Altered cytokine production and impaired antimycobacterial immunity in protein-malnourished guinea pigs

Protein malnutrition leads to multiple detrimental alterations of host immune responses to mycobacterial infection. In this study, we demonstrated that splenocytes from low-protein (LP) guinea pigs vaccinated 6 weeks previously with attenuated Mycobacterium tuberculosis H37Ra failed to control the accumulation of virulent M. tuberculosis H37Rv in cocultured autologous peritoneal macrophages, despite the fact that they were able to control the accumulation of virulent tubercle bacilli in cocultured syngeneic peritoneal macrophages from normally nourished guinea pigs as successfully as did those from high-protein (HP) counterparts. Vaccine-induced growth control of virulent M. tuberculosis H37Rv in these cocultures appeared to be mediated by CD4 lymphocytes but not CD8 cells. Tuberculin (purified protein derivative [PPD])-induced lymphoproliferation was markedly impaired in vaccinated LP guinea pigs, and the depletion of CD4 lymphocytes significantly decreased lymphocyte proliferation whereas CD8 cell depletion did not. Protein malnutrition also impaired the abilities of cells from vaccinated LP guinea pigs to produce cytokines, including interferon, tumor necrosis factor alpha (TNF-alpha) and transforming growth factor beta (TGF-beta), in response to PPD, despite the demonstration of higher serum levels of TNF-alpha and TGF-beta after an intravenous injection of PPD into LP guinea pigs. In contrast, peritoneal macrophages from protein-malnourished guinea pigs produced a higher level of TGF-beta 4 days after infection in vitro with M. tuberculosis H37Rv than did those from protein adequate controls. These results suggest that dietary protein malnutrition impairs vaccine-induced resistance to M. tuberculosis, in part, by altering the cytokine profile to favor macrophage deactivation.     

Protection by CD4 or CD8 T cells against pulmonary Mycobacterium bovis bacillus Calmette-Guérin infection

Mice deficient in CD8 T cells demonstrated levels of Th1 cytokines and granulomatous responses in the lungs very similar to those demonstrated by normal control mice and were fully capable of controlling pulmonary mycobacterial infection by Mycobacterium bovis BCG as assessed at day 37 postinfection. In comparison, mice deficient in CD4 T cells had similar levels of interleukin-12 (IL-12) and tumor necrosis factor alpha but lower levels of gamma interferon in the lungs and were still able to mount tissue granulomatous responses and control pulmonary mycobacterial infection. In contrast, IL-12(-/-) mice with impaired CD4 and CD8 T-cell responses had a markedly weakened control of infection, whereas SCID mice deficient in all T cells succumbed to such pulmonary mycobacterial infections.     

Lymphocyte-dependent inhibition of growth of virulent Mycobacterium tuberculosis H37Rv within human monocytes: requirement for CD4+ T cells in purified protein derivative-positive, but not in purified protein derivative-negative subjects

Protective human immunity to Mycobacterium tuberculosis (M. tb) has proven difficult to characterize, in part because of technical obstacles to in vitro infection of human cells with virulent M. tb. We established a reproducible method of infecting human monocytes (MN) with the virulent M. tb strain H37Rv that did not reduce MN viability. TNF-alpha had no effect on replication of H37Rv within MN, and IFN-gamma mediated only a 1.9-fold reduction in bacterial growth. In contrast, nonadherent cells (NAC) from purified protein derivative (PPD)-positive and PPD-negative subjects reduced intracellular growth of H37Rv by 6- and 10.6-fold, respectively (p = 0.007 and p = 0.005). CD4+ T cells were essential to growth inhibition mediated by NAC of PPD-positive subjects, whereas containment of M. tb by NAC of PPD-negative subjects did not require CD4+ cells. CD8+ T cells did not contribute to protection mediated by NAC of either group. Supernatants of cocultured H37Rv-infected MN and NAC only partially reduced intracellular growth of M. tb despite containing nanogram concentrations of TNF-alpha and IFN-gamma. Neutralizing antibodies to TNF-alpha, IFN-gamma, and IL-12 failed to affect the NAC-mediated growth limitation. NAC treated with emetine retained approximately 40% of their capacity to contain intracellular H37Rv, however. These studies indicate that protective human recall responses to M. tb are mediated primarily by CD4+ T cells, whereas CD4-CD8- lymphocytes may contribute to innate immunity to M. tb. The ability of NAC to activate M. tb-infected MN is only partly attributable to soluble mediators and may also involve contact-mediated mechanisms.     

Functional diversity of the CD8(+) T-cell response to Epstein-Barr virus (EBV): implications for the pathogenesis of EBV-associated lymphoproliferative disorders

Epstein-Barr virus (EBV)-specific CD8(+) cytotoxic T cells are thought to be critical for the control of EBV, which persists in healthy individuals as a latent infection of B cells. However, recent observations have indicated that CD8(+) T-cell responses are not uniformly cytotoxic and that CD8(+) T cells may be subdivided into type 1 and type 2 subsets that parallel the classically described Th1 and Th2 subsets of CD4(+) T cells. Using two-color flow cytometric analysis of intracellular cytokine expression at the single-cell level, we have identified two distinct but overlapping subsets of EBV-specific CD8(+) T cells, the first of which expressed high levels of interferon gamma (IFNgamma), but little or no interleukin-4 (IL-4), whereas the second subset was IFNgamma+/IL-4(+) double-positive. A significant proportion of EBV-specific CD8(+) T cells also expressed IL-13. Subsequent analysis of a panel of 27 EBV-specific CD8(+) T-cell clones showed inverse relationships between EBV-specific cytotoxicity and secretion of IL-4, IL-10, and IFNgamma, respectively. IL-10 was not secreted by the 11 most strongly cytotoxic clones, suggesting that IL-10 secretion may provide a functional definition of an EBV-specific type 2 CD8(+) T-cell subset with reduced EBV-specific cytotoxicity. Finally, we have demonstrated that EBV-specific CD8(+) T cells that express type 2 cytokines possess the ability to activate resting B cells. EBV-specific CD8(+) T cells thus have the potential to reactivate latent EBV infection in vivo and may contribute to the development of EBV-associated lymphoproliferative disorders and lymphoma.     

Preparation of recombinant gilthead seabream (Sparus aurata) growth hormone and its use for stimulation of larvae growth by oral administration

Optimal protective immunity against babesial infection is postulated to require both complement-fixing and opsonizing antibodies in addition to gamma interferon (IFN-gamma)-mediated macrophage activation. The rhoptry-associated protein 1 (RAP-1) of Babesia bigemina induces partial protective immunity and is a candidate vaccine antigen. Previous studies demonstrated that cattle immunized with native protein that were subsequently protected against challenge had a strong IFN-gamma and weaker interleukin-4 (IL-4) response in immune lymph node lymphocytes that reflected the cytokine profile of the majority of CD4(+) T-cell clones obtained from peripheral blood. RAP-1-specific T helper (Th) cell clones that coexpress IFN-gamma and IL-4 are typical of numerous parasite-specific clones examined. However, the function of such cells as helper cells to enhance immunoglobulin secretion by bovine B cells has not been reported. In cattle, both immunoglobulin G1 (IgG1) and IgG2 can fix complement, but IgG2 is the superior opsonizing subclass. Therefore, studies were undertaken to ascertain the functional relevance of RAP-1-specific, CD4(+) Th0 cells as helper cells to enhance IgG1 and/or IgG2 production by autologous B lymphocytes. For comparison, Th0 clones specific for the metazoan parasite Fasciola hepatica that expressed relatively more IL-4 than the B. bigemina-specific Th cells were similarly assayed. B. bigemina RAP-1-specific clones could enhance production of both IgG1 and IgG2 by autologous B cells, whereas Th cell clones specific for F. hepatica enhanced predominantly IgG1 production. The capacity to enhance IgG2 production was associated with production of IFN-gamma by Th cells cocultured with B cells, antigen, and IL-2. The in vitro helper T-cell activity of these T-cell clones was representative of the in vivo serologic responses, which were composed of a mixed IgG1-IgG2 response in B. bigemina RAP-1 immune cattle and a biased IgG1 response in F. hepatica-immune cattle.     

Interleukin-12 primes human CD4 and CD8 T cell clones for high production of both interferon-gamma and interleukin-10

Interleukin-12 (IL-12) induces differentiation of T helper 1 (Th1) cells, primarily through its ability to prime T cells for high interferon-gamma (IFN-gamma) production. We now report that the presence of IL-12 during the first several days of in vitro clonal expansion in limiting dilution cultures of polyclonally stimulated human peripheral blood CD4+ and CD8+ T cells also induces stable priming for high IL-10 production. This effect was demonstrated with T cells from both healthy donors and HIV+ patients. Priming for IL-4 production, which requires IL-4, was maximum in cultures containing both IL-12 and IL-4. IL-4 modestly inhibited the IL-12-induced priming for IFN-gamma, but almost completely suppressed the priming for IL-10 production. A proportion of the clones generated from memory CD45RO+ cells, but not those generated from naive CD45RO- CD4+ T cells, produced some combinations of IFN-gamma, IL-10, and IL-4 even in the absence of IL-12 and IL-4, suggesting in vivo cytokine priming; virtually all CD4+ clones generated from either CD45RO(-) or (+) cells, however, produced high levels of both IFN-gamma and IL-10 when IL-12 was present during expansion. These results indicate that each Th1-type (IFN-gamma) and Th2-type (IL-4 and IL-10) cytokine gene is independently regulated in human T cells and that the dichotomy between T cells with the cytokine production pattern of Th1 and Th2 cells is not due to a direct differentiation-inducing effect of immunoregulatory cytokines, but rather to secondary selective mechanisms. Particular combinations of cytokines induce a predominant generation of T cell clones with anomalous patterns of cytokine production (e.g., IFN-gamma and IL-4 or IFN-gamma and IL-10) that can also be found in a proportion of fresh peripheral blood T cells with "memory" phenotype or clones generated from them and that may identify novel Th subsets with immunoregulatory functions.     

Predominant Th1 cell infiltration in acute rejection episodes of human kidney grafts

T-cells and their cytokines are thought to play a major role in the genesis of cellular infiltration and rejection in human kidney allografts. Production of Th1 (IFN-gamma) and Th2-type (IL-4 and IL-5) cytokines was assessed in a large series of T-cell clones, derived from core biopsies of kidney grafts in 10 patients with acute interstitial grade I/II rejection (AIR), 6 patients with a histology of "borderline rejection" (BLR) and 3 with cyclosporine A (CsA) toxicity, all receiving standard maintenance immunosuppression. Biopsies were pre-cultured in IL-2 in order to preferentially expand T-cells activated in vivo, and T-cell blasts were cloned with phytohemagglutinin (PHA) and IL-2 using a highly efficient (23 to 98%) cloning technique. A total of 483 T-cell clones obtained from AIR episodes were compared with 346 and 132 clones derived from patients with BLR episodes and CsA toxicity, respectively. In two series of 22 AIR and 77 BLR T-cell clones, alloreactivity against donor cells was shown by 25 and 14% of CD8+ and 21 and 4% of CD4+ clones, respectively. When stimulated by donor-derived EBV B-cells, all these alloreactive clones produced IFN-gamma, but not IL-4 or IL-5 (Th1 clones). Upon stimulation with PHA, the principal qualitative and quantitative differences between AIR- and BLR-derived T-cell clones were that cells derived from AIR patients: (i) showed significantly higher proportions (80 +/- 15 vs. 55 +/- 13%) of Th1 clones in their progeny; (ii) included smaller proportions (3 +/- 4 vs. 20 +/- 17%) of clones incapable of producing IFN-gamma, IL-4 or IL-5 ('null' clones); and (iii) produced significantly higher quantities of IFN-gamma (100 +/- 50 vs. 36 +/- 7 U/10(6) cells/ml), these quantities also being significantly correlated (r = 0.83) with the degree of interstitial graft infiltration (item 'i' in the Banff histological grading). The clones derived from CsA toxicity biopsies exhibited a pattern very similar to that found in BIR cases. These data lead us to conclude that the powerful inflammatory response elicited in acute rejection of a kidney graft recruits and activates both allospecific and non-specific Th1 effector cells, which are primed to high IFN-gamma production. Our results also suggest that IFN-gamma could contribute, at least in part, to the degree of graft infiltration and to the severity of the rejection episode.     

The theory and practice of health education applied to nursing: a bi-polar approach

Because CD1-restricted T cells lack CD4 but produce IFN-gamma in response to nonpeptide mycobacterial antigens, they could play a unique role in immunity to tuberculosis. We studied CD1-restricted T cells in the context of HIV infection by expanding CD4(-) T cell lines from 10 HIV-infected patients. Upon stimulation with Mycobacterium tuberculosis antigen or upon exposure to macrophages infected with M. tuberculosis, these T cell lines proliferated, produced IFN-gamma, and showed cytolytic T cell (CTL) activity against macrophages pulsed with mycobacterial antigen, findings consistent with a protective role against M. tuberculosis. Anti-CD1b antibodies abrogated T cell proliferation, IFN-gamma production, and CTL activity, demonstrating that these T cells are CD1 restricted. IFN-gamma production in response to M. tuberculosis was enhanced by antitransforming growth factor-beta in 8/10 lines, and by IL-15 in 2/10 lines. IFN-gamma production was augmented in a nonantigen-specific manner by IL-12 in 4/8 lines. When live HIV was cocultured with CD1-restricted T cell lines, p24 antigen and proviral DNA were not detected, indicating that the T cells were not infectable with HIV. Vaccination strategies aimed at activation and expansion of M. tuberculosis-reactive CD1-restricted T cells in HIV-infected patients may constitute a novel means to provide protection against tuberculosis, while minimizing the risk of enhancing HIV replication through stimulation of CD4(+) cells.     

Gastrointestinal T lymphocytes retain high potential for cytokine responses but have severe CD4(+) T-cell depletion at all stages of simian immunodeficiency virus infection compared to peripheral lymphocytes

Gastrointestinal complications in human immunodeficiency virus (HIV) infection are indicative of impaired intestinal mucosal immune system. We used simian immunodeficiency virus (SIV)-infected rhesus macaques as an animal model for HIV to determine pathogenic effects of SIV on intestinal T lymphocytes. Intestinal CD4(+) T-cell depletion and the potential for cytokine responses were examined during SIV infection and compared with results for lymphocytes from lymph nodes and blood. Flow cytometric analysis demonstrated severe depletion of CD4(+)CD8(-) single-positive T cells and CD4(+)CD8(+) double-positive T cells in intestinal lamina propria lymphocytes (LPL) and intraepithelial lymphocytes (IEL) during primary SIV infection which persisted through the entire course of SIV infection. In contrast, CD4(+) T-cell depletion was gradual in peripheral lymph nodes and blood. Flow cytometric analysis of intracellular gamma interferon (IFN-gamma) and interleukin-4 (IL-4) production following short-term mitogenic activation revealed that LPL retained same or higher capacity for IFN-gamma production in all stages of SIV infection compared to uninfected controls, whereas peripheral blood mononuclear cells displayed a gradual decline. The CD8(+) T cells were the major producers of IFN-gamma. There was no detectable change in the frequency of IL-4-producing cells in both LPL and peripheral blood mononuclear cells. Thus, severe depletion of CD4(+) LPL and IEL in primary SIV infection accompanied by altered cytokine responses may reflect altered T-cell homeostasis in intestinal mucosa. This could be a mechanism of SIV-associated enteropathy and viral pathogenesis. Dynamic changes in intestinal T lymphocytes were not adequately represented in peripheral lymph nodes or blood.     

A tumor-specific Th2 clone initiating tumor rejection via primed CD8+ cytotoxic T-lymphocyte activation in mice

We established a CD4+ T-cell clone specific for syngeneic methylcholanthrene-induced sarcoma, S1509a raised in an A/J mouse, involved in tumor regression. The phenotype of the T-cell clone was CD3+, TCR-beta+, CD4+, CD45RB+, LFA-1+, ICAM-1+, CD44+, and VLA-4+. The CD4+ T-cell clone specifically proliferated through antigen stimulation with attenuated S1509a in the presence of syngeneic accessory cells, and this antigen-induced proliferation was inhibited with anti-CD4 and anti-I-Ek monoclonal antibodies. The CD4+ T-cell clone designated YS1093 secreted interleukin (IL) 4, IL-5, and IL-6, but not IFN-gamma, tumor necrosis factor alpha, or IL-2, thus indicating that the clone belongs to the Th2 type. YS1093 cells and their culture supernatant after antigen stimulation augmented the primed cytotoxic T lymphocyte killing activity at the effector phase. YS1093 cells having Th2-type characteristics made the homologous growing tumor regress in the tumor-bearing syngeneic mice when YS1093 cells were transferred into the tumor-bearing mice i.v. The in vivo tumor regression initiated by YS1093 cell transfer essentially required the presence of CD8+ T cells in the tumor-bearing hosts, thus suggesting that some specific Th2 cells are positively involved in tumor regression by activating primed CD8+ cytotoxic T lymphocytes against the homologous tumor in situ.     

Mapping of specific and promiscuous HLA-DR-restricted T-cell epitopes on the Plasmodium falciparum 27-kilodalton sexual stage-specific antigen

We have characterized HLA-DR-restricted T-cell epitopes on the 27-kDa protein (Pfg27), a sexual stage-specific antigen, of the human malaria parasite Plasmodium falciparum in subjects with a history of malaria. Pfg27, expressed early in the sexual stages, is recognized by monoclonal antibodies capable of reducing the infectivity of gametocytes in mosquitoes. By using 16 Pfg27-specific CD4(+)-T-cell clones derived from three donors, seven different T-cell epitopes were identified. Among them, P11 (amino acids 191 to 210 of the Pfg27 sequence, IDVVDSYIIKPIPALPVTPD) was found to contain a previously described binding motif for multiple HLA-DR allotypes. Indeed, P11 was found to be promiscuous in that it could be recognized by T cells in the context of at least five different HLA-DR molecules. The cytokine profile of the clones was mixed. Seven of nine T-cell clones exhibited a Th0-like cytokine profile, producing high levels of gamma interferon (IFN-gamma) and interleukin-4 (IL-4) upon stimulation with specific peptides and mitogens. The other two clones had a Th1-like cytokine profile with high expression of IFN-gamma and no IL-4. Identification of a promiscuous epitope in Pfg27 could play a significant role in the design of a subunit vaccine for suppressing malaria transmission.     

Activation of human CD8+ alpha beta TCR+ cells by Mycobacterium tuberculosis via an alternate class I MHC antigen-processing pathway

Human immune responses to M. tuberculosis are characterized by activation of multiple T cell subsets including CD4+, CD8+, and gammadelta T cells, and the role of CD8+ alphabeta TCR+ T cells in this response is poorly understood. Stimulation of T cells from healthy tuberculin skin test-positive persons with live M. tuberculosis-H37Ra or soluble M. tuberculosis Ags readily up-regulated IL-2Ralpha (CD25) expression on CD8+ T cells. Purified resting and activated CD8+ T cells produced IFN-gamma and proliferated to both M. tuberculosis bacilli and soluble mycobacterial Ags with monocytes as APC. Precursor frequency of mycobacterial Ag-specific CD8+ T cells by IFN-gamma enzyme-linked immunospot was 5-10-fold lower than the precursor frequency of CD4+ T cells, and IFN-gamma secretion by CD8+ T cells was 50-100-fold lower. CD8+ T cells secreted approximately 10-fold less IFN-gamma per cell than CD4+ T cells in response to mycobacterial Ags. CD8+ T cell responses to M. tuberculosis bacilli were blocked by anti-MHC class I antibody and required Ag processing. Processing of M. tuberculosis bacilli by monocytes for presentation to MHC class I-restricted CD8+ T cells was insensitive to brefeldin A treatment, which blocks the conventional MHC class I Ag-processing pathway. These results represent the first demonstration that human cells can process pathogen Ags via an alternate Ag-processing pathway for MHC class I and suggest a mechanism for participation of IFN-gamma-secreting CD8+ T cells in the human immune responses to M. tuberculosis.     

Interleukin-12 preserves the graft-versus-leukemia effect of allogeneic CD8 T cells while inhibiting CD4-dependent graft-versus-host disease in mice

We have recently demonstrated that a single injection of 4,900 IU of interleukin-12 (IL-12) on the day of bone marrow transplantation (BMT) markedly inhibits acute graft-versus-host disease (GVHD) in a fully major histocompatibility complex plus minor antigen-mismatched BMT model (A/J --> B10, H-2(a) --> H-2(b)), in which donor CD4(+) T cells are required for the induction of acute GVHD. We show here that donor CD8-dependent graft-versus-leukemia (GVL) effects against EL4 (H-2(b)) leukemia/lymphoma can be preserved while GVHD is inhibited by IL-12 in this model. In mice in which IL-12 mediated a significant protective effect against GVHD, marked GVL effects of allogeneic T cells against EL4 were observed. GVL effects against EL4 depended on CD8-mediated alloreactivity, protection was not observed in recipients of either syngeneic (B10) or CD8-depleted allogeneic spleen cells. Furthermore, we analyzed IL-12-treated recipients of EL4 and A/J spleen cells which survived for more than 100 days. No EL4 cells were detected in these mice by flow cytometry, tissue culture, adoptive transfer, necropsies, or histologic examination. Both GVL effects and the inhibitory effect of IL-12 on GVHD were diminished by neutralizing anti-interferon-gamma (IFN-gamma) monoclonal antibody. This study demonstrates that IL-12-induced IFN-gamma production plays a role in the protective effect of IL-12 against GVHD. Furthermore, IFN-gamma is involved in the GVL effect against EL4 leukemia, demonstrating that protection from CD4-mediated GVHD and CD8-dependent anti-leukemic activity can be provided by a single cytokine, IFN-gamma. These observations may provide the basis for a new approach to inhibiting GVHD while preserving GVL effects of alloreactivity.     

The lantibiotic mersacidin inhibits peptidoglycan biosynthesis at the level of transglycosylation

BACKGROUND & AIMS: The cytokine pattern secreted by T cells at the site of viral replication may influence the final outcome of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections. The aim of this study was to assess whether a cytokine imbalance oriented toward T helper (Th) 1 or Th2-type responses may play a role in chronic hepatitis B or C. METHODS: Production of interferon (IFN)-gamma, interleukin (IL)-4, and IL-5 by wide series of T-cell clones derived from the liver of 6 patients with chronic hepatitis B (291 clones) and 9 patients with chronic hepatitis C (260 clones) was studied. T-cell clones were generated by limiting dilution from freshly isolated mononuclear cells derived from liver tissue to give a reliable representation of the intrahepatic inflammatory infiltrates. RESULTS: The majority of liver-infiltrating T cells in chronic hepatitis C were Th1 cells able to secrete IFN-gamma but unable to secrete IL-4 or IL-5, whereas in hepatitis B, most CD4+ and CD8+ liver T cells were ThO-like cells able to produce not only IFN-gamma but also IL-4 and IL-5. CONCLUSIONS: The different cytokine profiles of T cells within the liver in chronic HBV and HCV infections illustrate a different behavior of the local immune response in these two infections that may have pathogenetic implications.     

In vivo relevance of CD30 in atopic dermatitis

CD30 expression was evaluated by immunohistochemistry in lesional skin biopsies of eight patients with active atopic dermatitis (AD) and three patients with allergic contact (nickel-induced) dermatitis (ACD). CD30 expression was also assessed in a large panel of CD4+ and CD8+ T-cell clones generated from the skin biopsies of four patients with AD. Finally, the levels of soluble CD30 (sCD30) were measured in the serum of 41 patients with AD, 19 patients with ACD, and 60 healthy controls. In all specimens of lesional AD skin, where the great majority of infiltrating cells were CD4+ T cells, remarkable numbers of cells were CD30+, whereas virtually no CD30+ cells were found in the skin of patients with ACD. In CD4+ T-cell clones generated from the lesional AD skin, most of which produced both interleukin (IL)-4 and interferon-gamma (IFN-gamma) (Th0-like cells) or IL-4 and IL-5, but not IFN-gamma (Th2-like cells), CD30 expression directly correlated with the ability to produce IL-4 and IL-5, but was inversely related to IFN-gamma production. High levels of sCD30 (correlated with disease activity: r = 0.618) were detected in the serum of most AD patients, whereas there was no increase of sCD30 levels in the serum of patients with ACD. These data support the view that Th0/Th2-type responses predominate in the skin of patients with AD and suggest that the presence of CD30+ T cells in tissues and/or increased levels of sCD30 in biologic fluids are indicative of Th2-dominated responses.     

Expansion of Philadelphia chromosome-negative CD3(+)CD56(+) cytotoxic cells from chronic myeloid leukemia patients: in vitro and in vivo efficacy in severe combined immunodeficiency disease mice

We have developed culture conditions for the efficient expansion of cytotoxic effector cells from peripheral blood mononuclear cells (PBMNCs) by the timed addition of interferon-gamma (IFN-gamma), interleukin-2 (IL-2), and the monoclonal antibody (MoAb) OKT3. These cells, termed cytokine-induced killer (CIK) cells, are composed primarily of T cells, and the population of cells with the greatest cytotoxic activity is an otherwise rare population of CD3(+)CD56(+) cells that expand dramatically under these culture conditions. CIK cells were expanded from PBMNCs from 13 patients with chronic myeloid leukemia (CML). These cultures contained a variable number of T cells at the start of the culture (median 44%, range 1% to 64%), yet after 21 to 28 days of culture, virtually all of the cells were CD3(+) T cells (median 97%, range 90% to 99%). The CD3(+)CD56(+) subset of cells expanded significantly (median 25-fold, range 2.2- to 525-fold). CIK cells from all patients showed cytotoxicity against the tumor cell lines OCI-LY8 and K562. In four patients the expanded CIK cells suppressed colony growth of autologous CML blast cells and myeloid progenitor cells. Allogeneic CIK cells from normal donors also suppressed CML colony growth but did not inhibit growth of normal hematopoietic colonies. Twelve of the 13 cultures were exclusively composed of Philadelphia (Ph)-negative cells and one culture had 1 out of 20 Ph-positive metaphases after 4 weeks in culture. Intracellular cytokine production was assayed by fluorescence-activated cell sorter (FACS), and the expanded T-cell cultures produced IL-2, IFN-gamma, and tumor necrosis factor-alpha (TNF-alpha), but not IL-4. Both the CD4(+) and CD8(+) subsets secreted this cytokine profile. To test the in vivo activity of the expanded CIK cells, CML was engrafted into severe combined immunodeficiency disease (SCID) mice using matrigel. After 4 weeks, 4 x 10(7) autologous CIK cells were injected intravenously by tail vein injection into groups of mice, and the animals were sacrificed after a total of 18 weeks. Bcr-abl was detected in the bone marrow or spleen of 5 out of 6 control mice and only 2 out of 13 mice who received the autologous CIK cells (P = .02). In an additional series of animals, the mice did not engraft with CML but instead developed large human Epstein-Barr virus-associated lymphomas by 12 weeks. The mice who received autologous CIK cells at 4 weeks had either no tumor (5) or small tumors (5), whereas all 10 mice that received CIK cells at week 8 developed lymphomas; however, these were not as large as in the 10 control mice who did not receive CIK cells (P = . 03). This study shows that CIK cells, which are Ph chromosome-negative, can be expanded from patients with CML and have potent in vitro and in vivo efficacy against autologous tumor cells.     

Mechanisms associated with defective TH1 cytokine production in HIV infection

Qualitative and quantitative changes in immune functions of different T-cell subsets associated with infection by human immunodeficiency virus type 1 (HIV-1) were analyzed by flow cytometric assessment of intracytoplasmic cytokines. The T(H)1 cytokines, interleukin-2 (IL-2) and interferon-gamma (IFN-gamma), were produced by both CD4 and CD8 T-cell subsets. When normal peripheral blood mononuclear cells (PBMC) were activated in culture, both cytokines were produced predominantly by CD4 (CD4) cell and only a minor fraction of normal CD8 cells produced these cytokines. In the cultures of PBMC from HIV-1-infected individuals (HIV+PBMC), more HIV+CD8 cells produced IL-2 and IFN-gamma. Production of IFN-gamma by HIV+CD4 cells was markedly reduced, while IL-2nd tumor necrosis factor-alpha (TNF-alpha) production by HIV+CD4 remained relatively intact until the disease progressed further. Normal CD4 cells which were isolated by using a cell sorter, FACSCalibur was still able to produce IL-2 and TNF-alpha. But for full production of IFN-gamma, normal CD4 required some accessory cells, the identity of which could not yet be established.     

Monocyte chemotactic peptide-1 expression and monocyte infiltration in acute renal transplant rejection

Disseminated infection caused by organisms of Mycobacterium avium complex is common in acquired immune deficiency syndrome (AIDS) patients. M. avium is an intracellular bacterium that multiplies within macrophages. We examined the effect of M. avium infection on the T-helper cell response in C57/BL/6 black mice. At weekly intervals, CD4+ T-cells were isolated from spleens and lines were created. T-cell lines were exposed to sonicated M. avium in the presence of feeder cells and macrophages and the supernatant were collected to measure the concentrations of interferon-gamma (IFN-gamma and interleukin-10 (IL-10). Production of IFN-gamma in CD4+ T-cells obtained from uninfected mice did not vary significantly during the 5 weeks. Levels of IFN-gamma produced by T-cell lines of infected mice were similar to the control mice during the first 2 weeks but significantly reduced (approximately 30 ng/ml) thereafter. In contrast, production of IL-10 by T-cell lines of infected mice was in a range of 190 to 342 pg/ml in weeks 1, 2 and 3, but increased to an average of 1300 pg/ml at weeks 4 and 5. Pre-immunized mice, when infected with M. avium strain 101, showed a different profile of T-cell cytokines, with high IFN-gamma and low IL-10 production. Proteins purified from a number of disease-associated (D-A) and non-D-A strains of M. avium were tested for the ability to induce IL-10. 65,000 MW and 60,000 MW proteins of M. avium induced significantly more IL-10 than 45,000 MW, 33,000 MW and 27,000 MW proteins. These results showed that M. avium predominantly stimulates either Th1 or Th2 T-helper cells according to the phase of the infection.     

Enhancing effect of interleukin-4 on the secretion of interferon-gamma by alpha s1-casein-specific CD8+ T cells

alpha s1-Casein-specific CD8+ T cell clones expressed the interleukin (IL)-4 receptor, although they did not secrete detectable IL-4. We found that IL-4 significantly enhanced the secretion of interferon (IFN)-gamma by these CD8+ T cell clones. IL-4 also enhanced the secretion of IFN-gamma induced by stimulating the immobilized anti-CD3 antibodies of polyclonal CD8+ T cells which had been isolated from lymph nodes and were stimulated in vitro with the immobilized anti-CD3 antibody and IL-2. In addition, IL-4 added at the time of this first in vitro stimulation induced strong IFN-gamma productivity, as well as IL-4 and IL-10 productivity, which were detectable upon restimulation of these cells. Results are discussed in relation to the inhibitory effects of IFN-gamma production on IL-4-producing cells.