Effect of antigen/antibody ratio on macrophage uptake, processing, and presentation to T cells of antigen complexed with polyclonal antibodies.

Activation of a galactosidase-specific murine T hybridoma clone and of a human tetanus toxoid-specific T clone by antigen-presenting cells (APC) was used to evaluate the regulatory function of antibodies complexed with the relevant antigen. Complexed antigen, in fact, is taken up with high efficiency thanks to Fc receptors borne by APC. Antibody/antigen ratio in the complexes proved to be a critical parameter in enhancing antigen presentation. Complexes in moderate antibody excess provided optimal T cell activation independently of the physical state of the complexes (precipitated by a second antibody or solubilized by complement). Complexes in extreme antibody excess, on the contrary, did not yield T cell activation although taken up by APC efficiently. The effect of antibodies at extreme excess was observed with substimulatory dose of antigen (loss of potentiation) and with optimal dose of antigen (loss of stimulation). An excess of specific polyclonal antibodies hampers proteolytic degradation of antigen in vitro, supporting the view that a similar mechanism may operate within the APC that have internalized immune complexes in extreme antibody excess. The possibility that immune complex forming in extreme antibody excess may turn off the T cell response is proposed as a regulatory mechanism.

Relevance of CD38 expression on CD8 T cells to evaluate antiretroviral therapy response in HIV-1-infected youths.

Surrogate markers for monitoring immuno-virological discordant responders, in addition to plasma viral load and CD4 cells, are still lacking. We assessed the diagnostic utility of CD38 expression on CD8 T cell assay, alone or in association with lymphocyte proliferation to mycotic antigens, in evaluating antiretroviral response. 28 vertically HIV-infected youths, 21 HAART- and seven 2 nucleotide reverse transcriptase inhibitors-treated, were enrolled in a retrospective study. Responders (57.1%) and non-responders (42.9%) to stable antiretroviral therapy for a minimum of 6 months, on the basis of viral load and CD4 T cells, comprehensively evaluated by CD38 expression on CD8 T lymphocytes [measured as CD38 antibody bound per CD8 T cell (CD38 ABC) and %CD38+ of total CD8 T cells (%CD38/CD8)] and lymphocyte proliferation to P. jiroveci, C. albicans, C. neoformans, A. fumigatus at a single time point after treatment, were selected. CD38 expression > or =2401 CD38 ABC and > or =85% CD38/CD8 cut-off points, accurately discriminates responders versus non-responders, both measures resulting in 75.0% (CI 42.8-94.5) sensitivity (identification of non-responder) and 93.8% (CI 69.8-99.8) specificity (identification of responder), when considered as single assays. The association '> or =2401 CD38 ABC or > or =85% CD38/CD8' improved sensitivity to 83.3% (CI 51.6-97.9), while the association '<2401 CD38ABC (or <85% CD38/CD8) and lymphoproliferative response positive to > or =2 tested organisms' improved specificity to 100% (CI 79.4-100). In conclusions, CD38 expression and mycotic antigen-specific T-cell proliferation may be used as additional parameters to existing criteria to evaluate antiretroviral response in immuno-virological discordant patients.

The use of Apostain in identifying early apoptosis.

Irradiated human peripheral blood lymphoid cells undergo apoptosis and progressively exhibit typical changes in light scatter and plasma membrane integrity that can be easily tracked by flow cytometry. Using this model, we assessed the capacity of a newly developed fluorochrome, Apostain, in identifying early apoptosis in unfixed samples. This probe is a plasma membrane permeant DNA dye that can be conveniently excited at 488 nm and has an emission wavelength > 650 nm. To identify apoptotic cells, Apostain relies on the transient changes of chromatin texture that allow to accommodate more of a DNA dye occurring in early apoptosis. As early as 4 h after irradiation a proportion of cells showed an enhanced Apostain uptake. Consistent with their initial apoptotic nature, these cells had a still integer plasma membrane, as assessed by ethidium bromide, and unaltered light scatter. With time, cells showing the enhanced Apostain uptake started to bind dimly Annexin-V and, later, reduced their forward scatter. After 18 h from irradiation, cells exhibiting a reduced forward scatter exhibited a bright staining with Annexin-V with a concomitant reduction in Apostain uptake, reflecting the gross chromatin disruption characterising the endpoint of apoptosis.

A simple procedure for freezing and storing lymphocyte panels in trays.

Lymphocytes from different donors may be frozen in Terasaki trays by a simple procedure. This method provides the tissue typing laboratory with a readily available panel that can be routinely used to test for lymphocytotoxic antibodies sera from patients waiting for kidney transplant, transplanted patients and polytransfused individuals. The advantage of this procedure is that expensive programmed freezing apparatus is not required.

Inhibition of the accessory function of murine macrophages in vitro by cyclosporine.

The accessory function of macrophages, which is strictly related to the induction of T cell activation, has been studied to determine whether it is affected by cyclosporine (CsA). Irradiated spleen cells, used as a source of macrophages, were pulsed overnight with beta-galactosidase (GZ) in the presence of CsA. After washing of the pulsed macrophages, cells from a GZ-specific T cell line were added to cultures and 3H-thymidine incorporation was measured 72 hr later. We found that 500 ng/ml CsA present during macrophage pulsing with GZ reduced T cell proliferation to 5%. On the other hand, 100 ng/ml CsA almost completely abrogated the proliferative response when present for the duration of the culture. Similar results were also obtained using antigen-pulsed peritoneal-adherent macrophages to stimulate the T cell line to proliferate, or a T hybridoma clone to produce interleukin-2 (IL-2). The possibility that CsA actually affects interleukin-1 (IL-1) production by macrophages by inhibiting uninvolved T cells could be ruled out. We conclude that CsA-induced inhibition of T cell functions (proliferation and IL-2 production) is partially due to the effect of the drug on the accessory function of macrophages. This immunosuppressive mechanism of action of CsA on macrophages has not been previously described.

Effects of cyclosporine on the immune system of the mouse. II. Cyclosporine inhibits the effector function of primary T helper cells, but not helper cell priming.

This paper describes various effects of cyclosporine on T helper (TH) cells for antibody production in the mouse. Immunosuppressive doses of cyclosporine abolish the help provided by virgin (primary) TH cells, but allow the priming of normal (and under certain conditions supranormal) numbers of TH cells as assayed by adoptive transfer experiments. The helper function of primed (secondary) TH cells is normally resistant to cyclosporine. However, if T cells are exposed to cyclosporine during priming their helper function remains totally cyclosporine-sensitive as long as the drug therapy is continued. The mechanisms involved in these selective effects are unknown, but the results indicate that in vivo cyclosporine can dissociate effector function (primary help) from TH cell proliferation (priming).

Effect of cyclosporine on the antigen-presenting function of human and murine accessory cells.

Recent reports have challenged the belief that accessory cells are resistant to cyclosporine. Such a tenet was based on the observation that several functions of accessory cells, such as IL-1 production and phagocytosis, are resistant to the drug. On the other hand, when a less primitive, more refined function of accessory cells was examined--i.e., the capacity to take up, process, and present antigen in an MHC-restricted fashion to antigen-specific T lymphocytes, CsA proved to be an effective inhibitor. In contrast to this finding, when antigen was provided in the form of an immune complex prepared with a monoclonal antibody, uptake of antigen--likely mediated by the Fc receptors--and subsequent processing and presentation were not affected by CsA. These results suggest that, depending on whether the antigen is taken up by constitutive or by receptor-mediated endocytosis, accessory cells can be functionally defined as resistant or sensitive to CsA.

Difference in sensitivity to cyclosporine in vitro of human alloreactive lines and clones.

The effectiveness of cyclosporine (CsA) as immunosuppressive agent in human kidney graft rejection is well established. However, in spite of efforts to maintain optimal plasma levels, a fraction of transplanted patients undergo rejection episodes and/or irreversible chronic rejection. This suggests that immunosuppression by CsA cannot control the alloreactive response if there is a high degree of histoincompatibility for HLA or non-HLA antigens, or it has little effect on the "high responder" patient. Both possibilities are difficult to test in the human system. A third hypothesis, the existence of individual CsA resistance, was tested by evaluating the in vitro inhibitory activity of CsA on alloreactive T cell lines from several individuals. A different degree of in vitro sensitivity to the drug was observed among alloreactive lines generated from different individuals and among clones obtained from the same bulk line. The variability at the individual level and at the clonal level may account for the onset of CsA-resistant rejection assuming that in vivo a positive selection in the presence of the drug occurs and allows for the resistant clones, if present, to dominate the sensitive ones.

Recognition of donor fibroblast antigens by lymphocytes homing in the human grafted kidney.

A human transplanted kidney, surgically removed because of untreatable chronic rejection, was used as the source of lymphocytes (K-L) of recipient origin that were expanded with interleukin-2 (IL-2), and of kidney fibroblasts (K-F) of donor origin that were maintained as an established line. Cytotoxicity assays were performed using K-L and peripheral blood lymphocytes (PBL) as effectors, and K-F and donor PBL as targets. From the results the following conclusions can be drawn: (1) cytotoxic lymphocytes, presumably involved in the process of chronic graft rejection, home in the kidney (from which they can be recovered) but are not detected in the circulation; (2) cytotoxic lymphocytes can be generated from peripheral lymphocytes by mixed lymphocyte culture (MLC) and further expansion in vitro with IL-2 (MLC-L); and (3) although both K-L and MLC-L are cytotoxic toward K-F, the former are not cytotoxic toward donor PBL. This suggests that although MLC-L recognize antigens shared by K-F and PBL, K-L recognize antigens specific for K-F only. These results, if confirmed, indicate that antigens not present on PBL, and possibly tissue-restricted are important in graft rejection. Thus, while monitoring transplanted patients, a lack of cytotoxicity in the recipient PBL may be misleading because the relevant cytotoxic effector cells may have disappeared from the periphery and the appropriate antigenic target may be absent on donor PBL.

Analysis of the antigen specific T cell repertoires in HIV infection.

In addition to HIV infection, several acquired immunodeficiencies lead to depletion of CD4 lymphocytes. These include immunosuppression resulting from high dose cancer chemotherapy or induced to control graft rejection, as well as in autoimmune diseases. The consequence of this depletion is an increased susceptibility to opportunistic infections or the inability to control primary infection in the case of HIV infection. In all instances a full or partial immunoreconstitution is desirable. In order to monitor the cellular immune state of a patient, rational information cannot be simply derived from phenotypic quantification of T lymphocytes. Instead loss or recovery of CD4 cells should be monitored by defining the specificity, the function and the clonality of the relevant cell population. Several methods are now available for this type of investigation. Here we describe an approach for the definition of clonal heterogeneity of antigen specific CD4 lymphocytes, a parameter that may help monitor loss or reconstitution in acquired immunodeficiencies. As examples of antigen specific CD4 T cell responses we focused on Pneumocystis carinii and on cytomegalovirus, as prototypic opportunistic pathogens which are responsible for severe infections in AIDS and in other immunosuppressive conditions which arise for instance following transplantation. Specific CD4 T cell lines were generated from normal controls and from seropositives in order to select antigen specific lymphocytes. The cells were subsequently analyzed for clonal diversity according to TCR BV gene family usage and according to TCR CDR3 size heterogeneity (spectratyping).

Repertoire breadth of human CD4+ T cells specific for HIV gp120 and p66 (primary antigens) or for PPD and tetanus toxoid (secondary antigens)

Antigen derived peptides bound on MHC class II molecules on presenting cells stimulate specific CD4 lymphocytes that are in a naive state if antigen is given for the first time, or in a memory state if antigen has been previously encountered. In order to compare clonal heterogeneity of the human CD4+ T helper repertoire in primary vs. recall responses, we have generated T cell lines in vitro by repeated stimulation of peripheral lymphocytes with primary or with recall antigens. Clonal heterogeneity was broad in the case of recall response to tetanus toxoid or PPD, with a high frequency of specific precursors (> 100 cells/10(6) lymphocytes). In contrast, T cell lines responsive to primary antigens (HIV gp120 or HIV p66) were oligoclonal as defined by TCR V beta gene usage and by spectratyping, and the precursor frequency was low (< 2 cells/10(6) lymphocytes). Primary T cell lines generated from blood samples drawn at different times from the same donor showed that clones with identical TCR CDR3 region coding sequences were expanded, suggesting that in these individuals a large progeny derived from one single precursor is present, even though a previous encounter with the antigen was not documented. Assuming an even in vivo distribution of such cells, the presence of one precursor every 10(6) CD4 lymphocytes (within the CD4 T repertoire that comprises roughly 10(11) CD4 T cells) indicates that approximately 10(5) identical T cells from the same clonal precursor account for the primary response against the model antigens we have studied.

Requirement for different presenting cells and for different processing mechanisms by human CD4 T helper clones specific for M. tuberculosis antigens.

Human T helper cells specific for mycobacterial antigens have been extensively investigated. Differences have been detected according to antigen specificity and to fine epitope specificity. In this work we have analyzed two additional parameters that allow discrimination among antigen specific T helper cells: requirement for certain types of antigen presenting cells (APC) and requirement for protease-sensitive antigen processing pathways. We used T cell clones from peripheral blood or from pleural exudates, and specific for different antigenic fractions of M. tuberculosis. APC were autologous peripheral blood mononuclear cells, adherent monocytes, adherent pleural monocytes, EBV transformed B lymphocytes and dendritic cells. Seven clones out of twelve were stimulated by all APC irrespective of their specificity, whereas other clones had more selective requirements. When protease inhibitors were used during antigen pulsing of APC, the production of certain epitopes, and thus T cell activation, was impaired with six clones out of sixteen. These results demonstrate that the human T helper repertoire specific for mycobacterial antigens is highly diverse also according to APC populations needed for presentation and to processing mechanisms required for production of the relevant T epitopes.

Analysis of cellular populations in peritoneal effluents of children on CAPD.

The characterization of cellular populations in peritoneal effluents (PE) of CAPD patients has been the subject of few studies. In order to establish a possible correlation between PE cells and clinical parameters, we studied the immunocompetent cells that are found in uncomplicated patients. In this study we used cytofluorimetric analysis to characterize phenotypically immunocompetent PE cells. We studied 17 children with a mean age of 8.7 +/- 5.3 years, who had been on CAPD for a mean duration of 16 months. The patients never suffered from peritonitis. The effluents were collected after overnight dialysis and centrifuged to concentrate cells. Surface phenotype of PE cells was tested with a panel of monoclonal antibodies. The analysis of 40 PE samples showed that the various cell types were present with different frequencies, suggesting that a dominant phenotype cannot be defined. The ratio between M3 positive cells (monocytic-macrophagic lineage) and T3 positive cells (T lymphocytes) showed a tendency to decrease after initiation of CAPD. T4/T8 ratio in all samples did not differ from that of peripheral lymphocytes. A high frequency of activated T cells (41% +/- 13), defined by the presence of DR antigens on T3+ M3- cells, was seen in PE and confirmed by the high frequency of T cells expressing the IL-2 receptor.

Detection of T cell CD4 epitopes in HIV-infected individuals.

HIV antigens can be detected in the circulation of HIV-infected patients and are associated with active virus production. Free virions and shedded gp120 bind CD4 with high affinity. We have studied the expression of Leu3a and OKT4 epitopes on a CD4+ T cell line (HPB-ALL), pretreated with HIV rgp120, and on CD4+ pheripheral blood T lymphocytes of HIV-infected patients. The associated determination of these epitopes (the Leu3a mapping at the gp120 binding site of CD4 and the OKT4 mapping at a site independent of gp120 binding) allowed to monitor binding of gp120 to surface CD4 and maintenance of CD4 expression. The comparison of MFI of gp120-treated versus untreated Leu3a+ HPB-ALL cells suggested that the Leu3a epitope was masked by treatment with 20 micrograms/ml rgp120, while with 1 microgram/ml rgp120 masking was undetectable, although gp120 was bound to cells. The determination of the Leu3a and OKT4 epitopes in 105 HIV-infected individuals and in 50 normal controls, showed that the Leu3a epitope is detected equally well in HIV-infected and in normal subjects, provided the anti-Leu3a is used at saturation. Therefore the binding to epitopes distinct from the gp120-binding site does not seem to be a requisite for the selection of anti-CD4 mAbs for immunophenotyping. To optimize the detection of CD4 masking, a limiting amount of conjugated anti-Leu3a has to be used. Measurements of CD4 binding by gp120 in terms of gp120-free CD4 molecules, as detected by reactivity with anti-Leu3a, may be used to monitor disease progression in HIV-infected subjects.

Characterization of multipotent cells from human adult hair follicles.

Recent works demonstrated the presence of a multipotent epithelial cell population in the bulge region of adult human hair follicles. These cells can be cultured in vitro, thus leading to the preparation of dermal-epidermal substitutes which are applicable in the treatment of burns and ulcers. We evaluated the main marker expression in cells obtained from stripped human hair follicles. A pool of hair follicles were incubated at 37 degrees C and 5% CO(2) in a growth medium. The cells were then labelled with antibodies (anti-CD34, anti-CD38, anti-CD45, anti-CD90, anti-CD133, anti-CD146) and analysed by cytometry. We also used hair follicles for immunohistochemical studies, employing antibodies such as CD34, Actin Smooth Muscle, Filaggrin, Desmin, Vimentin, Glial Fibrillary Acidic Protein, Ki-67, PanCytokeratin, CK15, CK19. The cytometry results revealed that a part of bulge cells were CD34+ (1-2%). CD34+ population comprises both large, CD45-, CD133-, CD146- cells and small, CD45+, CD133+, CD146+ cells. Thus, a part of CD34+ cells present a mature endothelial marker (CD146). An expression of the proliferation marker Ki-67 and the stem cell marker CD34 is present in the follicle bulge region. In conclusion, we observed that the stripped hair follicle has the same multipotent cell population as adult and fetal scalp hair follicles.