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.

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.

The Rapid Ex Vivo Generation of an Immunodominant Antigen-Specific Memory CD8 Population and Its Subsequent Homeostatic Expansion in Ablatively-Conditioned HCT Recipients.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3172-3172
Author(s):  
Melinda Roskos ◽  
Robert B. Levy
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Ex Vivo ◽  
Cell Populations ◽  
Cd8 Cells ◽  
Immunodominant Antigen ◽  
Transfer Strategies

Abstract There is currently significant interest in the transplant field to develop adoptive-transfer strategies utilizing T cell populations to provide immediate immune function as well as long-term immune reconstitution following hematopoietic cell transplantation (HCT). Presumably, these pre-selected T cell populations could then be further expanded in the transplant recipient as a consequence of lymphopenia-induced proliferation. However, clinical application of adoptive transfer strategies has been limited by practical (time, expense) and technical (isolation and expansion of antigen-specific T cell populations) difficulties, hence more efficient approaches need to be identified. Recent reports have demonstrated the feasibility for the rapid ex vivo generation of transgenic memory CD8 populations. We investigated the potential of applying this ex vivo approach to generate and expand an immunodominant antigen-specific memory population from primary CD8 T cells. CD8 cells recognizing the mouse H60 epitope were selected as the antigen-specific CD8 population. The H60 glycoprotein is the ligand for NKG2D and the LTFNYRNL peptide is an immunodominant minor transplantation antigen. H60 is expressed by BALB.B (H2b) hematopoietic cells and recognized by C57BL/6 (B6) CD8 cells within the context of the H2Kb molecule. CD8 T cells from normal B6 spleens were positively selected using Miltenyi beads. The purified CD8 cells (97%) were then cultured with bone marrow-derived B6 DC, rmIL-2, and H60 peptide (1μM) for 3 days. Cells were harvested and re-cultured with rmIL-15 for 2–4 days. The resultant CD8 population was enriched 10 fold for tetramer-stained H60+ CD8 T cells (average: 3.0% of CD8 T cells). The H60+ CD8 cells displayed a memory phenotype as characterized by CD44+, Ly6C+, CD62Lintermed, and CD25lo expression. We hypothesized these H60+ CD8 T cells could be further expanded in transplant recipients by lymphopenia-induced proliferation. To determine the expansion and persistence of H60+ TM post-HCT, H60+-enriched CD8 cells were co-transplanted with T cell-depleted B6 bone marrow into 9.0Gy-conditioned syngeneic recipients. The phenotype and number of H60+ cells in recipient spleens and bone marrow were assessed beginning 3 days post-HCT. Notably, the H60+ CD8 cells maintained their memory phenotype and persisted at least 2 months post-transplant. The ex vivo-generated H60+ TM underwent a relative expansion of 1.5–2 fold as assessed in recipient spleens, similar to the post-transplant expansion of H60+ CD8 TM derived in vivo from B6 mice primed to BALB.B cells. Moreover, this post-HCT expansion was also similar to that by an ex vivo-generated, transgenic CD8 TM population. Both (ex vivo and in vivo generated) H60+ TM populations also exhibited expansion (1.5–2 fold) in the bone marrow. In total, an immunodominant antigen-specific CD8 TM population was selectively generated and enriched ex vivo and found to undergo expansion following transplant into ablatively conditioned HCT recipients. The similarities in expansion and persistence between ex vivo generated H60 and in vivo primed H60 populations suggest this approach may have useful applications towards the development of successful adoptive transfer strategies.

Dasatinib-Induced Reduction of Tumor Growth Is Accompanied By the Changes in the Immune Profile in a Melanoma B16.OVA Mouse Model

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1408-1408
Author(s):  
Mette Matilda Ilander ◽  
Can Hekim ◽  
Markus Vähä-Koskela ◽  
Paula Savola ◽  
Siri Tähtinen ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Growth ◽  
Cd8 T Cells ◽  
Research Funding ◽  
K562 Cells ◽  
Cell Cytotoxicity ◽  
Cd8 Cells

Abstract Background: Dasatinib is a 2nd generation tyrosine kinase inhibitor (TKI) used in the treatment of chronic myeloid leukemia (CML). Its kinase inhibition profile is broad and includes several kinases important in the immune cell function such as SRC kinases. Furthermore, it is known that dasatinib has immunomodulatory effects in vivo. Recently, we observed that dasatinib induces a rapid and marked mobilization of lymphocytes, which closely follows the drug plasma concentration. The phenomenon is accompanied by an increase of NK-cell cytotoxicity. In addition, we have shown that dasatinib alters T-cell responses long-term favoring Th1 type of responses. Interestingly, the dasatinib induced immune effects have been associated with better treatment responses. We now aimed to characterize the dasatinib-induced antitumor immune responses in a syngeneic murine melanoma model to address whether dasatinib-induced immunoactivation affects tumor growth. Methods: Direct cytotoxic effect of dasatinib on B16.OVA melanoma cells in vitro was assessed with an MTS cell viability assay. T-cell cytotoxicity was assessed by preincubating splenocytes isolated from naïve and OT-I mouse spleen with 100 nM dasatinib and measured their cytotoxic capacity against B16.OVA cells. To further evaluate the dasatinib induced antitumor immune effects in vivo, B16.OVA cells were implanted subcutaneously in C57BL/6J mice. The mice (n=6/group) were treated daily i.g. either with 30 mg/kg dasatinib or vehicle only. Blood was collected before tumor transplantation, before treatment, and on treatment days 4, 7 and 11. Tumor volumes were measured manually and specific growth rate was calculated based on the first and the last day of the treatment. In addition to white blood cell differential counts, immunophenotyping of blood and tumor homogenate was performed by flow cytometry using antibodies against CD45.1, CD3, CD4, CD8b, NK1.1, CTLA4, PD-1 and CD107. Immunohistochemical staining of CD8+ T-cells was performed from the paraffin embedded tumor samples. Results: In vitro incubation of B16.OVA cells with dasatinib showed only a moderate unspecific cytotoxicity with the two highest concentrations of dasatinib (1- and 10 µM), whereas in K562 cells (a CML blast crisis cell line) almost complete killing was observed already with the 100nM concentration. The cell viability of B16.OVA cells was 90% with at 100 nM of dasatinib concentration (as compared to 21% of K562 cells) suggesting that there was no direct dasatinib sensitive target oncokinase in this cell line. In contrast, a significant enhancement in the cytotoxic capacity of splenocytes was observed when they were pretreated with 100nM dasatinib (60% of target cells were alive when incubated with dasatinib pretreated naïve splenocytes compared to 100% with control treated splenocytes, p=0.004). The in vivo tumor experiments demonstrated that the tumor volumes were smaller in dasatinib group, and there was a significant decrease in the specific tumor growth rate (0.06 vs. 0.18, p=0.01) on the 11th day of treatment. Interestingly, dasatinib treated mice had increased proportion of CD8+cells in the circulation (17.9% vs. 14.4%, p=0.005) and the CD4/CD8 ratio was significantly decreased (1.39 vs. 1.52, p= 0.04). During the tumor growth the mean CTLA-4 expression on CD8+ cells in PB increased from 1.2% to 9% in the control group, whereas, in dasatinib group the increase was more modest (1.2% to 5.7%). When the tumor content was analyzed, dasatinib treated mice had significantly more tumor infiltrated CD8+ T-cells (median 17 vs. 4/counted fields, p=0.03). In dasatinib group 80% of the tumor infiltrating CD8+ cells expressed PD-1 antigen compared to <5% of PD1 positive CD8+ cells in the peripheral blood suggesting either tumor induced CD8 T-cell exhaustion or the presence of tumor-reactive effector cells. Lastly, when CD4 and CD8 cells were depleted before tumor inoculation, dasatinib was no longer able to slow down the tumor growth. Conclusions: Dasatinib treatment slowed the tumor growth in a B16.OVA mouse model. The growth retardation was due to immunomodulatory properties of dasatinib as the drug was not directly cytotoxic and depletion of T-cells abolished the effect. Dasatinib may be a therapeutically useful immunomodulatory agent for targeting tumor-associated anergy, particularly in combination with novel checkpoint inhibitors and tumor-targeting drugs. Disclosures Hemminki: Oncos Therapeutics Ltd: Shareholder Other; TILT BioTherapeutics Ltd: Employment, Shareholder, Shareholder Other. Porkka:BMS and Novartis: Honoraria, Research Funding; Pfizer: Research Funding. Mustjoki:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

Enforced Co-Stimulation and Co-Inhibitory Blockade Synergize to Enhance the Functions of Exhausted CTL

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1911-1911
Author(s):  
Barry Flutter ◽  
Noha Edwards ◽  
Lei Zhang ◽  
Shivajanani Sivakumaran ◽  
Michael Croft ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd8 T Cell ◽  
Cd4 Cells ◽  
Isotype Control ◽  
Cd8 Cells ◽  
T Cell Transfer

Abstract Abstract 1911 A major limitation of adoptive T cell therapies for cancer is the failure to maintain durable anti-tumor immunity. Graft-versus-tumor responses following bone marrow transplantation (BMT) may only be short-lived due to 1) defects in memory precursor generation and 2) exhaustion of surviving CTL that results from direct recognition of alloantigen upon non-hematopoietic cells {Flutter et al. JCI 2010}. In this study, we have explored the potential for enhancing co-stimulatory signals either alone, or in combination with co-inhibitory PD-1-PD-L1 blockade to improve the long term CTL response. Signalling through OX40, a TNF-receptor family member, has been shown to have an important role in long-term immunity, including an enhancement in the generation of CD8 T cell memory precursors. The mechanisms of action are complex and may include both direct effects on CD8 cells and indirect effects on CD4 helper cells or via inhibition of Treg. In initial experiments, we evaluated the effects of early enforced OX40 co-stimulation following delayed transfer of donor T cells to haplo MHC-mismatched chimeras, 10 weeks following nonmyeloablative BMT. OX40 expression peaked on transferred CD4 and CD8 T cells in the first 1–2 weeks following transfer and was sustained thereafter, especially in the CD4 subset. 48 hours after T cell transfer, recipient mice were treated with agonistic anti-OX40 antibody (OX86) or isotype control. OX86 treatment led to a 9-fold increase in the expansion of CTL in comparison to isotype control treated mice, enhanced production of Granzyme B and IFNγ and led to more rapid eradication of host hematopoietic targets or host tumor cells. Moreover, OX86 antibody acted directly on CD8 T cells and bypassed the requirement for help from donor CD4 cells. However, although enforced OX40 co-stimulation boosted the primary effector response, it did not increase numbers of memory precursor cells, as assessed by survival and recall responses following transfer to antigen free hosts, and was unable to prevent eventual exhaustion of surviving donor CTL as tested at 60 days following transfer. Similarly, OX86 was unable to prevent exhaustion of CD8 cells transgenic for the male antigen-specific Matahari (Mh) TCR following adoptive transfer to male BMT recipients reconstituted with female BM. We have shown previously that the functions of exhausted donor CD8 cells are partially restored by blockade of the co-inhibitory PD-1 pathway in both haplo mismatched and MHC-matched mHAg mismatch models. We hypothesized that provision of co-stimulatory signals when exhaustion had become established would increase the effectiveness of co-inhibitory blockade. Therefore, 6 weeks after Mh CD8 T cell transfer to male BMT recipients, we examined the effect of OX86, with or without additional blockade of the PD-1 pathway. Only a minority of Mh CD8 cells from animals receiving isotype control antibody were proliferating in vivo as measured by BrdU incorporation over a 7 day pulse (20 +/−3% BrdU+) and few cells were able to produce IFNγ following antigen stimulation in vitro (3.5+/−1.4 x104 IFNγ+ cells/spleen). OX86 alone offered no restoration of function (15 +/− 2% BrdU+; 3.3+/−0.4 x104 IFNγ+ cells; p=ns). Blockade of PD-L1 modestly increased turnover of cells (37 +/− 6 % BrdU+; p<0.01 vs isotype), but in the absence of CD4 cells, did not significantly increase production of IFNγ (4.4+/−0.9 x104 IFNγ+ cells; p=ns). However, in vivo administration of OX86 combined with anti-PD-L1 blockade dramatically increased turnover of Mh CD8s (77 +/− 8% BrdU+; p<0.001 vs anti-PD-L1 alone, OX86 alone or Isotype) and enhanced their effector function ∼ 9-fold (27.4 +/− 6.8 x104 IFNγ+ cells/spleen; p<0.01 vs all others). In conclusion, forced co-stimulation via OX40 alone is unable either to prevent CTL exhaustion or restore CD8 T cell function when exhaustion has become established. In contrast, the marked synergy observed when agonistic OX40 signals are combined with co-inhibitory blockade, is consistent with a model in which the PD-1 pathway acts at a critical checkpoint that regulates the response to co-stimulation. Thus, these data suggest a novel approach to restoring the functions of exhausted anti-tumor CTL by modulating co-stimulatory and co-inhibitory pathways simultaneously. Disclosures: No relevant conflicts of interest to declare.

Effects of rhIL-7 administration in humans on in vivo expansion of naïve, memory and effector subsets of CD4+ & CD8+ T-cells

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 2504-2504
Author(s):  
C. Sportes ◽  
F. Hakim ◽  
M. Krumlauf ◽  
R. Babb ◽  
T. Fleisher ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
T Cell Subsets ◽  
Down Regulation ◽  
Cd8 Cells ◽  
Interleukin 7 ◽  
Circulating Cells

2504 Background: IL-7 has a critical and non-redundant role in T-cell lymphopoiesis and peripheral T-cell homeostasis. IL-7 administration may prove clinically valuable in conditions of disease induced (HIV) or iatrogenic T-cell depletion and for modulation of vaccine immune responses. In the first phase I study in humans, recombinant human interleukin-7 (“CYT 99–007”, Cytheris Inc., Rockville, MD) was administered subcutaneously every other day for two weeks in adults with refractory malignancies at 3, 10, 30 and 60 μg/kg/dose. Biologic activity, defined as a 50% increase over baseline of peripheral blood CD3+ T-cells, was seen at and above the 10μg/kg/dose in all patients. The kinetics of proliferation and expansion of peripheral blood T-cell subsets were analyzed. Methods: Multicolor flow cytometry was performed at baseline, 1, 2 and 3 weeks. Among CD4+ cells, the most naïve were defined as CD45RA+ /CD31+. Among CD4+ & CD8+ cells, the main naïve, memory and effector populations were defined respectively as CD45RA+/CD27+, CD45RA-/CD27+ and CD45RA-/CD27-. Within each subset, the number of cells in cycle was defined by Ki67 staining. Results: Following IL-7 therapy, there was marked proliferation of all T-cells subsets, peaking at week 1, most striking for the naive subsets with 30–70% of circulating cells induced to cycle. Proliferation rates were halved by week 2 despite continuation of treatment, coincident with the observed down-regulation of the IL-7 receptor. Cycling returned to baseline by week 3. Significant proliferation was also induced in effector and memory CD4+ and CD8+ T-cells but to a lesser magnitude, resulting in a greater net expansion of the naïve subsets, still ongoing one week after the end of treatment. Conclusions: IL-7 administration induces marked expansion of naïve, memory and effector CD4+ & CD8+ T-cells in humans. Consistent with the known down-regulation of the IL-7 receptor upon IL-7 exposure, proliferation rates decrease during the second week of treatment. rhIL-7 induced T-cell expansion may prove clinically valuable in adoptive immunotherapy as an adjunct to tumor vaccination and / or immunorestorative agent. [Table: see text]

scholarly journals Effect of presenilins in the apoptosis of thymocytes and homeostasis of CD8+ T cells

Blood ◽  
2007 ◽  
Vol 110 (9) ◽  
pp. 3218-3225 ◽  
Author(s):  
Antonio Maraver ◽  
Carlos E. Tadokoro ◽  
Michelle L. Badura ◽  
Jie Shen ◽  
Manuel Serrano ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Notch Signaling ◽  
Cd8 T Cells ◽  
Double Positive ◽  
Cd8 Cells ◽  
Notch Receptors ◽  
Secretase Activity

Abstract Many studies have positioned Notch signaling at various critical junctions during T-cell development. There is, however, debate regarding the role of Notch in the CD4 versus CD8 lineage commitment. Because there are 4 Notch receptors and RBP-Jκ–independent Notch signaling has been reported, we decided to eliminate γ-secretase activity once its activity is required for all forms of Notch signaling. T-cell–specific elimination of γ-secretase was carried out by crossing presenilin-1 (PS1) floxed mice with CD4-Cre mice and PS2 KO mice, generating PS KO mice. Thymic CD4+CD8+ double-positive (DP) cells from these mice were strikingly resistant to apoptosis by anti-CD3 treatment in vivo and expressed more Bcl-XL than control thymocytes, and deletion of only one allele of Bcl-XL gene restored wild-type levels of sensitivity to apoptosis. In addition, these PS KO animals displayed a significant decrease in the number of CD8+ T cells in the periphery, and these cells had higher level of phosphorylated p38 than cells from control littermates. Our results show that ablation of presenilins results in deficiency of CD8 cells in the periphery and a dramatic change in the physiology of thymocytes, bringing to our attention the potential side effects of presenilin inhibitors in ongoing clinical trials.

scholarly journals CD8 is required during positive selection of CD4-/CD8+ T cells.

1990 ◽  
Vol 171 (2) ◽  
pp. 427-437 ◽  
Author(s):  
J C Zúñiga-Pflücker ◽  
L A Jones ◽  
D L Longo ◽  
A M Kruisbeek
Keyword(s):  
T Cells ◽  
T Cell ◽  
Positive Selection ◽  
Cd8 T Cells ◽  
Selection Process ◽  
Cell Repertoire ◽  
Cd8 Cells ◽  
Mhc Molecules ◽  
Single Positive

Interactions between self-MHC molecules and T cells are necessary for the proper development of mature T cells, in part due to an absolute requirement for self-MHC-TCR interactions. Recently, we showed that CD4-mediated interactions also participate in shaping the T cell repertoire during thymic maturation. We now examine the possible role of the CD8 molecule during in vivo T cell development. Our results demonstrate that perinatal thymi treated with intact anti-CD8 mAb fail to generate CD8 single-positive T cells, while the generation of the other main phenotypes remains unchanged. Most importantly, the use of F(ab')2 anti-CD8 mAb fragments gave identical results, i.e., lack of generation of CD4-/CD8+ cells, with no effect on the generation of CD4+/CD8+. Furthermore, selective blocking of one CD8 allele with F(ab')2 mAbs in F1 mice expressing both CD8 alleles did not interfere with the development of CD4-/CD8+ cells, demonstrating that the absence of CD8+ T cells in homozygous mice is not due to depletion, but rather is caused by a lack of positive selection. This is most likely attributable to a deficient CD8-MHC class I interaction. Our findings strongly advocate that CD8 molecules are vital to the selection process that leads to the development of mature single-positive CD8 T cells.

CLL Cells in TCL1 Transgenic Mice Induce Similar Defects in CD4 and CD8 T Cells to Those Observed in Patients with CLL.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 50-50
Author(s):  
Gullu Gorgun ◽  
Tobias A.W. Holderried ◽  
Rifca Ledieu ◽  
David Zahrieh ◽  
John G. Gribben
Keyword(s):  
T Cells ◽  
T Cell ◽  
Transgenic Mice ◽  
Cd8 T Cells ◽  
Cell Function ◽  
Cd4 Cells ◽  
Cd8 Cells ◽  
The Impact ◽  
Young Mice

Abstract Deregulation of the TCL1 pathway plays a crucial role in B-CLL pathogenesis and targeted expression of TCL1 results in the development in older mice of a B cell lymphoproliferative disorder resembling human B-CLL. CLL patients develop progressively impaired immunity and gene expression profiling of CD4 and CD8 T cells in B-CLL patients revealed defects in genes regulating critical pathways for T cell effector function. The onset of CLL in TCL1-transgenic mice also results in defects similar to those observed in CLL patients. Therefore, this murine model mimics the impact of CLL on the normal immune system, suggesting this may be an appropriate model to examine in vivo the impact of steps taken to repair T cell defects. In this study we examined whether infusion of CLL cells obtained from older mice induced similar changes in T cells of young mice, providing direct demonstration in vivo of interactions of CLL cells with the host immune system which result in development of immune deficiencies. Global gene expression profiling was performed using the Mouse 430_2 Affymetrix chip on highly purified CD4 and CD8 T cells from 6 non-transgenic mice and 16 TCL1 transgenic mice of different ages and at different stages in disease development and compared to that of cells from 6 TCL1 transgenic mice without CLL injected one week previously with 50 x 106 CLL cells. On unsupervised analysis using DNA-Chip Analyzer CD4 and CD8 T cells of young mice without CLL clustered with non-transgenic mice of different ages, whereas CD4 and CD8 cells from mice with developing or established CLL clustered with the young mice injected with CLL cells. Supervised analysis using Permax identified significant differences in expression for 628 genes (125 genes upregulated and 503 downregulated) in CD4 cells and 620 genes (320 genes upregulated and 300 genes downregulated) in CD8 cells in T cells from CLL bearing mice and CLL cell injected mice compared to non-transgenic mice and non-tumor bearing TCL1 mice. Comparison of pathways perturbed in the mice using GenMAPP finder compared to that observed in our previous studies in patients with CLL demonstrates similar alteration in many pathways, including regulation of cell proliferation and cell cycle control, cell differentiation, cytoskeleton formation, intracellular transportation and vesicle formation and transport. Examining these pathways functionally, we observed significantly decreased T cell proliferation, cytotoxicity and helper T cell function, increased numbers of CD4+CD25+CTLA4+ regulatory T cells and increased IL-4 amd IL-13 and decreased IL-12, IFNγ, sTNFRI, sTNFRII in CD4 cells and decreased IL-12p40, TIMP1 and TIMP2 in CD8 cells in both CLL bearing mice or mice injected with CLL cells compared to mice without CLL. These similar findings in human and murine CLL are in keeping with the hypothesis that interaction of the CLL cells with the normal immune function induces changes that result in decrease in T cell differentiation and effector function. It is intriguing to postulate that this effect diminishes autologous anti-tumor responses. We conclude that development of CLL in these transgenic mice induces T cell defects that mimic the defects that occur in CLL patients and that the TCL1 transgenic mouse model will serve as an ideal model to study steps to repair T cell function and their impact on CLL.

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.

scholarly journals Both intrathymic and peripheral selection modulate the differential expression of V beta 5 among CD4+ and CD8+ T cells.

1992 ◽  
Vol 176 (6) ◽  
pp. 1733-1738 ◽  
Author(s):  
P J Fink ◽  
K Swan ◽  
G Turk ◽  
M W Moore ◽  
F R Carbone
Keyword(s):  
T Cells ◽  
T Cell ◽  
Transgenic Mice ◽  
Cd8 T Cells ◽  
Cell Receptor ◽  
T Cell Subsets ◽  
Gene Encoding ◽  
Cd8 Cells ◽  
Peripheral T Cells

Murine T cells expressing V beta 5 are characterized by (a) intrathymic deletion in the presence of I-E and products of endogenous mouse mammary tumor viruses, and (b) a greater representation in CD8+ relative to CD4+ peripheral T cells, thought to be due to more efficient intrathymic positive selection on class I rather than class II major histocompatibility complex antigens. We have engineered mice that are transgenic for a rearranged gene encoding a V beta 5+ beta chain of the T cell receptor for antigen. Deletion is not predicted in I-E- V beta 5+ transgenic mice, and until the age of 2 wk, the CD4/CD8 ratio of peripheral T cells is &gt; 3:1 and indistinguishable between transgenic and nontransgenic mice. Transgenic mice then show a rapid, age-dependent decline in the ratio of CD4+ to CD8+ T cells in the lymphoid periphery, reaching a low of 1:10 by 7 mo of age. Furthermore, the percent of peripheral CD4+ cells that express the transgene drops with age, reaching a low of about 60% at 7 mo, while the percent of CD8+ cells that express V beta 5 remains greater than 95% at all ages. The lymphoid periphery is implicated in this selection against CD4+ V beta 5+ T cells as it occurs more rapidly in thymectomized transgenic mice, and can be delayed in mice whose peripheral T cells are replaced by recent thymic emigrants after depletion by in vivo treatment with anti-Thy-1 antibodies. These results indicate that the relative expression of V beta 5 in T cell subsets can be influenced not only intrathymically in I-E+ V beta 5+ transgenic mice, but also by events in the periphery, in the absence of I-E expression.

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