Cord blood T cells retain early differentiation phenotype suitable for immunotherapy after TCR gene transfer to confer EBV specificity.

Adoptive T cell therapy can be effective for Epstein-Barr virus (EBV)-associated posttransplant lymphoproliferative disease and melanoma. Transducing high-affinity TCR genes into T lymphocytes is an emerging method to improve potency and specificity of tumor-specific T cells. However, both methods necessitate in vitro lymphocyte proliferation, generating highly differentiated effector cells that display reduced survival and antitumor efficacy postinfusion. TCR-transduction of naive lymphocytes isolated from peripheral blood is reported to provide superior in vivo survival and function. We utilized cord blood (CB) lymphocytes, which comprise mainly naive cells, for transducing EBV-specific TCR. Comparable TCR expression was achieved in adult and CB cells, but the latter expressed an earlier differentiation profile. Further antigen-driven stimulation skewed adult lymphocytes to a late differentiation phenotype associated with immune exhaustion. In contrast, CB T cells retained a less differentiated phenotype after antigen stimulation, remaining CD57-negative but were still capable of antigen-specific polyfunctional cytokine expression and cytotoxicity in response to EBV antigen. CB T cells also retained longer telomeres and in general possessed higher telomerase activity indicative of greater proliferative potential. CB lymphocytes therefore have qualities indicating prolonged survival and effector function favorable to immunotherapy, especially in settings where donor lymphocytes are unavailable such as in solid organ and CB transplantation.

Antibody-dependent killing of tumor cells by polymorphonuclear leukocytes. Involvement of oxidative and nonoxidative mechanisms.

Antibody-dependent cellular cytotoxicity (ADCC) against Raji cells was used as a model system to investigate the polymorphonuclear leukocyte (PMN) mechanisms involved in tumor cell lysis. PMN killed target cells by nonoxidative means, as indicated by the following observations: PMN from patients with chronic granulomatous disease (CGD), defective in their metabolic burst, lysed Raji cells normally; impairment of the oxidative metabolism of normal PMN by phenylbutazone did not affect ADCC. Rosenthal's inhibitor of phospholipase A2 completely prevented the lysis, suggesting the involvement of this enzyme in the target cell damage. The inhibition of the Raji cell glutathione redox cycle by carmustine [1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)] increased cell susceptibility to PMN-mediated ADCC. This increment of lysis was related to oxidative killing systems. In fact, CGD PMN had an ADCC against BCNU-treated Raji cells lower than that mediated by normal PMN, but comparable to that observed with untreated targets, and phenylbutazone reduced the lysis of BCNU-treated Raji cells by normal PMN to the level observed with the use of untreated targets. The remaining ADCC against BCNU-treated Raji cells mediated by CGD PMN and by normal PMN in the presence of phenylbutazone was suppressed by Rosenthal's inhibitor. Thus PMN killed sensitized BCNU-treated Raji cells by use of both oxidative and nonoxidative means. The results indicate that the interaction of sensitized Raji cells with PMN triggers simultaneously oxidative and non-oxidative potentially lytic systems and the mediator(s) of Raji cell lysis actually operating may depend on the metabolic state of the target cells themselves. Therefore, the lysis of sensitized tumor cells might not reflect the simple effect of PMN tumoricidal systems; rather it should be regarded as a result of an inability of the target cells to escape the various PMN cytolytic mechanisms.

Spontaneous apoptosis in neutrophils is associated with downregulation of HLA Class I and is prevented by ligation of Class I.

In many types of cells, ligation of human leukocyte antigens (HLA) Class I molecules with specific mAbs results in the transduction of signals that trigger different cell functions. We have investigated the effects of Class I ligation in human neutrophils. After several hours in culture, neutrophils split spontaneously into two subpopulations, one with normal and the other with reduced levels of Class I. The latter subpopulation displayed high binding capacity for Annexin V, showed a hypodiploid peak, electrophoretic DNA fragmentation, and morphological features of apoptotic cells. The addition of drugs known to delay apoptosis (GM-CSF or cAMP) resulted in a reduction of Class I modulation. Furthermore, ligation of surface Class I with F(ab')2 fragments of the anti-Class I mAb W6/32 resulted in a delay in the progression of apoptosis. These data indicate that this surface Class I molecule is a marker of age-related apoptosis, and the ligation of these molecules results in the transduction of a signal that inhibits apoptosis. Thus, the downregulation of HLA Class I molecules in aging neutrophils prevents their halting the apoptotic process.

Extracellular cytotoxicity by phagocytosing polymorphonuclear neutrophilic leukocytes: enhancement by a chemotactic stimulus.

This study investigated the influence of a chemotactic stimulus on the extracellular cytotoxicity mediated by phagocytosing polymorphonuclear neutrophilic leukocytes (PMN). We used N-formyl-methionyl-leucyl-phenylalanine (FMLP) as chemotactic peptide, opsonized zymosan as phagocytosable particle, and ox red blood cells (ORBC) as extracellular bystander targets. Phagocytosing PMN were found to kill ORBC efficiently, as determined by the 51Cr-release assay. FMLP, at the concentration of 100 nM, significantly enhanced the target cell lysis. PMN from two patients with chronic granulomatous disease and normal PMN plus catalase or free radical scavengers (mannitol, benzoate, histidine) were completely devoid of cytolytic activity both in the presence and in the absence of FMLP. The results indicate that the target cell lysis by phagocytosing PMN as well as the chemotactic peptide-related amplification of the lysis itself depend on the expression of the PMN oxidative cytotoxic potential. A similar response to a chemotactic stimulus in vivo could provide a mechanism for regulating PMN-dependent cytotoxic and inflammatory processes.

Role of the oxidative metabolic burst in the antibody-dependent cellular cytotoxicity mediated by neutrophil polymorphonuclears.

The purpose of the present study was to investigate the mechanisms by which neutrophil polymorphonuclears (PMN) mediate antibody-dependent cellular cytotoxicity (ADCC). Under experimental conditions which allow target cell phagocytosis, PMN were found to efficiently kill IgG-sensitized ox erythrocytes, as determined by the 51Cr release assay. Inhibition of the target cell ingestion by colchicine did not affect the PMN cytotoxic activity, suggesting that target cell phagocytosis does not represent an essential step in the PMN-mediated ADCC against erythrocytes. PMN from patients with Chronic Granulomatous Disease, who have defective oxidative metabolic burst, displayed an impaired ADCC activity, which was unaffected by changes in the phagocytic capacities induced by colchicine. The results indicate that, under the experimental conditions employed, both the intracellular and the extracellular target cell destruction by PMN involve oxygen-dependent mechanisms.

Immune complex stimulation of neutrophil apoptosis: investigating the involvement of oxidative and nonoxidative pathways.

Neutrophils are involved in the pathogenesis of various inflammatory diseases. One of the mechanisms by which neutrophilic inflammation is generated is immune complex (IC) deposition in tissue. As the clearance of apoptotic neutrophils from inflamed sites is considered a crucial determinant for the resolution of inflammation, we investigated the effects of IC-induced neutrophil activation on apoptosis and the mechanisms regulating neutrophil survival. Our results show that IC stimulated apoptosis efficiently. The percentage of apoptotic neutrophils was reduced by the anti-FcgammaRII mAb IV.3, but not by anti-FcgammaRIII mAb 3G8. The spontaneous apoptosis was completely inhibited by the antioxidant compound catalase, which in turn prevented only partially the apoptosis in presence of IC. The oxidative metabolism triggered by IC was inhibited only blocking both FcgammaRII and FcgammaRIII. Neutrophils from patients with chronic granulomatous disease, congenitally incapable of producing oxidants, showed low level of spontaneous apoptosis, but underwent a nearly 3-fold increment in the apoptosis rate when incubated with IC. In conclusion, neutrophil apoptosis appears to be a process governed by multiple pathways, some of which are strictly ROS-dependent, others acting in a nonoxidative manner. In particular, the herein shown FcgammaRII-dependent, ROS-independent, signal-inducing neutrophil apoptosis may uncover new pharmacological targets for the promotion of cell removal from sites of inflammation, thereby favoring the resolution of the inflammatory process.

Characteristics of the cytosol-synthesized proteins generating class-I-bound peptides.

The proteins synthesized in the cytosol are several thousand, and the number of peptides potentially able to be bound by class I molecules they can generate is therefore huge. On the other hand, the actual number of peptide-class I complexes required for CTL activation is around 200. We focused on the peptides bound by B27 molecules and by the whole class I. By comparing our results with analogous data from other laboratories, we found that 31 peptides matched protein sequences in data bases; in four cases, two peptides are derived from the same protein. The finding of four pairs of identical samples in a sampling of 31 peptides from a pool of unknown magnitude suggests that this pool is quite small. We have estimated the size of this pool by combinatorial analysis and by computer simulation, and we have found a most probable distribution of about 100 to the number of self-proteins that can actually generate peptides bound by class I molecules.

HLA-G, -E, -F preworkshop: tools and protocols for analysis of non-classical class I genes transcription and protein expression.

Non-classical MHC class I HLA-E, -F, and -G molecules differ from classical class I histocompatibility antigens by specific patterns of transcription, protein expression, and immunological functions. Restriction of the expression pattern of these non-classical antigens may play a key role in modulation of immune responses during pregnancy and diseases but remains to be additionally defined. A specific component of the second International Conference on HLA-G and the 13th HLA-G Histocompatibility Workshop will be dedicated to the analysis of transcription and expression of non-classical class I genes in normal and pathological tissues. In a first step, referred to as the preworkshop, we here report the analysis and conclusions of a working group which was constituted to gather and validate optimal reagents and protocols allowing RT-PCR analysis of HLA-E, -F, -G transcript levels and flow cytometry and immunochemistry analysis of HLA-G expression in cells and tissues. As a result of this work, use of specific primers and probes detecting alternative transcripts of HLA-E, -F, and G have been validated in transfected cells expressing differential pattern of HLA class I antigens. Analysis of the specificity and affinity of collected antibodies has allowed definition of reagents to be proposed for immunochemistry and flow cytometry analysis of HLA-G expression in normal and pathological tissues during the workshop. This work has allowed constitution of an extended workshop group which is now initiating analysis of non-classical class I transcription and expression in various cells and tissues, a collective contribution that will additionally refine our view of the expression of these antigens in normal and pathological situations.

Defective neutrophil mobilization to skin chambers in cancer patients.

The in vivo migration of neutrophils was evaluated in patients affected by epithelial carcinoma using the quantitative skin-chamber technique. The results demonstrated a significant impairment of the patients' neutrophil migration, which was reduced to approximately 4% of that of the controls. Patients' sera were able to inhibit the chemotactic responsiveness of normal neutrophils in vitro. It is therefore suggested that the defective in vivo migration of neutrophils in cancer patients is related to the presence of humoral cell-directed inhibitory activity. This defect of neutrophil function might contribute to the host-defense impairment of carcinoma patients.

Cellular mechanisms of artificial peptides binding to HLA.

On the basis of the consideration that cell-free models cannot precisely mimic the complexity of the intracellular environment, we used a system to investigate the mechanisms that enable antigen-presenting cells (APC) to bind exogenous peptides through their human leukocyte antigen (HLA) molecules. We evaluated the uptake of the radiolabeled peptide 17-29-Tyr of influenza virus matrix protein by B-EBV cell lines, under various conditions. The results can be summarized as follows: a) the kinetics of peptide binding and release are very fast in living, fully competent cells; b) the peptide-HLA complexes are short-living and the DR molecules continuously undergo peptidic exchange; c) using glutaraldehyde-fixed cells, the kinetics of the two phenomena are slow, closely resembling those observed with the same peptide and purified, immobilized DR molecules. The data suggest that in APC, cellular mechanisms are operative that increase the efficiency of both loading and unloading of Class II HLA with exogenous peptides. This is likely to be related to the recycling of Class II molecules to intracellular compartments, were binding takes place. The observation that the HLA-peptide complex is a dynamic structure, suggests the possibility of replacing natural peptides with synthetic ones at this level, in order to regulate the immune response.

MHC immunoevasins: protecting the pathogen reservoir in infection.

Alteration of antigen recognition by T cells as result of insufficient major histocompatibility complex (MHC)-dependent antigen-presenting function has been observed in many cases of infections, particularly in in vitro systems. To hide themselves from an efficient immune response, pathogens may act on MHC-related functions at three levels: (i) by limiting the number of potential antigens that can be presented to naïve T cells; (ii) by synthesizing proteins which directly affect MHC cell-surface expression; and (iii) by altering the normal intracellular pathway of peptide loading on MHC. Here, we review examples of pathogens' action on each single step of MHC function and we suggest that the result of these often synergistic actions is both a limitation of the priming of naïve T cells and, more importantly, a protection of the pathogen's reservoir from the attack of primed T cells. The above mechanisms may also generate a skewing effect on immune effector mechanisms, which helps preserving the reservoir of infection from sterilization by the immune system.

Mechanisms of tumour cell destruction by PMA-activated human neutrophils.

Phorbol myristate acetate (PMA)-activated neutrophils were found to destroy B lymphoblast tumour cells (Raji) as determined by the 51Cr release assay. The target cell lysis was prevented by azide, suggesting the involvement of the myeloperoxidase enzyme. Catalase and cytochrome c caused a marked impairment of the neutrophil-mediated cytolysis, whereas superoxide dismutase significantly enhanced the target cell destruction. These data indicate that hydrogen peroxide plays a key role in the target cell injury; superoxide anion appears to be devoid of direct cytotoxic activity, despite its requirement as a precursor of hydrogen peroxide. The target cell destruction required the presence of the iodide ion as oxidizable co-factor for the myeloperoxidase-hydrogen peroxide system. The chloride ion alone was uneffective. Inhibition of target cell metabolic pathways, involved in the cellular defences against oxidative injury, by the anti-neoplastic agent 1,3-bis-(2-chloroethyl)-1-nitrosurea (BCNU) resulted in an increased neutrophil-mediated cytolysis. Under the experimental conditions employed, PMA-activated neutrophils incubated with BCNU-treated Raji cells became cytotoxic also in the presence of the chloride ion alone as myeloperoxidase co-factor. Our results suggest that Raji target cell destruction by PMA-activated neutrophils depends on the myeloperoxidase-hydrogen peroxide-halide system. The cytolytic event is influenced by target cells themselves, which should be regarded as an active component of the cytotoxic system, capable of interfering with the lytic mediators of the effector cells.

Interference of target cell catalase with an early step of the neutrophil cytolytic pathway.

The hypochlorous acid (HOCL)-dependent lysis of human red blood cells (HRBC) targets by neutrophils, activated with opsonized zymosan particles (OPZ), was increased by inhibiting HRBC catalatic activity with aminotriazole (AT; HRBCAT). The inhibition of HRBC glutathione cycle activity with carmustine (BCNU; HRBCBCNU) had no effect. In addition, the recovery of hydrogen peroxide (H2O2) and HOCL from neutrophils, activated under conditions similar to those used for cytotoxicity assay, was reduced by the presence of HRBC and restored by replacing HRBC with HRBCAT, but not with HRBCBCNU. Linear relationships were found between the increments in the neutrophil-mediated lysis, observed by using HRBCAT instead of HRBC, and the increments in the H2O2 or HOCL recovery, detected by replacing HRBC with HRBCAT. Together these data, coupled with the results obtained by probing neutrophil cytolysis with chemical agents, suggest that the increased cytolytic efficiency displayed by neutrophils against HRBCAT, inhibited in their catalatic activity, is due to an enhanced availability of neutrophil-derived H2O2, with a consequent enhancement in the HOCL production (according to the following reaction: (formula; see text). Thus it appears that HRBC catalase restrains the neutrophil cytolytic activity, by interfering with an early step of the pathway through which neutrophils generate cytotoxins.

Role of hypochlorous acid and chloramines in the extracellular cytolysis by neutrophil polymorphonuclear leukocytes.

The lysis of human red blood cells (HRBC) by neutrophil polymorphonuclear leukocytes (PMN), triggered with opsonized zymosan (OPZ) particles, was inhibited by azide, catalase, Cl- -free medium and amino acids indicating the involvement of myeloperoxidase (MPO), hydrogen peroxide (H2O2), Cl- ions and hypochlorous acid (HOCl) respectively. Thus, the cytolytic process depends on the following reaction: (Formula: see text). Because the oxidizing agent HOCl is also the precursor of the chloramines, a group of oxidants formed by the reaction between HOCl and PMN-derived ammonia (NH4+) or amines (R-NH2), the observed HRBC lysis can be theoretically due to HOCl and/or chloramines. Nevertheless, we found that PMN-mediated cytotoxicity occurs as an unidirectional process, being HRBC targets lysed and PMN unaffected. This finding indicates that the cytotoxin must be relatively more efficient against HRBC as compared with PMN. In fact, reagent HOCl (used at concentrations comparable to those generated by PMN) but not chloramines displayed such a type of property. Taken together, the data suggest that HRBC are killed by PMN-derived HOCl without the requirement for chloramines: this implies that NH4+ and R-NH2, released by PMN, act as down-modulators of the cytotoxic process, serving as HOCl trapping agents.

Expression of cytolytic functions in HL-60 leukaemic cells after induction of polymorphonuclear leukocyte differentiation.

Mature polymorphonuclear leukocytes (PMN) are capable of mediating phorbol myristate acetate (PMA)- and antibody (A)-dependent cellular cytotoxicity (DCC) against ox red blood cells (ORBC) by using oxidative means. The purpose of the present study was to investigate the acquirement of these cytotoxic functions during PMN ontogeny, using the promyelocytic HL-60 cell line as a model for PMN differentiation. HL-60 cells were induced to differentiate along the PMN pathway by exposure to dimethyl sulfoxide (DMSO). Uninduced HL-60 cells were found to be completely devoid of PMA-DCC and ADCC activity. DMSO-induced cells progressively acquired the capacity to kill ORBC and to undergo the activation of oxidative metabolic burst when triggered by PMA. Despite approximately 40% of them also were capable of binding IgG-sensitized ORBC, no ADCC activity and respiratory burst activation was observed: this finding indicates that maturing HL-60 cells require a more complete maturation than that induced by DMSO to actually exert ADCC. Together the results suggest that: a. the acquirement of both PMA-DCC and ADCC potential is a post-promyelocytic event; b. the cytotoxicity activating stimuli, PMA and IgG-coated targets, follow different post-receptor transductional pathways to trigger the effector cell lytic systems: only the PMA receptor-linked pathway develops during DMSO-driven differentiation of HL-60 cells.

Augmentation of neutrophil-mediated erythrocyte lysis by cells derived in vitro from human monocytes.

Neutrophilic polymorphonuclear leukocytes (PMNs) were incubated with opsonized zymosan and lysed human erythrocytes (RBCs) as measured by a 51Cr release method. Conversely, myeloperoxidase (MPO)-negative hydrogen peroxide (H2O2)-generating cells, derived in vitro from human monocytes (monocyte-derived cells (MDCs), were ineffective per se but capable of augmenting the lysis by PMNs. The lysis by PMNs and PMNs plus MDCs was inhibited by catalase, azide, taurine, and alanine, consistent with the requirement for hypochlorous acid (HOCl). As detected under conditions similar to those used for lytic assays, MDCs failed to produce HOCl but augmented the HOCl recovery from the PMN-RBC system. Moreover, when the extent of the lysis was plotted as a function of the HOCl recovery, a positive linear relationship was found. Although the actual size of the H2O2 extracellular pool could not be measured because of the inexistence of a reliable assay to probe our cytolytic model without perturbing the equilibrium of the system, the results presented suggest that MDCs enhance the PMN-mediated lysis by improving the HOCl production, presumably by supplying extra amounts of H2O2 to be handled by PMN MPO. In fact, the events mediated by MDCs could be reproduced by using an appropriate H2O2-generating enzymatic system (glucose-glucose oxidase). The present study provides direct evidence for the possibility of cooperation between MPO-positive and MPO-negative phagocytes in exerting functions (HOCl production and, in turn, cytolysis) possibly relevant to the outcome of inflammatory processes.