stimulation and 9 of 11 of the lines also proliferated in response to splenic stimulator cells of SCID mice. These data demonstrate that the human T cells are in a state of reversible anergy in the murine host and that xenoreactivity might play a critical role in hu-PBL-SCID mice. Mechanisms that may determine repopulation of SCID mice with human peripheral blood mononuclear cells are discussed.