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Blood dendritic cell antigen 2 (BDCA2) is a receptor that is exclusively expressed on plasmacytoid dendritic cells, which are implicated in the pathogenesis of lupus erythematosus. Whether treatment with litifilimab, a humanized monoclonal antibody against BDCA2, would be efficacious in reducing disease activity in patients with cutaneous lupus erythematosus has not been extensively studied.In this phase 2 trial, we randomly assigned adults with histologically confirmed cutaneous lupus erythematosus with or without systemic manifestations in a 1:1:1:1 ratio to receive subcutaneous litifilimab (at a dose of 50, 150, or 450 mg) or placebo at weeks 0, 2, 4, 8, and 12. We used a dose-response model to assess whether there was a response across the four groups on the basis of the primary end point, which was the percent change from baseline to 16 weeks in the Cutaneous Lupus Erythematosus Disease Area and Severity Index-Activity score (CLASI-A; scores range from 0 to 70, with higher scores indicating more widespread or severe skin involvement). Safety was also assessed.A total of 132 participants were enrolled; 26 were assigned to the 50-mg litifilimab group, 25 to the 150-mg litifilimab group, 48 to the 450-mg litifilimab group, and 33 to the placebo group. Mean CLASI-A scores for the groups at baseline were 15.2, 18.4, 16.5, and 16.5, respectively. The difference from placebo in the change from baseline in CLASI-A score at week 16 was -24.3 percentage points (95% confidence interval [CI] -43.7 to -4.9) in the 50-mg litifilimab group, -33.4 percentage points (95% CI, -52.7 to -14.1) in the 150-mg group, and -28.0 percentage points (95% CI, -44.6 to -11.4) in the 450-mg group. The least squares mean changes were used in the primary analysis of a best-fitting dose-response model across the three drug-dose levels and placebo, which showed a significant effect. Most of the secondary end points did not support the results of the primary analysis. Litifilimab was associated with three cases each of hypersensitivity and oral herpes infection and one case of herpes zoster infection. One case of herpes zoster meningitis occurred 4 months after the participant received the last dose of litifilimab.In a phase 2 trial involving participants with cutaneous lupus erythematosus, treatment with litifilimab was superior to placebo with regard to a measure of skin disease activity over a period of 16 weeks. Larger and longer trials are needed to determine the effect and safety of litifilimab for the treatment of cutaneous lupus erythematosus. (Funded by Biogen; LILAC ClinicalTrials.gov number, NCT02847598.).

Antibody-binding of blood dendritic cell antigen 2 (BDCA2), which is expressed exclusively on plasmacytoid dendritic cells, suppresses the production of type I interferon that is involved in the pathogenesis of systemic lupus erythematosus (SLE). The safety and efficacy of subcutaneous litifilimab, a humanized monoclonal antibody that binds to BDCA2, in patients with SLE have not been extensively studied.We conducted a phase 2 trial of litifilimab involving participants with SLE. The initial trial design called for randomly assigning participants to receive litifilimab (at a dose of 50, 150, or 450 mg) or placebo administered subcutaneously at weeks 0, 2, 4, 8, 12, 16, and 20, with the primary end point of evaluating cutaneous lupus activity. The trial design was subsequently modified; adults with SLE, arthritis, and active skin disease were randomly assigned to receive either litifilimab at a dose of 450 mg or placebo. The revised primary end point was the change from baseline in the total number of active joints (defined as the sum of the swollen joints and the tender joints) at week 24. Secondary end points were changes in cutaneous and global disease activity. Safety was also assessed.A total of 334 adults were assessed for eligibility, and 132 underwent randomization (64 were assigned to receive 450-mg litifilimab, 6 to receive 150-mg litifilimab, 6 to receive 50-mg litifilimab, and 56 to receive placebo). The primary analysis was conducted in the 102 participants who had received 450-mg litifilimab or placebo and had at least four tender and at least four swollen joints. The mean (±SD) baseline number of active joints was 19.0±8.4 in the litifilimab group and 21.6±8.5 in the placebo group. The least-squares mean (±SE) change from baseline to week 24 in the total number of active joints was -15.0±1.2 with litifilimab and -11.6±1.3 with placebo (mean difference, -3.4; 95% confidence interval, -6.7 to -0.2; P = 0.04). Most of the secondary end points did not support the results of the analysis of the primary end point. Receipt of litifilimab was associated with adverse events, including two cases of herpes zoster and one case of herpes keratitis.In a phase 2 trial involving participants with SLE, litifilimab was associated with a greater reduction from baseline in the number of swollen and tender joints than placebo over a period of 24 weeks. Longer and larger trials are required to determine the safety and efficacy of litifilimab for the treatment of SLE. (Funded by Biogen; LILAC ClinicalTrials.gov number, NCT02847598.).

To monitor antigen-specific CD4+T cells during a recall immune response to tetanus toxoid (TT), a sequential analysis including ex vivo phenotyping and cytokine flow cytometry, followed by cloning and T-cell-receptor (TCR) spectratyping of cytokine-positive CD4+T cells, was performed. Grossly, twice as many TT-specific CD4+T-cell clones, ex vivo derived from the CCR7+/−CD69+interleukin-2-positive (IL-2+) CD4+subsets, belonged to the central memory (TCM; CD62L+CD27+CCR7+) compared to the effector memory population (TEM; CD62L−CD27−CCR7−). After the boost, a predominant expansion of the TCMpopulation was observed with more limited variations of the TEMpopulation. TCR beta-chain-variable region (BV) spectratyping and sequencing confirmed a large concordance between most frequently expressed BV TCR-CDR3 from ex vivo-sorted CCR7+/−CD69+IL-2+CD4+subsets and BV usage of in vitro-derived TT-specific CD4+T-cell clones, further demonstrating the highly polyclonal but stable character of the specific recall response to TT. Taken together, ex vivo flow cytometry analysis focused on the CCR7+/−CD69+IL-2+CD4+subsets appears to target the bulk of antigen-specific T cells and to reach an analytical power sufficient to adequately delineate in field trials the profile of the antigen-specific response to vaccine.

The aim of T cell vaccines is the expansion of antigen-specific T cells able to confer immune protection against pathogens or tumors. Although increase in absolute cell numbers, effector functions and TCR repertoire of vaccine-induced T cells are often evaluated, their reactivity for the cognate antigen versus their cross-reactive potential is rarely considered. In fact, little information is available regarding the influence of vaccines on T cell fine specificity of antigen recognition despite the impact that this feature may have in protective immunity. To shed light on the cross-reactive potential of vaccine-induced cells, we analyzed the reactivity of CD8(+) T cells following vaccination of HLA-A2(+) melanoma patients with Melan-A peptide, incomplete Freund's adjuvant and CpG-oligodeoxynucleotide adjuvant, which was shown to induce strong expansion of Melan-A-reactive CD8(+) T cells in vivo. A collection of predicted Melan-A cross-reactive peptides, identified from a combinatorial peptide library, was used to probe functional antigen recognition of PBMC ex vivo and Melan-A-reactive CD8(+) T cell clones. While Melan-A-reactive CD8(+) T cells prior to vaccination are usually constituted of widely cross-reactive naive cells, we show that peptide vaccination resulted in expansion of memory T cells displaying a reactivity predominantly restricted to the antigen of interest. Importantly, these cells are tumor-reactive.

CD8(+) cytolytic T lymphocytes (CTL) play a fundamental role in the clearance of malaria parasites from the liver in mouse models. In humans, however, only low levels of parasite-specific CD8(+) T lymphocytes have been observed in individuals living in endemic areas. In the present study, we identified high levels of circulating CD8(+) T lymphocytes specific for a previously described HLA-A2-restricted CTL epitope of the circumsporozoite (CS) protein of Plasmodium falciparum in an adult living in Burkina Faso, as evidenced by IFN-gamma ELISPOT assay and MHC-tetramer technology. After cloning by limiting dilution culture, T cell recognition of natural CS variants of P. falciparum was studied. The results demonstrate that naturally occurring variations drastically affect residues critical for T cell recognition as only two out of nine sequences analyzed were efficiently recognized by the CTL clones. These clones were also used to analyze T cell recognition of the endogenously presented cognate antigen. We observed efficient antigen recognition of both HLA-A*0201-transfected murine antigen presenting cells and liver cells from HLA-A*0201/K(b)-transgenic mice upon infection with recombinant vaccinia virus encoding the CS protein (WR-CS). More importantly, we demonstrate for the first time efficient recognition of WR-CS-infected human liver cells.

We have studied the alterations in CD45R phenotypes of CD4(+)CD45RA(-)RO(+) T cells in recipients of T cell-depleted bone marrow grafts. These patients are convenient models because early after transplantation, their T cell compartment is repopulated through expansion of mature T cells and contains only cells with a memory phenotype. In addition, re-expression of CD45RA by former CD4(+)CD45RA(-) T cells can be accurately monitored in the pool of recipient T cells that, in the absence of recipient stem cells, can not be replenished with CD45RA(+) T cells through the thymic pathway. We found that CD4(+)CD45RA(-)RO(+) recipient T cells could re-express CD45RA but never reverted to a genuine CD4(+)CD45RA(+)RO(-) naive phenotype. Even 5 years after transplantation, they still co-expressed CD45RO. In addition, the level of CD45RA and CD45RC expression was lower ( approximately 35 %) than that of naive cells. In contrast, the level of CD45RB expression was comparable to that of naive cells. We conclude that CD4(+)CD45RA(-)RO(+) T cells may re-express CD45(high) isoforms but remain distinguishable from naive cells by their lower expression of CD45RA / RC and co-expression of CD45RO. Therefore, it is likely that the long-lived memory T cell will be found in the population expressing both low and high molecular CD45 isoforms.

Background. To determine the extent to which major histoincompatibilities are recognized after bone marrow transplantation, we characterized the specificity of the cytotoxic T lymphocytes isolated during graft-versus-host disease. We studied three patients transplanted with marrow from donors who were histoincompatible for different types of HLA antigens. Methods. Patient 1 was mismatched for one ABDR-antigen (HLA-A2 versus A3). Two patients were mismatched for antigens that would usually not be taken into account by standard selection procedures: patient 2 was mismatched for an HLA-A subtype (A * 0213 versus A * 0201), whereas patient 3 was mismatched for HLA-C (HLA-C * 0501 versus HLA-C * 0701). All three HLA class I mismatches were detected by a pretransplant cytotoxic precursor test. Results. Analysis of the specificity of the cytotoxic T lymphocyte clones isolated after transplantation showed that the incompatibilities detected by the pretransplant cytotoxic precursor assay were the targets recognized during graft-versus-host disease. Conclusions. Independent of whether the incompatibility consisted of a full mismatch, a subtype mismatch, or an HLA-C mismatch, all clones recognized the incompatible HLA molecule. In addition, some of these clones had undergone antigen selection and were clearly of higher specificity than the ones established before transplantation, indicating that they had been participating directly in the antihost immune response.

Background: Intranasal administration of high amount of allergen was shown to induce tolerance and to reverse the allergic phenotype. However, mechanisms of tolerance induction via the mucosal route are still unclear. Objectives: To characterize the therapeutic effects of intranasal application of ovalbumin (OVA) in a mouse model of bronchial inflammation as well as the cellular and molecular mechanisms leading to protection upon re-exposure to allergen. Methods: After induction of bronchial inflammation, mice were treated intranasally with OVA and re-exposed to OVA aerosols 10 days later. Bronchoalveolar lavage fluid (BALF), T cell proliferation and cytokine secretion were examined. The respective role of CD4(+)CD25(+) and CD4(+)CD25(-) T cells in the induction of tolerance was analysed. Results: Intranasal treatment with OVA drastically reduced inflammatory cell recruitment into BALF and bronchial hyperresponsiveness upon re-exposure to allergen. Both OVA- specific-proliferation of T cells, T(h)1 and T(h)2 cytokine production from lung and bronchial lymph nodes were inhibited. Transfer of CD4(+)CD25(-) T cells, which strongly expressed membrane-bound transforming growth factor beta (mTGF beta), from tolerized mice protected asthmatic recipient mice from subsequent aerosol challenges. The presence of CD4(+)CD25(+)(Foxp3(+)) T cells during the process of tolerization was indispensable to CD4(+)CD25(-) T cells to acquire regulatory properties. Whereas the presence of IL-10 appeared dispensable in this model, the suppression of CD4(+)CD25(-)mTGF beta(+) T cells in transfer experiments significantly impaired the down-regulation of airways inflammation. Conclusion: Nasal application of OVA in established asthma led to the induction of CD4(+)CD25(-)mTGF beta(+) T cells with regulatory properties, able to confer protection upon allergen re-exposure.

Invariant NKT cells (iNKT cells) recognize glycolipid Ags via an invariant TCR α-chain and play a central role in various immune responses. Although human CD4+ and CD4− iNKT cell subsets both produce Th1 cytokines, the CD4+ subset displays an enhanced ability to secrete Th2 cytokines and shows regulatory activity. We performed an ex vivo analysis of blood, liver, and tumor iNKT cells from patients with hepatocellular carcinoma and metastases from uveal melanoma or colon carcinoma. Frequencies of Vα24/Vβ11 iNKT cells were increased in tumors, especially in patients with hepatocellular carcinoma. The proportions of CD4+, double negative, and CD8α+ iNKT cell subsets in the blood of patients were similar to those of healthy donors. However, we consistently found that the proportion of CD4+ iNKT cells increased gradually from blood to liver to tumor. Furthermore, CD4+ iNKT cell clones generated from healthy donors were functionally distinct from their CD4− counterparts, exhibiting higher Th2 cytokine production and lower cytolytic activity. Thus, in the tumor microenvironment the iNKT cell repertoire is modified by the enrichment of CD4+ iNKT cells, a subset able to generate Th2 cytokines that can inhibit the expansion of tumor Ag-specific CD8+ T cells. Because CD4+ iNKT cells appear inefficient in tumor defense and may even favor tumor growth and recurrence, novel iNKT-targeted therapies should restore CD4− iNKT cells at the tumor site and specifically induce Th1 cytokine production from all iNKT cell subsets.

Human cancer vaccines are often prepared with altered “analog” or “heteroclitic” antigens that have been optimized for HLA class I binding, resulting in enhanced immunogenicity. Here, we take advantage of CpG oligodeoxynucleotides as powerful vaccine adjuvants and demonstrate the induction of high T cell frequencies in melanoma patients, despite the use of natural (unmodified) tumor antigenic peptide. Compared with vaccination with analog peptide, natural peptide induced T cell frequencies that were approximately twofold lower. However, T cells showed superior tumor reactivity because of (i) increased functional avidity for natural antigen and (ii) enhancement of T cell activation and effector function. Thus, novel vaccine formulations comprising potent immune stimulators may allow to circumvent the need for modified antigens and can induce highly functional T cells with precise antigen specificity.

Activated CD8 T cells develop cytotoxicity against autologous cells bearing foreign Ags and self/tumor Ags. However, self-specific cytolysis needs to be kept under control to avoid overwhelming immunopathology. After peptide vaccination of melanoma patients, we studied molecular and functional properties of T cell subsets specific for the self/tumor Ag Melan-A/MART-1. Ex vivo analysis revealed three Ag-specific effector memory (EM) populations, as follows: CD28-negative EM (EM28−) T cells strongly expressing granzyme/perforin, and two EM28+ subsets, one with high and the other with low level expression of these cytotoxic proteins. For further functional characterization, we generated 117 stable CD8 T cell clones by ex vivo flow cytometry-based sorting of these subsets. All EM28−-derived clones lysed target cells with high efficacy. In contrast, EM28+-derived clones were heterogenous, and could be classified in two groups, one with high and the other with low killing capacity, correlating with granzyme/perforin expression. High and low killer phenotypes remained surprisingly stable for several months. However, strongly increased granzyme expression and cytotoxicity were observed after exposure to IL-12. Thus, the data reveal a newly identified subset of CD28+ conditional killer T cells. Because CD28 can mediate strong costimulatory signals, tight cytotoxicity control, as shown in this study through IL-12, may be particularly important for subsets of T cells expressing CD28.

CD45 engagement by monoclonal antibodies on human activated T cells triggers tumour necrosis factor-alpha (TNF-alpha) gene transcription in an epitope-specific manner. To dissect the early signalling events leading to TNF-alpha gene expression, we established that CD45 crosslinking resulted in tyrosine phosphorylation of p56lck, ZAP-70, CD3-zeta, LAT and Vav. This was accompanied by down-regulation of membrane-associated protein tyrosine phosphatase activity in the absence of demonstration of enhanced p56lck, p72syk and ZAP-70 kinase activity, which remained constitutive. These early events eventually triggered an intracellular Ca(2+) rise and phosphoinositide turnover. We conclude that down-regulation of membrane-associated tyrosine phosphatase activity by CD45 extracytoplasmic domain multimerization led, in an epitope-specific fashion, to unopposed tyrosine kinase activity and to the activation of the T-cell receptor/CD3 complex signalling cascade, resulting in TNF-alpha gene expression. This model strongly suggests that CD45 extracytoplasmic tail multimerization may contribute to the modulation T-cell functions.

Background Nasal administration of major peptide T cell epitopes gives contradictory data on the induction of peripheral tolerance. Objective To compare the prophylactic effect of intranasal treatment (INT) on the development of an allergic response, using either ovalbumin (OVA) or its major T cell epitope OVA 323-339 (OVAp). Methods BALB/c mice were treated intranasally with OVA or OVAp and subsequently immunized s.c. with OVA. Anti-OVA-specific antibody, T cell proliferation and cytokine responses were analysed. In an adoptive transfer model using OVAp specific TCR transgenic (Tg) T cells from D011.10 mice, in vivo tracking and characterization of transferred T cells in the cervical, inguinal and bronchial lymph nodes (BLN) and in the spleen were determined by FACS analysis. Results Prophylactic INT with OVA induced T cell tolerance towards subsequent OVA s.c. immunizations, inhibiting OVA specific T cell proliferation, IgE and IgG1 production, in contrast to INT with OVAp, which was unable to induce tolerance. In vivo analysis of transferred OVA-specific TCR Tg T cells showed that INT with OVA induced a preferential activation of T cells in BLN, as opposed to a broad, systemic activation with OVAp. In vivo, OVAp INT led to faster and more sustained cell division cycles than OVA INT. Ex vivo, tolerance to OVA was associated with the generation of IL-10 secreting CD4(+) T cells in BLN of OVA-treated mice only. Conclusion INT with OVA but not with OVAp led to regional (as opposed to systemic) T cell activation and the induction of IL-10 secreting CD4(+) T cells in BLN, potentially critical steps in the induction of T cell-specific tolerance via the nasal route.

We have developed an in vitro assay for tetanus toxin (tt) C fragment (C-fr) degradation. Purified endosomes (abbreviated endosomes 1101 or 1104) and lysosomes (abbreviated lysosomes 1101 or 1104) from the DRB1*1101 (Gly 86) and DRB1*1104 (Val 86) B cell lines were used to degrade 125I-labeled C-fr in vitro. Using three distinct methods of analysis, we show that the capacity of endosomes and lysosomes to degrade the tt C-fr or tt synthetic Y-P30 peptide differed. Using sodium dodecylsulfate-polyacrylamide gel electrophoresis, 125I-labeled C-fr degradation patterns observed either with endosomes 1101/1104 or lysosomes 1101/1104 are distinct both in terms of the number of fragments and the kinetics of generation of the fragments. These results were confirmed by high-performance liquid chromatography analysis, where we observed that the elution profiles of the 125I-labeled Y-P30 peptide digested by endosomes 1101/1104 were different compared to those obtained with lysosomes 1101/1104. Furthermore, the kinetics of degradation of 125I-labeled Y-P30 were faster with lysosomes 1104 than with lysosomes 1101. This difference in activity of the 1101 and 1104 organelles was also found in a functional assay where we showed that the activation capacity of the P30 peptide was diminished when digested by lysosome 1104, regardless of the antigen-presenting cell (APC) used, whereas endosomes 1101 or lysosomes 1101 modified P30 peptide in a form that discriminated between presentation by 1101 or 1104 APC. Taken together, these results suggest that the differential processing and presentation displayed by the DRB1*1101 and DRB1*1104 APC is due partly to a different enzymatic content and partly to the dimorphism at position DR beta 86.

SUMMARY The capacity of peripheral blood lymphocytes (PBL) or Epstein-Barr virus (EBV)-transformed B cell lines from CGD patients to process and present tetanus toxin (tt)-specific epitopes was assessed using various tt preparations and human tt-specific T cell clones. PBL from all of the donors were able to process and present either native tt and/or denatured tt to human T cell clones specific for various tt epitopes. Furthermore, no difference was found in the antigen requirement when normal or CGD EBV-B cell lines were used as antigen-presenting cells (APC). These results suggest that the deficiency in oxygen metabolism in CGD cells does not affect tt processing and presentation.

Asthma Limits Eosinophils Recruitment and IgE Production Via CD4-Dependent Mechanisms C. Longaretti, C. Barbey, E. Pradervand, F. Spertini; Division of Immunology and Allergy, Centre Hospitalier Universitaire Vaudois, Lausanne, SWITZERLAND. RATIONALE: We have recently shown that prophylactic induction of tolerance via the nasal route in ovalbumin (OVA) allergic mice was generating IL-10 producing regulatory CD4+ T cells. The aim of this study was to characterize, in an established model of asthma, the mechanisms leading to effective immune tolerance via the mucosal route. METHODS: OVA-sensitized asthmatic mice were intranasally treated with 1.5 mg OVA for 3 days. Mice were challenged by OVA aerosol 10 days later. Cells implicated in protection were characterized by transfer experiments. RESULTS: Upon challenge, intranasal OVA treatment (INT) drastically reduced inflammatory cell recruitment into bronchoalveolar lavage fluid (BALF), including eosinophils, inhibited OVA specific IgE rise and decreased bronchial hyperreactivity. T cell proliferation of ex vivo purified bronchial lymph node cells was inhibited as well as TH2 and TH1 cytokine production. Protection was effective for an extended period of time and was not associated with eotaxin reduction. Cell transfer experiment of CD4+ cells from INT OVA animals limited recruitment of inflammatory cells, including eosinophils, into BALF of recipient mice. CONCLUSIONS: INT with OVA was able to markedly limit asthmatic inflammation upon allergen challenge. CD4+ regulatory T cells appears to play a crucial role in this protection via mechanisms to be defined. Funding: Swiss National Science Foundation 1049 Preventive Use of the Co-administration of Immunostimulatory DNA Sequences (ISS-ODN) With Olive Pollen Proteins in a Mouse Model of Allergic Asthma L. Conejero1,2, Y. Higaki1,2, M. J. Fernandez-Bohorquez1, I. VarelaNieto2, M. L. Baeza1, J. M. Zubeldia1; 1Allergy Department, Hospital Gregorio Maranon, Madrid, SPAIN, 2Instituto Investigaciones Biomedicas Alberto Sols-CSIC, Madrid, SPAIN. RATIONALE: ISS-ODNs are potent immunomodulators and stimulators of innate immunity. We studied the effects of the co-administration of these sequences with olive pollen proteins (Olea) given prior to Olea sensitization in a mouse model of allergic asthma. METHODS: BALB/c mice were distributed in different groups and immunized by intradermal injections of ISS-ODN and Olea, ISS-ODN, Olea, or saline (SS) on days 0, 7 and 14. Mice were then sensitized by subcutaneous injections with Olea in alum twice. Blood samples were collected weekly to measure immunoglobulin levels. Mice were sacrificed and splenocytes were cultured with and without the allergen for 3 days. Supernatants were assayed to determine the level of different cytokines. Finally, bronchial reactivity to methacholine was determined by Whole Body Plethysmography. RESULTS: Concomitant administration of ISS-ODN with Olea before the mice were sensitized resulted in significant changes in the immunological and functional parameters: a) Production of Olea-specific IgE was greatly reduced (p= 0.05); b) Significant higher levels of specific IgG2a, IFNand IL-18 production were detected (p< 0.05); and c) Pulmonary function test revealed a greater reduction in the bronchial hypereactivity to methacholine (p< 0.05). CONCLUSIONS: The co-administration of ISS-ODN with Olea prior to sensitization prevents the development of an allergic phenotype in our mouse model of bronchial asthma. Funding: FIS 01/0598

As compared to bone marrow, transplantation with cord blood (CB) is associated with a lower risk of GVHD. Although some cases of GVHD III have been reported, usually the grade of GVHD remains low, even when donor and recipient are not fully HLA matched. To investigate whether this is due to an intrinsic property of CB T cells, we studied the conditions under which CB CD8+ cells would be able to generate an alloresponse. In addition, we measured the cytokines secreted by the alloreactive cytotoxic T lymphocyte (CTL) clones generated. We show that: (1) the capacity of CB cells to generate alloreactive CTLs in vitro is diminished as compared to adult cells (AB); (2) in the presence of exogenous IL-2, CB cells do generate a normal alloreactive response; (3) although CB-derived CD8+ allospecific clones showed the same T1/T0 cytokine profile as the clones from AB, they secreted a lower amount of IFN-gamma; and (4) in addition, cloned CD4+ CB cells are mainly T2/T0 type, whereas AB CD4+ T cells were mainly of T1/T0 type. These data suggest that the reduced GVHD observed after transplantation with CB might be the result of the naiveness of CB T cells. After allostimulation, CB cytotoxic T cells differ from AB T cells by a higher activation threshold, a lower secretion of IFN-gamma by both CD4+ and CD8+ CB cells and their bias towards a T2 type CD4 response.