Repifermin (keratinocyte growth factor-2) reduces the severity of graft-versus-host disease while preserving a graft-versus-leukemia effect.

Graft-versus-host disease (GVHD) is the principal complication after allogeneic bone marrow transplantation (BMT). Reductions in systemic GVHD are frequently associated with a corresponding diminishment of the graft-versus-leukemia (GVL) response. In this study, we tested the effects of a novel recombinant human keratinocyte growth factor, repifermin (keratinocyte growth factor-2), on the induction of GVHD in a well-defined murine BMT model (B6 --> B6D2F1). Administration of repifermin (5 mg/kg/d) to allogeneic BMT recipients resulted in a significant decrease in both systemic GVHD and target organ histopathology. Repifermin treatment also reduced serum levels of tumor necrosis factor alpha and lipopolysaccharide compared with control mice. In contrast, repifermin did not affect T-cell proliferation, cytokine production, or cytotoxic responses to host antigens. When 2000 host-derived P815 (H-2(d)) leukemia cells were added to the bone marrow inoculum, repifermin preserved GVL effects and resulted in significantly delayed mortality compared with control-treated allogeneic BMT recipients. Collectively, these data suggest that repifermin administration may represent a novel strategy to separate the toxicity of GVHD from the beneficial GVL effects after allogeneic BMT.     

Intervention of MAdCAM-1 or fractalkine alleviates graft-versus-host reaction associated intestinal injury while preserving graft-versus-tumor effects

Coincidence of the beneficial graft-vs.-tumor (GVT) effects and the detrimental graft-vs.-host disease (GVHD) remains the major obstacle against the widespread use of allogeneic bone marrow transplantation (BMT) as tumor immunotherapy. We here demonstrate that intervention of MAdCAM-1 (mucosal vascular addressin cell adhesion molecule-1) or fractalkine/CX3CL1 after the expansion of allo-reactive donor CD8 T cells selectively inhibits the recruitment of effector donor CD8 T cells to the intestine and alleviates the graft-vs.-host reaction (GVHR) associated intestinal injury without impairing GVT effects. In a nonirradiated acute GVHD model, donor CD8 T cells up-regulate the expression of intestinal homing receptor alpha4beta7 and chemokine receptors CXCR6 and CX3CR1, as they differentiate into effector cells and subsequently infiltrate into the intestine. Administration of anti-MAdCAM-1 antibody or anti-fractalkine antibody, even after the expansion of alloreactive donor CD8 T cells, selectively reduced the intestine-infiltrating donor CD8 T cells and the intestinal crypt cell apoptosis without affecting the induction of donor derived anti-host CTL or the infiltration of donor CD8 T cells in the hepatic tumor. Moreover, in a clinically relevant GVHD model with myeloablative conditioning, these antibodies significantly improved the survival and loss of weight without impairing the beneficial GVT effects. Thus, interruption of alpha4beta7-MAdCAM-1 or CX3CR1-fractalkine interactions in the late phase of GVHD would be a novel therapeutic approach for the separation of GVT effects from GVHR-associated intestinal injury.     

Post-hematopoietic cell transplantation control of graft-versus-host disease by donor CD425 T cells to allow an effective graft-versus-leukemia response.

After allogeneic hematopoietic cell transplantation (HCT), the high inverse correlation between graft-versus-host disease (GVHD) and leukemic relapse requires that calculated measures be taken to reduce GVHD pathology while retaining the graft-versus-leukemia (GVL) effect. We sought to determine whether donor CD4(+)CD25(+) regulatory T cells could control ongoing GVHD, thereby providing an initial window of time in which the alloreactive anti-host response is permitted to begin, with the intent of most effectively eliminating residual leukemia cells. Prevention of lethal GVHD by infusion of donor CD4(+)CD25(+) cells early after HCT (day 2) was achieved across a major histocompatibility complex barrier in the haploidentical C3H-->(B6xC3H)F(1) model. However, in vitro expansion of donor CD4(+)CD25(+) T cells, stimulated by recipient cells in the presence of high-dose interleukin-2, was required for successful regulation. In contrast, in the major histocompatibility complex-matched, minor histocompatibility antigen-disparate, CD8-mediated B10.BR-->CBA GVHD model, lethal disease could be completely prevented by a single infusion of freshly isolated donor CD4(+)CD25(+) cells administered as late as 10 days after HCT. Of importance, this late regulatory effect required only a 3:1 ratio of effector CD8:CD4(+)CD25(+) T cells, indicating a strong potential for the delayed infusion of CD4(+)CD25(+) cells to control GVHD across minor histocompatibility antigen barriers. Furthermore, this regulation did not interfere with complete and lasting donor engraftment of the hematopoietic compartment. Of most significance, the day 10 infusion of donor CD4(+)CD25(+) cells into CBA HCT recipients that had been challenged with the MMCBA6 myeloid leukemia cell line did not block an effective GVL response, despite reducing lethal GVHD. These results suggest that donor CD4(+)CD25(+) T cells infused soon after transplantation can ameliorate the development of GVHD without sacrificing a sufficient GVL effect.     

Leucyl-leucine methyl ester-treated haploidentical donor lymphocyte infusions can mediate graft-versus-leukemia activity with minimal graft-versus-host disease risk.

L-leucyl-L-leucine methyl ester (LLME) prevents GVHD in several animal models by depleting dipeptidyl peptidase I (DPPI)-expressing cytotoxic cellular subsets. However, clinical application has been hampered by difficulties in stem cell engraftment following treatment of donor bone marrow inocula with LLME at the concentrations necessary to purge DPPI-expressing T-cells. Noting that T-cells can mediate graft-versus-leukemia (GVL) responses via both perforin (usually co-expressed in cytotoxic granules with DPPI) and Fas ligand pathways in a murine model, we hypothesized that LLME might be useful for treatment of delayed DLIs for potential GVL activity with a decreased risk of GVHD induction. In regard to the clinical setting, the ex vivo use of LLME for this purpose would circumvent any toxicity issues for donor stem cells, because by that time patients would have already achieved successful engraftment. For our preclinical studies, we used the haploidentical C57BL/6 (B6) (H2b) --> ((B6 x DBA/2)F1 (H(2b/d)) murine model with lethally irradiated hosts that had received transplants of T-cell-depleted bone marrow cells and were challenged with the MMD2-8 myeloid leukemia line (H2d) of DBA/2 origin. A DLI of LLME-treated donor splenocytes, from B6 mice presensitized to recipient alloantigens, was administered in varying doses 14 days post-marrow transplantation, and the potential for both GVHD and GVL activity was assessed. All mice that received any dose of LLME-treated DLI survived indefinitely, without evidence of cachexia nor B-cell hypoplasia, in contrast to the severe and lethal GVHD induced by mock-treated DLI. Histological analysis largely correlated with the symptomatic findings and revealed no GVHD-like lesions in the spleens of LLME-treated DLI recipients, although some mice displayed various degrees of hepatic mononuclear infiltration. Most notably, mice given LLME-treated DLI also experienced DLI dose-dependent increases in survival against the challenge with the MMD2-8 leukemia. LLME-treated splenocytes remained immunocompetent, as these cells could proliferate in response to mitogens and to restimulation with ovalbumin when used as a recall antigen. In conclusion, LLME-treated DLI possesses immune potential and, in particular, GVL activity without inducing clinically evident GVHD.     

Amotosalen-treated donor T cells have polyclonal antigen-specific long-term function without graft-versus-host disease after allogeneic bone marrow transplantation.

We have previously shown that amotosalen HCl (S-59 psoralen)-treated donor splenocytes, which have limited proliferative capacity in vitro, can protect major histocompatibility complex-mismatched bone marrow transplant (BMT) recipients from lethal murine cytomegalovirus infection without causing graft-versus-host disease. In this study, we further investigated the effects of amotosalen-treated donor T cells on immune reconstitution after allogeneic BMT. We were surprised to find that amotosalen-treated donor T cells persisted long-term in vivo, comprising 6% to 10% on average of the T-cell compartment of transplant recipients at 4 months after transplantation. Donor T cells derived from amotosalen-treated splenocytes were predominantly polyclonal CD44 hi/int CD8 + memory T cells and were functionally active, synthesizing interferon gamma in response to stimulation with murine cytomegalovirus antigen. Amotosalen-treated donor T cells, reisolated from BMT recipients' spleens >/=4 months after transplantation, proliferated in vitro, thus indicating repair of amotosalen-mediated DNA cross-links. Compared with infusion of untreated donor splenocytes, amotosalen-treated cells enhanced thymopoiesis by bone marrow-derived stem cells in BMT recipients. However, amotosalen treatment abrogated the thymopoietic activity of lymphoid progenitor cells among the donor splenocytes. Thus, infusion of amotosalen-treated donor T cells produced rapid immune reconstitution after major histocompatibility complex-mismatched BMT by transferring long-lived polyclonal memory T cells with antiviral activity and also by enhancing bone marrow-derived thymopoiesis. This is a novel approach to adoptive immunotherapy in allogeneic BMT.     

Role of tumor necrosis factor-alpha in graft-versus-host disease and graft-versus-leukemia responses.

Tumor necrosis factor-alpha (TNF-alpha) antagonist therapy has proven effective in inflammatory conditions such as rheumatoid arthritis and Crohn's disease. There is substantial evidence that TNF-alpha also plays a role in the development of graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation, which along with leukemia relapse remains one of the 2 major impediments to success of the approach. Using a recently developed potent rat/mouse chimeric monoclonal antibody directed against murine TNF-alpha (CNTO2213), the authors investigated the effect of TNF-alpha blockade on GVHD mediated by either CD4(+) or CD8(+) donor T cells. The results indicated that the treatment had only a moderate effect on both a CD8(+) T cell-mediated major histocompatibility complex-matched GVHD model involving multiple minor histocompatibility antigens and a p-->F(1) acute GVHD model directed against a haplo-mismatched major histocompatibility complex barrier involving both CD4(+) and CD8(+) T cells. In contrast, treatment with the anti-TNF-alpha antibody had a highly significant effect (100% survival rate) on the CD4(+) T cell-mediated component of this latter model. Importantly, anti-TNF-alpha antibody did not block the development of a graft-versus-leukemia effect against a murine myeloid leukemia challenge in either a syngeneic or allogeneic p-->F(1) setting. This suggests that the inhibition of TNF-alpha during allogeneic hematopoietic cell transplantation may be able to diminish the inflammatory GVHD reaction without hindering effective graft-versus-leukemia responses.     

Infusion of select leukemia-reactive TCR Vbeta+ T cells provides graft-versus-leukemia responses with minimization of graft-versus-host disease following murine hematopoietic stem cell transplantation.

T-cell receptor (TCR) Vbeta-expression analysis by complementarity-determining region 3 (CDR3)-size spectratyping can identify the reactive populations in an immunologic response. This analysis was used in this study to characterize the Vbeta responses of C57BL/6 (B6) CD4+ and CD8+ T cells directed to either alloantigen (against [B6xDBA/2]F1; anti-H2d) or the syngeneic myeloid leukemia MMB3.19. Vbeta families exhibiting reactivity to the leukemia cells were then enriched for and administered in both syngeneic and allogeneic hematopoietic stem cell transplantation (HSCT) models to assess in vivo graft-versus-leukemia (GVL) potential. In syngeneic transplants, enrichment for pools of selected Vbeta families (Vbeta7, -11, and -13) of T cells or for a single Vbeta family (Vbeta7) of CD4+ T cells conveyed a beneficial GVL response to the recipients. Furthermore, in the haploidentical allogeneic model, both Vbeta6,7-enriched donor B6 T cells and Vbeta7-enriched CD4+ T cells exhibited significant GVL responses with concomitant minimization of graft-versus-host disease (GVHD) development compared with equal numbers of unfractionated T cells. These results suggest that CDR3-size spectratype analysis of and subsequent selection from donor T-cell repertoires can be an effective approach to separate GVL and GVHD potential following allogeneic HSCT.     

Ex vivo expansion of regulatory T cell and T helper 2 cells using a hematosphere culture

Regulatory T cells (T-reg) are important components of immune system required to understand the mechanistic details of cancer immunity and autoimmune diseases. However, reliable and efficient methods of regulatory T cell expansion have not been established yet. Here, we show that a human peripheral blood mononuclear cell (PBMNC) derived blood-born hematosphere (BBHS) culture without cytokine treatment increased T-reg and T helper 2 cell (Th2) populations in the cell suspension. We found that the isolated cells suspended around the hematospheres expressed T helper cell markers. Importantly, the Foxp3+/CD25+ T-reg and IL-4+/CD25+ Th2 cell populations in the cell suspension were greatly expanded during hematosphere culture. Through ELISA analysis of the supernatant, we showed that secretion of Th2-related cytokines such as IL-5, IL-10, and IL-13 also increased. Taken together, hematosphere culture is a good method for ex vivo expansion of T-reg and Th2, which can provide therapeutic benefits for the treatment of immune disorders.     

Ex vivo expanded dendritic cells home to T-cell zones of lymphoid organs and survive in vivo after allogeneic bone marrow transplantation

Little is known about adoptive transfer of allogeneic ex vivo expanded dendritic cells (eDCs). We investigated the trafficking pattern of eDCs in mice after allogeneic bone marrow transplantation by using bioluminescence imaging. eDCs were expanded from bone marrow precursors in the presence of GM-CSF, interleukin-4, and Flt3L and retrovirally transduced to express luciferase (luc) and green fluorescence protein (gfp). Flow cytometry showed polyclonal DC populations after expansion that consisted of CD11c+CD11b+ and CD11c-CD11b+ cells that co-expressed CD40, CD80, CD86, and MHCII. eDCs were functional in mixed lymphocyte reactions and produced tumor necrosis factor-alpha on phytohemagglutinin stimulation. The eDCs were then injected intravenously into BALB/c recipient mice that had received allogeneic bone marrow transplantation 6 weeks previously. On day 1 after transfer, eDCs were detected by bioluminescence imaging throughout the lungs and spleen. In the later course, signals were observed throughout thymus, lower abdomen, and spleen throughout a period of more than 42 days. Immunofluorescence microscopy confirmed CD11c positivity on the gfp+ donor cells, which localized in T-cell zones of mesenteric lymph nodes, Peyer's patches, spleen, and thymus. These findings are important for adoptive immunotherapies because they indicate that eDCs migrate efficiently in vivo and are capable of surviving long term.     

Linomide inhibits programmed cell death of peripheral T cells in vivo

Programmed cell death (PCD) is involved in the physiological regulation of lymphocyte turnover, as well in the antigen-driven selection of T and B cells. Here it is shown that the immunomodulator linomide (quinoline-3-carboxamide) inhibits the apoptotic decay of peripheral T lymphocytes in response to three different stimuli. First, linomide reduces the superantigen-mediated apoptosis and deletion of specific T lymphocytes of both the CD4⁺ and the CD8⁺ subsets without affecting other superantigen-triggered phenomena such as T cell expansion and anergy. Second, linomide abolishes the T lymphopenia and inhibits PCD of splenic CD4⁺ and CD8⁺ T cells induced by exogenous glucocorticoids. This effect is restricted to peripheral T lymphocytes and does not concern thymocytes. Finally, linomide abolishes the development of lymphopenia that follows infection with vaccinia virus, while reducing PCD of CD4⁺ and CD8⁺ peripheral T cells. The anti-apoptotic effect of linomide could account for its immunostimulatory properties and might be relevant to the treatment of immunodeficiencies associated with an increased apoptotic decay of T lymphocytes.     

Ubiquitin-like polypeptide inhibits the proliferative response of T cells in vivo

The monoclonal nonspecific suppressor factor (MNSF), a lymphokine produced by murine T cell hybridoma, possesses pleiotrophic Ag-nonspecific suppressive functions. Recently, we demonstrated that the recombinant form of the ubiquitin-like segment (rUbi-L) of MNSFbeta, a 15.6 kDa-protein consisting of a polypeptide with 36% homology with ubiquitin fused to the ribosomal protein S30, presented an antigen-nonspecific immunoregulatory action in a manner similar to native MNSF. Although this cytokine has been characterized in vitro, little is known about its effects in vivo. Thus, we investigated whether rUbi-L shows a suppressor activity in vivo. The proliferative response of Con A (5 microg/ml)-stimulated splenocytes of mice treated with rUbi-L (500 ng/body) was notably decreased in a dose-dependent manner (max. 57+/-20%). In contrast, administration of high dose ubiquitin (50 microg/body) showed a little, but significant, effect (30+/-7%). Interestingly, concomitant addition of ubiquitin inhibited Ubi-L-induced suppression. Mice injected with rUbi-L without gelatin did not show any suppressive effect. NA4 (1microg/body), a neutralizing monoclonal antibody against rUbi-L, abolished the Ubi-L-mediated suppression. Therefore, ubiquitin-like polypeptide may be implicated in the immune responses in vivo.     

Aberrations of suppressor T cells in human graft-versus-host disease.

To determine whether imbalances in immunoregulatory T-cell subsets exist in patients with graft-versus-host disease, we analyzed T cells in three patients with acute and in six patients with chronic graft-versus-host disease after bone-marrow transplantation. The normal human peripheral-blood T-cell compartment is composed of 80 per cent TH2-and 20 per cent TH2+ T cells, and defined by reactivity with subset-specific heteroantiserums. Human suppressor cells are TH2+, whereas helper cells are TH2-. Patients with acute and chronic graft-versus-host disease had abnormalities in these populations, and their T cells frequently bore la-like antigens. Patients with acute disease lacked TH2+ cells, and the reappearance of this subset preceded the cessation of disease activity. Chronic disease, in contrast, was more heterogeneous. Suppressor cells were lacking in two patients but increased in the other four. Two of these four patients had TH2+, la+ T cells, suggesting in vivo activation of suppressor cells. Studies showing that these TH2+, la+ cells actively suppressed the in vitro immune response support this hypothesis and suggest that the immunoregulatory cells may profoundly affect the overall immune response.     

Ex vivo measurement of the cytotoxic capacity of human primary antigen-specific CD8 T cells

The major function of CD8 T cells is to kill specifically target cells. Moreover in certain incurable diseases, antigen-specific human CD8 T cells are impaired, and assessment of their cytolytic activity could bring insights into their physiopathological role and ways to restore immune dysfunctions for immunotherapeutic purposes. Despite this, T cell cytolytic function has been seldom analyzed thoroughly in humans, due to the lack of approaches well suited for ex vivo assessment of T cell cytotoxicity. Current techniques require prior in vitro expansion of antigen-specific CD8 T cell populations and the use of immortalized cells as targets to measure the cell-mediated killing. Furthermore, bulk cytotoxic activity is frequently measured using percentage of specific lysis calculations that do not quantify actual target cell death and effector numbers at the single cell level. Here we established a new flow cytometry-based assay that allows accurate single-cell analysis of cytotoxic capacity of primary antigen-specific CD8 T cells generated in vivo in humans after antigenic exposure without in vitro amplification that can be used for specificities restricted by different HLAs as target cells are autologous cells. We show that this assay is robust, highly sensitive irrespective of the frequency of antigen-specific CD8 T cells, and allows accurate calculation of the index of cytotoxic capacity in lytic units. This new assay provides a sensitive method to measure the intrinsic cytotoxic activity of antigen-specific CD8 T cells directly ex vivo on human primary cells.     

Ex vivo isolation protocols differentially affect the phenotype of human CD4+ T cells

Leukemic T cell lines have facilitated signal transduction studies but their physiological relevance is restricted. The use of primary T lymphocytes overcomes this limitation but it has long been speculated that methodological aspects of blood collection and the isolation procedure modify the phenotype of the cell. Here we demonstrate that several characteristics of human peripheral T cells are affected by the selection conditions. A significantly higher induction of the chemokine receptor CXCR4 was observed on CD4+ lymphocytes isolated by sheep red blood cell (SRBC) rosetting and CD4 MicroBeads as compared with positively selected CD4+ cells where the antibody/bead complex was immediately detached. These latter cells expressed CXCR4 at levels equivalent to that observed on CD4+ lymphocytes obtained by negative antibody-mediated selection. Furthermore, CD4+ cells isolated by SRBC rosetting and CD4 MicroBeads formed aggregates upon in vitro culture. CD4+ lymphocytes obtained by SRBC rosetting as well as those isolated following positive selection demonstrated basal phosphorylation of the extracellular signal-regulated kinase (ERK)-2. Altogether these data suggest that certain discrepancies concerning signal transduction in primary human T cells can be attributed to the selection conditions. Thus, it is essential to establish the parameters influenced by the isolation protocol in order to fully interpret T cell responses to antigens, chemokines, and cytokines.     

Interleukin-10 inhibits the primary allogeneic T cell response to human epidermal Langerhans cells

In this study, we analyzed the effect of interleukin-10 (IL-10) on the primary allogeneic T cell response induced by human Langerhans cells (LC), the dendritic cells from epidermis. We showed that IL-10 strongly inhibited the T cell response, provided it was added at the beginning of the mixed epidermal cell lymphocyte reaction (MELR). Proliferation of both CD4⁺ and CD8⁺ T cell subsets was affected by the cytokine. An inhibitory effect of IL-10 on human LC allostimulatory function was evidenced by the fact that IL-10-preincubated LC, but not IL-10-preincubated T cells, can display inhibitory effect. LC treatment with IL-10 partially inhibited the increase of HLA-DR expression on cultured LC as the percentage of highly positive HLA-DR cells was lower than that observed in the absence of the cytokine. IL-10 inhibited T cell alloreaction induced by 2-day-cultured human LC which constitutively display high levels of HLA class II, as well as ICAM-1 and LFA-3 antigens. This suggests that the suppressive effect of the cytokine was not merely related to an impaired up-regulation of these molecules. Addition of IL-1 during the MELR potentiated the allogeneic T cell proliferation and could reverse, at least partly, the inhibitory effect of IL-10. Collectively, these data indicate that IL-10 can prevent the alloreaction induced by human dendritic cells, providing new insights into the potential clinical use of this cytokine.     

Ex vivo expansion of functional T lymphocytes from HIV-infected individuals

This study was designed to define the conditions for expansion of functional T lymphocytes from human immunodeficiency virus (HIV)-infected subjects, with the ultimate goal of using these cells for immunotherapy. The most appropriate culture conditions for good T cell proliferation included stimulation with anti-CD3 and anti-CD28 coated microspheres, and propagation in Aim V serum-free media with 20 U/ml interleukin-2 (IL-2), supplemented with decreasing concentrations of serum for the initial 8 days. Under these conditions, a 14-day culture period yielded approximately a 10,000-fold expansion of T lymphocytes from HIV-infected donors. The cultured cells comprised approximately 15% CD4+ cells and 70% CD8+ cells. These cells retained functional capacity as assessed by cytotoxicity towards HIV proteins, and production of IL-2 and interferon-gamma (IFN-gamma). Viral replication within the culture system was controlled, but not eliminated, without the requirement for antiviral agents. These culture conditions were demonstrated to be suitable for larger scale expansion of cells in hollow fibre bioreactors. This methodology provides a suitable means of producing large quantities of functional T cells for use in autologous immunotherapy protocols.     

Deletion of self-reactive T cells in the donor-derived T cells but not in the host-derived T cells in fully allogeneic radiation chimeras. Mls-reactive T cells in allogeneic radiation chimeras

Kinetics of T cells bearing V beta 6 capable of recognizing Mls-1a were examined in the thymus and peripheral lymphoid organs of two allogeneic bone marrow chimeras; AKR/J(H-2k, Thy1.1,Mls-1a)----C3H/He(H-2k, Thy1.2,Mls-1b) and AKR/J----C57BL/6(H-2b,Thy1.2, Mls-1b). Sequential appearance of host- and donor-derived T cells occurred in the thymus and the peripheral lymphoid organs of both AKR----C3H and AKR----B6 chimeras. The first cells to repopulate the thymus were Thy1.2+ host-derived radioresistant cells, which were synchronized in their development. The host-derived cells in thymus of AKR----B6 chimeras differentiate more rapidly than those in AKR----C3H chimeras. An almost complete replacement from host-derived cells to donor-derived cells occurred by day 21 after reconstitution in AKR----C3H and AKR----B6 chimeras. In the donor-derived thymocytes, none of CD4- or CD8-single positive thymocytes expressed high density of V beta 6 in either AKR----C3H or AKR----B6 chimeras, whereas the host-derived thymocytes in AKR----B6 chimeras contained an appreciable number of CD4-single positive thymocytes bearing V beta 6. In the peripheral lymphoid organs, T cells bearing V beta 6 were virtually abolished in Thy1.1+ cell pool of both AKR----C3H and AKR----B6 chimeras. While V beta 6+ T cells of host-origin were detected in the peripheral lymphoid organs in AKR----B6 chimeras. These result indicated that the donor-derived mature T cells showed deletion of V beta 6 in the thymus and the peripheral lymphoid organs in both AKR----C3H and AKR----B6 chimeras, whereas lack of V beta 6 deletion was observed in the host-derived mature T cells in the AKR----B6 chimeras. These results suggested that the host-derived thymocytes may likely to escape undergoing a negative selection against donor-phenotype in the radiation bone marrow chimeras.     

T cells in hypersensitivity pneumonitis: effects of in vivo depletion of T cells in a mouse model.

C57BL/6 mice were given intranasal instillation of optimal doses of the actinomycete Faeni rectivirgula 150 micrograms/mouse 3 days/wk), an important offending agent causing hypersensitivity pneumonitis. This instillation was associated with a very significant increase in the lung weight of the mice and also a large increase (10-fold) in the number of cells recovered from the bronchoalveolar lavage (BAL) of instilled mice. Also, this instillation was associated with a very significant fibrosis at 4 and 8 wk (2-fold increase in hydroxyproline levels in the lungs). We determined the effect of depleting certain T-cell subsets on the progression of this inflammatory disease. Elimination of the L3T4 subset did not significantly affect the increase in the lung index, the lung cellular influx, or its profile. Fibrosis was also unaffected by this depletion of L3T4+ cells. Similarly, depletion of Lyt2+ (CD8+) cells did not lead to significant changes in these disease parameters. Depletion of all T cells (Thyl+) was also ineffective at modifying the number of infiltrating cells and the lung index score. However, identification of cell types in BAL showed that mice depleted of Thyl+ cells had a cellular influx that was almost exclusively neutrophilic throughout the instillation period, whereas control mice developed only a transient neutrophilic response to F. rectivirgula instillation, which was replaced by a recruitment of mononuclear cells, mostly macrophages. Also, depletion of Thyl+ cells before and during F. rectivirgula challenge had no effect on tumor necrosis factor-alpha levels in the BAL of treated mice (63 +/- 13 U/ml in anti-Thy1. 2 antibodies treated versus 52 +/- 10 U/ml in the BAL of control mice given F. rectivirgula).(ABSTRACT TRUNCATED AT 250 WORDS)     

Ex vivo monitoring of antigen-specific CD4+ T cells after recall immunization with tetanus toxoid

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 (T(CM); CD62L+ CD27+ CCR7+) compared to the effector memory population (T(EM); CD62L- CD27- CCR7-). After the boost, a predominant expansion of the T(CM) population was observed with more limited variations of the T(EM) population. 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.