Expression of the Human Mucosal Lymphocyte Antigen, HML-1, by T Cells Activated with Mitogen or Specific Antigen In Vitro

Expression of the human mucosal lymphocyte antigen, HML-1 (CD103), recently identified as a novel ä^Eβ₇ integrin, was studied on peripheral blood lymphocytes activated with mitogen or specific antigen. HML-1 was up-regulated on PHA activated T-lymphoblasts cultured in 100IU/ml interleukin-2 (IL-2), reaching a peak of >50% positive cells at day 7, and expression was maintained at this level throughout the 28-day culture period. Following a transient decrease in the percentage of L-selectin cells, expression of this molecule was maintained on most PHA T-lymphoblasts. Cells activated by purified protein derivative of M. tuberculosis (PPD) or in mixed lymphocyte culture also up-regulated and maintained HML-1 expression for 14 days. In contrast, in all cases the percentage of CD25⁺ cells rose initially but subsequently declined over the same time periods. When freshly isolated cells from tonsil, spleen, mesenteric lymph node and lung were analysed, only lung contained significant numbers (39 × 6%) of HML-1⁺ cells. In both freshly isolated and activated cell populations the great majority of HML-1⁺ cells co-expressed CD8 although some HML-1⁺CD8^? cells were also present. Production of TGF-β₁ peaked early during T-lymphoblast and MLR cultures and was not related to induction of HML-1 expression. Immunoprecipitation studies showed that the HML-1 molecule expressed on 10-day PHA T-lymphoblasts was indistinguishable from that found on intestinal intraepithelial lymphocytes and that no α⁴β₇ integrin was expressed by these cells. Although HML-1 expression is essentially restricted to mucosal leucocytes in vivo, these experiments show that it is readily induced and maintained along with co-expression of L-selectin following CD8⁺ T-lymphocyte activation in vitro.     

Effects of Purified Protein Derivative (PPD)-Activated Syngeneic Epidermal Cells on a PPD-Specific Rat T-Helper Cell Line

An important question in local immune regulation in the skin is how keratinocytes at inflammatory sites can modify a local T-cell response to antigens introduced via the skin. In the present study we investigated the effects of rat epidermal cells obtained from the site of a tuberculin reaction, on the proliferative response of a syngeneic purified protein derivative (PPD)-specific CD4+ T-cell line. Epidermal cell suspensions from the tuberculin-reactive ears contained 23-37% cells expressing class II transplantation antigens as judged by immunocytochemistry compared with 2-3% in normal epidermis. When comparing the capacity of these two different epidermal cell populations to induce a PPD-specific T-cell response in vitro, it was found that the PPD-reactive epidermal cells induced a lower T-cell response than did normal epidermal cells. This discrepancy cannot be explained by an infiltration of inflammatory cells into the epidermis of tuberculin-reactive ears. Our data indicate that epidermal cells modified during a delayed-type hypersensitivity reaction in vivo may suppress an antigen-specific T-cell proliferation.     

In Vitro Sensitization of Human T Lymphocytes to Hapten (TNP)-Conjugated and Non-treated Autologous Cells is Restricted by Self-HLA-D

By co-culturing human T lymphocytes with TNP-treated autologous B lymphocytes and macrophages for 10 days in vitro, sensitization to TNP-treated autologous cells could be detected in a proliferative assay. By restimulation with different types of allogeneic cells and with cells from donors compatible or incompatible for the HLA-ABC or -D determinants, results were obtained suggesting that the TNP-specific response was restricted by the HLA-D but not the -ABC antigens of the autologous priming cells. Furthermore, our experiments demonstrate that T lymphocytes can also be primed against non-treated autologous cells in vitro and suggest that the HLA-D determinants may be involved also in this auto-sensitization.     

In Vitro Lymphocyte Response to Purified Protein Derivative, BCG, and Mycobacterium leprae in a Population Not Exposed to Leprosy

Lymphocytes from 14 BCG-vaccinated donors, seven tuberculin positive and seven tuberculin negative by skin testing, were stimulated in vitro with four mycobacterial antigens, purified protein derivative (PPD), PPD/BCG, whole BCG bacilli, and whole Mycobacterium leprae and also with Candida antigen and phytohemagglutinin. The response was measured by incorporation of ³H-labeled thymidine. The response to PPD, PPD/BCG, and BCG was found to correlate with the result of skin testing with turberculin. The turberculin-positive group also responded more strongly to M. leprae, whereas the two groups did not differ significantly in their response to Candida antigen or phytohemagglutinin. These findings indicate a certain degree of cross-reactivity between BCG and M. leprae. The use of the lymphocyte transformation test to measure antigenic cross-reactivity is discussed.     

Bystander T Cells Participate in Specific Response to Cockroach Antigen (CR) In Vitro

Allergic reactions due to whole body, body parts and fecal products of cockroach (CR) are characterized by inflammatory reaction that may lead to symptoms of rhinitis or asthma in atopic individuals. Although the majority of T cells at the site of CR hypersensitivity are not antigen specific, the cellular subset and cytokine receptors that participate and control the outcome of the reaction have not been fully studied. In this study, we have used fluorescent activated cell sorter (FACS) analysis to characterize the activation marker and cytokine profile of antigen specific and bystander T cells after in vitro stimulation of peripheral blood lymphocytes with whole body extract of CR antigen. There was significant enhancement of CD69 on blast and bystander T cells in all atopic subjects compared to non‐atopics. Both antigen specific and bystander T cells showed increased expression of HLA‐DR, CD25 and CD71 in 9 of 11 atopic patients compared to control. There was also an increase in CD45RA + and a decrease in CD45RO + cells following antigen stimulation. These results correlated with the increase in the early apoptotic cells observed in patients as measured by Annexin V stain. Our data revealed that there was no difference in the expression of CD95 in both stimulated and bystander T cells. However, there was enhancement of FasL by CR antigen, suggesting that the increased apoptosis that was observed was probably due to the Fas/FasL interaction. Positive intracellular IL2, IL‐4 and IFN‐γ in T cells were observed in only the antigen specific blast cells in 83% of patients studied. These results suggest interplay of memory T cell response, apoptosis, and activated bystander T cells activities in maintaining cellular homeostasis during allergic reaction in cockroach sensitive atopic subjects.     

Bystander T Cells Participate in Specific Response to Cockroach Antigen (CR) In Vitro

Allergic reactions due to whole body, body parts and fecal products of cockroach (CR) are characterized by inflammatory reaction that may lead to symptoms of rhinitis or asthma in atopic individuals. Although the majority of T cells at the site of CR hypersensitivity are not antigen specific, the cellular subset and cytokine receptors that participate and control the outcome of the reaction have not been fully studied. In this study, we have used fluorescent activated cell sorter (FACS) analysis to characterize the activation marker and cytokine profile of antigen specific and bystander T cells after in vitro stimulation of peripheral blood lymphocytes with whole body extract of CR antigen. There was significant enhancement of CD69 on blast and bystander T cells in all atopic subjects compared to non-atopics. Both antigen specific and bystander T cells showed increased expression of HLA-DR, CD25 and CD71 in 9 of 11 atopic patients compared to control. There was also an increase in CD45RA + and a decrease in CD45RO + cells following antigen stimulation. These results correlated with the increase in the early apoptotic cells observed in patients as measured by Annexin V stain. Our data revealed that there was no difference in the expression of CD95 in both stimulated and bystander T cells. However, there was enhancement of FasL by CR antigen, suggesting that the increased apoptosis that was observed was probably due to the Fas/FasL interaction. Positive intracellular IL2, IL-4 and IFN-γ in T cells were observed in only the antigen specific blast cells in 83% of patients studied. These results suggest interplay of memory T cell response, apoptosis, and activated bystander T cells activities in maintaining cellular homeostasis during allergic reaction in cockroach sensitive atopic subjects.     

Suppressor Cells Induced by Purified Protein Derivative of Tuberculin (PPD): the Suppression is Mediated by Cells that Proliferate in Response to Stimulation with PPD

Lymphocytes stimulated with purified protein derivative of tuberculin (PPD) were found to inhibit the PPD stimulation of fresh, autologous lymphocytes. This suppressor effect was exerted after preincubation with both high and low concentrations of PPD. Optimal suppression occurred after preincubation with PPD in concentrations of 5 micrograms/ml and higher, the same concentrations that gave optimal stimulation of DNA synthetsis in primary cultures. The suppressor effect was abolished completely by 'hot pulse' treatment and partly by treatment with colchicine during PPD preincubation, showing that the PPD-induced suppressr cells are generated by cell division. When fresh lymphocytes were incubated together with PPD-pretreated cells in cultures that were not stimulated with PPD, the PPD-stimulated lymphocytes exerted a stimulatory effect on the fresh lymphocytes. This effect was maximal for cells preincubated for 1 h with PPD, decreasing with increasing duration of preincubation with PPD. Possible explanations of this observation are discussed.     

Efficient Propagation and Cloning of Human T Cells in the Absence of Antigen by Means of OKT3, Interleukin 2, and Antigen-Presenting Cells

The analysis of T lymphocytes infiltrating tissues afflicted by autoimmune diseases may provide major clues towards understanding the pathogenesis of such diseases. Currently the best approach to studying heterogeneous populations such as T lymphocytes involves long-term culture and cloning. In order to grow and clone T lymphocytes, regular restimulation with the specific antigen is essential, otherwise growth will stop and/or specificity may be lost. In autoimmune diseases the antigens involved in triggering the immunological reaction of T cells are usually unknown. Therefore an alternative way of stimulating T lymphocytes without loss of specificity is clearly needed. Here we describe the cloning and expansion of antigen-specific T cell clones from the blood of a healthy donor to sizeable numbers of cells (greater than 10(8)) by means of anti-CD3 monoclonal antibody and recombinant IL-2. The results obtained showed that this approach can be used to clone and 'expand' T lymphocytes that retain antigen specificity over a prolonged period, in this case over 10 weeks. This technique has been used to clone and expand T lymphocytes infiltrating the affected tissues in a variety of autoimmune disorders such as Hashimoto's thyroiditis, Graves' disease, and rheumatoid arthritis, and is an efficient method of propagating T cells, by mimicking the antigenic stimulus.     

Lymphocyte Specificity to Protein Antigens. IV. In Vivo and In Vitro Activation of Cytochrome-Specific T Cells Is Dependent on Protein Conformation*

ABSTRACT: Data are presented that suggest that the activation of cytochrome c-specific T lymphocytes is dependent on the integrity of the three-dimensional structure of the protein antigen. These results were obtained both at the population level and at the clonal level. The capacity of the native and denatured forms of cytochrome c to activate a different set of T cell clones was observed both in vivo and in vitro. These results could be explained by the differential capacity of macrophages to process and/or present the two forms of cytochrome c .     

Similar Effects of Treatment with α Interferon on the Protein Synthesis of Human Large Granular Lymphocytes, T Cells, and Monocytes

Preparations of human large granular lymphocytes (LGL), T cells, and monocytes (MC) were obtained through centrifugation on Percoll gradients and preparative E-rosetting. The different preparations contained more than 80% of the appropriate cell type, as judged by their ability to lyse 51Cr-labelled K562 cells, cell morphology, and the presence of cell surface structures recognized by the OKT3, OKT10, Leu 7 and OKM1 monoclonal antibodies. The protein synthesis is unstimulated and alpha interferon (IFN-alpha)-treated cells of the different types was studied by subjecting 35S-methionine-labelled cell extracts to two-dimensional gel electrophoresis. The general pattern of protein synthesis in LGL and T cells was virtually identical, whereas at least 7 major proteins were synthesized at a higher rate in monocytes. The effects of IFN-alpha on the protein synthesis of LGL and T cells were identical, IFN-alpha increasing the rate of synthesis of 9 proteins. These proteins were also expressed, but not always IFN-augmentable, in monocytes. No additional, cell-type associated, IFN-inducible proteins were found. This suggests that the augmenting effect of IFN-alpha on the cytotoxic capacity of LGL, T cells, and monocytes may be to affect common steps in their lytic machineries.     

Human Purified CD8+ T Cells: Ex vivo Expansion Model to Generate a Maximum Yield of Functional Cytotoxic Cells

CD8⁺ T cells are a critical component of the cellular immune response. They play an important role in the control of viral infection and eliminating cells with malignant potential. However, attempts to generate and expand human CD8⁺ T cells in vitro for an adoptive immunotherapy have been conducted with limitation of the very low frequency of CD8⁺ T cells in blood. Therefore, several expansion protocols have been developed to obtain large and efficient numbers of human CD8⁺ T cells for use in adoptive immunotherapies. In this study various common culture conditions using different cytokines IL-2, IL-4, IL-7, IL-10, IL-12 and IL-15 and autologous feeders and sera were investigated to expand human purified CD8⁺ T cells. The importance and the influence of these factors on the growth and phenotype of CD8⁺ T cell were assessed by serially sampling cultures using flow cytometry. We demonstrated that combination of IL-2 (50U/ml) and autologous feeders induced maximal CD8⁺ T cell proliferation (40–50 folds) compared to other cytokines. Immunophenotypic analysis of cultured cells showed that expanded CD8⁺ T cells were activated and differentiated. Furthermore our expansion model also demonstrated that expanded CD8⁺ T cells are functionally cytotoxic active by killing Allogeneic LCLs cells. In conclusion, we have developed a reliable, simple method that uses minimal cell numbers to generate a high yield of functional cytotoxic CD8⁺ T cells, which can be used for the development of cellular immunotherapies.     

Priming to Mycobacterial Antigen In Vivo Using Antigen-Pulsed Antigen Presenting Cells Generated In Vitro is Influenced by the Dose and Presence of IL-4 in APC Cultures

Antigen presenting cells (APC) similar to immature dendritic cells can be generated in vitro from bone marrow precursors. The authors have compared the yield, the phenotype and the function of murine bone marrow cells cultured for 7 or 11 days in either granulocyte macrophage colony stimulating factor alone (GM BMAPC) or in combination with interleukin-4 (GM/IL-4 BMAPC). The results showed that GM/IL-4 BMAPC expressed the highest levels of MHC Class 2 molecules, CD86 /B7-2 and CD80/B7-1 co-stimulatory molecules and the lowest levels of F4/80 macrophage marker. However, when these APC were pulsed with BCG culture filtrate antigen or PPD they were not correspondingly more effective at stimulating activated T lymphocytes in vitro or priming naive T lymphocytes in vivo . Also, in contrast to GM BMAPC, high backgrounds recorded following injections of GM/IL-4 BMAPC without antigen were not consistently reduced by lowering the dose and irradiating the cells prior to administration. The authors conclude that the degree of maturity of BMAPC varies with culture conditions and that this may be an important consideration where BMAPC are to be used in vivo in immunotherapeutic regimens.     

Long-Term Maintenance In Vitro of Human T Cells by Repeated Exposure to the Same Stimulator Cells

Cells from one-way human mixed leukocyte cultures (MLC) which had reverted to small lymphocytes after 2 weeks' incubation responded with accelerated kinetics and higher thymidine incorporation on restimulation with lymphocytes or lymphoblastoid cell line (LCI.) cells having relevant antigens. In contrast to fresh lymphocytes, they did not respond to autologous LCL cells. Cultures could be restimulated every second week with relevant allogeneic lymphocytes and could thus be maintained for periods of up to 4 months. Almost all these cultured cells had T-cell characteristics, during stimulation as well as in their reverted phase. The response to phytohemagglutinin (PHA) successively disappeared with repeated allogeneic restimulation. whereas the response to the relevant lymphocytes and cells of related donors was maintained. When lymphocytes had been, stimulated with autologous LCL cells, the restimulation response was accelerated, although lower than after the primary stimulation Restimulated cultures could not be maintained by further restimulation. Allogeneic and autologous LCL were equally efficient restimulators. A low level of stimulation was also achieved with allogeneic lymphocytes. The PHA response was usually reduced.     

Utilization of the MHC Class I (H-2Kb) Purified Molecule and its Synthetic Peptides for Inhibition of Kb-Specific Suppressor T Cells and their Induction In Vivo by the MHC Peptides

Six synthetic peptides of the MHC class I molecule corresponding to individual H-2K^b participants in amino acid sequences of domains α₁ (peptide 1 and 2) and α₂ (peptides 3,4, 5,6) were selected. K^b-specific suppressor T cells (Ts) were induced in vivo in mice, then pretreated with a set of peptides and assayed by proliferation decrease in a three-cell lymphocyte culture (MLC). The effector function of Ts was abolished by the complex of the α₂-domain peptides (but nol by the α₁-domain peptides) and decreased by particular peptides separately (4, 5, 6) of the α₂-domain. Both α₁- and α₂-domain peptides. added in high concentration, decreased otherwise efficient enrichment of Ts during the absorption-elution procedure on the syngeneie macrophage (Mφ) monolayers. A similar significant effect was observed using the purified K^b molecule (100μg/ml) in the allogeneic Mφ monolayer. Interaction between Ts receptors and some MHC peptides indicates in effector Ts activation in vivo by induction with peptides 5 and 6 of the α₂-domain. The fine mechanisms of interaction between MHC class I molecule epitopes and T-cell receptors of each of the T-cell subsets separately are presently being studied.     

In Vivo Collaboration between Precursor T Cells and Helper T Cells in the Development of Delayed-Type Hypersensitivity Reaction to Influenza Virus in Mice

Nude, athymic mice do not mount a delayed-type hypersensitivity (DTH) response to influenza A virus. A single injection of T helper cells (gamma-irradiated, 2-day immune spleen cells) or three injections over 3 days of a concanavalin-A-activated spleen cell supernatant to virus-sensitized nude mice resulted in a 'normal' DTH response when the mice were challenged with the virus. It was previously shown that the cells responsible for the reaction were T cells and required I-region compatibility. Injection of T helper cells into normal mice did not affect the level of the subsequent DTH response. However, injection of such cells into mice pretreated with anti-thymocyte serum (ATS) restored the ability of the mice to mount a DTH response. The results show that (1) nude mice contain precursor T cells for influenza virus antigen; (2) an I region-restricted response can be generated in the absence of a thymus; and (3) in vivo collaboration between DTH T-cell precursors and helper T cells can be shown to occur in congenitally nude mice and ATS-treated mice.     

The Effect of Antigen Stimulation on the Migration of Mature T Cells from the Peripheral Lymphoid Tissues to the Thymus

Although the maturation and export of T cells from the thymus has been extensively studied, the movement of cells in the opposite direction has been less well documented. In particular, the question of whether T cells which have been activated by antigen in the periphery are more likely to return to the thymus had been raised but not clearly answered. We examined this issue by activating T cells present in the periphery with their cognate antigen, and assessing migration to the thymus. TCR-transgenic cells from OT-I mice (Thy1.2⁺), which recognise the ovalbumin peptide OVA_257–264 in the context of H-2K^b, were transferred into otherwise unmanipulated Thy1.1⁺ C57BL/6 mice. Recipient mice were injected i.v. with 5 μg peptide (SIINFEKL) approximately 24 hours later. The numbers of donor-derived (Thy1.2⁺) cells in the thymus and peripheral lymphoid tissue were determined. The results clearly show increased numbers of transgenic cells in the thymus 3 days after antigenic stimulation. However, since numbers of transgenic cells increased in the spleen and LN in about the same proportion, the data do not support the notion that there is highly increased selective migration of activated T cells to the thymus. Rather, they suggest that a sample of peripheral cells enters the thymus each day, and that the mature immigrants detected in the thymus merely reflect the contents of the peripheral T cell pool.     

In Vitro Effect of Inosiplex on T Lymphocytes. I Influence on T Cells with Receptors for IgG (T γ)

Inosiplex, a complex of inosine and 2-hydroxypropyldimethyl ammonium-4-(acetylamino) benzoate, 1:3 molar ratio, originally developed for antiviral use, is now under wider investigation because of its immunopotentiating properties.

This compound can have some actions on T cells at various stages of differentiation, thus promoting an enhancement of their blastogenic responses to varied mitogenic agents (PHA, Con A, PWM, MLC, tetanus toxoid, and viral antigens).

Our studies demonstrate that under the influence of inosiplex human peripheral blood T lymphocytes bearing Fc IgG receptors have an augmented receptor avidity for SRBC which result in an increased E active rosette formation, and that T cells preincubated with the drug at the appropriate concentrations express more Fc IgG receptors.

Even though Tγ cells exert “in vitro” immunoregulatory properties, the increase in percentage of Tγ lymphocytes do not correlate with a potentiation of the Con A-induced suppressor activity of T cells.

Moreover, the lymphocytes treated with the substance in the absence of Con A exert helper functions, increasing the mitogenic responses of the second culture PHA - treated lymphocytes.

These data appear to suggest a pro-proliferative inosiplex-induced effect which could mask a concomitant suppressor cell induction.     

The Absence of a Human Thymus Lymphocyte Antigen (HTLA) on Basophils and Mast Cells

Basophilic granulocytes and mast Cells of different species have been reported to originate from thymocytes and other lymphocytes. These observations were recently confirmed when evidence was given that thymic antigen is present on rabbit basophilic. In the study reported here, human leukocytes were tested by the immunofluorescence technique and the immunoelectron-microscopy technique to ascertain whether a human lymphocyte antigen (HTLA) could be detected on their surface, We could demonstrate the presence of HTLA on lymphocytes but not on basophilic granulocytes, nor on mast cells in Cryostat secretion of various tissues.