Selective expansion and partial activation of human NK cells and NK receptor-positive T cells by IL-2 and IL-15.

IL-2 and IL-15 are lymphocyte growth factors produced by different cell types with overlapping functions in immune responses. Both cytokines costimulate lymphocyte proliferation and activation, while IL-15 additionally promotes the development and survival of NK cells, NKT cells, and intraepithelial lymphocytes. We have investigated the effects of IL-2 and IL-15 on proliferation, cytotoxicity, and cytokine secretion by human PBMC subpopulations in vitro. Both cytokines selectively induced the proliferation of NK cells and CD56(+) T cells, but not CD56(-) lymphocytes. All NK and CD56(+) T cell subpopulations tested (CD4(+), CD8(+), CD4(-)CD8(-), alphabetaTCR(+), gammadeltaTCR(+), CD16(+), CD161(+), CD158a(+), CD158b(+), KIR3DL1(+), and CD94(+)) expanded in response to both cytokines, whereas all CD56(-) cell subpopulations did not. Therefore, previously reported IL-15-induced gammadelta and CD8(+) T cell expansions reflect proliferations of NK and CD56(+) T cells that most frequently express these phenotypes. IL-15 also expanded CD8alpha(+)beta(-) and Valpha24Vbeta11 TCR(+) T cells. Both cytokines stimulated cytotoxicity by NK and CD56(+) T cells against K562 targets, but not the production of IFN-gamma, TNF-alpha, IL-2, or IL-4. However, they augmented cytokine production in response to phorbol ester stimulation or CD3 cross-linking by inducing the proliferation of NK cells and CD56(+) T cells that produce these cytokines at greater frequencies than other T cells. These results indicate that IL-2 and IL-15 act at different stages of the immune response by expanding and partially activating NK receptor-positive lymphocytes, but, on their own, do not influence the Th1/Th2 balance of adaptive immune responses.     

Reversal of coccidioidal anergy in vitro by dendritic cells from patients with disseminated coccidioidomycosis

Coccidioides immitis is a pathogenic, dimorphic fungus found in the southwestern United States and is the causative agent of coccidioidomycosis. Extrathoracic dissemination of coccidioidomycosis is associated with a lack of cellular immunity. Dendritic cells (DCs) have been shown to initiate and modulate cellular immune responses. To determine whether DCs could modulate or initiate the immune response in this disease, monocyte-derived DCs were generated from coccidioidal Ag nonresponsive patients with disseminated coccidioidomycosis and healthy nonimmune individuals. DCs generated from both groups demonstrated phenotypes characteristic of DCs and stimulated strong allogeneic MLR. DCs from patients and healthy nonimmune individuals pulsed with the coccidioidal Ag preparation T27K induced lymphocyte proliferation. Mature DCs were much more efficient than immature DCs in these stimulations. Furthermore, restimulation of T27K-primed PBMC with Ag-pulsed DCs generated a C. immitis-specific cellular immune response in PBMC from patients with disseminated coccidioidomycosis as well as healthy nonimmune individuals. These results show that 1) DCs have the capacity to stimulate specific cellular immune responses from patients with disseminated coccidioidomycosis who are nonresponsive to coccidioidal Ag and healthy nonimmune individuals in vitro; 2) DCs can be used to screen coccidioidal Ags as candidates for human vaccine development; and 3) DC therapy may be useful in the treatment of disseminated coccidioidomycosis.     

Interferon is able to reduce tumor cell susceptibility to human lymphokine-activated killer (LAK) cells

It is known that IL-2 induces lymphocytes to produce interferon-gamma (IFN-gamma) and this IFN type is particularly efficient in inducing tumor cell resistance to natural killer (NK) cell-mediated lysis. We have investigated the effect of IFN on tumor cell sensitivity to LAK cell-mediated cytotoxicity. Pretreatment of the human K562 leukemia and HHMS melanoma with IFN-gamma and the Daudi lymphoma with IFN-alpha caused a significant reduction in sensitivity to lysis by human LAK cells generated in vitro in the presence of human recombinant IL-2 (100 U/ml). The LAK activity was mediated by cells expressing NK cell markers (CD16,NKH1) as well as by cells with T cell markers (CD3, CD5). IFN-treated K562 cells were protected from lysis mediated by all these populations. Supernatants from LAK cultures containing IFN-gamma were able to induce NK and LAK resistance when used to pretreat K562 overnight. Antibodies to IFN-gamma but not to IFN-alpha were able to neutralize this activity. Taken together, these results indicate that the production of IFN-gamma by LAK cells may be of importance in induction of tumor cell resistance to LAK cell-mediated lysis.     

CD25+ regulatory T cell depletion augments immunotherapy of micrometastases by an IL-21-secreting cellular vaccine

IL-21 is an IL-2-like cytokine, signaling through a specific IL-21R and the IL-2R gamma-chain. Because the TS/A mammary adenocarcinoma cells genetically modified to secrete IL-21 (TS/A-IL-21) are strongly immunogenic in syngeneic mice, we analyzed their application as vaccine. In mice bearing TS/A-parental cell (pc) micrometastases, vaccination with irradiated TS/A-IL-21 cells significantly increased the animal life span, but cured only 17% of mice. Spleen cells from cured mice developed CTL activity and produced IFN-gamma in response to stimulation by the AH1 epitope of the gp70env Ag of TS/A-pc. We tested whether the low therapeutic outcome might be due to CD4+CD25+ regulatory T cells (Treg) present in TS/A-pc tumors and draining lymph nodes and whether IL-21 had any effect on these cells. Indeed, CD4+CD25+ cells suppressed IFN-gamma production by splenocytes from immune mice in response to stimulation by the AH1 peptide. Low concentrations of IL-21 (10 ng/ml) failed to reverse the inhibitory activity of CD4+CD25+ cells in an allogeneic MLR, whereas 60 ng/ml rIL-21 partially restored responder T cell proliferation. IL-21R expression on CD25- lymphocytes suggested that IL-21 could be more effective in mice depleted of CD25+ cells. Depletion of Treg cells by a single dose of anti-CD25 mAb combined with TS/A-IL-21 cell vaccine cured >70% of mice bearing micrometastases, whereas anti-CD25 mAb treatment alone had no effect. Successful combined immunotherapy required NK cells, CD8+ T cells, and IFN-gamma. In conclusion, immunotherapy of micrometastases by an IL-21-based cellular vaccine is strongly potentiated by CD25+ cell depletion.     

Loss of IFN-gamma production by invariant NK T cells in advanced cancer

Invariant NK T cells express certain NK cell receptors and an invariant TCRalpha chain specific for the MHC class I-like CD1d protein. These invariant NK T cells can regulate diverse immune responses in mice, including antitumor responses, through mechanisms including rapid production of IL-4 and IFN-gamma, but their physiological functions remain uncertain. Invariant NK T cells were markedly decreased in peripheral blood from advanced prostate cancer patients, and their ex vivo expansion with a CD1d-presented lipid Ag (alpha-galactosylceramide) was diminished compared with healthy donors. Invariant NK T cells from healthy donors produced high levels of both IFN-gamma and IL-4. In contrast, whereas invariant NK T cells from prostate cancer patients also produced IL-4, they had diminished IFN-gamma production and a striking decrease in their IFN-gamma:IL-4 ratio. The IFN-gamma deficit was specific to the invariant NK T cells, as bulk T cells from prostate cancer patients produced normal levels of IFN-gamma and IL-4. These findings support an immunoregulatory function for invariant NK T cells in humans mediated by differential production of Th1 vs Th2 cytokines. They further indicate that antitumor responses may be suppressed by the marked Th2 bias of invariant NK T cells in advanced cancer patients.     

A distinct IL-18-induced pathway to fully activate NK T lymphocytes independently from TCR engagement

NK T lymphocytes are characterized by their ability to promptly generate IL-4 and IFN-gamma upon TCR engagement. Here, we demonstrate that these cells can also be fully activated in the absence of TCR cross-linking in response to the proinflammatory cytokine IL-18 associated with IL-12. NK T cells stimulated with IL-18 plus IL-12 proliferated, killed Fas+ target cells, and produced high levels of IFN-gamma without IL-4. In these conditions, IFN-gamma production was at least 10-fold higher than that upon TCR cross-linking. Interestingly, a 2-h pretreatment with IL-12 plus IL-18 sufficed to maintain the high IFN-gamma-producing potential during subsequent stimulation with anti-TCR mAbs or with the specific Ag alpha-galactosylceramide. Similar effects were observed in vivo, because splenic CD4+ NK T cells from MHC class II-deficient mice secreted IFN-gamma without further stimulation when removed 2 h after a single injection of IL-12 plus IL-18. In conclusion, our evidence for activation of NK T lymphocytes in response to IL-18 plus IL-12 in the absence of TCR engagement together with the maintenance of preferential IFN-gamma vs IL-4 production upon subsequent exposure to specific Ags is consistent with the active participation of this cell population in innate as well as acquired cellular immune responses.     

Interleukin-10 suppresses natural killer cell but not natural killer T cell activation during bacterial infection

Interleukin (IL)-10 is an anti-inflammatory cytokine known to modulate the outcome of sepsis by decreasing pro-inflammatory cytokine production, including IL-12, a main activator of natural killer (NK) cells. We hypothesized that neutralization of IL-10 would increase NK and natural killer T (NKT) cell activation through increased IL-12 in a mouse model of bacterial peritonitis. NK and NKT cell activations were measured by CD69 expression on NK1.1+/CD3- and NK1.1+/CD3+ cells after cecal ligation and puncture (CLP). NK cells were significantly more activated in mice treated with anti-IL-10 antibodies, whereas no such effect was observed in NKT cells. Similarly, intracellular interferon gamma (IFN-gamma) levels were increased in NK cells of anti-IL-10-treated mice, but not in NKT cells. IL-12 and IL-18 levels were increased in both CLP groups, but in anti-IL-10-treated mice, early IL-12 and late IL-18 levels were significantly higher than in controls. Survival at 18 h after CLP was lower in anti-IL-10 mice, which was associated with increased liver neutrophil accumulation. In summary, these data show an activating effect of IL-10 on NK, but not on NKT cells after CLP, which corresponded with decreased survival, higher IFN-gamma production, and increased remote organ neutrophil accumulation. These effects were not mediated by IL-12 and IL-18 alone, and reinforce a role for NK cells in remote organ dysfunction following peritonitis.     

Exposure to Leishmania major modulates the proportion of CD4+ T cells without affecting cellular immune responses

The immune responses of individuals exposed to Leishmania major were evaluated and compared with those of non-exposed volunteers. Forty-one patients with active lesion(s), 43 healed individuals, 15 vaccinees 1 month or 1 year post vaccination, and 15 non-exposed volunteers were studied. Leishmanin skin test (LST) response, proliferative response of lymphocyte (PRL) to L. major antigen, IFN-gamma and IL-4 production, and percentage of L. major-specific CD4+, CD8+ and CD16+/CD56+ cells in peripheral blood mononuclear cells were assessed. Data showed positive LST (>5 mm) in 92% of patients, 98% of healed, and 80% or 43% of vaccinees 1 month and 1 year post vaccination, respectively. Positive PRL (SI>2.5) was displayed in 90%, 84%, 46% and 7% of patients, healed, vaccinated (post 1 year) and non-exposed donors, respectively. The mean +/-S.E. of IFN-gamma was 924 +/- 149, 1,278 +/- 185, 470 +/- 282 or 258 +/- 82 pg/ml in patients, healed cases and vaccinees after 1 month or 1 year, respectively. Positive IFN-gamma responders (>300 pg/ml) were shown in 72% of patients, 81% of healed cases, 31% or 39% of vaccinees and 0% of non-exposed donors. A reduced percentage of CD4+ T-cells and an increased percentage of NK cells were found in exposed individuals compared to non-exposed donors. The data indicated that exposure to L. major modulates the proportion of CD4+ T cells and increases NK cells percentage. However, the cellular immune responses including induction of LST, and IFN-gamma production are increased in exposed individuals.     

The human liver contains multiple populations of NK cells, T cells, and CD3+CD56+ natural T cells with distinct cytotoxic activities and Th1, Th2, and Th0 cytokine secretion patterns.

The human liver contains significant numbers of T cells, NK cells, and lymphocytes that coexpress T and NK cell receptors. To evaluate their functional activities, we have compared the cytotoxic activities and cytokines produced by normal adult hepatic CD3+CD56- (T) cells, CD3-CD56+ (NK) cells, and CD3+CD56+ (natural T (NT)) cells. In cytotoxicity assays using immunomagnetic bead-purified NK cell, T cell, and NT cell subpopulations as effectors, fresh hepatic NK cells lysed K562 targets, while NT cells could be induced to do so by culturing with IL-2. Both NT and T cells were capable of redirected cytolysis of P815 cells using Abs to CD3. Flow cytometric analysis of cytokine production by fresh hepatic lymphocyte subsets activated by CD3 cross-linking or PMA and ionomycin stimulation indicated that NT cells and T cells could produce IFN-gamma, TNF-alpha, IL-2, and/or IL-4, but little or no IL-5, while NK cells produced IFN-gamma and/or TNF-alpha only. The majority of NT cells produced inflammatory (Th1) cytokines only; however, approximately 6% of all hepatic T cells, which included 5% of Valpha24 TCR-bearing NT cells and 2% of gammadeltaTCR+ cells, simultaneously produced IFN-gamma and IL-4. The existence of such large numbers of cytotoxic lymphocytes with multiple effector functions suggests that the liver is an important site of innate immune responses, early regulation of adaptive immunity, and possibly peripheral deletion of autologous cells.     

Immunomodulating effects in vitro of interleukin-2 and interferon-gamma on human blood and bone marrow mononuclear cells

Mononuclear cells (MNC) derived from peripheral blood (PBMNC) of 23 normal donors and 4 AIDS patients, and from bone marrow (BMMNC) of 15 normal donors were incubated at 37 degrees C in culture medium alone or in the presence of either natural or recombinant human interleukin-2 (IL-2) or recombinant human interferon-gamma (IFN-gamma; 1-1,000 U/ml). The cultured cells were washed on days 1, 4 or 7 and tested for various immune functions in vitro and for cell surface phenotype. IL-2, but not IFN-gamma, was found mitogenic for both PBMNC and BMMNC. The natural killer (NK) activity of both PBMNC and BMMNC was the only function tested that was markedly augmented (over 100-fold compared to medium control) by both lymphokines. Pretreatment of PBMNC with IL-2 at greater than or equal to 10 U/ml profoundly suppressed (up to 90%) various functions, such as mitogenic responses (phytohemmagglutinin, concanavalin A, pokeweed mitogen), allogeneic mixed leukocyte reaction, antibody production and T cell colony formation in agar. In contrast, some BMMNC functions were elevated at low doses of IL-2 and IFN-gamma, and significant suppression of BMMNC was seen only with high doses of IL-2 (greater than or equal to 100 U/ml) and IFN-gamma (1,000 U/ml). IL-2 was by far more effective than IFN-gamma in both the amplification of NK activity and the suppression of most of the other functions. IL-2, but not IFN-gamma, was found to activate/induce suppressor cells and increased the proportion of Leu-2+ (CD8) cells in PBMNC; the suppressive effect was time- and dose-dependent. The IL-2-induced suppression could be diminished by inclusion of anti-IL-2 antibody during the pretreatment phase. Similar suppressive effects were noted in PBMNC from AIDS patients. These findings suggest that: (a) high-dose IL-2 may elicit immunosuppression which can be mediated by nondiscriminative highly cytotoxic cells (i.e. lymphokine-activated killer cells) and/or by noncytotoxic, nonspecific suppressor cells, and (b) that PBMNC respond differently to the lymphokines than do BMMNC.     

IL-27 mediates complete regression of orthotopic primary and metastatic murine neuroblastoma tumors: role for CD8+ T cells

We have shown previously that IFN-gamma-inducing cytokines such as IL-12 can mediate potent antitumor effects against murine solid tumors. IL-27 is a newly described IL-12-related cytokine that potentiates various aspects of T and/or NK cell function. We hypothesized that IL-27 might also mediate potent antitumor activity in vivo. TBJ neuroblastoma cells engineered to overexpress IL-27 demonstrated markedly delayed growth compared with control mice, and complete durable tumor regression was observed in >90% of mice bearing either s.c. or orthotopic intra-adrenal tumors, and 40% of mice bearing induced metastatic disease. The majority of mice cured of their original TBJ-IL-27 tumors were resistant to tumor rechallenge. Furthermore, TBJ-IL-27 tumors were heavily infiltrated by CD8(+) T cells, and draining lymph node-derived lymphocytes from mice bearing s.c. TBJ-IL-27 tumors are primed to proliferate more readily when cultured ex vivo with anti-CD3/anti-CD28 compared with lymphocytes from mice bearing control tumors, and to secrete higher levels of IFN-gamma. In addition, marked enhancement of local IFN-gamma gene expression and potent up-regulation of cell surface MHC class I expression are noted within TBJ-IL-27 tumors compared with control tumors. Functionally, these alterations occur in conjunction with the generation of tumor-specific CTL reactivity in mice bearing TBJ-IL-27 tumors, and the induction of tumor regression via mechanisms that are critically dependent on CD8(+), but not CD4(+) T cells or NK cells. Collectively, these studies suggest that IL-27 could be used therapeutically to potentiate the host antitumor immune response in patients with malignancy.     

The effect of interleukin-15 on the expression of killer-cell immunoglobulin-like receptors on peripheral natural killer cells in human

Interleukin (IL)-15 has emerged as a key regulator of both natural killer (NK) cell differentiation and activation. The aim of the present study was to investigate the expansion of the population of cells expressing killer-cell immunoglobulin-like receptors (CD158a and CD158b) in human peripheral lymphocytes by treatment with IL-15. One million peripheral lymphocytes were cultured in RPMI1640 medium alone or in medium containing IL-2 at 100 U/ml or IL-15 at 0.1, 1.0, or 10.0 ng/ml for 48 h. After each incubation, we assessed the natural killing activity and the population of CD16(+)CD158a(+)/b(+) cells and CD8(+)CD158a(+)/b(+) cells. IL-15 increased the NK activity and expanded the populations of CD16(+)CD158a(+)/b(+) cells and CD8(+)CD158a(+)/b(+) cells. These actions were dose dependent, and the effects of IL-15 at 1.0 ng/ml were close to those of IL-2 at 100 U/ml. These findings suggest that IL-15 induces the effector functions of resting NK cells throughout the body, and thereby plays a critical role in the activation of tissue-associated immune responses.     

Differential regulation of IFN-gamma, TNF-alpha, and IL-10 production by CD4(+) alphabetaTCR+ T cells and vdelta2(+) gammadelta T cells in response to monocytes infected with Mycobacterium tuberculosis-H37Ra.

Mycobacterium tuberculosis bacilli readily activate CD4(+) and gammadelta T cells. CD4(+) and gammadelta T cells were compared for their ability to regulate IFN-gamma, TNF-alpha, and IL-10 production, cytokines with significant roles in the immune response to M. tuberculosis. PBMC from healthy tuberculin positive donors were stimulated with live M. tuberculosis-H37Ra. CD4(+) and gammadelta T cells were purified by negative selection and tested in response to autologous monocytes infected with M. tuberculosis. Both subsets produced equal amounts of secreted IFN-gamma. However, the precursor frequency of IFN-gamma secreting gammadelta T cells was half that of CD4(+) T cells, indicating that gammadelta T cells were more efficient producers of IFN-gamma than CD4(+) T cells. TNF-alpha production was markedly enhanced by addition of CD4(+) and gammadelta T cells to M. tuberculosis infected monocytes, and TNF-alpha was produced by both T cells and monocytes. No differences in TNF-alpha enhancement were noted between CD4(+) and gammadelta T cells. IL-10 production by M. tuberculosis infected monocytes was not modulated by CD4(+) or gammadelta T cells. Thus CD4(+) and gammadelta T cells had similar roles in differential regulation of IFN-gamma, TNF-alpha, and IL-10 secretion in response to M. tuberculosis infected monocytes. However, the interaction between T cells and infected monocytes differed for each cytokine. IFN-gamma production was dependent on antigen presentation and costimulators provided by monocytes. TNF-alpha levels were increased by addition of TNF-alpha produced by T cells and IL-10 production by monocytes was not modulated by CD4(+) or gammadelta T cells.     

Heterogeneous human NK cell responses to Plasmodium falciparum-infected erythrocytes

Human NK cells can respond rapidly to Plasmodium falciparum-infected RBC (iRBC) to produce IFN-gamma. In this study, we have examined the heterogeneity of this response among malaria-naive blood donors. Cells from all donors become partially activated (up-regulating CD69, perforin, and granzyme) upon exposure to iRBC but cells from only a subset of donors become fully activated (additionally up-regulating CD25, IFN-gamma, and surface expression of lysosomal-associated membrane protein 1 (LAMP-1)). Although both CD56dim and CD56bright NK cell populations can express IFN-gamma in response to iRBC, CD25 and LAMP-1 are up-regulated only by CD56dim NK cells and CD69 is up-regulated to a greater extent in this subset; by contrast, perforin and granzyme A are preferentially up-regulated by CD56bright NK cells. NK cells expressing IFN-gamma in response to iRBC always coexpress CD69 and CD25 but rarely LAMP-1, suggesting that individual NK cells respond to iRBC either by IFN-gamma production or cytotoxicity. Furthermore, physical contact with iRBC can, in a proportion of donors, lead to NK cell cytoskeletal reorganization suggestive of functional interactions between the cells. These observations imply that individuals may vary in their ability to mount an innate immune response to malaria infection with obvious implications for disease resistance or susceptibility.     

Robust NK cell-mediated human immunodeficiency virus (HIV)-specific antibody-dependent responses in HIV-infected subjects

Antibody-dependent cellular cytotoxicity (ADCC) is a potentially effective adaptive immune response to human immunodeficiency virus (HIV) infection. The study of ADCC responses has been hampered by the lack of simple methods to quantify these responses and map effective epitopes. We serendipitously observed that standard intracellular cytokine assays on fresh whole blood from a cohort of 26 HIV-infected subjects identified non-T lymphocytes expressing gamma interferon (IFN-gamma) in response to overlapping linear peptides spanning HIV-1 proteins. The effector cells were CD3(-) CD4(-) CD8(-) CD14(-) CD2(+) CD56(+/-) NK lymphocytes and degranulated granzyme B and perforin in response to antigen stimulation. Serum transfer assays demonstrated that the specific response was mediated by immunoglobulin G. Fresh blood samples from half of the HIV-infected cohort demonstrated robust HIV peptide-specific IFN-gamma expression by NK cells, predominately to Env, Pol, and Vpu HIV-1 proteins. Responses were readily mapped to define minimal epitopes utilizing this assay. Antibody-dependent, HIV-specific NK cell recognition, involving components of both innate and adaptive immune systems, represents a potentially effective immune response to induce by vaccination.     

IFN-gamma-mediated inhibition of human IgE synthesis by IL-21 is associated with a polymorphism in the IL-21R gene

IL-21 is a cytokine produced by CD4+ T cells that has been reported to regulate human, as well as, mouse T and NK cell function and to inhibit Ag-induced IgE production by mouse B cells. In the present study, we show that human rIL-21 strongly enhances IgE production by both CD19+ CD27- naive, and CD19+ CD27+ memory B cells, stimulated with anti-CD40 mAb and rIL-4 and that it promotes the proliferative responses of these cells. However, rIL-21 does not significantly affect anti-CD40 mAb and rIL-4-induced Cepsilon promoter activation in a gene reporter assay, nor germline Cepsilon mRNA expression in purified human spleen or peripheral blood B cells. In contrast, rIL-21 inhibits rIL-4-induced IgE production in cultures of PBMC or total splenocytes by an IFN-gamma-dependent mechanism. The presence of a polymorphism (T-83C), in donors heterozygous for this mutation was found to be associated not only with lower rIL-21-induced IFN-gamma production levels, but also with a lower sensitivity to the inhibitory effects of IL-21 on the production of IgE, compared with those in donors expressing the wild-type IL-21R. Taken together, these results show that IL-21 differentially regulates IL-4-induced human IgE production, via its growth- and differentiation-promoting capacities on isotype-, including IgE-, committed B cells, as well as via its ability to induce IFN-gamma production, most likely by T and NK cells, whereas the outcome of these IL-21-mediated effects is dependent on the presence of a polymorphism in the IL-21R.     

Melatonin fails to modulate immune parameters influenced by calorie restriction in aging Fischer 344 rats

The aim of this study was to determine if long-term treatment with melatonin (MEL), a purported anti-aging agent, was as effective as calorie restriction (CR) in modulating immune parameters in aging Fischer 344 male rats. Splenic lymphocytes were isolated from 17-month-old rats that, beginning at 6 weeks of age, were treated with MEL (4 or 16 microg/ml in drinking water) and from 17-month-old rats fed ad libitum (AL) or rats fed a CR diet (55% of AL intake). The number of splenic T cell populations and T cell subsets was measured by flow cytometry, the proliferative response of splenocytes to Concanavalin A (Con A) and lipopolysaccharide (LPS) was measured by [(3)H]thymidine incorporation, and the induction of cytokine production (IL-2 and IFN-gamma) was measured by ELISA assay. In addition, the level of the natural killer (NK) cell activity was assessed by fluorimetric assay. CR rats had a higher number of lymphocytes expressing the na?ve T cell marker (CD3 OX22) than AL rats (P < 0.05). CR rats also showed greater induction of proliferative response, IL-2 and IFN-gamma levels following Con A simulation, and NK cell activity than AL rats (P < 0.05). MEL-treated rats did not differ from AL rats in any of these parameters or in any other measurement. These results indicate that MEL treatment is unable to modulate immune function in a manner comparable with that of CR.     

Innate immune response to malaria: rapid induction of IFN-gamma from human NK cells by live Plasmodium falciparum-infected erythrocytes

To determine the potential contribution of innate immune responses to the early proinflammatory cytokine response to Plasmodium falciparum malaria, we have examined the kinetics and cellular sources of IFN-gamma production in response to human PBMC activation by intact, infected RBC (iRBC) or freeze-thaw lysates of P. falciparum schizonts. Infected erythrocytes induce a more rapid and intense IFN-gamma response from malaria-naive PBMC than do P. falciparum schizont lysates correlating with rapid iRBC activation of the CD3(-)CD56(+) NK cell population to produce IFN-gamma. IFN-gamma(+) NK cells are detectable within 6 h of coculture with iRBC, their numbers peaking at 24 h in most donors. There is marked heterogeneity between donors in magnitude of the NK-IFN-gamma response that does not correlate with mitogen- or cytokine-induced NK activation or prior malaria exposure. The NK cell-mediated IFN-gamma response is highly IL-12 dependent and appears to be partially IL-18 dependent. Exogenous rIL-12 or rIL-18 did not augment NK cell IFN-gamma responses, indicating that production of IL-12 and IL-18 is not the limiting factor explaining differences in NK cell reactivity between donors or between live and dead parasites. These data indicate that NK cells may represent an important early source of IFN-gamma, a cytokine that has been implicated in induction of various antiparasitic effector mechanisms. The heterogeneity of this early IFN-gamma response between donors suggests a variation in their ability to mount a rapid proinflammatory cytokine response to malaria infection that may, in turn, influence their innate susceptibility to malaria infection, malaria-related morbidity, or death from malaria.