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.

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.

Synovial fluid from patients with rheumatoid arthritis inhibits neutrophil apoptosis: role of adenosine and proinflammatory cytokines.

OBJECTIVE: In synovial fluid (SF) from patients with rheumatoid arthritis (RA), neutrophils are exposed to proinflammatory mediators endowed with either anti-apoptotic or pro-apoptotic properties. We investigated neutrophil apoptosis in the presence of SF from 11 RA patients. METHODS: SF was obtained from affected knees of 11 patients with RA. Human neutrophil apoptosis was evaluated by light microscopic examination and flow-cytometric analysis of annexin V binding. Immune complex-induced neutrophil activation was evaluated as superoxide anion production. Adenosine levels in SF were detected by chromatographic analysis and cytokine levels were studied by enzyme-linked immunosorbent assay. RESULTS: Spontaneous and immune complex-triggered neutrophil apoptosis was reduced by SF from eight out of 11 patients. Immune complex-induced neutrophil activation was unaffected by SF. The cytokines tested had no role in promoting the anti-apoptotic activity of SF. On the contrary, the anti-apoptotic activity of SF was found to depend on the presence of adenosine. Adenosine levels detected in the various samples of SF correlated significantly with the anti-apoptotic activity of the fluids and with the number of apoptotic neutrophils detected in the articular exudate. CONCLUSION: The microenvironment of rheumatoid SF is a proinflammatory milieu responsible for the in loco persistence of activated and long-surviving neutrophils. Adenosine plays a crucial role in this phenomenon, which is related to anti-apoptotic activity.

Bone marrow transplantation from unrelated donors: the impact of mismatches with substitutions at position 116 of the human leukocyte antigen class I heavy chain.

The hypothesis was tested that amino acid substitutions in specific positions within human leukocyte antigen class I heavy chain would have different impacts on transplant-related mortality (TRM) in patients receiving transplanted bone marrow from unrelated donors. One hundred patients and their unrelated donors were typed by sequence-based typing for the human leukocyte antigen (HLA)-A, -B, and -C loci. All pairs were matched for DRB1, DRB3, DRB4, DRB5, DQA1, and DQB1 loci. Forty pairs were also matched at class I, and 60 pairs had one or more mismatches at class I loci. It was found that substitutions at positions 116 and 114 of class I heavy chain significantly increased the risk for TRM in univariate and bivariate Cox analyses. Conversely, no association between number of multiple mismatches or number of amino acid substitutions and TRM was seen when positions 116 and 114 were adjusted for. Variables predictive of TRM in multivariate Cox analysis were number of cells infused, diagnosis (chronic myeloid leukemia [CML] or non-CML), and amino acid substitution at position 116 or 152. The only variable predictive of severe acute graft-versus-host disease (GVHD) in multivariate Cox analysis was substitution at position 116. Actuarial risk for acute GVHD grade III-IV, TRM, and relapse in pairs with substitutions at position 116 (n = 37) compared to other pairs (n = 63) was, respectively, 36% versus 14% (P =.01), 59% versus 28% (P =.001), and 25% versus 31% (P =.4). In conclusion these data suggest that substitutions at position 116 of class I heavy chain increase the risk for acute GVHD and TRM in patients who receive transplanted bone marrow from unrelated donors.

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.

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.

Natural killer cells lyse autologous herpes simplex virus infected targets using cytolytic mechanisms distributed clonotypically.

Natural killer (NK) cells have the capability of lysing targets that have down-regulated the expression of HLA class I molecules. Herpes simplex virus (HSV) infection results in a profound reduction of HLA class I molecules on the surface of infected cells. For this reason, NK cell populations kill efficiently HSV-infected cells. The recent availability of a panel of monoclonal antibodies directed to NK receptors for HLA class I (CD158a, CD158b, anti-p70, anti-p140, and CD94) allowed an accurate dissection of the NK cell subpopulations. Using this approach, the relationship between the expression of NK cell receptors and the capability of lysing HSV-infected cell targets was analyzed at the clonal level. NK cell clones were derived from healthy donors, and cytolytic properties were assayed against HSV-infected autologous fibroblasts. NK cell clones, classified according to the expression of natural killer-cell receptors on their surface, displayed a great heterogeneity of cytolytic properties against HSV-infected cells. Nevertheless, a more accurate functional analysis demonstrated not only that HSV infection downregulated the expression of HLA-A and HLA-B and did not modify the expression of HLA-C, but also that NK cell clones expressing the "activating" form of the anti HLA-C NK cell receptor were more cytolytic than other clones. This finding suggests that two different and clonally distributed mechanisms of NK cell activation may be employed by NK cells to kill HSV-infected autologous target cells.

Melanomas and melanoma cell lines do not express HLA-G, and the expression cannot be induced by gammaIFN treatment.

HLA-G is an effective ligand of natural killer (NK) inhibitory receptors, HLA-G transcripts have been detected in several human tumors, and cytokines like gamma interferon (IFN) enable HLA-G molecules to be expressed. These findings are particularly upsetting in case of melanomas: IFN treatment is frequently included in melanoma therapeutic protocols, and downregulation of classical class I molecules occurs in nearly half of these tumors. Therefore, a melanoma cell downregulating classical class I and de novo expressing HLA-G, either constitutively or upon IFN treatment, is probably a stealthy target for the immune system, having inhibited both the cytotoxic T lymphocyte (CTL) and the NK activity. To elucidate this point we have investigated the expression of HLA-G molecules in 45 melanoma cell lines before and after gammaIFN treatment. Analysis was performed by immunofluorescence and flow cytometry, using the anti-HLA-G MoAbs 87G and G233, by Western blot, using the anti-HLA-G MEM/G1 MoAb and PAG1 antiserum, and by RT-PCR analysis. In addition, 8 melanoma tissues from patients free from therapy and 6 nevi were studied by immunohistochemistry using the 87G MoAb. No evidence was gathered of HLA-G expression, neither constitutive nor, in cell lines, after gammaIFN treatment. We therefore conclude that HLA-G expression is an uncommon event in melanomas, and that a therapy including IFNs cannot harm the patient by inducing the de novo expression of HLA-G molecules at least in its G1 isoform.

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.

Activation-related differences in HLA class I-bound peptides: presentation of an IL-1 receptor antagonist-derived peptide by activated, but not resting, CD4+ T lymphocytes.

We have compared by reverse phase HPLC the set of peptides eluted from HLA class I molecules in resting and activated CD4+ Jurkat cells. Two peptides were identified that are presented de novo upon activation. After sequencing, one of these peptides turned out to derive from IL-1R antagonist (IL-1Ra). In keeping with this observation, we found that activated, but not resting, Jurkat cells express IL-1Ra. These data indicate that activation of a CD4+ T cell line may result in presentation of peptides derived from proteins expressed de novo after activation. Since IL-1Ra was not known to be expressed by cells of the T lineage, we also investigated its pattern of expression in normal T lymphocytes. Reverse transcriptase-PCR analyses allowed us to demonstrate that IL-1Ra is expressed upon activation by normal CD4+ lymphocytes from peripheral blood and by thymocytes, but not by CD8+ T cells. Interestingly, of the two forms of IL-1Ra that have been detected in different cell lineages, the intracellular one and the secreted one, only the former is expressed by activated CD4+ T cells.

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.

Cellular mechanisms of exogenous peptide binding to HLA class II molecules in B cells.

We have investigated the ability of APC Class II molecules to bind and release exogenous peptides, two phenomena that are still poorly understood. In order to investigate the half-life of the complex of an exogenous peptide with DR molecules we have evaluated the uptake and release of the radiolabeled peptide 17-29-Tyr of influenza virus matrix protein (MA 17-29-Y) by a B-EBV cell line at different times and under different conditions. We have found that the kinetics of both binding and release of the peptide are very fast in living cells; 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. As confirmed by the study of a specific T-cell clone activation, the Class II-MA 17-29-Y complexes are short-living ones, with an average half-life of 55 min, and the DR molecules that bind exogenous peptides continuously undergo peptidic exchange. These data, taken together, suggest that the APC are endowed with cellular mechanisms that increase the efficiency of both the loading and the unloading of Class II HLA with exogenous peptides. These mechanisms do not appear to require ATP or to involve newly synthesized Class II molecules, intracellular acidic compartments, or the microtubule-microfilament system. On the other hand, an undamaged cell membrane appears to be crucial for an efficient binding.

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.

Binding of labelled influenza matrix peptide to HLA DR in living B lymphoid cells.

T cells recognize protein antigens as fragments (peptides) held in a defined binding site of class I or class II major histocompatibility (MHC) molecules. The formation of complexes between various immunologically active peptides and different MHC molecules has been demonstrated directly in binding studies between the peptides and solubilized, purified molecules of class II MHC. Studies with intact cells, living or fixed, have not directly demonstrated the binding of the peptides to MHC molecules on antigen-presenting cells, but the formation of such complexes has been shown indirectly through the capacity of antigen-presenting cells to stimulate specific T cells. Here we report evidence that supports directly the binding of radiolabelled influenza matrix peptide 17-29 to products of the human class II MHC locus HLA-DR, on living homozygous B-cell lines, and we show that the kinetics of such binding is much faster with living cells than with fixed cells. Furthermore, whereas the peptide reacts with HLA-DR molecules of all alleles, it binds preferentially to DR1, the restricting element in antigen presentation.

Monocyte-derived macrophages as helper cells in monocyte-mediated cytolysis.

Human peripheral blood monocytes (M), incubated with opsonized zymosan particles (OPZ), lysed human erythrocyte (RBC) targets, as detected by a 51Cr release method. Conversely, cells derived in vitro from M (monocyte-derived macrophages, MDM) were ineffective. When added to the M-RBC system, MDM enhanced the lysis. The lysis by M and M plus MDM was prevented by catalase, azide and amino acids (alanine, taurine), consistent with the requirement for hypochlorous acid (HOCl). Moreover, MDM per se incapable of generating HOCl augmented the HOCl recovery from the M-RBC system. The results provide evidence for a previously unrecognized form of interaction between two distinct populations of mononuclear phagocytes.

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.