Cellular origin of cytotoxic effectors and secondary educated lymphocytes in human mixed leukocyte reaction

Human lymphocytes sensitized in vitro during a mixed leucocyte reaction (MLR) against an allogeneic-stimulating cell respond by blast transformation and generation of specific cytotoxic effector cells. Both proliferation and cytotoxicity are maximum on Days 6 and 7 of culture. On Day 14, no more dividing cells or cytotoxic cells are detected in such primary cultures. Restimulation by the specific priming cell triggers a secondary proliferative response and rapid reappearance of specific cytotoxic effector cells. The velocity sedimentation cell separation method which separates cells according to their size was applied to human lymphocytes sensitized in vitro during an MLR on Day 7 of culture. Blast cells were separated from nondividing small lymphocytes. It was shown that: (1) cytotoxic effectors generated at the peak of a primary response are exclusively present in the isolated blast population; (2) highly cytotoxic secondary effector cells are induced to reappear mainly from the blast-derived population upon restimulation; and (3) secondary educated proliferative cells mainly derive from the blast population. Conversely, the blast-depleted small lymphocyte population is operationally depleted of cells able to respond by proliferation to the priming cell while responding normally against third party control cells. HLA-D region specificity of the secondary proliferative response is suggested.     

Antigenic requirements for the generation of secondary cytotoxicity.

The in vitro role of products of the H-2 complex in restimulation of secondary proliferative and cytotoxic responses has been studied. In this paper we have studied in particular the role of CD antigens, the target antigens for cytotoxic T lymphocytes. We have used UV-treated cells that express their CD antigens but no LD. Like others, we demonstrate that CD antigens alone are able to generate a secondary cytotoxic response and furthermore that these CD antigens must be the same as those used for primary sensitization. However, in contrast to the results of others, in some cases UV-treated cells were unable to restimulate such a response even when appropriate controls showed that the CD antigens on UV-treated cells were functional. Furthermore, in these cases the addition of an LD stimulus in the presence of UV-treated cells results in a secondary cytotoxic response that is significantly greater than that elicited by LD restimulation alone. Such potentiation is not observed if the CD antigens used for restimulation are different from those used for the primary sensitization. In addition, a non-H 2 differences(s), presumably the M1s locus, appears to be able to generate a secondary response. The implication of these results are discussed in terms of "memory" T lymphocytes in cell-mediated immunity.     

Specific selection of cytotoxic effector cells: the generation of cytotoxic T cells in rat thoracic duct lymphocyte populations positively or negatively selected for reactivity to specific strong histocompatibility alloantigens.

These studies consider the generation of cytotoxic T lymphocytes (CTL) from precursors (CTLP) present in rat thoracic duct lymphocytes after stimulation with strong alloantigens. Also, they explore the relationship between CTLP and "initiator" (I) lymphocytes responsible for specific GVH and MLI reactions. Positively selected TDL populations prepared in bulk MLI cultures show enriched GVH and MLI reactivity for the selecting major histocompatibility complex (MHC) haplotype, but no cytotoxic activity, raising the possibility that I and CTLP may belong to different subpopulations, and the latter failed to differentiate or to survive under these culture conditions. Restimulation of these cells in Marbrook culture vessels with the original priming haplotype under conditions suitable for generating killer cells in vitro resulted in greatly increased specific CTL activity with accelerated kinetics soon after priming and normal kinetics later. These findings indicate that "memory" killer cells can be generated in a previously stimulated lymphocyte population that had no overt cytotoxic activity. Restimulation with third party haplotypes failed to give CTL activity either to specific or to third party targets. Negatively selected TDL populations prepared by "filtration" through x-irradiated F1 rats, depleted of specific GVH and MLI responses, were also depleted of the ability to generate CTL in Marbrook cultures stimulated with the selecting haplotype. Stimulation with third party haplotypes, or with both third party and specific haplotypes together, gave CTL effective only against the third party target.     

Secondary response of in vitro-primed human lymphocytes to allogeneic cells

In vitro-primed human lymphocytes proliferate in a secondary mixed lymphocyte reaction (MLR) under the control of MLR-S specificities. HL-A antigens are unable to induce a secondary Proliferation. In familial haploidentical combinations, the secondary proliferation is specific for the priming MLR-S specificity, i.e., as early as 24 to 48 hours after the re-stimulation, a clearcut response is observed toward the sensitizing MLR-S specificity. The secondary response is reflected in acceleration of the reaction rather than in the peak of (³H) TdR uptake. However, when either haploidentical familial primed responding cells or unrelated cells primed toward MLR-S homozygous cells were used, no early typing response was observed against unrelated cells. The level of (³H) TdR incorporation toward cells which possessed and those which did not possess the priming specificity was identical until day 3–4. Noneless, the peak response toward cells possessing the priming MLR-S specificity occurs regularly 24 to 48 hours prior to the peak response toward the cells negative for the priming specificity (day 3–4 as opposed to day 5). Technical improvements are therefore needed before such a technique will provide a clearcut MLR-S typing methodology.     

Altered T Lymphocyte Proliferation upon Lipopolysaccharide Challenge Ex Vivo

Context

Sepsis is characterized by the development of adaptive immune cell alterations, which intensity and duration are associated with increased risk of health-care associated infections and mortality. However, pathophysiological mechanisms leading to such lymphocyte dysfunctions are not completely understood, although both intrinsic lymphocyte alterations and antigen-presenting cells (APCs) dysfunctions are most likely involved.

Study

The aim of the current study was to evaluate whether lipopolysaccharide (LPS, mimicking initial Gram negative bacterial challenge) could directly impact lymphocyte function after sepsis. Therefore, we explored ex-vivo the effect of LPS priming on human T lymphocyte proliferation induced by different stimuli.

Results

We showed that LPS priming of PBMCs reduced T cell proliferative response and altered IFNγ secretion after stimulation with OKT3 but not with phytohaemagglutinin or anti-CD2/CD3/CD28-coated beads stimulations. Interestingly only LPS priming of monocytes led to decreased T cell proliferative response as opposed to LPS priming of lymphocytes. Importantly, LPS priming was associated with reduced expression of HLA-DR, CD86 and CD64 on monocytes but not with the modification of CD3, CTLA4, PD-1 and CD28 expressions on lymphocytes. Finally, IFNγ stimulation restored monocytes accessory functions and T cell proliferative response to OKT3.

Conclusion

We conclude that LPS priming does not directly impact lymphocyte functions but reduces APC’s capacity to activate T cells. This recapitulates ex vivo indirect mechanisms participating in sepsis-induced lymphocyte alterations and suggests that monocyte-targeting immunoadjuvant therapies in sepsis may also help to improve adaptive immune dysfunctions. Direct mechanisms impacting lymphocytes being also at play during sepsis, the respective parts of direct versus indirect sepsis-induced lymphocyte alterations remain to be evaluated in clinic.     

In vitro secondary MLR. I. Kinetics of proliferation and specificity of in vitro primed responder cells.

We have examined the kinetics and specificity of secondary in vitro mixed lymphocyte reactions (MLR). With limited numbers of primed responder cells (PRC) in the presence of "excess antigen" it was possible to obtain proliferative responses that were proportional to the number of PRC initially placed in culture. The responding cells, after an initial lag period, seem to grow exponentially until day 3 of culture. The responses of PRC (with the strain combinations and culture conditions described in this report) seemed to be directed toward stimulator cell determinants whose expression was determined by genes in the I region of the MHC. In one case, the relevant incompatibilities could be further restricted to the I-A region. Although PRC responded best to stimulator cells sharing the I region with the priming stimulator cell, apparent cross-reactivity could be observed by restimulating PRC with stimulator cells that did not carry the MHC haplotype of the priming stimulator cell. The rate of proliferation (measured as 3H-thymidine incorporation) in these apparent cross-reactions was reproducible and comparable to the rate observed in response to the priming stimulator cell. It was possible, therefore, to estimate the proportion of PRC that reacted in the presence of third party stimulator cells compared to the response of these PRC to the priming stimulator cells. We have estimated that the response of A (B6) PRC against H-2d and H-2s haplotype stimulator cells is about half of the response of these PRC to H-2b, the priming stimulator cell.     

Interaction of reovirus with cell surface receptors. II. Generation of suppressor T cells by the hemagglutinin of reovirus type 3

In studying reovirus interactions with lymphocytes, we have found that reovirus type 3, but not type 1, inhibits the in vitro proliferative response of murine splenic lymphocytes to concanavalin A (Con A). By analyzing recombinant clones containing genes from both reovirus types 1 and 3, we found that the S1 gene, the gene that encodes the viral hemagglutinin, is responsible for the inhibitory effect. In addition we found that type 3, but not type 1, generates suppressor T cells in vitro capable of suppressing Con A proliferation. By analyzing recombinant clones, we also found that the viral hemagglutinin is responsible for the generation of suppressor T cells by reovirus type 3. These effects were observed whether UV-inactivated or live virus was used. Reovirus type 3 inhibition of the proliferative response of murine splenic lymphocytes to Con A was blocked by anti-reovirus type 3 antibody but not by anti-reovirus type 1 antibody. Antiviral antibody had no effect on the ability of reovirus type 3 induced suppressor cells to inhibit Con A proliferation. We have previously demonstrated a receptor on murine lymphocytes for the hemagglutinin of reovirus type 3, and our results suggest that the in vitro suppression of Con A proliferation of murine lymphocytes by reovirus type 3 is secondary to the interaction of the viral hemagglutinin with a receptor on the surface of murine lymphocytes, which results in the generation of functionally active suppressor T cells.     

Specific absorption of T lymphocytes committed to soluble protein antigens by incubation on antigen-pulsed macrophage monolayers.

Monolayers of macrophages (Mphi) pulsed with antigen were used as immunosorbents for T lymphocytes from guinea pigs primed to soluble protein antigens. T lymphocytes were cultured on the Mphi monolayers for 4 hr, then aspirated and reincubated on a fresh monolayer pulsed with the same antigen for a second and a third step. T lymphocytes so treated were selectively deprived of cells responding in assay for antigen-dependent proliferation against the antigen used for pulsing the absorbing monolayer, but maintained their response to other antigens. The lymphocytes adhering to the Mphi of the absorbing monolayer were capable of giving a full response to the antigen used for pulsing the Mphi of the monolyers. The proliferative response of F1 T lymphocytes to antigen in association with Mphi of either parental strain could be absorbed leaving the response to antigen in association with Mphi of the other parental strain. The absorption of the proliferative response was not inhibited by addition of excess soluble antigen to the medium of the absorption culture. Our results indicate that specific guinea pig T lymphocytes responding by proliferation to soluble protein antigens recognize and bind specifically to a complex of Ia antigen and protein antigen at the surface of the Mphi.     

C2 deficiency in man. genetic relationship to a mixed lymphocyte reaction determinant (7a*)

Three unrelated individuals with, respectively, lupus erythematosus, polyarteritis, and membranoproliferative glomerulonephritis and totally deficient in the second component of complement are demonstrated to be mutually poorly reactive in mixed lymphocyte culture and homozygous for the mixed lymphocyte reaction determinant (MLR-S or LD) short 7a (7a^*). The gene controlling the elaboration of C2 in man is shown to be separate from, and probably to map outside of, the second locus ofHL-A and theMLR-S locus. Genetic linkage disequilibrium is strongly suggested between HL-A 10, W18, 7a^*, and C2 deficiency.     

The role of cellular proliferation in the induction of experimental autoimmune thyroiditis (EAT) in mice

Experimental autoimmune thyroiditis (EAT) can be induced in susceptible strains of mice by injection of mouse thyroglobulin (MTg) and adjuvant. Lymphocytes from immunized mice develop a proliferative response to MTg which generally correlates with the development of EAT. We utilize a cell transfer system wherein spleen cells from CBA/J mice primed with MTg and lipopolysaccharide (LPS) in vivo are activated by culture with MTg in vitro to transfer EAT to naive recipients. In vivo priming of CBA/J mice is required to develop an antigen specific proliferative response to MTg. This response is optimal between 48 and 90 hr of culture at an MTg concentration of 125-250 micrograms/ml. The correlation between proliferation and transfer of EAT is not absolute as primed Balb/c X CBA/J F1 and AKR lymphocytes do not proliferate detectably in response to MTg but can be activated to transfer EAT; primed Balb/c lymphocytes neither proliferate nor transfer EAT. Proliferation per se is not sufficient to activate cells to transfer EAT as culture with nonspecific mitogens is not effective in activating primed CBA/J spleen cells to transfer EAT. However, lymphoblasts generated during in vitro culture of primed CBA/J spleen cells with MTg are responsible for transfer of EAT; small lymphocytes are ineffective. We conclude that antigen specific proliferation in response to MTg is essential in activating lymphocytes in vitro to transfer EAT.     

The plaque-forming cell response of human blood lymphocytes. I. PFC response of lymphocytes to formalin-treated staphylocci.

The plaque-forming cell and proliferative responses of human peripheral blood lymphocytes induced by formalin-treated Staphylococcus aureus of the Cowan strain were studied in vitro. Human blood mononuclear cells were incubated for 6 days with staphylococci in culture medium RPMI 1640 supplemented with 10% human AB serum. The number of anti-sheep erythrocyte plaque-forming cells was determined by the Jerne technique. Lymphocyte proliferation was measured by [³H]thymidine incorporation. Individual lymphocyte donors could be classified as high or low responders to staphylococci. Lymphocyte proliferation appeared necessary for the generation of plaque-forming cells. The plaque-forming cell response was greatly influenced by the source of the human AB serum used in the culture medium. The addition of hydrocortisone to the culture medium augmented the plaque-forming cell response. Human B lymphocytes prepared by passage through a column containing Sepharose 4B conjugated to anti-human F(ab)₂ generated plaque-forming cells when incubated with staphylococci. However, the addition of T lymphocytes to these B-lymphocyte preparations augmented the plaque-forming cell response to staphylococci.     

Synergy among rat T cells in the proliferative response to alloantigen.

A synergistic interaction in the proliferative response to alloantigen is described for mixtures of rat thymus and lymph node cells. The optimal conditions for synergy are quantitatively defined. Regression analysis of the slope of the dose-response curve has been utilized to estimate the degree of interaction in thymus-lymph node cell mixtures. The slope of the response of cell mixtures was noted to be significantly greater than the slope for the response of lymph node cells alone. Irradiation was shown to have a differential effect on the response of thymus and lymph node cells in mixtures. Irradiated thymus cells retained the capacity for synergy in mixtures, whereas irradiated lymph node cells did not. Additional studies have demonstrated that both de novo protein synthesis and specific antigen recognition by both responding cell populations in mixtures was required for maximal synergy. These studies demonstrate that synergy cannot be explained as an artifact of altered cell density in vitro. They establish that thymus cells and lymph node cells represent distinct subsets which manifest qualitatively different functions in the proliferative response to alloantigen. Thymus cells can respond directly to alloantigen by proliferation but also have the capacity to amplify the proliferative response of lymph node cells—a capacity which is resistant to X irradiation but requires recognition of alloantigen and de novo protein synthesis. Lymph node cells may similarly respond by proliferation to alloantigen but lack the amplifier activity of thymus cells. Synergy for rat lymphoid cells, like mouse lymphoid cells, has been shown to involve an interaction of thymus-derived lymphocytes.     

Human cellular immune response to the saliva of Phlebotomus papatasi is mediated by IL-10-producing CD8+ T cells and Th1-polarized CD4+ lymphocytes

Background

The saliva of sand flies strongly enhances the infectivity of Leishmania in mice. Additionally, pre-exposure to saliva can protect mice from disease progression probably through the induction of a cellular immune response.

Methodology/Principal Findings

We analysed the cellular immune response against the saliva of Phlebotomus papatasi in humans and defined the phenotypic characteristics and cytokine production pattern of specific lymphocytes by flow cytometry. Additionally, proliferation and IFN-γ production of activated cells were analysed in magnetically separated CD4+ and CD8+ T cells. A proliferative response of peripheral blood mononuclear cells against the saliva of Phlebotomus papatasi was demonstrated in nearly 30% of naturally exposed individuals. Salivary extracts did not induce any secretion of IFN-γ but triggered the production of IL-10 primarily by CD8+ lymphocytes. In magnetically separated lymphocytes, the saliva induced the proliferation of both CD4+ and CD8+ T cells which was further enhanced after IL-10 blockage. Interestingly, when activated CD4+ lymphocytes were separated from CD8+ cells, they produced high amounts of IFN-γ.

Conclusion

Herein, we demonstrated that the overall effect of Phlebotomus papatasi saliva was dominated by the activation of IL-10-producing CD8+ cells suggesting a possible detrimental effect of pre-exposure to saliva on human leishmaniasis outcome. However, the activation of Th1 lymphocytes by the saliva provides the rationale to better define the nature of the salivary antigens that could be used for vaccine development.     

Antigen-specific suppressor cell activity in man: I. Temporal, antigenic, and proliferative requirements

The temporal, antigenic, and proliferative requirements of antigen-specific suppression of the in vitro plaque-forming cell (PFC) response of human peripheral blood mononuclear cells (PBM) were studied. Suppressor cell activity (SCA) was generated by priming PBM with high doses of the T-cell-dependent antigen ovalbumin (OA) and measured by adding washed primed cells to target PFC cultures. Priming with high doses of OA was shown to induce a population of antigen-specific T lymphocytes which interfered with the anti-OA PFC response of optimally stimulated target cultures. The generation of SCA was demonstrated following as little as 24 hr of high-dose OA priming and could be abrogated by prolonged priming (72 hr) or by pretreatment with mitomycin prior to priming. The expression of optimal SCA required the addition of primed PBM at the initiation of the target culture and could be directly correlated to both the OA concentration used for priming and the number of primed cells added. Higher priming doses of OA (up to 100 μg) generated increasing numbers of cells capable of antigen-specific SCA as measured via a cell dilution protocol. Our data suggest that an antigen-driven and dose-dependent expansion of an antigen-specific T-suppressor cell pool is an early regulative event limiting the in vitro PFC response of human lymphocytes to OA.     

Induction and properties of DNP-specific T cells in mice sensitized to DNP-coupled mycobacterium.

Splenic T lymphocytes from mice sensitized to 100 microgram of DNP-coupled mycobacterium (DNP-Tbc) showed in vitro proliferative response against DNP- or TNP-conjugated protein antigens. The increased uptake of 3H-thymidine induced by DNP-HSA was partially inhibited by the addition of 10(-4)M DNP-EACA. DNP-AECM-Ficoll did not induce any significant proliferative responses in DNP-Tbc-primed T cell population. However, priming with DNP-Tbc augmented anti-DNP IgG antibody response induced with DNP-Ficoll. The augmentation of IgG response was not due to the presence of DNP-primed B cells or anti-DNP antibody. The results showed that the priming with DNP-Tbc induced DNP-reactive T helper cells which could be triggered with DNP-Ficoll. The possible role of mycobacterium in the induction of hapten-specific T helper cells is discussed.     

Human macrophage-lymphocyte interaction in proliferation to soluble antigen: II. Characterization of lymphocyte binding to antigen-pulsed macrophage monolayers

Human peripheral blood T lymphocytes from individuals immune to tetanus toxoid (TT) or mumps soluble antigen can be depleted of cells capable of proliferating in response to these antigens by specific adsorption to antigen-pulsed macrophage (Mφ) monolayers. Specific adsorption is manifest by depletion of proliferative activity (measured by tritiated thymidine incorporation) in the fraction of T lymphocytes nonadherent to Mφ monolayers pulsed with the appropriate antigen. The occurrence of T lymphocyte-Mφ binding is supported by experiments which failed to detect cell-free factors or cells capable of inhibiting lymphocyte proliferation in the nonadherent lymphoid population. A quantitative analysis of the immunoadsorption assay suggests that proliferative activity representing at least 90% of that displayed by reciprocal control lymphocytes can be depleted on Mφ monolayers. Experiments employing varying numbers of adsorbing Mφ relative to T lymphocytes have established practical limits which predict the efficiency of adsorption. Kinetic studies indicate that specific adsorption occurs within 1 hr, with maximal levels or binding evident by 4–6 hr; binding remains stable for at least 16 hr. Specific adsorption is temperature dependent, occurring maximally at 37 °C, but not at all at 4 °C. T lymphocyte-Mφ interaction is complex and includes more than a recognition of pulsed antigen alone. This conclusion is supported by the finding that antigen-pulsed Mφ fail to absorb MLC-nonidentical allogeneic lymphocytes and that a heterologous antiserum directed against human Ia-like molecules (p23,30) inhibits specific T cell-Mφ binding.     

Cellular immunity in the mouse. I. In vitro lymphocyte reactivity

The mixed lymphocyte culture (MLC) and antigen-mediated proliferative response represent important correlates to the in vivo phenomena of allograft rejection and delayed hypersensitivity. This study defines an in vitro model to measure mouse lymphocyte responsiveness to allogeneic cells, antigen (tuberculoprotein), and nonspecific mitogens. Results describe optimal cells concentration, time and conditions of culture. Optimal conditions include the use of high cell concentration, flat-bottomed vials, RPMI-1640 medium, and fresh human serum. Peripheral blood lymphocytes demonstrated greater proliferation than lymph node lymphocytes, which in turn demonstrated greater activity than splenic lymphocytes. Significant proliferation occurred in serum-free media, dialyzed against fresh serum and supplemented with hydrocortisone and carrier protein. The MLC response in the mouse appears dependent on multiple subpopulations of cells and on soluble substances produced by them.     

An antibody cross-reacting with LA and FOUR antigens of theHL-A system

A volunteer was immunized by planned blood transfusions from an LA and FOUR incompatible donor. Among the several antibodies produced, an antibody population was found that cross-reacts with both LA and FOUR antigens; both these crossreacting determinants were present in the immunizing donor. Monospecificity of the antibody has been proven by adsorptions and elutions. Resistance to lysis experiments excluded the possibility that this antibody does not react against LA and FOUR, but reacts instead against an antigen belonging to another independentHL-A locus with alleles in strong linkage disequilibrium withLA andFOUR alleles. The following hypotheses are therefore formulated: a)LA andFOUR factors have structural analogies; b) LA and FOUR molecules lie in close proximity on the cell surface so that the appearance of a “hybrid” antigenic determinant becomes possible.     

In vitro glucocorticoid inhibition of steroid-treated residual circulatory human lymphocytes

Various doses of glucocorticoids given in vivo caused a similar degree of maximal lymphopenia. The sensitivities of mitogen-induced proliferation to the suppressive effects of glucocorticoid added in vitro were studied in residual lymphocytes obtained after steroid injection. Methylprednisolone (MP) administered intravenously depleted circulatory lymphocytes and reduced markedly the proliferative responses of residual cells to mitogens (phytohemagglutinin and concanavalin A) 4 to 8 hr after the injection. The addition of MP in vitro to the residual cells further inhibited the cell proliferation. The degrees of proliferation inhibition induced by in vitro MP were compared in cells obtained at various intervals after MP injection. At each specific mitogen concentration, lymphocytes obtained at various intervals were inhibited to a similar degree by MP in the cultures. There was no evidence that cells obtained at the period of maximal lymphopenia, 4 to 8 hr after MP injection, were more resistant to the inhibition of glucocorticoid added in vitro. Hence, the residual lymphocytes were not “steroid-resistant” in the sense of proliferative responses to T-cell mitogens. These results indicate the mechanism of lymphocyte sequestration is unrelated to the suppressive effects of glucocorticoid on cell proliferation.     

Human immune responses to hapten-conjugated cells. II. The roles of autologous and allogeneic histocompatibility determinants in proliferative responses in vitro.

Proliferative responses of human lymphocytes primed in vitro to autologous TNP-cells were found to be associated with autologous D-region determinants irrespective of HLA-B locus antigens. Family studies of secondary TNP-conjugate proliferative responses demonstrated a gene dosage effect in this phenomenon. Moreover, co-culture with allogeneic cells did not affect the net TNP-conjugate proliferative responses of primed responder cells, suggesting that HLA-D region preference was due to a requirement for representation of TNP-molecules in association or combination with autologous MHC structures. Alloantigens were found to influence the sensitization of lymphocytes to autologous hapten-conjugated cells. Co-culture of allogeneic and TNP-modified autologous stimulator cells in primary cultures enhanced the secondary TNP proliferative response. Sensitization of human lymphocytes to allogeneic cells alone did not prime responses to autologous modified cells. However, priming lymphocytes to modified autologous cells potentiated responses to allogeneic cells. The data suggest a complex relationship between responses to alloantigens and modified autologous cells.