Mechanisms of Cross-Presentation in Graft-Vs-Host Disease.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 687-687
Author(s):  
Xiaojian Wang ◽  
Derry Roopenian ◽  
Catherine Martone ◽  
Ning Li ◽  
Hongmei Li ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cell Expansion ◽  
Type I ◽  
Cd8 Cells ◽  
Cross Presentation ◽  
Ifn Γ ◽  
Cd4 Help

Abstract Abstract 687 Graft-versus-host disease (GVHD) remains a major cause of morbidity and mortality in allogeneic stem cell transplantation. We previously showed that recipient antigen-presenting cells (APCs) are required for CD8-dependent GVHD, while donor APCs promote GVHD in a MHC matched (C3H.SW (right arrow) B6; both H-2b) model (Shlomchik et al, Science 1999; Matte et al., Nat Med 2004). However, how donor APCs promote maximal GVHD was not addressed. The LTFNYRNL peptide from H60 is a dominant minor histocompatibility antigen (miHA) presented by H-2Kb. To study cross-presentation of H60, we crossed B6 mice congenic for H60 (CH60; hematopoietically restricted) or transgenic for H60 driven by an actin promoter (actH60; H60 is ubiquitously expressed) with B6 Kb-/- mice. These mice express H60 but cannot directly present it to donor CD8 cells as they do not express Kb. CH60C*Kb-/-and actH60*Kb-/- were irradiated and reconstituted with C3H.SW (H60-) bone marrow,106(6 superscript) CD8 T cells and 2*105( 5 superscript) CD4 (to promote the CD8 response to H60). Using H60-MHC tetramers, we detected H60-specific CD8 T cell expansion as early as day 8, with a peak at day14, demonstrating cross-priming by donor C3H.SW APCs. Intracellular IFN-γ staining and an in vivo CTL assay showed that these cross-primed CD8 T cells had effector functions. Surprisingly, accumulation of H60-tetramer+ cells was greater when it was exclusively cross-presented. SIINFEKL, a peptide derived from ovalbumin (OVA), is also presented by Kb. Therefore to confirm our findings we used B6 mice transgenic for ovalbumin crossed to Kb-/- mice (ova*Kb-/-) as recipients in the same model. SIINFEKL-tetramer+ T cells expansion was also observed in ova*Kb-/- recipients, demonstrating cross-priming. The source of miHA did not affect the cross-priming as similar SIINFEKL-reactive T cell expansion occurred in retransplanted (right arrow)ova*Kb-/-, ova*Kb-/-(right arrow) Kb-/- bone marrowγKb-/- chimeras. Cross-priming of SIINFEKL-reactive CD8 cells even occurred when BALB/c mice transgenic for OVA (BALB/c-ova; (H-2d)) were transplanted with B6 BM and a mix of B6 CD4 and CD8 cells. SIINFEKL-reactive cells produced IFN-γ and killed SIINFEKL-pulsed B6 cells in vivo. Because of the availability of knockout/transgenic mice backcrossed to B6, we used this system to explore mechanisms of cross-presentation. Donor CD11c+ dendritic cells (DCs) were required as cross-priming was abrogated when BALB/c-OVA mice were transplanted with BM from mice constitutively lacking CD11c+ DCs (Birnberg et al, Immunity 2008). CD4 help has been reported to be important for cross-priming. Surprisingly, however, cross-priming by donor APCs was unaffected when BALB/c-OVA mice were transplanted with B6 MHCII-/- BM but was greatly reduced in recipients of B6 CD40-/- BM. Thus, while CD40L activation of cross-priming DCs is important, CD4 cells which are likely the source of the CD40L need not actually make T cell receptor:MHC contacts with the cross-presenting DC. CD40L-conditioning of donor APCs is not required to cross-prime memory cells, as sort-purified memory CD8 cells from SIINFEKL-vaccinated mice expanded robustly in actOVA*Kb-/- but not Kb-/- mice. Cross-priming also occurred in recipients of IFNAR1-/- BM, indicating that Type I IFN activation of donor APCs is not required as has been reported in nontransplant settings. Taken together, our data demonstrate that cross-presentation by donor DCs occurs in MHC-matched and -mismatched transplants, and this cross-presentation likely explains the reduced GVHD we observed in recipients of MHCI- donor bone marrow. That T cells can be cross-primed to nonhematopoietic antigens provides a basis for persistent GVHD and for the generation of CD8 responses against antigens not initially targeted. We also found transplantation to be a permissive environment for cross-priming in that CD4 help could be delivered in trans, type I IFN APC activation was not required and memory cells could be activated without CD4 help. These data provide further rationale for targeting donor DCs and pathways required for cross-presentation to prevent and treat GVHD. Disclosures: No relevant conflicts of interest to declare.

R-DOTAP (Versamune): A novel enantiospecific cationic lipid nanoparticle that induces CD4 and CD8 cellular immune responses to whole protein and tumor-specific peptide antigens.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e15211-e15211
Author(s):  
Lauren Virginia Wood ◽  
Siva K Gandhapudi ◽  
Karuna Sundarapandiyan ◽  
Frank K Bedu-Addo ◽  
Gregory Conn ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cationic Lipid ◽  
Cd8 T Cell ◽  
Lipid Nanoparticles ◽  
Type I ◽  
T Cell Epitopes ◽  
Ifn Γ

e15211 Background: Immunotherapy approaches are limited in their ability to induce antigen-specific CD8+ T cells in vivo able to recognize and kill tumor cells. We developed a novel immunotherapy approach using enantiomerically pure, R-DOTAP cationic lipid nanoparticles and tumor-derived T cell antigens, and previously demonstrated that R-DOTAP formulations efficiently prime cytotoxic T cells through enhanced cross presentation and induction of type I interferons.[1] A phase I clinical trial of a R-DOTAP HPV16 peptide formulation confirmed induction of strong in vivo HPV-specific CD8+ cytolytic T-cells without associated systemic toxicities. In this study, we assessed R-DOTAP nanoparticle formulations containing whole protein (ovalbumin) or long multi-epitope peptides from the tumor antigen TARP (T-cell alternate reading frame protein): a 58-residue protein overexpressed in prostate and breast cancers, documented to be immunogenic in humans. Methods: R-DOTAP formulations were prepared containing ovalbumin (OVA) or TARP peptides. C57BL/6K mice were immunized with 10 μg/mouse of OVA plus R-DOTAP, CFA or sucrose on Days 0, 15 and 30. OVA-specific cellular and humoral responses following vaccination were assessed by measuring splenic CD4 and CD8 T cell IFN-γ production and circulating OVA-specific antibodies in serum. HLA-A2 transgenic mice (AAD mice) were vaccinated with long, multi-epitope TARP peptides delivered as an R-DOTAP admixture or with CFA or sucrose on Days 0 and 7. Antigen-specific T cell responses were measured by IFN-γ ELISpot assay. Results: OVA R-DOTAP formulations induced strong antigen-specific effector CD4 and CD8 immune and memory responses detected 7 and 30 days, respectively, following vaccination as well as OVA-specific antibody responses. In TARP peptide vaccinated mice, R-DOTAP formulations were able to present multiple CD8 T cell epitopes and stimulate responses that were superior to CFA. Conclusions: Our results suggest that R-DOTAP is a versatile immune activating therapy that can be formulated with long, multi-epitope tumor-derived peptides or whole proteins. R-DOTAP formulations induce quantitatively robust antigen-specific CD4 and CD8 T cells in vivo compared to well-established immune stimulants. Reference: 1.Gandhapudi SK, Ward M, Bush JP et al. Antigen Priming with Enantiospecific Cationic Lipid Nanoparticles Induces Potent Antitumor CTL Responses through Novel Induction of a Type I IFN Response. J Immunol 2019;202:3524-3536

scholarly journals Improved Expansion and Function of CAR T Cell Products from Cultures Initiated at Defined CD4:CD8 Ratios

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3334-3334 ◽  
Author(s):  
Dong Hoon Lee ◽  
Francisco Cervantes-Contreras ◽  
Sang Yun Lee ◽  
Damian J. Green ◽  
Brian G. Till
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Cultures ◽  
Cd8 T Cells ◽  
Cell Expansion ◽  
Cd8 Cells ◽  
Car T Cell ◽  
Car T ◽  
Cd8 Cell

Abstract BACKGROUND: Current manufacturing paradigms for chimeric antigen receptor (CAR) modified T cells require ex vivo T cell activation, genetic modification, and expansion in cytokine-containing cell cultures. Most CAR T cell studies infuse cell products generated from unselected cells, in which the CD4:CD8 ratio is determined by what is collected during leukapheresis. The proportion of each subset can vary greatly in these products, reflecting the high heterogeneity of the CD4:CD8 ratio present in patients' (pts) blood at the time of treatment. Preclinical data demonstrate superior in vivo anti-tumor efficacy of cell products consisting of equal numbers of CD4+ and CD8+ T cells, and a recent clinical trial, in which CD19-targeted CAR T cell products were infused at a 1:1 ratio of CD4:CD8 T cells, showed clear dose-response and dose-toxicity relationships. However, CAR T cell manufacturing using parallel CD4+ and CD8+ CAR T cell cultures adds significant cost and complexity compared with a single-stream culture. Additionally, we have found that CD8+ T cell cultures from heavily treated pts often exhibit suboptimal expansion. We therefore evaluated whether CAR T cell products with approximately equal ratios of CD4+ to CD8+ cells could be generated by mixing CD4+ and CD8+ cells at a defined ratio at culture initiation, and whether the presence of ex vivo CD4+ help can improve CD8+ cell expansion. METHODS: CD4+ and CD8+ T cells were isolated from apheresis peripheral blood mononuclear cell products of lymphoma pts (n=15) treated in one of three CD20-targeted CAR T cell trials or healthy donors (n=5) using positive (CD4) and negative (CD8) immunomagnetic bead selection. Cell cultures (1x106 cells each) were established by activating CD4+ and CD8+ cells with anti-CD3/CD28-coated paramagnetic beads, mixing them at various ratios in 24-well plates, and transducing 24 hours later with a lentivirus encoding the 1.5.3-NQ-28-BB-z CD20 CAR. Beads were removed at day 4, and cells were expanded in IL-2 containing medium. At day 7, cells were counted and analyzed by flow cytometry for CD4, CD8, and CAR expression, and then restimulated 1:1 with irradiated CD20+ lymphoblastoid cells to boost growth. On day 14 cells were again counted and analyzed by flow cytometry for CD4, CD8, and CAR expression, as well as immunophenotype. In some cases CAR+CD4+ and CAR+CD8+ cells were flow-sorted from either CD4-only, CD8-only, and mixed cultures, stained with CFSE, and restimulated with irradiated CD20+ Raji tumor cells. Proliferation and cytokine secretion were measured by flow cytometry and Luminex, respectively. In vivo T cell activity was assessed using an NSG mouse model in which T cells were infused 7 days after i.v. injection of Raji-ffLuc cells. Suboptimal doses of T cells were used to distinguish differences between conditions, since higher doses cure most mice. Mice received either: (1) 70:30 mixed CD4:CD8 CAR+ cultures (n=8), (2) 1:1 ratio of CD4:CD8 CAR+ cells grown separately (n=8), (3) Mixed CD4:CD8 empty vector cells (n=5), or (4) no treatment (n=5). RESULTS: An initial CD4:CD8 ratio of 70:30 yielded a median CD4/CD8 ratio of 1.1 for pts (Figure 1A) and 1.3 for donors at day 7, and a ratio of 0.6 and 1.0 for pts and donors, respectively, at day 14. Mixed cultures resulted in CD8+ cell expansion that was significantly higher than in separate cultures. At day 7, mean CD8+ cell expansion was 12.1-fold vs. 4.6-fold for 70:30 mixed vs. separate cultures for donors, and 2.9-fold vs. 0.7-fold for pts (Figure 1B). At day 14 CD8+ mean cell expansion was 105-fold (70:30 mixed) vs. 25-fold (separate) for donors and 40-fold vs. 1.9-fold for pts. CD8+ cells grown in mixed cultures also exhibited higher expression of memory markers (CD62L and CCR7), lower levels of exhaustion markers (Lag-3 and PD-1), and better proliferation compared with CD8 cells grown in separate cultures. In the mice we found that tumor growth inhibition was superior in the 70:30 mixed culture group than in mice receiving a 1:1 ratio of CD4:CD8 cells grown separately (Figure 1C). CONCLUSIONS: A single-stream CAR T cell culture initiated at a defined CD4:CD8 ratio of 70:30 yielded on average approximately equal numbers of CD4+ and CD8+ cells in the final cell product. Mixed CD4+ and CD8+ cultures generated CD8+ T cells with a less differentiated phenotype, and superior expansion, proliferative capacity, and in vivo activity compared with CD4+ and CD8+ cells manufactured in parallel. Disclosures Green: Juno Therapeutics: Patents & Royalties, Research Funding. Till:Mustang Bio: Patents & Royalties, Research Funding.

scholarly journals CD28 Costimulation Is Required for In Vivo Induction of Peripheral Tolerance in CD8 T Cells

2002 ◽  
Vol 197 (1) ◽  
pp. 19-26 ◽  
Author(s):  
Melanie S. Vacchio ◽  
Richard J. Hodes
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Tolerance Induction ◽  
Peripheral Tolerance ◽  
Cd8 Cells ◽  
Costimulatory Signal ◽  
Cd28 Costimulation

Whereas ligation of CD28 is known to provide a critical costimulatory signal for activation of CD4 T cells, the requirement for CD28 as a costimulatory signal during activation of CD8 cells is less well defined. Even less is known about the involvement of CD28 signals during peripheral tolerance induction in CD8 T cells. In this study, comparison of T cell responses from CD28-deficient and CD28 wild-type H-Y–specific T cell receptor transgenic mice reveals that CD8 cells can proliferate, secrete cytokines, and generate cytotoxic T lymphocytes efficiently in the absence of CD28 costimulation in vitro. Surprisingly, using pregnancy as a model to study the H-Y–specific response of maternal T cells in the presence or absence of CD28 costimulation in vivo, it was found that peripheral tolerance does not occur in CD28KO pregnants in contrast to the partial clonal deletion and hyporesponsiveness of remaining T cells observed in CD28WT pregnants. These data demonstrate for the first time that CD28 is critical for tolerance induction of CD8 T cells, contrasting markedly with CD28 independence of in vitro activation, and suggest that the role of CD28/B7 interactions in peripheral tolerance of CD8 T cells may differ significantly from that of CD4 T cells.

scholarly journals IL-1 enhances expansion, effector function, tissue localization, and memory response of antigen-specific CD8 T cells

2013 ◽  
Vol 210 (3) ◽  
pp. 491-502 ◽  
Author(s):  
Shlomo Z. Ben-Sasson ◽  
Alison Hogg ◽  
Jane Hu-Li ◽  
Paul Wingfield ◽  
Xi Chen ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Granzyme B ◽  
Interleukin 1 ◽  
Cd8 T Cell ◽  
Cell Receptor ◽  
In Vivo Models ◽  
Ifn Γ

Here, we show that interleukin-1 (IL-1) enhances antigen-driven CD8 T cell responses. When administered to recipients of OT-I T cell receptor transgenic CD8 T cells specific for an ovalbumin (OVA) peptide, IL-1 results in an increase in the numbers of wild-type but not IL1R1−/− OT-I cells, particularly in spleen, liver, and lung, upon immunization with OVA and lipopolysaccharide. IL-1 administration also results in an enhancement in the frequency of antigen-specific cells that are granzyme B+, have cytotoxic activity, and/ or produce interferon γ (IFN-γ). Cells primed in the presence of IL-1 display enhanced expression of granzyme B and increased capacity to produce IFN-γ when rechallenged 2 mo after priming. In three in vivo models, IL-1 enhances the protective value of weak immunogens. Thus, IL-1 has a marked enhancing effect on antigen-specific CD8 T cell expansion, differentiation, migration to the periphery, and memory.

Identification of the AAV2 Capsid CD8+ T Cell Epitope in C57BL/6 Mice.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3188-3188
Author(s):  
Denise E. Sabatino ◽  
Federico Mingozzi ◽  
Haifeng Chen ◽  
Peter Colosi ◽  
Hildegund C.J. Ertl ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Capsid Protein ◽  
Cd8 T Cells ◽  
Cell Epitope ◽  
T Cell Response ◽  
Cd8 Cells ◽  
Media Control ◽  
Ifn Γ

Abstract Recently, a clinical trial for adeno-associated virus serotype 2 (AAV2) mediated liver directed gene transfer of human Factor IX to subjects with severe hemophilia B revealed that two patients developed transient asymptomatic transaminitis following vector administration. Immunology studies in the second patient demonstrated a transient T cell response to AAV2 capsid peptides suggesting that the immune response to the AAV capsid may be related to the transient transaminitis. We hypothesized that the observations made in the human subjects were due to a CD8 T cell response to AAV2 capsid protein. Preclinical studies in mice and dogs, which are not naturally infected by wild type AAV2 viruses, did not predict these findings in the clinical study. Thus, we developed a mouse model in which we were able to mimic this phenomenon (Blood 102:493a). In an effort to further characterize the immune responses to AAV2 capsid proteins in this mouse model, we identified the T cell epitope in the AAV capsid protein recognized by murine C57Bl/6 CD8 T cells. A peptide library of AAV2 VP1 capsid peptides (n=145) that were synthesized as 15mers overlapping by 10 amino acids were divided into 6 pools each containing 24–25 peptides. C57Bl/6 mice were immunized intramuscularly with an adenovirus expressing AAV2 capsid protein. Nine days later the spleen was harvested and intracellular cytokine staining (ICS) was used to assess release of IFN-γ from CD8 T cells in response to 6 AAV2 capsid peptide pools. ICS demonstrated CD8 cells from mice immunized with Ad-AAV2 produced IFN-γ (3.5% of the CD8 cells) in response to Pool F (amino acid 119–145) while no IFN-γ release in CD8 cells was detected with Pool A to E (mean 0.28%±0.25%) compared to the media control (0.16%). This detection of IFN-γ release from CD8 T cells indicates a specific proliferation to a peptide(s) within this peptide pool (Pool F). A matrix approach was used to further define which peptide(s) contained the immunodominant epitope. Eleven small peptide pools of Pool F were created in which each peptide was represented in 2 pools. ICS of splenocytes from immunized (Ad-AAV2 capsid) C57Bl/6 mice demonstrated IFN-γ response from CD8 cells to 3 of the matrix pools corresponding to peptide 140 (PEIQYTSNYNKSVNV) and 141 (TSNYNKSVNVDFTVD) compared with media controls. To determine the exact peptide sequence that binds to the MHC Class I molecule, 9 amino acid peptides (n=7) were created that overlap peptide 140 and 141. Peptide SNYNKSVNV showed positive staining for both CD8 and IFN- γ(3.2%) compared with the six other peptides (0.14%±0.08%), media control (0.08%) and mice that were not immunized (0.11%). This epitope lies in the C terminus of the AAV2 VP1 capsid protein. Current studies using strains of mice with different MHC H2 haplotypes will allow us to determine which of the C57Bl/6 MHC alleles the epitope binds. These findings will provide us with a powerful tool for assessing immune responses to AAV capsid in the context of gene therapy. Specifically, they will allow us to determine how long immunologically detectable capsid sequences persist in an animal injected with AAV vectors. This in turn will provide a basis for a clinical study in which subjects are transiently immunosuppressed, from the time of vector injection until capsid epitopes are no longer detectable by the immune system.

Effects of Amotosalen Hydrochloride and Ultraviolet a Light on CD4 and CD8 Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4981-4981
Author(s):  
Catherine T. Jordan ◽  
James C. Zimring ◽  
John D. Roback
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Differential Sensitivity ◽  
Ultraviolet A ◽  
Differential Effects ◽  
Cd8 Cells ◽  
Uva Light ◽  
In Vivo Effects

Abstract Background: Graft versus host disease (GvHD) and infections by opportunistic pathogens, such as cytomegalovirus (CMV), are causes of significant morbidity and mortality in recipients of allogeneic bone marrow transplants (BMT). Thus, there is a need for methods of graft engineering that inhibit the alloreactive T cells responsible for lethal GvHD without compromising the activity of antiviral T cells. In a murine MHC-mismatched BMT model, we have previously demonstrated that adoptive transfer of polyclonal T cells from donors immunized to murine CMV (MCMV) can decrease viral load but cause lethal GvHD. However, pretreatment of these cells with the psoralen, amotosalen hydrochloride, and ultraviolet A (UVA) light prevents GvHD without compromising antiviral response. We have previously hypothesized that these effects were due to differential sensitivity of naïve and memory T cells to amotosalen/UVA. Recent investigations have demonstrated that CD4 T cells are most directly responsible for lethal GvHD in this model. This observation suggested an alternative hypothesis, equally consistent with previous data, that the observed in vivo effects of amotosalen/UVA treatment are due to differential effects on CD4 and CD8 T cells. The assessment of this new hypothesis is the focus of the current studies. Methods: Two models of T cell activation/proliferation were utilized to test the effects of amotosalen/UVA treatment on CD4 and CD8 cells: stimulation of B6.PL (H-2b) cells with concavalinA, and primary mixed lymphocyte reaction (MLR) between MHC-mismatched B6.PL (H-2b) and BALB/c (H-2d). Responder cells were pretreated with 2nM amotosalen and varying doses of UVA light (0–5 minutes). Proliferation of CD4 and CD8 cells was measured by flow cytometric analysis of CFSE-labeled responder cells. Results: In both systems, CD4 proliferation was effectively eliminated by immediately prior treatment with amotosalen and UVA doses of 1 minute or higher. CD8 proliferation was eliminated by amotosalen and UVA doses of 2 minutes and higher. Both the amount of division on a per cell basis and the overall number of cells that initiated division followed similar trends. Conclusions: These data demonstrate that both CD4 and CD8 T cells are sensitive to treatment with amotosalen/UVA and suggest a subtle difference in sensitivity of CD4 and CD8 populations. Since division of both CD4 or CD8 cells is inhibited at doses of amotosalen/UVA that prevent GvHD but allow anti-viral activity in vivo, it is unlikely that the observed differential sensitivity of T-cell subsets is sufficient to explain the in vivo effects of amotosalen/UVA treatment in this model. Using similar methodologies, ongoing studies are assessing the hypothesis that amotosalen/UVA has differential effects on naïve and mature T cells.

Pentostatin Is Advantageous Relative to Fludarabine for in Vivo Murine T Cell Depletion, Suppression of T Cell Cytokine Secretion, and Inhibition of HVGR

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3483-3483
Author(s):  
Jacopo Mariotti ◽  
Jason Foley ◽  
Kaitlyn Ryan ◽  
Nicole Buxhoeveden ◽  
Daniel Fowler
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cytokine Secretion ◽  
Cell Depletion ◽  
T Cell Depletion ◽  
T Cell Suppression ◽  
Ifn Γ ◽  
Cell Suppression

Abstract Although fludarabine and pentostatin are variably utilized for conditioning prior to clinical allogeneic transplantation, limited data exists with respect to their relative efficacy in terms of host immune T cell depletion and T cell suppression. To directly compare these agents in vivo in a murine model, we compared a regimen of fludarabine plus cyclophosphamide (FC) similar to one that we previously developed (Petrus et al, BBMT, 2000) to a new regimen of pentostatin plus cyclophosphamide (PC). Cohorts of mice (n=5–10) received a three-day regimen consisting of P alone (1 mg/kg/d), F alone (100 mg/kg/d), C alone (50 mg/kg/d), or combination PC or FC. Similar to our previous data, administration of P, F, or C alone yielded minimal host T cell depletion (as measured by enumeration of splenic CD4+ and CD8+ T cells) and minimal T cell suppression (as determined by CD3, CD28 co-stimulation of a constant number of remaining splenic T cells and measuring resultant cytokine secretion by multi-analyte assay). The PC and FC regimens were similar in terms of myeloid suppression (p=.2). However, the PC regimen was more potent in terms of depleting host CD4+ T cells (remaining host CD4 number [× 10^6/spleen], 2.1±0.3 [PC] vs. 4.4±0.6 [FC], p<0.01) and CD8+ T cells (remaining host CD8 number, 1.7±0.2 [PC] vs. 2.4±0.5 [FC], p<0.01). Moreover, the PC regimen yielded greater T cell immune suppression than the FC regimen (cytokine values are pg/ml/0.5×10^6 cells/ml; all comparisons p<0.05) with respect to capacity to secrete IFN-γ (13±5 [PC] vs. 48±12 [FC]), IL-2 (59±44 [PC] vs. 258±32 [FC]), IL-4 (34±10 [PC] vs. 104±12 [FC]), and IL-10 (15±3 [PC] vs. 34±5 [FC]). In light of this differential in both immune T cell depletion and suppression of T cell effector function, we hypothesized that T cells from PC-treated recipients would have reduced capacity to mediate a host-versus-graft rejection response (HVGR) relative to FC-treated recipients. To directly test this hypothesis, we utilized a host T cell add-back model of rejection whereby BALB/c hosts were lethally irradiated (1050 cGy; day -2), reconstituted with host-type T cells from PC- or FC-treated recipients (day -1; 0.1 × 10^6 T cells transferred), and finally challenged with fully MHC-disparate transplantation (B6 donor bone marrow cells, 10 × 10^6 cells; day 0). In vivo HVGR was quantified by the following method at day 7 post-BMT: harvest of splenic T cells, stimulation with host- or donor-type dendritic cells, and use of six-color flow cytometry to detect host T cells, CD4 and CD8 subsets, and cytokine secretion by capture method. Consistent with our hypothesis, PC-treated cells acquired greatly reduced alloreactivity in vivo relative to FC-treated cells: the percentage of host CD4+ T cells secreting IFN-γ in an allospecific manner was 2.3±0.8% in recipients of PC-treated T cells and 62.7±13.4% in recipients of FC-treated cells (p<0.001). Similarly, the percentage of host CD8+ T cells secreting IFN-γ in an allospecific manner was 8.6±2.8% in recipients of PC-treated T cells and 92.7±4.1% in recipients of FC-treated T cells (p<0.001). We therefore conclude that at similar levels of myeloid suppression, the PC regimen is superior to the FC regimen in terms of murine T cell depletion, suppression of global T cell cytokine secretion, and inhibition of in vivo capacity to acquire allospecificity in response to fully genetically disparate marrow allografts. These data provide a rationale to develop PC regimens as an alternative to currently utilized FC regimens.

Tissue Parenchymal Cell Expression of B7-H1 Inhibits Infiltrating T Cell Expansion and Prevents Persistence of Graft-Versus-Host Disease

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2974-2974
Author(s):  
Xiaofan Li ◽  
Wei He ◽  
Ruishu Deng ◽  
Can Liu ◽  
Miao Wang ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cell Expansion ◽  
Cd8 T Cell ◽  
Target Tissue ◽  
T Cell Expansion ◽  
Parenchymal Cells

Abstract Abstract 2974 Alloreactive donor CD8+ T cells facilitate engraftment and mediate graft versus leukemia (GVL) effects but also cause graft versus host disease (GVHD) in murine and human recipients after allogeneic hematopoietic cell transplantation (HCT). B7-H1 (PD-L1) expression by antigen-presenting cells has an important role in tolerizing activated T cells by binding to PD-1. We and others previously reported that disruption of binding between B7-H1 and PD-1 augments acute GVHD. Parenchymal cells do not usually express B7-H1 but can be induced by inflammatory cytokines (i.e. IFN-g) to express B7-H1. The role of B7-H1 expression by parenchymal tissue cells in regulating the expansion and persistence of donor CD8+ cells in tissues of mice with GVHD has not yet been evaluated. In the current studies, we evaluated the role of B7-H1 expression by GVHD target tissues in regulating donor CD8+ T cell function in 3 different experimental GVHD systems, using in vivo bioluminescent imaging (BLI), in vivo BrdU-labeling, and in vitro proliferation assays. The first system evaluated the role of B7-H1 expression in TBI-conditioned recipients. In these recipients, injected donor CD8+ T cells showed two waves of expansion that correlated with two phases of clinical GVHD. The first wave of donor CD8+ T cell expansion was associated with upregulated expression of B7-H1 in GVHD target tissues and only weak clinical GVHD. The second wave of donor CD8+ T cell expansion was associated with loss of B7-H1 expression, vigorous donor CD8+ T proliferation and expansion in the GVHD target tissues, and lethal GVHD. In a gain-of-function experiment, B7-H1 expression was induced in hepatocytes by hydrodynamic injection of B7-H1 cDNA during the second wave of T cell expansion in mice with GVHD; this subsequently decreased T cell expansion in the liver and ameliorated GVHD. The second system evaluated the role of B7-H1 expression in anti-CD3-conditioned recipients. In wild-type recipients, injected donor CD8+ T cells had only a single wave of expansion, and the mice had no signs of GVHD. B7-H1 expression by tissue cells (i.e. hepatocytes) was up-regulated, and the tissue infiltrating donor CD8+ T cells were anergic. In B7-H1−/− recipients, injected donor CD8+ T cells proliferated vigorously in GVHD target tissues and caused lethal GVHD.The third system evaluated the role of B7-H1 in unconditioned Rag-2−/− recipients after administration of blocking anti-B7-H1 and in the B7-H1−/−Rag-2−/− chimeras with B7-H1 sufficient Rag-2−/− bone marrow cells, in which B7-H1 deficiency was only in tissue parenchymal cells. Both blockade of B7-H1 and B7-H1 deficiency in parenchymal cells resulted in vigorous donor CD8+ T proliferation in GVHD target tissues and caused lethal GVHD. Taken together, these results show that expression of B7-H1 in GVHD target tissue parenchymal cells plays an important role in regulating the proliferation of infiltrating donor CD8+ T cells and preventing the persistence of GVHD. Our studies also indicate that TBI but not anti-CD3 conditioning can lead to loss of GVHD target tissue cell expression of B7-H1 and persistence of GVHD. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Retrovirally induced CTL degranulation mediated by IL-15 expression and infection of mononuclear phagocytes in patients with HTLV-I–associated neurologic disease

Blood ◽  
2008 ◽  
Vol 112 (6) ◽  
pp. 2400-2410 ◽  
Author(s):  
Yoshimi Enose-Akahata ◽  
Unsong Oh ◽  
Christian Grant ◽  
Steven Jacobson
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Spastic Paraparesis ◽  
Cd8 T Cell ◽  
Mononuclear Phagocytes ◽  
Type I ◽  
Human T Cell ◽  
Ifn Γ

AbstractCD8+ T cells contribute to central nervous system inflammation in human T-cell lymphotropic virus type I (HTLV-I)–associated myelopathy/tropical spastic paraparesis (HAM/TSP). We analyzed CD8+ T-cell dysfunction (degranulation and IFN-γ production) and have demonstrated that CD8+ T cells of patients with HAM/TSP (HAM/TSP patients) spontaneously degranulate and express IFN-γ in ex vivo unstimulated culture. CD8+ T cells of HTLV-I asymptomatic carriers and healthy donors did not. Spontaneous degranulation was detected in Tax11-19/HLA-A*201 tetramer+ cells, but not in CMV pp65 tetramer+ cells. Interestingly, degranulation and IFN-γ production in CD8+ T cells was induced by coculture with autologous CD14+ cells, but not CD4+ T cells, of HAM/TSP patients, which correlated with proviral DNA load in CD14+ cells of infected patients. Moreover, the expression of IL-15, which induced degranulation and IFN-γ production in infected patients, was enhanced on surface of CD14+ cells in HAM/TSP patients. Blockade of MHC class I and IL-15 confirmed these results. Thus, CD8+ T-cell dysregulation was mediated by both virus infection and enhanced IL-15 on CD14+ cells in HAM/TSP patients. Despite lower viral expression than in CD4+ T cells, HTLV-I–infected or –activated CD14+ cells may be a heretofore important but under recognized reservoir particularly in HAM/TSP patients.

Double Loading of Dendritic Cell MHC Class I and MHC Class II with an AML Antigen Repertoire Enhances Primary and Secondary T-Cell Responses In Vitro.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2529-2529
Author(s):  
William K. Decker ◽  
Dongxia Xing ◽  
Sufang Li ◽  
Simon N. Robinson ◽  
Hong Yang ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mhc Class Ii ◽  
Cd8 T Cells ◽  
Class Ii ◽  
T Cell Response ◽  
Class I ◽  
Cross Presentation ◽  
Ifn Γ

Abstract Despite improvements in therapy for acute myelogenous leukemia (AML), a significant percentage of patients still relapse and succumb to their disease. Dendritic cell immunotherapy offers the promise of potentially effective supportive therapy for a variety of neoplastic conditions; and the use of DCs loaded with tumor antigens is now recognized as an important investigational therapy. Though a variety of methods have been used to load DC vaccines, the loading of the MHC class II compartment with tumor lysate has predominated. The priming of a class II-mediated (CD4) T-cell response may be crucial to the success of DC immunotherapy as such a response is likely required for the development of memory CD8+ T-cells. DC cross-presentation is credited with the ability of lysate-loaded DCs to prime both CD4 and CD8 T-cell responses, enabling the generation of CD8+ CTLs without the loading of the MHC class I compartment (i.e. the cytoplasm). Recently, however, several reports have raised doubts as to the efficiency of cross-presentation as a mechanism for CTL priming in vivo. To examine this issue, we have loaded human DCs with both AML tumor lysate and mRNA. This technique allows the full repertoire of class I antigens to be presented without dependence upon cross-presentation; and, moreover, provides a full complement of class II antigens necessary for CD4 T-cell priming and the generation of memory responses. Methods: CD14+ precursors were isolated from normal donor PBPCs by magnetic separation. Immature DCs were then generated by culturing precursors for six days in GM-CSF and IL-4. Lysate was produced by three successive freeze/thaw cycles of blasts. mRNA was extracted from blasts using Trizol and oligo-dT separation. Immature DCs were pulsed for three hours with AML lysate and subsequently electroporated with AML mRNA. Loaded DCs were matured for 48 hours with IL-1β, TNF-α, IL-6, and PGE2 and then used to prime autologous T-cells. Short-term responses were assayed on day 5 of the 1st stimulation. Memory responses were assayed on day 10 of a tertiary stimulation. Results: Doubly-loaded DCs can prime a superior T-cell response in vitro in comparison to that of singly-loaded DCs, demonstrating a 30–70% increase in IFN-γ ELISpots over lysate-loaded DCs (p&lt;0.001) and a 3–4 fold increase in ELISpots in comparison to mRNA loaded DCs (p&lt;0.001). These results were verified by flow cytometry which showed 35% of CD8+ T-cells primed by doubly-loaded DCs were CD69+/IFN-γ+ vs. 14% of CD8+ T-cells primed by lysate-loaded DCs (p&lt;0.001). This enhancement may be based upon both an upregulation of CD83 surface expression (p&lt;0.0019) of doubly-loaded DCs and/or the upregulation of B7.1/B7.2 that accompanies elevated CD40L signaling. Memory responses were also greatly improved, with a 126% increase in total ELISpots (double loaded DCs versus lysate loaded DCs; p&lt;0.03) and a 187% increase in total IFN-γ secretion (p&lt;0.03). Unloaded (p&lt;0.01) and mRNA (p&lt;0.007) loaded DCs exhibited a virtual inability to generate memory T-cells in vitro, suggesting that the perpetuation of the memory response is reliant upon T-cell help. Conclusion: DCs doubly-loaded with lysate and mRNA are more efficient in the generation of primary and secondary immune responses than are singly-loaded DCs. The clinical administration of such doubly-loaded DCs may offer an important therapeutic option to patients with AML.

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