Inhibition by interferon-gamma of human mononuclear cell-mediated low density lipoprotein oxidation. Participation of tryptophan metabolism along the kynurenine pathway

In this study we examined the potential inhibition by interferon-gamma (IFN gamma) of the early stages of low density lipoprotein (LDL) oxidation mediated by human peripheral blood mononuclear cells (PBMC) and monocyte-derived macrophages (MDM) in Ham's F-10 medium supplemented with physiological amounts of L-tryptophan (Trp). We assessed LDL oxidation by measuring the consumption of LDL's major antioxidant (i.e., alpha-tocopherol) and targets for oxidation (cholesteryllinoleate and cholesterylarachidonate), together with the accumulation of cholesterylester hydroperoxides and the increase in relative electrophoretic mobility of the lipoprotein particle. Exposure of PBMC or MDM to IFN gamma induced the degradation of extracellular Trp with concomitant accumulation of kynurenine, anthranilic and 3-hydroxyanthranilic acid (3HAA) in the culture medium. Formation of 3HAA, but neither Trp degradation nor formation of kynurenine and anthranilic acid, was inhibited by low amounts of diphenylene iodonium (DPI) in a concentration-dependent manner. In contrast to oxidative Trp metabolism, exposure of human PBMC or MDM to IFN gamma failed to induce degradation of arginine, and nitrite was not detected in the cell supernatant, indicating that nitric oxide synthase was not induced under these conditions. Incubation of LDL in Trp-supplemented F-10 medium resulted in a time-dependent oxidation of the lipoprotein that was accelerated in the presence of PBMC or MDM but inhibited strongly in the presence of both cells and IFN gamma, i.e., when Trp degradation and formation of 3HAA were induced. In contrast, when IFN gamma was added to PBMC or MDM in F-10 medium that was virtually devoid of Trp, inhibition of cell-accelerated LDL oxidation was not observed. Exogenous 3HAA added to PBMC or purified monocytes in the absence of IFN gamma also strongly and in a concentration-dependent manner inhibited LDL oxidation. Selective inhibition of IFN gamma-induced formation of 3HAA by DPI caused reversion of the inhibitory action of this cytokine on both PBMC- and MDM-mediated LDL oxidation. These results show that IFN gamma treatment of human PBMC or MDM in vitro attenuates the extent of LDL oxidation caused by these cells, and indicate that Trp degradation with formation of 3HAA is a major contributing factor to this inhibitory activity.

Human basophils and eosinophils are the direct target leukocytes of the novel IL-1 family member IL-33

In mice, interleukin-18 (IL-18) regulates Th1- or Th2-type immune responses depending on the cytokine environment and effector cells involved, and the ST2-ligand, IL-33, primarily promotes an allergic phenotype. Human basophils, major players in allergic inflammation, constitutively express IL-18 receptors, while ST2 surface expression is inducible by IL-3. Unexpectedly, freshly isolated basophils are strongly activated by IL-33, but, in contrast to mouse basophils, do not respond to IL-18. IL-33 promotes IL-4, IL-13 and IL-8 secretion in synergy with IL-3 and/or FcepsilonRI-activation, and enhances FcepsilonRI-induced mediator release. These effects are similar to that of IL-3, but the signaling pathways engaged are distinct because IL-33 strongly activates NF-kappaB and shows a preference for p38 MAP-kinase, while IL-3 acts through Jak/Stat and preferentially activates ERK. Eosinophils are the only other leukocyte-type directly activated by IL-33, as evidenced by screening of p38-activation in peripheral blood cells. Only upon CD3/CD28-ligation, IL-33 weakly enhances Th2 cytokine expression by in vivo polarized Th2 cells. This study on primary human cells demonstrates that basophils and eosinophils are the only direct target leukocytes for IL-33, suggesting that IL-33 promotes allergic inflammation and Th2 polarization mainly by the selective activation of these specialized cells of the innate immune system.

In vitro characterization of immune-related properties of human fetal bone cells for potential tissue engineering applications

We describe herein some immunological properties of human fetal bone cells recently tested for bone tissue-engineering applications. Adult mesenchymal stem cells (MSCs) and osteoblasts were included in the study for comparison. Surface markers involved in bone metabolism and immune recognition were analyzed using flow cytometry before and after differentiation or treatment with cytokines. Immunomodulatory properties were studied on activated peripheral blood mononuclear cells (PBMCs). The immuno-profile of fetal bone cells was further investigated at the gene expression level. Fetal bone cells and adult MSCs were positive for Stro-1, alkaline phosphatase, CD10, CD44, CD54, and beta2-microglobulin, but human leukocyte antigen (HLA)-I and CD80 were less present than on adult osteoblasts. All cells were negative for HLA-II. Treatment with recombinant human interferon gamma increased the presence of HLA-I in adult cells much more than in fetal cells. In the presence of activated PBMCs, fetal cells had antiproliferative effects, although with patterns not always comparable with those of adult MSCs and osteoblasts. Because of the immunological profile, and with their more-differentiated phenotype than of stem cells, fetal bone cells present an interesting potential for allogeneic cell source in tissue-engineering applications.

Immune response deviation and enhanced expression of chemokine receptor CCR4 in TBI patients due to unknown serum factors

BACKGROUND: Severe brain trauma leads to an activation of the immune system. To this date, neither the exact perturbation of the specific immune reaction induced by the traumatic brain injury (TBI), nor the interactions leading to the infiltration of peripheral immune cells into the brain are fully understood. PATIENTS AND METHODS: Serum was collected from 17 patients with TBI and a long bone fracture, 24 patients with an isolated long bone fracture and from healthy individuals. The effect of the serum on normal human monocytes and T-lymphocytes was tested in vitro by assessing proliferation and expression of surface markers, chemokine receptors and cytokines. RESULTS: Serum collected from patients with a TBI and a long bone fracture increased the expression of the chemokine receptor CCR4 in monocytes when compared to patients with an isolated long bone fracture. Extending this comparison to T-lymphocytes, the serum from TBI patients induced lower proliferation rates and decreased expression of the pro-inflammatory cytokine TNF-alpha, while simultaneously increasing the secretion of immune-modulatory cytokines (IL-4, IL-10 and TGF-beta) (p<0.05). CONCLUSION: Patients with a TBI release currently unknown soluble factors into the circulating blood that up regulate expression of chemokine receptor CCR4 in peripheral blood monocytes whilst concurrently inducing expression of immunosuppressive cytokines by activated T-lymphocytes.

JC virus-specific immune responses in human immunodeficiency virus type 1 patients with progressive multifocal leukoencephalopathy

Progressive multifocal leukoencephalopathy (PML) is a frequently fatal disease caused by uncontrolled polyomavirus JC (JCV) in severely immunodeficient patients. We investigated the JCV-specific cellular and humoral immunity in the Swiss HIV Cohort Study. We identified PML cases (n = 29), as well as three matched controls per case (n = 87), with prospectively cryopreserved peripheral blood mononuclear cells and plasma at diagnosis. Nested controls were matched according to age, gender, CD4(+) T-cell count, and decline. Survivors (n = 18) were defined as being alive for >1 year after diagnosis. Using gamma interferon enzyme-linked immunospot assays, we found that JCV-specific T-cell responses were lower in nonsurvivors than in their matched controls (P = 0.08), which was highly significant for laboratory- and histologically confirmed PML cases (P = 0.004). No difference was found between PML survivors and controls or for cytomegalovirus-specific T-cell responses. PML survivors showed significant increases in JCV-specific T cells (P = 0.04) and immunoglobulin G (IgG) responses (P = 0.005). IgG responses in survivors were positively correlated with CD4(+) T-cell counts (P = 0.049) and negatively with human immunodeficiency virus RNA loads (P = 0.03). We conclude that PML nonsurvivors had selectively impaired JCV-specific T-cell responses compared to CD4(+) T-cell-matched controls and failed to mount JCV-specific antibody responses. JCV-specific T-cell and IgG responses may serve as prognostic markers for patients at risk.

EXPRESSION AND IMMUNOLOGICAL CHARACTERIZATION OF HERV-K TRANSMEMBRANE ENVELOPE PROTEIN IN HUMAN BREAST CANCER

The human endogenous retrovirus K (HERV-K) env gene encodes envelope protein comprising surface (SU) and transmembrane (TM) domains. Having shown the exclusive expression of SU in human breast cancer and the stimulation of SU-specific immune responses in patients with breast cancer, our research here confirmed and extended the data by investigating the expression of HERV-K TM envelope domain and the induction of specific immune responses against TM in breast cancer patients. We found HERV-K TM mRNA and protein expression only in human breast cancer cells but not in normal controls. The specific immune responses against TM domain were induced in mice determined by enzyme-linked immunosorbent assay (ELISA) and IFN-γ enzyme-linked immunosorbent spot (ELISPOT) assay. Furthermore, ELISA detected higher titers of anti-HERV-K TM Env IgG antibodies in sera of breast cancer patients. In addition, the magnitude of the anti-HERV TM B cell response was correlated with the disease stage. Peripheral blood mononuclear cells (PBMCs) before and after in vitro stimulation (IVS) with HERV-K TM from patients with breast cancer as well as healthy controls were tested for T cell responses against HERV-K TM domain by ELISPOT assay. Breast cancer patients (n=21) had stronger HERV-K TM-specific cellular responses than healthy controls (n=12) (P < 0.05). These findings suggest, for the first time, that HERV-K TM expression was enhanced in human breast cancer cells and was able to induce specific B cell and T cell immune responses in breast cancer patients. This study provides support for HERV-K TM as a promising source of antigen for anti-tumor immunotherapy, prevention, diagnosis, and prognosis.

Nuclear proteins interacting with an AP-1/CRE-like promoter sequence in the human TNF$\alpha$ gene

Monocyte developmental heterogeneity is reflected at the cellular level by differential activation competence, at the molecular level by differential regulation of gene expression. LPS activates monocytes to produce tumor necrosis factor-$\alpha$ (TNF). Events occurring at the molecular level necessary for TNF regulation have not been elucidated, but depend both on activation signals and the maturation state of the cell: Peripheral blood monocytes produce TNF upon LPS stimulation, but only within the first 72 hours of culture. Expression of c-fos is associated with monocytic differentiation and activation; the fos-associated protein, c-jun, is also expressed during monocyte activation. Increased cAMP levels are associated with down regulation of macrophage function, including LPS-induced TNF transcription. Due to these associations, we studied a region of the TNF promoter which resembles the binding sites for both AP-1(fos/jun) and CRE-binding protein (or ATF) in order to identify potential molecular markers defining activation competent populations of monocytic cells.^ Nuclear protein binding studies using extracts from THP-1 monocytic cells stimulated with LPS, which stimulates, or dexamethasone (Dex) or pentoxyfilline (PTX), which inhibit TNF production, respectively, suggest that a low mobility doublet complex may be involved in regulation through this promoter region. PTX or Dex increase binding of these complexes equivalently over untreated cells; approximately two hours after LPS induction, the upper complex is undetectable. The upper complex is composed of ATF2 (CRE-BP1); the lower is a heterodimer of jun/ATF2. LPS induces c-jun and thus may enhance formation of jun-ATF2 complexes. The simultaneous presence of both complexes may reduce the amount of TNF transcription through competitive binding, while a loss of the upper (ATF2) and/or gain of the lower (jun-ATF2) allow increased transcription. AP-1 elements generally transduce signals involving PKC; the CRE mediates a cAMP response, involving PKA. Thus, this element has the potential of receiving signals through divergent signalling pathways. Our findings also suggest that cAMP-induced inhibition of macrophage functions may occur via down regulation of activation-associated genes through competitive binding of particular cAMP-responsive nuclear protein complexes. ^

Molecular analysis of the human $\gamma$-chain T cell receptor genes

In our studies we have focused on the issue of variability and diversity of the $\gamma$ (or $\delta)$ chain T cell receptor (TCR) genes by studying cDNA transcripts in peripheral blood mononuclear cells or $\gamma\delta$ TCR+ T cell clones. The significance of these studies lies in the better understanding of the molecular biology of the $\gamma\delta$ T cell receptor as well as in answering the question whether certain molecular forms predominate in $\gamma\delta$ T cells exhibiting specific immunologic functions. We establish that certain $\gamma$-chain TCR genes exhibit particular patterns of rearrangements in cDNA transcripts in normal individuals. V$\gamma$I subgroup were shown to preferentially rearrange to J$\gamma$2C$\gamma$2 gene segments. These preferential VJC rearrangements, may have implications regarding the potential for diversity and polymorphism of the $\gamma$-chain TCR gene. In addition, the preferential association of V$\gamma$I genes with J$\gamma$2C$\gamma$2, which encode a non-disulfide-linked $\gamma\delta$ TCR, suggests that $\gamma$ chains utilizing V$\gamma$I are predominantly expressed as non-disulfide-linked $\gamma\delta$ TCR heterodimers. The implications of this type of expression remain to be determined. We identified two alternative splicing events of the $\gamma$-chain TCR genes occurring in high frequency in all the normal individuals examined. These events may suggest additional mechanisms of regulation and control as well as diversification of $\gamma\delta$ TCR gene expression. The question whether particular forms of $\gamma$ or $\delta$-chain TCR genes are involved in HLA Class I recognition by specific $\gamma\delta$ cytotoxic T cell clones was addressed. Our results indicated that the T cell clones expressed identical $\gamma$ but distinct $\delta$-chains suggesting that the specificity for recognition of HLA-A2 or HLA-A3 may be conferred by the $\delta$-chain TCR. The issue of the degree of diversity and polymorphism of the $\delta$-chain TCR genes in a patient with a primary immunodeficiency (Omenn's syndrome) was addressed. A limited pattern of rearrangements in peripheral blood transcripts was found, suggesting that a limited $\gamma\delta$ TCR repertoire may be expressed in this particular primary immunodeficiency syndrome. Overall, our findings suggest that $\delta$-chain TCR genes exhibit the potential for significant diversity and that there are certain preferential patterns of expression that may be associated with particular immunologic functions. ^

Integrins in human T cell function: Transdominant regulation, conformational constraints, and intracellular effectors

Integrin adhesion molecules have both positive and negative potential in the regulation of peripheral blood T cell (PB T cell) activation, yet their mechanism of action in the mediation of human T lymphocyte function remains largely undefined. The goals of this study then were to elucidate integrin signaling mechanisms in PB T cells.^ By ligating $\beta$1 integrins with mAb 18D3, it was demonstrated that costimulation of PB T cell proliferation induced by coimmobilizing antibodies specific for $\beta$1, $\beta$2, and $\beta$7 integrin subfamilies in conjunction with the anti-CD3 mAb OKT3 was inhibited. Costimulation of T cell proliferation induced by non-integrins CD4, CD26, CD28, CD44, CD45RA, or CD45RO was unaffected. Inhibition of costimulation correlated with diminished IL-2 production. In his manner, $\beta$1 integrins could regulate heterologous integrins of the $\beta$2 and $\beta$7 subfamilies in a transdominant fashion. It was also demonstrated that integrin costimulation of T cell activation was acutely sensitive to the structural conformation of $\beta$1 integrins. Using the cyclic hexapeptide CWLDVC (TBC772, which is based on the $\alpha4\beta1$ integrin binding site in fibronectin) in soluble form, it was shown that integrins locked into a conformation displaying a neo-epitope called the ligand induced binding site (LIBS) recognized by mAb 15/7 were inhibited from sending mitogenic signals to T cells. When BSA-conjugated TBC772 was coimmobilized with anti-CD3 mAb OKT3, costimulation of proliferation occurred. This suggested that temporally uncoupling integrin receptor occupancy from receptor crosslinking inhibited $\beta$1 integrin signaling mechanisms. When subsets of PB T cells were examined to determine those initially activated by integrins within 6 hours of activation, costimulation induced intracellular accumulation of IL-2 predominantly in the CD4$\sp+$ and CD45RO$\sp+$ T cell subsets. This was similar to a number of PB T cell costimulatory molecules including CD26, CD43, CD44. Only CD28 costimulated IL-2 production from both CD45RA$\sp+$ and CD45RO$\sp+$ subpopulations.^ The GTPase Rho has been implicated in regulating integrin mediated stress fiber formation and anchorage dependent growth in fibroblasts, so studies were initiated to determine if Rho played a role in integrin dependent T cell function. In order to perform this, a technique based on scrape-loading was developed to incorporate macromolecules into PB T cells that maintained their functional activity. With this technique, C3 exoenzyme from Clostridium botulinum was incorporated into PB T cells. C3 ADP-ribosylates Rho proteins on Asn$\sp{41},$ which is in close proximity to the Rho effector domain, rendering it inactive. It was demonstrated that functional Rho is not required for basal or upregulated PB T cell adhesion to $\beta$1 integrin substrates, however PB T cell homotypic aggregation induced by PMA, which is an event mediated predominantly by the integrin $\rm\alpha L\beta2,$ was delayed. PB T cells lacking Rho function displayed altered cell morphology on $\beta$1 integrin ligands, producing stellate, dendritic-like pseudopodia. Rho activity was also found to be required for integrin dependent costimulation of proliferation. When intracellular accumulation of IL-2 was measured, inactivation of Rho prevented both integrin and CD28 costimulatory activity. Rho was identified to lie upstream of signals mediating PKC activation and Ca$\sp{++}$ fluxes, as PMA and ionomycin activation of PB T cells was unaffected by the inactivation of Rho. ^

Memory regulatory T cells reside in human skin.

Regulatory T cells (Tregs), which are characterized by expression of the transcription factor Foxp3, are a dynamic and heterogeneous population of cells that control immune responses and prevent autoimmunity. We recently identified a subset of Tregs in murine skin with properties typical of memory cells and defined this population as memory Tregs (mTregs). Due to the importance of these cells in regulating tissue inflammation in mice, we analyzed this cell population in humans and found that almost all Tregs in normal skin had an activated memory phenotype. Compared with mTregs in peripheral blood, cutaneous mTregs had unique cell surface marker expression and cytokine production. In normal human skin, mTregs preferentially localized to hair follicles and were more abundant in skin with high hair density. Sequence comparison of TCRs from conventional memory T helper cells and mTregs isolated from skin revealed little homology between the two cell populations, suggesting that they recognize different antigens. Under steady-state conditions, mTregs were nonmigratory and relatively unresponsive; however, in inflamed skin from psoriasis patients, mTregs expanded, were highly proliferative, and produced low levels of IL-17. Taken together, these results identify a subset of Tregs that stably resides in human skin and suggest that these cells are qualitatively defective in inflammatory skin disease.

Retinal differentiation of human bone marrow-derived stem cells by co-culture with retinal pigment epithelium in vitro.

The goal of this study was to assess the in vitro differentiation capacity of human bone marrow-derived stem cells (hBMSCs) along retinal lineages. Mononuclear cells (MNC) were isolated from bone marrow (BM) and mobilized peripheral blood (mPB) using Ficoll-Paque density gradient centrifugation, and were sorted by magnetic-activated cell sorting (MACS) for specific stem cell subsets (CD34(+)CD38(+)/CD34(+)CD38(-)). These cells were then co-cultured on human retinal pigment epithelial cells (hRPE) for 7 days. The expression of stem cell, neural and retina-specific markers was examined by immunostaining, and the gene expression profiles were assessed after FACS separation of the co-cultured hBMSCs by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Furthermore, in vitro functionality of the differentiated cells was analyzed by quantifying phagocytosis of CY5-labeled photoreceptor outer segments (POS). After 7 days of co-culture, hBMSCs adopted an elongated epithelial-like morphology and expressed RPE-specific markers, such as RPE65 and bestrophin. In addition, these differentiated cells were able to phagocytose OS, one of the main characteristics of native RPE cells. Our data demonstrated that human CD34(+)CD38(-) hBMSC may differentiate towards an RPE-like cell type in vitro and could become a new type of autologous donor cell for regenerative therapy in retinal degenerative diseases.

Differential influence of maternal and fetal pregnancy factors on the in-vitro induction of human regulatory T cells: a preliminary study.

PROBLEM Given the important role of regulatory T cells (Treg) for successful pregnancy, the ability of soluble maternal and fetal pregnancy factors to induce human Treg was investigated. METHOD OF STUDY Peripheral blood mononuclear cells (PBMCs) or isolated CD4+CD25‒ cells were cultured in the presence of pooled second or third trimester pregnancy sera, steroid hormones or supernatants from placental explants, and the numbers and function of induced CD4+CD25+FOXP3+ Treg were analysed. RESULTS Third trimester pregnancy sera and supernatants of early placental explants, but not sex steroid hormones, induced an increase of Tregs from PBMCs. Early placental supernatant containing high levels of tumour necrosis factor-α, interferon-γ, interleukins -1, -6 and -17, soluble human leucocyte antigen-G, and transforming growth factor-β1, increased the proportion of Treg most effectively and was able to induce interleukin-10-secreting-Treg from CD4+CD25‒cells. CONCLUSIONS Compared with circulating maternal factors, placental- and fetal-derived factors appear to exert a more powerful effect on numerical changes of Treg, thereby supporting fetomaternal tolerance during human pregnancy.

Blocking of LFA-1 enhances expansion of Th17 cells induced by human CD14(+) CD16(++) nonclassical monocytes.

Among human peripheral blood (PB) monocyte (Mo) subsets, the classical CD14(++) CD16(-) (cMo) and intermediate CD14(++) CD16(+) (iMo) Mos are known to activate pathogenic Th17 responses, whereas the impact of nonclassical CD14(+) CD16(++) Mo (nMo) on T-cell activation has been largely neglected. The aim of this study was to obtain new mechanistic insights on the capacity of Mo subsets from healthy donors (HDs) to activate IL-17(+) T-cell responses in vitro, and assess whether this function was maintained or lost in states of chronic inflammation. When cocultured with autologous CD4(+) T cells in the absence of TLR-2/NOD2 agonists, PB nMos from HDs were more efficient stimulators of IL-17-producing T cells, as compared to cMo. These results could not be explained by differences in Mo lifespan and cytokine profiles. Notably, however, the blocking of LFA-1/ICAM-1 interaction resulted in a significant increase in the percentage of IL-17(+) T cells expanded in nMo/T-cell cocultures. As compared to HD, PB Mo subsets of patients with rheumatoid arthritis were hampered in their T-cell stimulatory capacity. Our new insights highlight the role of Mo subsets in modulating inflammatory T-cell responses and suggest that nMo could become a critical therapeutic target against IL-17-mediated inflammatory diseases.

Expression and biological activities of bovine interleukin 4: effects of recombinant bovine interleukin 4 on T cell proliferation and B cell differentiation and proliferation in vitro

Interleukin 4 (IL-4) is a pleotropic cytokine affecting a wide range of cell types in both the mouse and the human. These activities include regulation of the growth and differentiation of both T and B lymphocytes. The activities of IL-4 in nonprimate, nonmurine systems are not well established. Herein, we demonstrate in the bovine system that IL-4 upregulates production of IgM, IgG1, and IgE in the presence of a variety of costimulators including anti-IgM, Staphylococcus aureus cowan strain I, and pokeweed mitogen. IgE responses are potentiated by the addition of IL-2 to IL-4. Culture of bovine B lymphocytes with IL-4 in the absence of additional costimulators resulted in the increased surface expression of CD23 (low-affinity Fc epsilon RII), IgM, IL-2R, and MHC class II in a dose-dependent manner. IL-4 alone increased basal levels of proliferation of bulk peripheral blood mononuclear cells but in the presence of Con A inhibited proliferation. In contrast to the activities of IL-4 in the murine system, proliferation of TH1- and TH2-like clones was inhibited in a dose-dependent manner as assessed by antigen-or IL-2-driven in vitro proliferative responses. These observations are consistent with the role of IL-4 as a key player in regulation of both T and B cell responses.

ANTIBODY-DEPENDENT AND ANTIBODY-INDEPENDENT CELLULAR CYTOTOXICITY AGAINST SHIGELLA

Diarrhea is a major cause of morbidity and mortality worldwide. Shigella causes up to 20% of all diarrhea. Gut-level immunity and breast-feeding of infants are important factors in protection against shigellosis. The lumen of the gut is lined with lymphocytes which mediate natural killer cytotoxicity, NKC, and antibody-dependent cellular cytotoxicity, ADCC. NKC and ADCC are extracellular, nonphagocytic leukocyte killing mechanisms, which occur in the absence of complement, without prior antigen stimulation, and without regard to the major histocompatibility complex. In this study, virulent and avirulent shigellae were used as the target cells. Leukocytes from peripheral blood, breast milk, and guinea pig gut-associated tissues were used as effector cells. Adult human peripheral blood mononuclear cells and lymphocytes, but not macrophages or polymorphonuclear leukocytes, mediated NKC and ADCC at an optimal effector to target cell ratio of 100:1 in a 60 minute bactericidal assay. An antiserum dilution of 1:10 was optimal for ADCC. Whole, viable lymphocytes were necessary for cytotoxicity. Lymphocyte NKC, but not ADCC, was greatly enhanced by interferon. Lymphocyte NKC occurred against several virulent strains of S. sonnei and a virulent strain of S. flexneri. ADCC (using immune serum directed against S. sonnei) occurred against virulent S. sonnei, but not against avirulent S. sonnei or virulent S. flexneri. Lymphocyte ADCC was not inhibited by the presence of phenylbutazone or by pretreatment of lymphocytes with anti-HNK serum plus complement. Both adherent and non-adherent breast milk leukocytes mediated NKC and ADCC. Mononuclear cells from young children demonstrated normal ADCC, when compared to ADCC of adult cells. Neonatal cord blood and a CGD patient's peripheral blood mononuclear and ploymorphonuclear cells demonstrated high ADCC compared to adult cells. Intraepithelial lymphocytes, spleen cells, and peritoneal cells from normal guinea pigs demonstrated NKC and ADCC. Animals which had been starved and opiated were made susceptible to infection by Shigella. The susceptible animals demonstrated deficient NKC and ADCC with all three leukocyte populations. High NKC and ADCC activity of gut-associated leukocytes from human breast milk and guinea pig tissues may correlate with resistance to infection. ^