Host T-cell Research Articles

Conformation of the Dileucine‐Based Sorting Motif in HIV‐1 Nef Revealed by Intermolecular Domain Assembly

The human immunodeficiency virus 1 (HIV-1) Nef protein is a pathogenicity factor required for effective progression to AIDS, which modulates host cell signaling pathways and T-cell receptor internalization. We have determined the crystal structure of Nef, allele SF2, in complex with an engineered SH3 domain of human Hck showing unnaturally tight binding and inhibitory potential toward Nef. This complex provides the most complete Nef structure described today, and explains the structural basis of the high affinity of this interaction. Intriguingly, the 33-residue C-terminal flexible loop is resolved in the structure by its interactions with a highly conserved hydrophobic groove on the core domain of an adjacent Nef molecule. The loop mediates the interaction of Nef with the cellular adaptor protein machinery for the stimulated internalization of surface receptors. The endocytic dileucine-based sorting motif is exposed at the tip of the acidic loop, giving the myristoylated Nef protein a distinctly dipolar character. The intermolecular domain assembly of Nef provides insights into a possible regulation mechanism for cargo trafficking.

Combination CTLA-4 Blockade and 4-1BB Activation Enhances Tumor Rejection by Increasing T-Cell Infiltration, Proliferation, and Cytokine Production

BackgroundThe co-inhibitory receptor Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4) attenuates immune responses and prevent autoimmunity, however, tumors exploit this pathway to evade the host T-cell response. The T-cell co-stimulatory receptor 4-1BB is transiently upregulated on T-cells following activation and increases their proliferation and inflammatory cytokine production when engaged. Antibodies which block CTLA-4 or which activate 4-1BB can promote the rejection of some murine tumors, but fail to cure poorly immunogenic tumors like B16 melanoma as single agents.Methodology/Principal FindingsWe find that combining αCTLA-4 and α4-1BB antibodies in the context of a Flt3-ligand, but not a GM-CSF, based B16 melanoma vaccine promoted synergistic levels of tumor rejection. 4-1BB activation elicited strong infiltration of CD8+ T-cells into the tumor and drove the proliferation of these cells, while CTLA-4 blockade did the same for CD4+ effector T-cells. Anti-4-1BB also depressed regulatory T-cell infiltration of tumors. 4-1BB activation strongly stimulated inflammatory cytokine production in the vaccine and tumor draining lymph nodes and in the tumor itself. The addition of CTLA-4 blockade further increased IFN-γ production from CD4+ effector T-cells in the vaccine draining node and the tumor. Anti 4-1BB treatment, with or without CTLA-4 blockade, induced approximately 75% of CD8+ and 45% of CD4+ effector T-cells in the tumor to express the killer cell lectin-like receptor G1 (KLRG1). Tumors treated with combination antibody therapy showed 1.7-fold greater infiltration by these KLRG1+CD4+ effector T-cells than did those treated with α4-1BB alone.Conclusions/SignificanceThis study shows that combining T-cell co-inhibitory blockade with αCTLA-4 and active co-stimulation with α4-1BB promotes rejection of B16 melanoma in the context of a suitable vaccine. In addition, we identify KLRG1 as a useful marker for monitoring the anti-tumor immune response elicited by this therapy. These findings should aid in the design of future trials for the immunotherapy of melanoma.

Conflicting selection pressures on T-cell epitopes in HIV-1 subtype B

Analysis of population-level polymorphism in eight coding genes of human immunodeficiency virus type 1 (HIV-1) subtype B revealed evidence not only of past purifying selection, but also of abundant slightly deleterious nonsynonymous variants subject to ongoing purifying selection. Both CD4 and CTL epitopes showed an excess of nonsynonymous variants that were singletons (occurring in just one sequence) in our dataset. Overall, median gene diversities at polymorphic nonsynonymous sites were highest at sites located in neither CD4 nor CTL epitopes, while polymorphic nonsynonymous sites in CD4 epitopes revealed the lowest median gene diversity. Our results support the hypothesis that there is an evolutionary conflict between immune escape and functional constraint on epitopes recognized by host T-cells, and suggest that amino acid sequences of CD4 epitopes are subject to particularly strong functional constraint.

Polyfunctional T-Cell Responses Are Disrupted by the Ovarian Cancer Ascites Environment and Only Partially Restored by Clinically Relevant Cytokines

BackgroundHost T-cell responses are associated with favorable outcomes in epithelial ovarian cancer (EOC), but it remains unclear how best to promote these responses in patients. Toward this goal, we evaluated a panel of clinically relevant cytokines for the ability to enhance multiple T-cell effector functions (polyfunctionality) in the native tumor environment.Methodology/Principal FindingsExperiments were performed with resident CD8+ and CD4+ T cells in bulk ascites cell preparations from high-grade serous EOC patients. T cells were stimulated with α-CD3 in the presence of 100% autologous ascites fluid with or without exogenous IL-2, IL-12, IL-18 or IL-21, alone or in combination. T-cell proliferation (Ki-67) and function (IFN-γ, TNF-α, IL-2, CCL4, and CD107a expression) were assessed by multi-parameter flow cytometry. In parallel, 27 cytokines were measured in culture supernatants. While ascites fluid had variable effects on CD8+ and CD4+ T-cell proliferation, it inhibited T-cell function in most patient samples, with CD107a, IFN-γ, and CCL4 showing the greatest inhibition. This was accompanied by reduced levels of IL-1β, IL-1ra, IL-9, IL-17, G-CSF, GM-CSF, Mip-1α, PDGF-bb, and bFGF in culture supernatants. T-cell proliferation was enhanced by exogenous IL-2, but other T-cell functions were largely unaffected by single cytokines. The combination of IL-2 with cytokines engaging complementary signaling pathways, in particular IL-12 and IL-18, enhanced expression of IFN-γ, TNF-α, and CCL4 in all patient samples by promoting polyfunctional T-cell responses. Despite this, other functional parameters generally remained inhibited.Conclusions/SignificanceThe EOC ascites environment disrupts multiple T-cell functions, and exogenous cytokines engaging diverse signaling pathways only partially reverse these effects. Our results may explain the limited efficacy of cytokine therapies for EOC to date. Full restoration of T-cell function will require activation of signaling pathways beyond those engaged by IL-2, IL-12, IL-18, and IL-21.

Allogeneic Hematopoetic Transplantation for CLL: Results of Combination Chemotherapy as Pre-Transplant Targeted Lymphocyte Depletion on Disease Response, Donor Chimerism, and Transplantation Outcomes.

AbstractAbstract 3476The development of reduced intensity conditioning (RIC) regimens has increased the use of allogeneic hematopoietic stem cell transplantation (alloHSCT) in patients with chronic lymphocytic leukemia (CLL). Despite decreased rates of transplant related mortality, RIC regimens confer slower rates of donor engraftment and increased relapse post-transplant. It has been reported that faster rates of conversion to donor chimerism after RIC alloHSCT result in lower rates of CLL relapse1. We hypothesized that systematic targeted host T-lymphocyte depletion (TLD) to a target level by serial administration of standard chemotherapy prior to RIC alloHSCT will result in more rapid donor engraftment and full donor chimerism, thereby enhancing graft versus leukemia activity. We evaluated the effects of TLD on host CD4 lymphocyte depletion, disease response, and toxicity as well as donor engraftment, non-relapse mortality, and disease control in 27 patients undergoing RIC alloHSCT for CLL from 1999 to 2010. All patients underwent TLD receiving 1–3 cycles of EPOCH-F±R (etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin, fludarabine ± rituximab) prior to RIC (fludarabine/cyclophosphamide) and matched-related (N=17) or unrelated (N=10) donor transplantation followed by calcineurin inhibitor-based GVHD prophylaxis. The number of cycles administered (max of three cycles) was titrated to achieve an absolute CD4 count ≤ 100 cells/μL prior to RIC. The median number of EPOCH-F±R cycles given was 2. The median CD3+, CD4+, and CD8+ lymphocyte counts at baseline were 819, 443, and 269 cells/μL, respectively. The median CD3+, CD4+ and CD8+ lymphocyte counts after TLD were 124, 97, and 52 cells/μL, respectively. The overall response rate to EPOCH-F±R was 50% (9% CR, 41% PR), including 44% of patients who were refractory to their last treatment. The incidence of grade 3 and 4 non-hematologic toxicity with TLD was 72% and 32%, respectively. No patients failed to engraft. The median days to full donor peripheral blood lymphoid and myeloid chimerism were 28 (14-60) and 21 (14-180), respectively. Development of full lymphoid chimerism by day 14 (first evaluation point) versus day 28 or greater trended towards association with development of acute GVHD (90% vs. 35%, P=0.014) but not chronic GVHD (50% vs. 41%, P=0.71). Achievement of full lymphoid chimerism on day 14 as compared to day 28 or greater trended towards association with decreased CLL cells identified in the bone marrow by multicolor flow cytometry at day 100 (median 0% vs. 8.5%, P=0.016) but was not associated with (P=0.077) improved overall survival (OS). There was no association between rates of development of full myeloid chimerism and acute or chronic GVHD or disease control. The rates of acute (grade 2–4) and chronic GVHD were 52% and 44%, respectively. Non-relapse related mortality at one and two years was 21% and 30%, respectively. The rate of complete remission was 54% and the rate of minimal residual disease negativity after transplantation as assessed by multicolor flow cytometry was 55%. The incidence of disease progression after transplant was 33%. The three year event-free and OS probabilities were 56% and 57%, respectively. The median OS was 5.99 years (95% CI, 2.11-NR). In summary, the use of TLD resulted in a high rate of early donor engraftement after RIC alloHSCT in patients with CLL. Rapid donor engraftment was associated with better disease control and occurence of acute GVHD, but it did not appear to alter overall survival. These data suggest that methods resulting in faster donor chimerism may improve CLL control after RIC alloHSCT. Separation of antileukemic effects from severe GVHD remain a challenge.1. Brown JR et al. Biol Blood Marrow Transplant. 2006 Oct;12(10):1056-64. Disclosures:No relevant conflicts of interest to declare.

Induction of tolerance to bone marrow allografts by donor-derived host nonreactive ex vivo–induced central memory CD8 T cells

AbstractEnabling engraftment of allogeneic T cell–depleted bone marrow (TDBM) under reduced-intensity conditioning represents a major challenge in bone marrow transplantation (BMT). Anti–third-party cytotoxic T lymphocytes (CTLs) were previously shown to be endowed with marked ability to delete host antidonor T cells in vitro, but were found to be less effective in vivo. This could result from diminished lymph node (LN) homing caused by the prolonged activation, which induces a CD44+CD62L− effector phenotype, and thereby prevents effective colocalization with, and neutralization of, alloreactive host T cells (HTCs). In the present study, LN homing, determined by imaging, was enhanced upon culture conditions that favor the acquisition of CD44+CD62L+ central memory cell (Tcm) phenotype by anti–third-party CD8+ cells. These Tcm-like cells displayed strong proliferation and prolonged persistence in BM transplant recipients. Importantly, adoptively transferred HTCs bearing a transgenic T-cell receptor (TCR) with antidonor specificity were efficiently deleted only by donor-type Tcms. All these attributes were found to be associated with improved efficacy in overcoming T cell–mediated rejection of TDBM, thereby enabling high survival rate and long-term donor chimerism, without causing graft-versus-host disease. In conclusion, anti–third-party Tcms, which home to recipient LNs and effectively delete antidonor T cells, could provide an effective and novel tool for overcoming rejection of BM allografts.

Encephalitogenic T-cells increase numbers of CNS T-cells regardless of antigen specificity by both increasing T-cell entry and preventing egress

This study utilized an adoptive transfer model of experimental autoimmune encephalomyelitis (EAE) induction in mice to characterize the mechanisms involved in CNS accumulation of transferred and host T-cells. Using a flow cytometric technique, we examined phenotypic characteristics of CNS T-cells following disease initiation and the role of T-cell activation in CNS invasion and retention. Host T-cell activation increased cell recruitment and EAE severity. CNS antigen specific T-cells were required to induce T-cell retention within the CNS. Once retention was initiated, CNS T-cells were retained regardless of specificity. This study characterizes mechanisms involved in CNS accumulation of T-cells during EAE pathogenesis.

Genotype 1 and global hepatitis C T-cell vaccines designed to optimize coverage of genetic diversity

Immunological control of hepatitis C virus (HCV) is possible and is probably mediated by host T-cell responses, but the genetic diversity of the virus poses a major challenge to vaccine development. We considered monovalent and polyvalent candidates for an HCV vaccine, including natural, consensus and synthetic 'mosaic' sequence cocktails. Mosaic vaccine reagents were designed using a computational approach first applied to and demonstrated experimentally for human immunodeficiency virus type 1 (HIV-Delta). Mosaic proteins resemble natural proteins, but are assembled from fragments of natural sequences via a genetic algorithm and optimized to maximize the coverage of potential T-cell epitopes (all 9-mers) found in natural sequences and to minimize the inclusion of rare 9-mers to avoid vaccine-specific responses. Genotype 1-specific and global vaccine cocktails were evaluated. Among vaccine candidates considered, polyvalent mosaic sequences provided the best coverage of both known and potential epitopes and had the fewest rare epitopes. A global vaccine based on conserved proteins across genotypes may be feasible, as a five-antigen mosaic cocktail provided 90, 77 and 70% coverage of the Core, NS3 and NS4 proteins, respectively; protein coverage diminished with increased protein variability, dropping to 38% for NS2. For the genotype 1-specific vaccine, the H77 prototype vaccine sequence matched only 50% of the potential epitopes in the population, whilst a polyprotein three-antigen mosaic cocktail increased potential epitope coverage to 83%. More than 75% coverage of all HCV proteins was achieved with a three-antigen mosaic cocktail, suggesting that genotype-specific vaccines could also include the more variable proteins.

Cross-immune tolerance: conception and its potential significance on transplantation tolerance

The diversity of alloreactive T cells in graft rejection and the presence of extensive crossreactivity among alloreactive T cells indicate that the induction of transplantation tolerance may fundamentally alter the size of host T-cell repertoire involved in protective immunity and immune surveillance, especially those that are crossreactive to conventional antigens. We herein highlight the crossreactive nature of alloreactive T cells and the potential risks of altered T-cell repertoire associated with the induction of transplantation tolerance. The possibility that T-cell tolerance to one set of antigens results in their tolerance to other unrelated antigens due to T-cell crossreactivity and/or heterogeneity is defined as 'cross-immune tolerance'. The definition and significance of this concept were discussed in details.

Targeted Lymphocyte Depletion Prior to Reduced Intensity Allogeneic Stem Cell Transplantation: A Personalized Approach to Facilitate Engraftment.

Abstract 865Reduced intensity allogeneic stem cell transplantation (RIST) is associated with decreased transplant-related mortality (TRM), broadening the pool of patients who could potentially benefit from allogeneic cellular therapy. However, RIST is typically associated with higher rates of mixed chimerism and graft rejection compared to myeloablative conditioning. Data from clinical studies have shown that the number of therapies prior to transplant, which inversely correlates with host T-cell immunity, is a statistically important predictor of graft rejection and mixed chimerism. In order to compensate for variability in host immune status and facilitate early full-donor chimerism (>95%), we developed a strategy of targeted lymphocyte depletion (TLD) which uses repetitive cycles of disease-specific conventional-dose chemotherapy to provide both tumor cytoreduction and lymphocyte depletion prior to RIST. The number of TLD cycles (0-3 maximum) was based on reaching a target CD4+ count <100 cells/μl; patients with higher pre-TLD CD4+ count required more cycles. We employed the TLD approach in 111 patients (mean age = 49 years, (19-71) with advanced hematologic malignancies. Median CD3+, CD4+, and CD8+counts at enrollment were: 673 cells/μl (5-3953), 286 cells/μl (5-3888), and 277 cells/μl (1-1763) respectively. Following TLD chemotherapy, median CD3+, CD4+, and CD8+ counts were: 164 cells/μl (1-1496), 82 cells/μl (0-508), and 52 cells/μl (1-1195) respectively. All patients then received an identical reduced intensity conditioning regimen (fludarabine/cyclophosphamide) followed by HLA-matched sibling peripheral blood stem cell allografts. All patients received cyclosporine as graft versus host disease (GVHD) prophylaxis (1) alone, (2) with methotrexate, (3) with TH2 cells, (4) with sirolimus or (5) with TH2 cells and sirolimus. Immediately prior to stem cell infusion (Day 0) median host CD3+, CD4+, and CD8+ counts were 4 cells/μl (0-69), 3 cells/μl (0-65) , and 1 cell/μl (0-44) respectively. 109 evaluable patients demonstrated 100% engraftment; there were no graft failures. At Day +14, median lymphocyte chimerism was 99% and median myeloid and whole blood chimerism were 100%. Patients were able to maintain chimerism as evidenced by median 100% chimerism in the myeloid, lymphoid and whole blood compartments at Day +28 and median 100% whole blood chimerism at Day +100. Full donor lymphocyte chimerism at Day +14 was associated with lower post-TLD CD4+ counts (p=0.012) and Day 0 CD3+, CD4+, and CD8+ counts (p<0.0005). Full donor myeloid chimerism at Day+14 and Day+28 was associated with lower Day 0 CD3+, CD4+, CD8+ counts (p<0.05). Full donor whole blood chimerism at Day +14, Day +28 and Day+100 was associated with lower Day 0 CD3+, CD4+, and CD8+ counts (p<0.05). The CD3+ and CD34+ dose contained in the allograft was associated with Day +14 lymphoid chimerism (p=0.04) and with Day +28 myeloid chimerism (p=0.03) respectively. CMV status was associated with both lymphoid and whole blood chimerism at Day +14 (p<0.05). Only lymphoid chimerism at Day+28 was associated with GVHD prophylaxis regimen (p<0.05). Patients with acute GVHD II-IV had significantly lower CD4+ counts post-TLD (p=0.01). Patients with acute GVHD III-IV had significantly lower CD4+ and CD3+ counts at Day 0 (p<0.05). These data demonstrate the tremendous variability in pre-transplant host T-cell numbers. By using the strategy of TLD, we were able to compensate for this variability while achieving levels of T-cell depletion comparable to myeloablative conditioning. We conclude that TLD provides a personalized approach to pre-transplant host immune status resulting in absence of graft rejection and rapid and full donor chimerism following RIST. Disclosures:No relevant conflicts of interest to declare.

Role of CCR7 in Facilitating Direct Allosensitization and Regulatory T-Cell Function in High-Risk Corneal Transplantation

Chemokine receptor 7 (CCR7) is a key homing molecule for immune cell trafficking, including corneal antigen-presenting cell (APC) migration from the inflamed cornea to draining lymph nodes (LNs). Here, the authors investigated the effect of CCR7-facilitated donor APC trafficking on allosensitization, regulatory T-cell (Treg) function, and graft survival in corneal transplantation. CCR7(-/-) or wild-type (WT) allogeneic corneal grafts were transplanted onto the neovascularized high-risk recipient beds. Two weeks later, the frequency of directly alloprimed host T cells was measured by the IFN-gamma ELISPOT assay. Treg function was tested by a coculture suppression assay and an IFN-gamma ELISPOT assay. Kaplan-Meier analysis was performed to evaluate graft survival. The recipients of CCR7(-/-) grafts had fewer migrated donor APCs and lower frequency of IFN-gamma-producing T cells in the draining LNs. However, there was no statistically significant difference in transplant survival between recipients of CCR7(-/-) and those of WT grafts. Tregs from the CCR7(-/-) graft recipient group showed reduced regulatory potential for the suppression of proliferation of naive T cells and direct alloprimed T cells and expressed lower Foxp3 levels. In vitro studies confirmed that mature CCR7(+) major histocompatibility complex class II(+) CD86(+) graft-derived dendritic cells were critical for Treg function. Not only is CCR7-mediated donor-derived APC trafficking to the draining LNs important in the initiation of host T-cell priming, it is crucial for Treg-mediated tolerance.

TLI and ATG conditioning with low risk of graft-versus-host disease retains antitumor reactions after allogeneic hematopoietic cell transplantation from related and unrelated donors

A hematopoietic cell transplantation regimen was adapted from a preclinical model that used reduced-intensity conditioning (RIC) and protected against graft-versus-host disease (GVHD) by skewing residual host T-cell subsets to favor regulatory natural killer T cells. One hundred eleven patients with lymphoid (64) and myeloid (47) malignancies received RIC using total lymphoid irradiation (TLI) and antithymocyte globulin (ATG) followed by the infusion of granulocyte colony-stimulating factor-mobilized grafts. Included were 34 patients at least 60 years of age, 32 patients at high risk of lymphoma relapse after disease recurrence following prior autologous transplantation, and 51 patients at high risk of developing GVHD due to lack of a fully human leukocyte antigen (HLA)-matched related donor. Durable chimerism was achieved in 97% of patients. Cumulative probabilities of acute GVHD (grades II-IV) were 2 and 10% of patients receiving related and unrelated donor grafts. Nonrelapse mortality (NRM) at 1 year was less than 4%. Cumulative incidence of chronic GVHD was 27%. The 36-month probability of overall and event-free survival was 60% and 40%, respectively. Disease status at start of conditioning and the level of chimerism achieved after transplantation significantly impacted clinical outcome. The high incidence of sustained remission among patients with active disease at time of transplantation suggests retained graft-versus-tumor reactions. Active trial registration currently at clinicaltrials.gov under IDs of NCT00185640 and NCT00186615.

Total lymphoid irradiation for graft-versus-host disease protection

The immunosuppressive effects of total lymphoid irradiation (TLI) for protection against graft-versus-host disease (GvHD) after allogeneic bone marrow transplantation have been studied for years in animal models. In preclinical models of bone marrow transplantation non-myeloablative TLI conditioning protects against GvHD by skewing host T-cell subsets to favor regulatory natural killer T cells that suppress GvHD by polarizing donor T cells towards secretion of non-inflammatory cytokines such as IL-4. These preclinical models have recently been adapted to human transplantation. Patients receiving allogeneic hematopoietic cell transplantation for hematological malignancies conditioned with TLI and depletive T-cell antibodies showed sustained donor chimerism, a reduced incidence of acute GvHD yet retained anti-tumor activity. As in the preclinical models, the low incidence of GvHD is associated with increased IL-4 secretion by chimeric donor T cells. This review summarizes the evolution of the preclinical TLI protocols and their recent translation to clinical trials, and discusses the mechanisms involved in protection from GvHD and the induction of tolerance following mixed chimerism.

Infectious agents and lymphoma development: molecular and clinical aspects

This review is focused on the role of infectious agents in the development of some lymphoma entities. Associations involving bacterial infections mostly regard marginal zone B-cell lymphomas of mucosa-associated lymphoid tissue (MALT)-type. Some paradigmatic examples of these associations include the Helicobacter pylori-related gastric MALT lymphoma and the more recently reported links between Chlamydophila psittaci and ocular adnexal MALT lymphomas and Borrelia burgdorferi and cutaneous MALT lymphomas. The well-documented association between Epstein-Barr virus infection and related lymphoproliferative disorders are analysed as an example of lymphotropic virus with tumourigenic activity. Molecular, biological and clinical features as well as therapeutic implications of these associations are analysed and future perspectives in this field are discussed.

Decreased T‐cell receptor excision DNA circles in peripheral blood mononuclear cells among benzene‐exposed workers

Benzene is a volatile aromatic hydrocarbon solvent which is widely used in many industries. The chronic exposure of humans to benzene in the workplace has been associated with blood disorders, as well as toxicity in lymphopoiesis, including aplastic anaemia and leukaemia. However, the mechanisms of benzene-induced haematotoxicity and leukaemogenesis remain unclear. In this study, we investigated the level of T-cell receptor excision DNA circles (TRECs) in peripheral blood mononuclear cells (PBMCs) in benzene-exposed workers. This would therefore be considered as a potential marker for estimates of thymic output and an evaluation of the content of naïve T-cells. It is hoped that the data will bring a comprehensive understanding on the influence of benzene exposure in the host T-cell immune function. Quantitative detection of TRECs in DNA of PBMCs from benzene-exposed workers was preformed by real-time polymerase chain reaction using the TaqMan technique. The benzene-exposed workers were divided into four groups, and 27 normal individuals were served as controls. The result indicated that the TRECs levels of all benzene-exposed groups were significantly decreased as compared with those of controls. In conclusion, the recent thymic output function and the T-cell immune function were apparently impaired in workers after benzene exposure.

Regulation of Dendritic- and T-Cell Fate by Injury-Associated Endogenous Signals

Two events characterize tissue injury and sterile inflammation: (1) generation/release of autoantigens, and (2) generation of homeostatic inflammatory signals. Homeostatic signals recruit leukocytes and promote cell migration and division to replace injured cells. Moreover, they activate antigen-presenting phagocytes, in particular, dendritic cells (DCs), in anticipation of microbial invasion. Activated DCs undergo a differentiation process, referred to as maturation, and migrate to secondary lymphoid organs. Maturing DCs upregulate the molecular machinery required for the priming of naive T cells, including T lymphocytes recognizing autoantigens, which represent a substantial fraction of the host T-cell repertoire. Recent data indicate that cues generated at sites of injury shape T-cell clonal expansion, regulating sensitivity to activation-dependent apoptosis and commitment towards a Th1, Th2, Th7, or regulatory T-cell fate. Endogenous signals of tissue injury, also called damage-associated molecular patterns (DAMPS) or alarmins, therefore provide a code for switching the outcome of the presentation of autoantigens towards results as diverse as T-cell-mcdiated protective immunity, tissue repair, persistent inflammation and autoimmunity, or tolerance.

Graft rejection as a Th1-type process amenable to regulation by donor Th2-type cells through an interleukin-4/STAT6 pathway

AbstractGraft rejection has been defined as the mirror image of graft-versus-host disease, which is biologically characterized primarily as a Th1-type process. As such, we reasoned that graft rejection would represent a Th1 response amenable to Th2 modulation. Indeed, adoptive transfer of host Th1-type cells mediated rejection of fully MHC-disparate murine bone marrow allografts more effectively than host Th2-type cells. Furthermore, STAT1-deficient host T cells did not differentiate into Th1-type cells in vivo and failed to mediate rejection. We next hypothesized that donor Th2 cell allograft augmentation would prevent rejection by modulation of the host Th1/Th2 balance. In the setting of donor Th2 cell therapy, host–anti-donor allospecific T cells acquired Th2 polarity, persisted posttransplantation, and did not mediate rejection. Abrogation of rejection required donor Th2 cell IL-4 secretion and host T-cell STAT6 signaling. In conclusion, T cell–mediated marrow graft rejection primarily resembles a Th1-type process that can be abrogated by donor Th2 cell therapy that promotes engraftment through a novel mechanism whereby cytokine polarization is transferred to host T cells.

Ex-Vivo Acquisition of Central Memory Phenotype Is Critical for Tolerance Induction by Donor Anti-3rd party CD8 T Cells in Allogeneic Bone Marrow Transplantation.

The robust immunity remaining after reduced intensity conditioning represents a major barrier to durable engraftment of MHC disparate T cell depleted BM transplants (TDBMT). One approach to address this challenge could be afforded by using donor CD8 CTLs, endowed with potent veto activity (the ability of a cell to specifically suppress CTL precursors directed against its antigens, while sparing other CTLs capable of affording protection against infections and malignancies). However, their use is limited due to their marked GVH reactivity. This problem can be addressed effectively by stimulation of donor CD8 T cells against 3rd party stimulators under exogenous IL2 deprivation. The anti-3rd party veto CTLs support engraftment of TDBMT without causing GVHD. Nevertheless, although these cells displayed highly efficient veto activity in vitro, they were less efficient in vivo, requiring administration in conjunction with Rapamycin. In our study we found, using in vivo imaging, that veto CTLs display poor homing to the host lymph nodes (LNs). Therefore, we hypothesized that the relatively low efficiency of veto CTLs in vivo could be attributed to their failure to co-localize with the host T cells (HTCs) at the LNs, during the critical time at which the HTCs are triggered against donor antigens. To enhance LNs homing we generated anti-3rd party CD8 T cells under culture conditions favoring the acquisition of central memory (Tcm) phenotype (CD44highCD62Lhigh). In order to monitor CTLs or Tcm cells adoptively transferred into irradiated recipients of BM allografts, we used (Host x Donor)F1 cells co-expressing host and donor MHC that can be distinguished by FACS. When CD62Lhigh Tcm cells, expanded in the presence of IL15, were administered, the number of cells located at the host LNs two days post transplant was 40-fold higher compared to that found for veto CTLs expanded in the presence of IL2, which exhibit a CD62Llow effector phenotype (p<0.05). Moreover, the total number of Tcm cells harvested from various organs was increased by 9 folds between days 2 to 6 post BMT (p<0.05) in sharp contrast to the CTLs which displayed a non significant proliferation, indicating that Tcm cells possess superior proliferative capabilities. In addition, the Tcm cells exhibited marked durable persistence in-vivo when tested 100 days post transplant, comprising 17%±9% of the total CD8 T cell compartment following infusion of 1x106 cells. When evaluated in a stringent mouse model for T cell mediated BM allograft rejection, Tcm cells displayed marked tolerizing activity. In this model, 3x106 Balb-Nude BM is transplanted into lethally irradiated C3H hosts that were previously inoculated with 1.25x104 HTC (a number sufficient to induce BM rejection and lethality). Thus, administration of 5x106 anti-3rd party Tcm cells derived from (C3H x BALB)F1 mice, with no further immunosuppression, led to overall survival of 83% (19/23) at 100 days post BMT, while administration of 107 anti-3rd party CTLs (bearing an effector phenotype) could only afford survival of 19% (3/16) (p<0.05) in the absence of Rapamycin. To evaluate the risk of GVHD, allogeneic Balb/c derived anti-3rd party Tcm cells or naive CD8 T cells were administered in conjunction with 5x106 Balb/c-Nude BM cells into lethally irradiated C3H hosts. As expected, naive CD8 cells caused lethal GVHD and only 6% (1/16) of the mice which received 2x106 naive cells survived 100 days post BMT. In contrast, the mice receiving Tcm cells displayed 83% survival (10/12 or 5/6 for 2x106 or 5x106 Tcm cells, respectively) with no signs of GVHD. Furthermore, when evaluated in the graft rejection model, 5x106 fully allogeneic donor Tcm cells induced survival, accompanied with complete donor chimerism at 100 days post transplant in 73% (11/15) of the recipients. The similar efficacy of overcoming rejection displayed by both fully allogeneic and F1 derived Tcm cells is in accordance with a veto mechanism, as opposed to potential ablation of host resistance by donor alloreactive clones. Collectively, the ability of anti-3rd party CD8 Tcm cells to expand and persist in vivo without GVHD, their marked LN homing and their capacity to overcome rejection of TDBMT in the absence of further immunosuppression, strongly suggest that these cells hold marked potential for tolerance induction in allogeneic hematopoietic stem cell transplantation.

T-cell responses to the trypanosome variant surface glycoprotein are not limited to hypervariable subregions.

Variable subregions within the variant surface glycoprotein (VSG) coat displayed by African trypanosomes are predicted sites for T- and B-cell recognition. Hypervariable subregion 1 (HV-1) is localized to an internal amphipathic alpha helix in VSG monomers and may have evolved due to selective pressure by host T-cell responses to epitopes within this subregion. The prediction of T-cell receptor-reactive sites and major histocompatibility complex class II binding motifs within the HV-1 subregion, coupled with the conservation of amino acid residues in other regions of the molecule sufficient to maintain secondary and tertiary VSG structure, prompted us to test the hypothesis that Th cells may preferentially recognize HV-1 subregion peptides. Thus, we examined the fine specificity of VSG-specific T-cell lines, T-cell hybridomas, and Th cells activated during infection. Our results demonstrate that T-cell epitopes are distributed throughout the N-terminal domain of VSG but are not clustered exclusively within HV-1 or other hypervariable subregions. In contrast, T-cell-reactive sites were not detected within the relatively conserved C-terminal domain of VSG. Overall, this study is the first to dissect the fine specificity of T-cell responses to the trypanosome VSG and suggests that evolution of a conserved HV-1 region may be unrelated to selective pressures exerted by host T-cell responses. This study also demonstrates that T cells do not recognize the relatively invariant C-terminal region of the VSG molecule during infection, suggesting that it could serve as a potential subunit vaccine to provide variant cross-specific immunity for African trypanosomiasis.

52 Defining host T-cell responses in oral candidiasis

52 Defining host T-cell responses in oral candidiasis