Induction of Endogenous Repair Mechanisms by Single-Stranded DNA Oligonucleotide Therapy for Correction of the DNA-PK Mutation in Murine Severe Combined Immune Deficiency.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 454-454
Author(s):  
Jakub Tolar ◽  
Angela Panoskaltsis-Mortari ◽  
Anton K. Yerich ◽  
Ron T. McElmurry ◽  
Georg A. Hollander ◽  
...  

Abstract Gene correction is an attractive strategy for gene therapy since it allows the corrected gene to remain regulated within its native genome location. We have explored gene correction of murine severe combined immunodeficiency (SCID) with single-stranded DNA oligonucleotides (SSO). Murine SCID is characterized by severe T- and B-cell lymphopenia and is caused by a point mutation in the DNA protein kinase subunit (DNA-PK). To correct the mutant missense sequence (T to A substitution), a silent mutation was introduced by synthesizing the SSO non-transcribed sequence (45 bp) surrounding the site of the SCID mutation and replacing the T nucleotide with a C nucleotide to permit production of wild-type (wt) DNA-PK protein. Since the fetus is potentially an ideal permissive environment for gene correction due to the high proliferative rate of its tissues, SSO were injected in utero either directly into the liver of the fetus or transplacentally (via hydrodynamic infusion to the pregnant dam). E15/16 BALB/c-SCID recipients (N = 78) were injected with SSO (20 mcg/fetus). Twenty nine mice survived to term and, when evaluated by peripheral blood (PB) FACS at 15–30 weeks of life, 11 had significant phenotypic evidence of immune restoration defined as ≥ 2% CD4+ or CD8+ T cells: 6 had both CD4+ and CD8+ T cells, 2 had CD4+ cells only and 3 had CD8+ T cells only. The highest level of CD4+ cells seen was 9%, the highest level of CD8+ cells was 2% and both had TCR rearrangement and 27% and 15% genotypic correction of the mutated bp by quantitative pyrosequencing (PSQ) of DNA isolated from whole blood. Since placental membranes are permeable to some molecules, SSO were hydrodynamically delivered to pregnant BALB/c-SCID dams (100 mcg). Two of 8 evaluable mice injected on day E5/6 had significant numbers of T cells, one of which had 20% CD8+ with 3% CD4+ cells at 13 weeks of life, and PSQ showed a 13% correction rate. Sixteen offspring injected at E13/14 were analyzed: 2 had 4% and 5% CD4+ cells and the latter also had 6% of CD8+ cells with PSQ correction rates of 22% and 11%, respectively. Of 40 mice evaluated after transplacental injections at age E15/16, 9 had >2% CD4+ or CD8+cells. The four with the highest T cell count had a genotypic correction of 12–25% of wt levels. Notably, littermates with no phenotypic correction had no evidence of gene correction at the DNA-PK mutation site. However, in all immune-restored animals that were analyzed for gene correction, (2/78 after in utero; 7/64 after transplacental delivery) an A to T rather than the anticipated A to C correction occurred. This is consistent with the hypothesis that SSO stimulated homologous recombination with a preferred utilization of the endogenous T rather than the exogenous C due to preferential pairing of two pyrimidines (A with T) than pyrimidine with purine (A with C). In summary, we show that SSO therapy for correction of DNA-PK mutation is possible when SSO are injected in utero at late gestation or are hydrodynamically delivered to the pregnant dam. These findings also suggest that while DNA homology around the mutation site is necessary for correction, the wt nucleotide is favored by the endogenous DNA repair pathway.

Blood ◽  
2003 ◽  
Vol 102 (1) ◽  
pp. 180-183 ◽  
Author(s):  
Carl E. Mackewicz ◽  
Baikun Wang ◽  
Sunil Metkar ◽  
Matthew Richey ◽  
Christopher J. Froelich ◽  
...  

Abstract In HIV infection, CD8+ cells show cytotoxic and noncytotoxic anti-HIV activity. The latter function is mediated, at least in part, by a secreted antiviral protein, the CD8+ cell antiviral factor (CAF). Because antiviral effector molecules, such as perforin and granzymes, reside in the exocytic granules of CD8+ T cells, we examined the possibility that granules contain CAF-like activity. CD8+ cells from HIV-infected individuals showing strong CAF-mediated antiviral activity were induced to release their granule constituents into culture media. Within 1 hour of stimulation, high levels of granzyme B (a primary granule constituent) were found in the culture fluids of previously activated CD8+ cells. The same culture fluids contained no or very low amounts of CAF activity, as measured with HIV-infected CD4+ cells. Maximal levels of CAF activity were not observed until 5 or 7 days after stimulation, consistent with typical CAF production kinetics. In addition, extracts of granules purified from antiviral CD8+ cells did not show any CAF activity, whereas the cytoplasmic fraction of these cells showed substantial levels of antiviral activity. These findings suggest that CAF does not reside at appreciable levels in the exocytic granules of antiviral CD8+ T cells. (Blood. 2003;102: 180-183)


Blood ◽  
2001 ◽  
Vol 98 (2) ◽  
pp. 467-474 ◽  
Author(s):  
Patricia A. Taylor ◽  
Christopher J. Lees ◽  
Herman Waldmann ◽  
Randolph J. Noelle ◽  
Bruce R. Blazar

The promotion of alloengraftment in the absence of global immune suppression and multiorgan toxicity is a major goal of transplantation. It is demonstrated that the infusion of a single modest bone marrow dosage in 200 cGy-irradiated recipients treated with anti-CD154 (anti-CD40L) monoclonal antibody (mAb) resulted in chimerism levels of 48%. Reducing irradiation to 100 or 50 cGy permitted 24% and 10% chimerism, respectively. In contrast, pan–T-cell depletion resulted in only transient engraftment in 200 cGy-irradiated recipients. Host CD4+ cells were essential for alloengraftment as depletion of CD4+ cells abrogated engraftment in anti-CD154–treated recipients. Strikingly, the depletion of CD8+ cells did not further enhance engraftment in anti-CD154 mAb–treated recipients in a model in which rejection is mediated by both CD4+ and CD8+ T cells. However, anti-CD154 mAb did facilitate engraftment in a model in which only CD8+ T cells mediate rejection. Furthermore, CD154 deletional mice irradiated with 200 cGy irradiation were not tolerant of grafts, suggesting that engraftment promotion by anti-CD154 mAb may not simply be the result of CD154:CD40 blockade. Together, these data suggest that a CD4+regulatory T cell may be induced by anti-CD154 mAb. In contrast to anti-CD154 mAb, anti-B7 mAb did not promote donor engraftment. Additionally, the administration of either anti-CD28 mAb or anti-CD152 (anti–CTLA-4) mAb or the use of CD28 deletional recipients abrogated engraftment in anti-CD154 mAb–treated mice, suggesting that balanced CD28/CD152:B7 interactions are required for the engraftment-promoting capacity of anti-CD154 mAb. These data have important ramifications for the design of clinical nonmyeloablative regimens based on anti-CD154 mAb administration.


Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3873-3873
Author(s):  
Yangqiu Li ◽  
Qingsong Yin ◽  
Shaohua Chen ◽  
Lijian Yang ◽  
Grzegorz Przybylski ◽  
...  

Abstract Thymic recent output function is characterized its importance of thymus to T-cell diversity in the periphery of both children and adults. The generation of TCR diversity occurs in the thymus through recombination of gene segments encoding the variable parts of the TCR α and β chains. During these processes, by-products of the rearrangements are generated in the form of signal joint T-cell receptor excision circles (sjTRECs), which is considered as a very valuable tool to estimate thymic function. Quantitative of δRec-ψJα sjTRECs can direct evaluate the recent thymic output function, but it is unable to analyze the particular thymic output function of different TCR Vβ subfamily naive T cells. The complexity of TCR Vβ repertoire is an important factor for immune reconstitution, quantitative analysis of series TCR Vβ-Dβ sjTRECs could be used to evaluate the levels of different Vβ subfamily naive T cells. In the present study, quantitative analysis of δRec-ψJα sjTRECs was performed in mononuclear cells, CD3+, CD4+ and CD8+T cells from peripheral blood of normal individuals and cord blood by real-time PCR(TaqMan). And the analysis of 23 TCR Vβ-Dβ1 sjTRECs was performed by semi-nested PCR. Different amounts of DNA (corresponding to 2*105, 5*104, 1*104 and 1*103 cells respectively) from all samples were amplified to estimate the frequency of TCR Vβ-Dβ sjTRECs. The mean value of δRec-ψJα sjTRECs was detected in 4.10±3.65/1000 PBMCs, 6.37±5.28/1000 CD3+cells, 3.28±1.24/1000 CD4+cells, 4.67±3.63/1000 CD8+cells from normal individuals (n=14) and 35.59±47.56/1000 CBMC, 71.48±86.42/1000 CD3+cells, 41.02±32.9/1000 CD4+ cells, 52.05±52.32/1000 CD8+cells from cord blood (n=9) (p=0.0208, p=0.0096, p=0.0003, p=0.0026, respectively). A part of Vβ subfamily sjTRECs could be detected in all samples from cord blood (Vβ2, 3, 4, 5, 10, 13, 14, 15, 19 and 22) and peripheral blood (Vβ10, 13 and 14) at 5*104 cells level, some of Vβ subfamily sjTRECs could be detected in 1*103 cells level. The frequencies of 23 Vβ-Dβ1 sjTRECs were different at the same cellular concentration. The number of detectable Vβ subfamily sjTRECs was 22.00±0.94/2×105, 18.8±1.87/5×104, 10.40±2.99/1×104 and 0.78±1.39/1×103 CBMCs, as compared with 18.70±2.45/2×105 (p=0.002), 13.7±2.67/5×104 (p<0.001), 5.5±2.07/1×104 (p=0.001) and 0.50±0.71/1×103 (p=0.739) in PBMCs from normal individuals. Similar results were found in CD4+ and CD8+ T cells which were sorted from both CBMCs and PBMCs, the number of detectable Vβ subfamily sjTRECs was 13.90±2.38/1×104 CD4+cells, 11.5±1.96/1×104CD8+cells from cord blood and 5.6±2.68/1×104 CD4+cells (p<0.001) and 8.2±2.57/1×104CD8+cells (p>0.005) from normal individuals. The results indicate that the number of detectable sjTRECs of Vβ subfamilies and the frequencies of most Vβ-Dβ1 sjTRECs in normal PBMCs, CD4+ and CD8+T cells were obviously lower than those in cord blood. In conclusions, the results provide the base data of naïve T cells levels and thymic recent output function in cord blood and peripheral blood of normail individuals in chinese.


1990 ◽  
Vol 171 (3) ◽  
pp. 953-958 ◽  
Author(s):  
S R Webb ◽  
J Sprent
Keyword(s):  
T Cells ◽  

Contrary to existing dogma, evidence is presented that proliferative responses of mature unprimed T cells to Mlsa antigens involve CD8+ cells as well as CD4+ cells. The response of CD8+ cells to Mlsa antigens proved to be heavily dependent on help from CD4+ cells, and responses were stronger in three I-E+ strain combinations than in an I-E- combination. In I-E+ combinations, CD8+ blast cells accounted for 20-25% of the blasts generated from unseparated T cells responding to Mlsa-bearing stimulator cells in vitro; similar findings applied to blast cells generated in vivo. The observation that the majority (greater than or equal to 50%) of Mlsa-stimulated CD8+ cells (and CD4+ cells) were V beta 6+ indicated that CD8+ cells respond to Mlsa antigens, per se, rather than to nonspecific stimuli. Whether CD4+ and CD8+ cells use the same or different H-2-restricting elements to respond to Mlsa antigens has yet to be resolved.


Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 180-180
Author(s):  
Vassiliki Rizouli ◽  
David Zahrieh ◽  
Gullu Gorgun ◽  
Tobias A.W. Holderried ◽  
Carlo Croce ◽  
...  

Abstract The TCL1 gene at 14q32.1 is involved in chromosomal translocations and inversions in T cell leukemias, but targeted expression of the TCL1 gene in mice results in the development of a CLL like disorder in older mice resembling human B-CLL, so that deregulation of the TCL1 pathway proved to play a crucial role in CLL pathogenesis in mice. Development and progression of B-CLL is associated with immune dysregulation and in particular T cell defects. Although patients with CLL often have normal or even increased T cell numbers, there is an abnormal CD4/CD8 ratio, impaired mitogen responses and defective effector function in response to antigenic stimulation. We have previously categorized defects in the gene expression profile of CD4 and CD8 T cells in patients with CLL. We therefore sought to determine if the onset of CLL in the TCL1-transgenic mice also resulted in defects similar to those observed in patients with CLL. The aim was to determine if this murine model would mimic the impact of CLL on the normal immune system, so that we could use this model to examine in vivo the impact of steps taken to repair T cell defects. To examine this, we highly purified CD4 and CD8 T cells by positive selection from non-transgenic mice and TCL1 transgenic mice of different ages and at different stages in disease development. Total RNA was extracted, cRNA synthesized, labeled and hybridized to the Mouse 430_2 Affymetrix chip. We used DNA-Chip Analyzer (dChip) to perform an unsupervised analysis, which consisted of gene filtering, excluding genes that lacked sufficient variability across groups, and hierarchical clustering of genes and samples. This type of analysis demonstrated that CD4 and CD8 T cells of young mice without CLL clustered with non-transgenic mice of different ages, but separately from CD4 and CD8 cells from mice with developing and established CLL. Supervised analysis using Permax of the gene expression profiles of T cells in non-transgenic mice and non-tumor bearing TCL1 mice compared to CLL bearing mice identified significant differences in expression for 348 in CD4 cells and 127 genes for CD8 cells. In CD4 cells from CLL mice 202 genes were upregulated and 146 were downregulated and in CD8 cells 32 genes were upregulated and 95 genes downregulated. Analysis of the genes observed to be altered in the CLL bearing mice revealed that the majority were involved in genes regulating cytoskeleton formation, intracellular transportation, vesicle formation and transport, cell cycle control and cell differentiation. Comparison of the pathways perturbed in the mice compared to that observed in our previous study in patients with CLL demonstrated alteration in many similar pathways. The 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 would diminish 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.


Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2186-2186
Author(s):  
Michiyo Ohyashiki ◽  
Junko H Ohyashiki ◽  
Ayako Hirota ◽  
Chiaki Kobayashi ◽  
Kazuma Ohyashiki

Abstract Abstract 2186 Background and Aim: MicroRNAs (miRNAs) consist of short noncoding RNA molecules of approximately 18–22 nucleotide that regulate post-transcriptional gene expression by degradation or repression of mRNA molecules. The miR-17-92a cluster is known as a regulator of the immune system and is critical for lymphoid cellular development and tumorigenesis in lymphoid tissue. Most knowledge of the miR-17-92 cluster in normal and abnormal conditions of the lymphoid system is based on mouse experiments. It is suggested that the accumulation of activated CD4+ T cells by higher mir-17-92 expression leads to a breakdown of T-cell tolerance in the periphery and may promote B-cell activation, germinal center reaction and autoantibody generation. However, only limited reports on in vivo human lymphocyte senescence exist. We therefore set out to determine miR-92a levels in circulating lymphocytes obtained from healthy participants to ascertain the possible association between immunological condition and the expression level of miR-17-92. Experimental design: We separated lymphocytes from 21 healthy volunteers, aged 23 to 58 years (13 men and 8 women), for surface marker and miR-92a level analyses. The CD4+ or CD8+ T-cell fractions were separated with an isolation kit for humans (Miltenyi Biotec, Bergisch Gladbach, Germany) and AutoMACS Pro Separator (Miltenyi Biotec), according to the supplier's instruction, and stored at −80°C until utilization. After separation of CD4+ or CD8+ cells, the miR-92a levels were measure, as reported previously (PLoS ONE. 2011, 24;6(2);e16408). Immunophenotyping was done with flow cytometry. Results: The miR-92a of separated CD8+ lymphocytes decreased significantly with age (P = 0.0002), and miR-92a in CD4+ cells tended to decrease with age (P = 0.0635). We found a positive correlation between CD8+ miR-92a expression level and the percentage of naive CD8+ T cells (RO−CD8+CD27+ cells (P = 0.0046)) with L-selectin antigen (CD3+CD8+CD62L+ (P = 0.0011)) in healthy subjects. This suggests that the miR-92a of a majority of CD8+ T is derived from naive CD8+ T cells with L-selectin antigen, and CD8+ miR-92a expression level declines progressively with age (P < 0.0001 and P < 0.0001, respectively). In CD4+ cells, we observed a trend of decreasing CD4+ miR-92a level with age, while no significant difference was notable with lymphocyte subset fraction as far as we tested. The index of CD8+ miR-92a values (CD8+ miR-92a×number of CD8 cells) was positively correlated with the index of CD4+ miR-92a values (P = 0.0101). Discussion: These results indicate an age-related reduction of naive T cells may link to miR-92a of T-lymphocytes and may influence immune dysfuction with age. In conclusion, our results suggest that the miR-92a level may represent attrition of naïve CD8+ T cells, possibly due to apoptosis of naïve T cells. Additionally down-regulation of the miR-92a level in individuals older than 40 years may indicate impairment or exhaustion of naïve T-cells linked to immune dysfunction and contributed disease states. Disclosures: No relevant conflicts of interest to declare.


Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1911-1911
Author(s):  
Barry Flutter ◽  
Noha Edwards ◽  
Lei Zhang ◽  
Shivajanani Sivakumaran ◽  
Michael Croft ◽  
...  

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.


1994 ◽  
Vol 180 (5) ◽  
pp. 1715-1728 ◽  
Author(s):  
M Croft ◽  
L Carter ◽  
S L Swain ◽  
R W Dutton

We have generated primary effector populations from naive CD8 T cells in response to antigen and determined their patterns of cytokine secretion upon restimulation. The effect of exogenous factors on the effector generation was examined and compared with responses of antigen-specific CD4 effectors generated under comparable conditions. CD8 cells from bm1 mice were stimulated with C57BL/6 (B6) antigen presenting cells (APCs) bearing allogeneic class I and CD8 cells from female severe combined immunodeficiency (SCID) B6 mice, transgenic for a T cell receptor alpha/beta (TCR-alpha/beta) that recognizes H-Y on Db, were stimulated with APCs from male mice. In parallel, CD4 cells from bm12 mice were stimulated with alloantigen and CD4 cells from V beta 3/V alpha 11 TCR transgenics were stimulated with a peptide of pigeon cytochrome c on IEk. T cells from both transgenic mice were of naive phenotype whereas normal mice contained 10-20% memory cells. Effector CD8 populations generated were L-selectin low, CD45RB high, and CD44 high. Naive CD8 cells from SCID anti-H-Y mice made little or no cytokine immediately upon stimulation in contrast to naive CD4 which produced large amounts of interleukin 2 (IL-2). Both populations, however, generated primary effectors over 4-5 d that made substantial quantities of many cytokines upon restimulation. Both CD8 and CD4 effectors produced similar patterns of cytokines with alloantigen or specific antigen. Cytokines present during naive CD8 stimulation influenced the cytokine secretion profile of the effectors, as previously shown for CD4 cells, although secretion by CD8 effectors was generally lower than that of CD4 effectors. CD8 cells cultured with IL-2 alone made predominantly interferon gamma (IFN-gamma) and no IL-4 or IL-5, similar to CD4 cells. Priming with IFN-gamma increased IFN-gamma secretion from CD4 effectors, but had little if any effect on CD8 cells. In contrast, priming with IL-12 generated CD8 effectors, as well as CD4 effectors, producing elevated quantities of IFN-gamma, with similar levels from both the CD4 and CD8 populations. The presence of IL-4 during effector cell generation promoted synthesis of IL-4 and IL-5 from both CD8 and CD4 cells while downregulating IFN-gamma secretion. CD8 cells made only small amounts of IL-4, more than 100-fold less than CD4 cells, whereas significant levels of IL-5 were induced, only 3-10-fold lower than from CD4.(ABSTRACT TRUNCATED AT 400 WORDS)


Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2542-2542
Author(s):  
Chiara Borsotti ◽  
Rouven Muller ◽  
Masao Yamasaki ◽  
Markus G. Manz

Abstract Abstract 2542 Graft-versus-host disease (GVHD) and opportunistic infections represent two major causes of morbidity and mortality following allogeneic hematopoietic stem cell transplantation (allo-HSCT). Both of these conditions are respectively mediated or controlled to a large extend by donor-derived T cells. Moreover T cells play a critical role in promoting stem cell engraftment and decreasing the probability of disease relapse. Therefore the separation of the effects of GVHD from the beneficial effects mediated by T cells has been a long-standing challenge of transplantation immunology. The contribution of different subsets of donor T cells to the pathogenesis of GVHD has been studied in different mouse models: both purified naïve CD4+ and CD8+ T cells were able to induce acute GVHD. On the contrary memory T cells failed to induce GVHD. In order to dissect the contribution of different human T cells, we sought to define a xenogeneic model for GVHD in RAG2−/−gc−/− mice. Newborn RAG2−/−gc−/− mice were intraperitoneally (i.p.) injected with either peripheral blood mononucleated cells (PBMC), immunomagnetically selected CD4+ or CD8+ cells, or FACS sorted CD45RA+CD4+ or CD45RO+CD4+ cells. Mice were weaned 3 weeks after birth and were scored weekly for signs of GVHD and bled for human engraftment evaluation. Both PBMC and CD4+ injected groups developed xenogeneic GVHD (x-GVHD) and the survival at 12 weeks was 42% and 53% respectively. The peak of peripheral blood (PB) engraftment was reached between week 6 and 7 and it was about 40% in both groups. Human cells were present in high percentage (>60%) in the spleen and liver of mice that displayed signs of x-GVHD. Moreover in these mice developed detectable thymi and mesenteric lymph nodes (MLN) which are not normally present in the RAG2−/−gc−/− mice and which were populated by human cells. In PBMC injected mice, CD4+ and CD8+ T cells were the only populations observed with no survival of myeloid, B or NK cells. PBMC or CD4+ groups developed GVHD in 55%, while mice adoptively transferred with CD45RA+CD4+ cells manifested a positive score for x-GVHD (30%) but displayed milder symptoms compared with the PBMC or CD4+ groups. The lesser morbidity and mortality correlated with a lower human engraftment in PB (25% at week 7) and in spleen (about 40%). Only 10% of mice injected with CD45RO+CD4+ cells showed x-GVHD symptoms and the human engraftment level in PB remained below 20%. Finally mice injected with CD8+ cells did not develop any x-GVHD symptoms. The human PB engraftment was low (<15%) in these mice and it decreased over time. In summary we established an easy and reliable xenogeneic mouse model suitable for dissecting the contribution of different human T cell populations to x-GVHD. Our results suggest that depletion of CD4+CD45RA+ naïve T cells and/or adoptive transfer of CD4+CD45RO+ memory T cells may find use in the allo-HSCT setting, allowing for rapid reconstitution of T cell-mediated immunity while minimizing GVHD. Disclosures: No relevant conflicts of interest to declare.


Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 952-952
Author(s):  
Sara Ghorashian ◽  
Ben Carpenter ◽  
Angelika Holler ◽  
Emma Nicholson ◽  
Maryam Ahmadi ◽  
...  

Abstract Abstract 952 Background: The efficacy of T cell therapies for cancer may be limited when targeting tumour-associated antigens (TAA) which are also self-antigens. Ongoing exposure to TAA on normal cells may lead to tolerance via anergy or exhaustion of antigen-specific T cells. Methods: We have designed a model of tolerance to TAA in which T cell receptor (TCR)-transduced CD8 T cells recognise pMDM2, a TAA that is also a ubiquitous self-antigen. CD8+ T cells were transduced with pMDM2-specific TCR (MDM-CD8) and transferred to sub-lethally irradiated B6 mice that express pMDM2 in the context of MHC Class I (H2-Kb). MDM-CD8 cells are detectable 4 weeks after transfer but show defective in vivo killing of target cells pulsed with MDM2 peptide. We have used this model to determine the mechanism of tolerance and to evaluate whether tolerant CD8+ T cells can be rescued by CD4 help. Results: To determine whether tolerance of MDM-CD8 cells was dependent upon recognition of cognate antigen, we transferred MDM-CD8 cells into mice of a different MHC background (BALB/c) which lack H2-Kb required for presentation of the TCR-recognised MDM2 peptide. When BALB/c MDM-CD8 cells were transferred to BALBc hosts their functions were preserved and they retained efficient antigen-specific cytolysis. To determine whether tolerance could be modified by provision of CD4+ T cell help, we co-transferred MDM-CD8 with transgenic OT-II CD4+ cells. OT-II cells were primed with dendritic cells (DCs) loaded with cognate pOVA323-339 or irrelevant peptide. When activated through their TCR, OT-II cells increased both the frequency of MDM2-specific CD8 cells and their cytotoxic functions, indicating that CD4 help can overcome CD8 tolerance to TAA. Ineffective antigen presentation to CD4 cells and lack of known MHC class II-restricted TAA are major limitations to providing CD4 help in T cell therapy for cancer. We therefore tested whether transfer of the MHC Class I-restricted MDM2 TCR into CD4 cells could provide help upon transfer to antigen-expressing hosts. Co-transfer of MDM2-TCR-transduced CD4 cells with CD8 cells improved antigen-specific killing of target cells when compared to single transfer of either TCR-transduced CD8 or CD4 cells. Conclusion: CD4 cells rendered capable of responding to an MHC class I restricted TAA by TCR transfer can rescue tolerance developing in a CD8 population with the same specificity. This is potentially a novel way to circumvent defective immune responses arising in adoptively transferred effector cells due to prolonged exposure to cognate antigen on normal host cells. Disclosures: Stauss: Cell Medica: Scientific Advisor Other.


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