scholarly journals Off-target effects of Cre recombinase reveal limits of adoptive T cell transfers and persistent proliferation of effector CD8 T-cells

2018 ◽  
Author(s):  
Lisa Borkner ◽  
Anja Drabig ◽  
Xiaoyan Zheng ◽  
Julia Drylewicz ◽  
Thomas Marandu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Adoptive Transfer ◽  
Memory T Cells ◽  
Murine Cytomegalovirus ◽  
Effector Memory ◽  
Latently Infected ◽  
The One

Effector-memory T-cells (TEM) are assumed to be short-lived cells that poorly proliferate upon antigenic restimulation, thus depending on central-memory T-cells (TCM) to replenish their numbers during homeostasis, largely depending on adoptive transfer evidence. Here we analyzed T cells in their natural environment and observed robust long-term in vivo cycling within the TEM subset that was stronger than the one in the TCM subset. We compared the non-persistent vaccinia virus and the persisting murine Cytomegalovirus (MCMV), which induces inflationary TEM responses that remain high during viral latency. We analyzed Ki67 expression during acute, resolved and latent infection and found Ki67hiBcl2lo TEM in acutely or latently infected mice, arguing for antigen-driven TEM proliferation. In vivo labeling with deuterium showed that TEM acquired deuterium more rapidly than TCM, and were rapidly lost during chase. Similarly, antibody-mediated depletion of primed CD8 T cells in latenly infected mice revealed that TEM replenished more rapidly than TCM, suggesting that TEM cycle faster than TCM. Finally, we utilized the ability of Tamoxifen-induced Cre-ERT2 recombinase to induce chromosomal translocations when large amounts of Tamoxifen are administered for an extended time, which resulted in a selective depletion of proliferating Ki67hi cells that hardly affected the TCM subset, but drove a selective loss of Ki67hiBcl2lo effector T-cells, and an increase in the death of TEM in the spleen, arguing that TEM preferentially proliferate in the spleen. Since our results contradicted previous evidence from adoptive transfer experiments, we tested T cell homing to the spleen upon adoptive transfer. TEM homing was substantially poorer than the one of TCM, likely explaining the previously reported expansions of TCM, but not TEM, upon transfer into latently infected mice. In conclusion, our data suggest that memory inflation is largely maintained by splenic proliferation of antigen-specific TEM, rather than by continued expansion and differentiation of TCM.

scholarly journals IL-7 and IL-21 are superior to IL-2 and IL-15 in promoting human T cell–mediated rejection of systemic lymphoma in immunodeficient mice

Blood ◽  
2010 ◽  
Vol 115 (17) ◽  
pp. 3508-3519 ◽  
Author(s):  
John C. Markley ◽  
Michel Sadelain
Keyword(s):  
T Cells ◽  
T Cell ◽  
Adoptive Transfer ◽  
Memory T Cells ◽  
Tumor Rejection ◽  
Effector Memory ◽  
Transfer Model ◽  
Human T Cell

Abstract The γc-cytokines are critical regulators of immunity and possess both overlapping and distinctive functions. However, comparative studies of their pleiotropic effects on human T cell–mediated tumor rejection are lacking. In a xenogeneic adoptive transfer model, we have compared the therapeutic potency of CD19-specific human primary T cells that constitutively express interleukin-2 (IL-2), IL-7, IL-15, or IL-21. We demonstrate that each cytokine enhanced the eradication of systemic CD19+ B-cell malignancies in nonobese diabetic/severe combined immunodeficient (NOD/SCID)/γcnull mice with markedly different efficacies and through singularly distinct mechanisms. IL-7– and IL-21–transduced T cells were most efficacious in vivo, although their effector functions were not as enhanced as IL-2– and IL-15–transduced T cells. IL-7 best sustained in vitro T-cell accumulation in response to repeated antigenic stimulation, but did not promote long-term T-cell persistence in vivo. Both IL-15 and IL-21 overexpression supported long-term T-cell persistence in treated mice, however, the memory T cells found 100 days after adoptive transfer were phenotypically dissimilar, resembling central memory and effector memory T cells, respectively. These results support the use of γc-cytokines in cancer immunotherapy, and establish that there exists more than 1 human T-cell memory phenotype associated with long-term tumor immunity.

Establishing CD8+ T Cell Immunity by Adoptive Transfer of Autologous, IL-15 Expanded, Anti-Tumor CTL with a Central/Effector Memory Phenotype Can Induce Objective Clinical Responses.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 782-782 ◽  
Author(s):  
Marcus Butler ◽  
Philip Friedlander ◽  
Mary Mooney ◽  
Linda Drury ◽  
Martha Metzler ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Adoptive Transfer ◽  
De Novo ◽  
Effector Memory ◽  
Large Numbers ◽  
Central Effector

Abstract Abstract 782 The goal of cellular immunotherapy is to build long-lasting anti-tumor immunologic “memory” in patients and reject tumors for a lifetime. Previously, we and others demonstrated that IL-15 promotes the generation of T cells with a central memory (CM) phenotype which have the capacity to persist and establish effective anti-tumor memory in vivo. Furthermore, it has been shown that CD83 delivers a CD80-dependent T cell stimulatory signal that allows T cells to be long-lived. Based on these findings, we developed a system to generate large numbers of long-lived antigen-specific CD8+ T cells with a memory phenotype. This in vitro culture system utilizes IL-15 and a standardized, renewable artificial antigen presenting cell (aAPC) which was produced by transducing CD80, CD83, and HLA-A*0201 to the human cell line, K562. This aAPC can uniquely support the priming and prolonged expansion of large numbers of antigen-specific CD8+ CTL which display a central/effector memory (CM/EM) phenotype, possess potent effector function, and can be maintained in vitro for >1 year without any feeder cells or cloning. We hypothesized that adoptive transfer of these CTL with a CM/EM phenotype should result in anti-tumor memory in humans even without lymphodepletion or high dose IL-2. For our “first-in-human” clinical study, we chose the melanoma antigen MART1 as a target antigen, since MART1-specific HLA-A*0201+-restricted precursor CTL are detectable in some melanoma patients and can be immunophenotyped pre-infusion. Autologous CD8+ T cells were stimulated weekly with peptide-pulsed human cell-based aAPC and expanded with low dose IL-2 and IL-15. After three weeks, polyclonal MART1 CTL were reinfused without additional lymphodepletion, chemotherapy, IL-2, or vaccination. Eight study participants have enrolled and received a total of 15 MART1 CTL infusions (31% MART1 multimer positivity, median). All but one subject received two reinfusions where the 2nd graft was produced from CD8+ T cells harvested two weeks after the 1st reinfusion. To date, ≥2×109 CTL with potent effector function and a CM/EM phenotype were successfully generated for all subjects. No dose limiting toxicities were observed at either Dose Level 1 (2×108/m2) or Dose Level 2 (2×109/m2). Clinical activity was observed with a response by RECIST criteria in 1 subject, which was confirmed by a negative PET/CT 100 days following the last CTL infusion. In addition, 1 patient experienced a mixed response, 1 had stable disease, 3 had progression, and 2 are currently on active therapy. Multimer staining showed that, immediately post infusion, the percentage of CD8+ T cells specific for MART1 temporarily increased in all subjects, with the highest (6.5%) observed in subject #7. In 4 subjects, sustained increases in the frequency of MART1 specific T cells by more than two-fold (range 2.0-10x) for ≥21 days were observed despite the fact that no exogenous cytokines or vaccination was administered. Moreover, an increase of detectable MART1 specific T cells which display a CM phenotype was observed in all evaluable subjects and was observed for ≥35 days in 6 of 8 subjects. In subject #2, the conversion of MART1 CTL immunophenotype from a naïve to a mixture of naïve/memory phenotypes was observed for more than 6 months. We identified 10 individual MART1 T cell clonotypes from peripheral CD45RA- memory T cells on day 21. Clonotypic TCR Vbeta CDR3 analysis revealed that CTL grafts contained 7 out of 10 of these clonotypes. Furthermore, 6 clonotypes persisted in the peripheral CD45RA- memory fraction on days 39, 67 and/or 132. In Subject #3, who showed a mixed clinical response, 5 individual MART1 T cell clonotypes were isolated from lung metastases. 4 out of 5 clones were included in the CTL grafts. This finding supports the possibility that infused CTL can traffic and localize to sites of disease. Intriguingly, in both subjects, we were able to identify MART1 CTL clonotypes that were not detectable in the CTL grafts but possibly emerged after CTL infusion, indicating that adoptive transfer of MART1-specific CTL may provoke a de novo antitumor response. Taken together, these results suggest that CM/EM MART1 CTL generated ex vivo using our cell-based artificial APC in the presence of IL-15 may persist in vivo and induce de novo anti-tumor responses. Further enhancement of anti-tumor activity may be achieved through vaccination, cytokine administration, and/or removal of cytokine sinks and inhibitory factors following appropriate lymphodepletion. Disclosures: No relevant conflicts of interest to declare.

A Comparison of Human CMVpp65-Specific Central Memory and Effector Memory CD8 T-Cells When Stimulated in-Vivo with IL-2 or IL-15/IL-15Rα Complex in a Murine Xenograft Model of Adoptive Cell Therapy

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4805-4805
Author(s):  
Tzu-Yun Kuo ◽  
Aisha Hasan ◽  
Richard J O'Reilly
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Adoptive Immunotherapy ◽  
Memory T Cells ◽  
Cd8 T Cell ◽  
Central Memory ◽  
Effector Memory ◽  
Cell Clones

Abstract Initial clinical trials of adoptive immunotherapy have shown that the efficacy of adoptively transferred T-cells in man is often limited by the failure of cultured T cells, particularly cloned CD8 T cells, to persist in vivo. These studies demonstrated that the transferred T cells induced only transient responses and that persistence of the transferred T-cell clonotypes correlated with disease regression. A previous study suggested that CMV virus-specific CD8 T cell clones derived from central memory T cells (TCM), but not effector memory T cells (TEM), persisted long-term in non-human primates. On the other hand, another study comparing TCM and TEM derived SIV virus specific CD8 T-cell clones that were adoptively transferred in non-human primates demonstrated limited persistence of both TCM and TEM derived transferred T cells, and failed to show any difference between the two cell types. Because of these conflicting data, we have reexamed the persistence of adoptively transferred viral antigen specific T-cells derived from TCM and TEM population. Accordingly, we developed a NOG mouse model for studying the ability of human CMVpp65-specific T cells derived from central memory and effector memory populations to migrate to and accumulate in human tumor xenografts expressing CMVpp65, to alter the growth of these tumors and to persist in the tumors. This model also allows us to test immunomodulating agents and their ability to enhance targeted T-cell accumulations, antitumor activity and persistence. We analyzed CMVpp65-specific CD8 T cells derived from TCM and TEM precursors in vitro and in vivo. To tract the T-cells in vivo, we transduced membrane-bound Gaussia luciferase into TCM and TEM populations and monitored T cell trafficking by in vivo bioluminescence. Contrary to expectation, our results initially showed no differences between TCM and TEM derived CMVpp65-specific T-cell in mice co-treated with IL-2 in the time to accumulation, ultimate level of accumulation, degree of CMVpp65+ tumor regression or T-cell persistence. However, in mice cotreated with IL-15/IL-15Rα complex, both TCM and TEM exhibited more sustained engraftment and more prolonged accumulation in both the targeted tumor and in the marrow. In mice treated with IL-15/IL-15Rα, TCM and TEM derived T cells showed a similar effector memory phenotype and a similar level of regression of tumor growth. Thus, adoptive transfer of CMVpp65 specific TCM or TEM when combined with IL-15/IL-15Rα complex may support better persistence of antigen-specific T-cells following adoptive immunotherapy. Studies comparing IL-15/IL-15Rα complex with IL-15 alone are in progress. Disclosures No relevant conflicts of interest to declare.

Allogeneic Memory T Cells Derived from Host DC-Activated CD44loCD8+ T Precursors Sustain Host Tissue Injury of Ongoing Graft-versus-Host Disease.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3040-3040
Author(s):  
Yi Zhang ◽  
Gerard Joe ◽  
Elizabeth Hexner ◽  
Jiang Zhu ◽  
Stephen G. Emerson
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Memory T Cells ◽  
Effector Memory ◽  
Memory Phenotype ◽  
Memory T Cell ◽  
Cell Responses ◽  
With Memory

Abstract Graft-versus-host disease (GVHD) directed against minor histocompatibility antigen (miHAs) evolves over weeks to months, suggesting a requirement for persistent alloreactive donor T cells. In patients with allogeneic bone marrow transplantation (allo-BMT), persistency of GVHD is accompanied with elevated allogeneic CD4+ T cells with memory phenotype in peripheral blood. In contrast, several other studies have recently shown that T cells with memory phenotype (CD44hiCD62Lhi/lo) from normal donor mice do not induce acute GVHD. While these T cells with memory phenotype may be induced by environmental antigenic stimulation or may represent cells undergoing homeostasis in vivo, we found that early activated donor CD44hiCD8+ T cells with effector/memory phenotype upon ex vivo host DC stimulation are also functional defective in GVHD induction in vivo. However, whether alloreactive memory T cells might develop in vivo in recipient with ongoing GVHD, and if this is the case, whether these in vivo generated alloreactive memory T cells may be responsbile for persistency of GVHD, remain unknown. Using the C3H.SW anti-C57BL/6 (B6) and B6 anti-BALB.B mouse models of human GVHD directed against miHAs, we found that alloreactive CD8+ T cells secreting high levels of IFN-γ in recipient mice receiving C3H.SW CD44loCD8+ T cells + T−BM peaked by day 14, declined by day 28, and increased again after 35 days of transplantation, corresponding to the kinetics of primary and memory T cell responses. Indeed, while donor C3H.SW CD8+ T cells recovered from these B6 mice receiving C3H.SW CD44loCD8+ T cells + T−BM 10 days after allo-BMT, at the peak time of primary allogeneic immune response, upregulated the expression of effector marker CD25, donor CD8+ T cells recovered 42 days after allo-BMT from B6 mice with ongoing GVHD, at the time of memory T cell development, expressed high levels of CD44 and CD122 but down-regulated CD25. However, both d10-CD8+ and d42-CD8+ T cells expressed identical levels of cytotoxic molecules including granzyme B, perforin and FasL and were able to kill B6 mouse-derived EL-4 leukemic cells. Compared to naïve CD44loCD8+ T cells that were lost after cultured in the presence of IL-2+IL-15 for 5 days, d42-donor CD8+ T cells recovered from B6 mice with ongoing GVHD survived over 5 days in the presence of IL-2+IL-15 alone and these surviving d42-CD8+ T cells were able to rapidly proliferate in responding to B6 DCs+IL-2+IL-15 in secondary culture. Flow cytometry analysis showed that d42-CD8+ T cells contained at least two distinct subsets: CD44hiCD62Llo (80% to 85%) and CD44hiCD62LhiCD8+(3% to 6%) T cells, resembling to the phenotype of effector memory and central memory CD8+ T cells, respectively. Administration of irradiated secondary B6 mice with either d42-CD44hiCD62Lhi or d42-CD44hiCD62Llo CD8+ T cell subset recovered at day 42 from primary B6 mice receiving C3H.SW CD44loCD8+ T cells + T−BM caused virulent GVHD. These results indicate that alloreactive memory T cells develop in vivo in recipient mice with acute GVHD where host mHAs persist and may be responsible for the persistence of GVHD. Accordingly, we suggest that in vivo blockade of both alloreactive effector and memory T cell responses will be necessary for GVHD prevention and treatment.

scholarly journals Tumor masses support naive T cell infiltration, activation, and differentiation into effectors

2010 ◽  
Vol 207 (8) ◽  
pp. 1791-1804 ◽  
Author(s):  
Elizabeth D. Thompson ◽  
Hilda L. Enriquez ◽  
Yang-Xin Fu ◽  
Victor H. Engelhard
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Adoptive Transfer ◽  
Cell Activation ◽  
Ex Vivo ◽  
Tumor Infiltrating Lymphocytes ◽  
T Cell Infiltration

Studies of T cell responses to tumors have focused on the draining lymph node (LN) as the site of activation. We examined the tumor mass as a potential site of activation after adoptive transfer of naive tumor-specific CD8 T cells. Activated CD8 T cells were present in tumors within 24 h of adoptive transfer and proliferation of these cells was also evident 4–5 d later in mice treated with FTY720 to prevent infiltration of cells activated in LNs. To confirm that activation of these T cells occurred in the tumor and not the tumor-draining LNs, we used mice lacking LNs. Activated and proliferating tumor-infiltrating lymphocytes were evident in these mice 24 h and 4 d after naive cell transfer. T cells activated within tumors acquired effector function that was evident both ex vivo and in vivo. Both cross-presenting antigen presenting cells within the tumor and tumor cells directly presenting antigen activated these functional CD8 effectors. We conclude that tumors support the infiltration, activation, and effector differentiation of naive CD8 T cells, despite the presence of immunosuppressive mechanisms. Thus, targeting of T cell activation to tumors may present a tool in the development of cancer immunotherapy.

Ex Vivo Fludarabine Selectively Depletes Human Naive T-Cell Subsets: A Step Toward Modifying Donor Lymphocyte Infusion.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1071-1071
Author(s):  
Melody M. Smith ◽  
Cynthia R. Giver ◽  
Edmund K. Waller ◽  
Christopher R. Flowers
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Memory T Cells ◽  
Ex Vivo ◽  
T Cell Subsets ◽  
Effector Memory ◽  
Naïve T Cell ◽  
Naive T Cell

Abstract Ex vivo modification of donor lymphocytes with purine analogs (mDL) may help to minimize graft versus host disease (GvHD) while providing beneficial graft versus leukemia (GvL) effects. In a murine model system, we have shown that allogeneic donor splenocytes, treated with fludarabine ex vivo have significantly reduced GvHD activity when transferred to irradiated recipient mice, and retain anti-viral and GvL activities (Giver, 2003). This effect appears to be mediated by relative depletion of donor CD4 CD44low, “naive” T-cells. As a first step toward developing mDL for use in patients, we sought to evaluate the effects of ex vivo fludarabine exposure on human T-cell subsets, and to determine the minimum dose of fludarabine required to achieve this effect. Methods: Peripheral blood mononuclear cell samples from 6 healthy volunteers were evaluated at 0, 24, 48, and 72 hour time points after ex vivo incubation in varying dosages of fludarabine: 2, 5, and 10(n=3) mcg/ml. Fludarabine incubated samples were compared to samples that received no fludarabine (untreated). The total viable cell number was determined and the fractions and absolute numbers of viable CD4 and CD8 naïve and memory T-cells were determined using flow cytometry after incubation with 7-AAD (dead cell stain), CD4, CD8, CD45RA, CD62L, and CCR7 antibodies, and measuring the total viable cells/ml. Results: The numbers of viable CD4 and CD8 T-cells remained relatively stable in control cultures. Without fludarabine, the average viability at 72 hr of naive and memory T-cells were 92% and 77% for CD4 and 86% and 63% for CD 8 (Fig. 1A). Naive CD4 T-cells were more sensitive to fludarabine-induced death than memory CD4 cells. At 72 hr, the average viability of fludarabine-treated naive CD4 T-cells was 33% at 2 mcg/ml (8.2X the reduction observed in untreated cells) and 30% at 5 mcg/ml, while memory CD4 T-cells averaged 47% viability at 2 mcg/ml (2.3X the reduction observed in untreated cells) (Fig. 1B) and 38% at 5 mcg/ml. The average viability of naive CD8 T-cells at 72 hr was 27% at 2 mcg/ml and 20% at 5 mcg/ml, while memory CD8 T-cell viability was 22% at 2 mcg/ml and 17% at 5 mcg/ml. Analyses on central memory, effector memory, and Temra T-cells, and B-cell and dendritic cell subsets are ongoing. The 5 and 10 mcg/ml doses also yielded similar results in 3 initial subjects, suggesting that 2 mcg/ml or a lower dose of fludarabine is sufficient to achieve relative depletion of the naive T-cell subset. Conclusions: Future work will determine the minimal dose of fludarabine to achieve this effect, test the feasibility of using ex vivo nucleoside analog incubation to reduce alloreactivity in samples from patient/donor pairs, and determine the maximum tolerated dose of mDL in a phase 1 clinical trial with patients at high risk for relapse and infectious complications following allogeneic transplantation. Figure Figure

Development of a Nonhuman Primate Model for Analysis of the Adoptive Transfer of Antigen-Specific T Cell Clones.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 770-770
Author(s):  
Carolina Berger ◽  
Michael Jensen ◽  
Stanley R. Riddell
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Adoptive Transfer ◽  
Cell Transfer ◽  
Primate Model ◽  
T Cell Clones ◽  
Cell Clones

Abstract In principle, the adoptive transfer of T cell clones specific for antigens expressed by pathogens or malignant cells could be therapeutically effective and allow precise control of the specificity, function, and magnitude of T cell immunity. However, the infusion of large numbers of cultured T cells or T cell clones in clinical trials has frequently failed to eradicate tumors or provide long-term control of infection. This may be due in part to the acquisition of an effector phenotype by the T cells during in vitro culture, which reduces their ability to survive in vivo and establish an immune response of sufficient magnitude for sustained efficacy. Several approaches including the administration of cytokines such as IL15, or lymphodepletion prior to cell transfer might promote the establishment of T cell memory after T cell transfer. To facilitate the rational development of clinical trials of T cell therapy, we have employed a nonhuman primate model of adoptive T cell transfer in which culture conditions and cell doses identical to those in human studies are utilized, and designed strategies to permit rigorous analysis of the persistence, function, phenotype, and migration of transferred cells. CD8+ CTL specific for macaque CMV were detected using an overlapping peptide panel and cytokine flow cytometry, isolated as individual T cell clones by limiting dilution, and propagated to large numbers in vitro. The T cell clones were transduced to express an intracellular truncated CD19 (ΔCD19) surface marker to allow tracking and functional assessment of T cells in vivo, and enriched by immunomagnetic selection to high purity (>98%) prior to transfer. The persistence of transferred ΔCD19+ T cells in the blood and their migration to the bone marrow and lymph nodes was determined by flow cytometry after staining with anti CD19, CD8, and CD3 antibodies. The infusion of ΔCD19+CD8+ CTL (3 x 108/kg) was safe and the cells remained detectable in vivo for >5 months. ΔCD19+CD8+ T cells were easily detected in the blood 1 day after transfer at a level of 2.7% of CD8+ T cells and gradually declined over 56 days to a stable population of 0.15–0.2% of CD8+ T cells. At the time of transfer the ΔCD19+CD8+ T cells had an effector phenotype (CD62L− CD127−), but gradually converted to a CD62L+CD127+ memory phenotype in vivo. The infused T cells were found at high levels in lymph node and bone marrow at day 14 after transfer (1.4% and 2.5%, respectively) and the cells at these sites were predominantly CD62L+. The ΔCD19+CD62L+ T cells lacked direct lytic function and expressed low levels of granzyme B, consistent with memory T cells. Sorting of these cells from post-transfer PBMC showed that in vitro activation restored lytic activity. The transferred ΔCD19+CD62L+ T cells in post-infusion PBMC produced IFNγ and TNFα comparable to endogenous CMV-specific CD8+ CTL. These results demonstrate that a subset (5–10%) of transferred CD8+ CTL clones can persist long-term as functional memory T cells. The macaque CD8+ T cell clones are responsive to IL15 in vitro and a safe regimen for administering IL15 to macaques that boosts endogenous T cells has been identified. Studies are now in progress to determine if IL15 can enhance the efficiency with which effector and memory CD8+ T cell responses can be augmented after adoptive transfer of T cell clones.

Host-Reactive Donor CD8+ T Memory Stem Cells but Not Mature T Memory Cells Primed Against Leukemic Cells Mediate Potent Graft-Versus-Leukemia Effect.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 190-190
Author(s):  
Elizabeth O. Hexner ◽  
Dale Frank ◽  
Stephen G. Emerson ◽  
Yi Zhang
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
T Cell ◽  
Adoptive Transfer ◽  
Memory T Cells ◽  
Leukemic Cells ◽  
Leukemia Cells ◽  
Effector Memory ◽  
Memory Cells ◽  
Alloreactive T Cell

Abstract The potent ability of allogeneic hematopoietic stem cell transplantation (Allo-HSCT) and donor leukocyte infusion (DLI) to cure leukemia remains the most striking example of the ability of the immune system to recognize and destroy tumors. Unfortunately, both allo-HSCT and DLI are often complicated by graft-versus-host disease (GVHD). In addition, durable responses to conventional DLI for acute leukemias have been disappointing. A better understanding of the mechanisms of alloreactive T cell-mediated anti-leukemia activity will be important for separating the GVL effect from GVHD. Adoptive transfer of selected subsets of T cells specific for miHA- or leukemia associated antigens might offer the chance to maximize GVL while minimizing GVHD. Using mouse models of human GVHD directed against miHAs, we recently demonstrated that antigen-experienced CD44loCD62LhiCD8+ T cells contain T memory stem cells that have greater ability than naïve T cells and mature memory T cells to proliferate and generate alloreactive effector cells and all memory T cell subsets (Nature Medicine, 2005, 11:1299). Using the same mouse model, we have now found that although B6/SJL mice receiving donor CD44hiCD8+ T cells (mature memory cells) primed against B6 mouse-derived myeloid leukemia C1498 cells do not develop clinical GVHD, most will die from C1498 leukemia by day 45 following injection of C1498 cells. Adoptive transfer of CD44loCD8+ T cells primed against C1498 leukemic cells caused clinical GVHD, but the majority of recipients (75%) survived long term free of C1498 leukemia. Surprisingly, the GVL effect of donor CD44loCD8+ T cells primed against C1498 leukemia cells was significantly inhibited when C1498 leukemia cell-primed CD44hiCD8+ T cells and CD44loCD8+ T cells were co-injected into B6/SJL mice, with only 25% of the mice surviving without leukemia. These results suggest that while the GVL effect is clearly mediated by antigen experienced CD44loCD8+ T cells, CD44hiCD8+ T memory cells primed against tumor cells are not only functionally defective in eliminating leukemia cells but are also potent inhibitors of alloreactive T cell-mediated GVL activity. We found that host-reactive effector memory CD8+ T cells produced 10-fold higher IL-10 than unstimulated naïve T cells and T memory stem cells, while CD8+ T memory stem cells expressed upregulated IL-10 receptors. These findings suggest that the inhibitory effect of mature memory T cells on alloreactive T cell-mediated GVL effect may be associated with increased production of IL-10 by mature memory cells and/or enhanced susceptibility of T memory stem cells to IL-10 secreted by mature memory cells. In addition, host dendritic cell activation of donor CD8+ naïve T cells progressively induced the generation of memory stem cells (CD44loCD62LhiSca-1hi), central memory cells (CD44hiCD62Lhi) and effector memory cells (CD44hiCD62Llo). CD8+ T memory stem cells displayed a TCR V-beta repertoire similar to that of unstimulated naive T cells. In contrast, both central memory and effector memory T cells showed a skewed TCR V-beta repertoire. Thus, selective elimination of suppressive CD44hiCD8+ T cells may represent an approach to augmenting GVL activity while preserving a diverse TCR V-beta repertoire.

T Large Granular Lymphocyte (LGL) Leukemia Characterized by Expansion of Terminal CD3+/CD8+/CD62L−/CD45RA+ Effector Memory T Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4962-4962
Author(s):  
JianXiang Zou ◽  
Jeffrey S. Painter ◽  
Fanqi Bai ◽  
Thomas P. Loughran ◽  
P.K. Epling-Burnette
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Memory T Cells ◽  
Effector Memory ◽  
T Cell Proliferation ◽  
Normal Controls ◽  
Terminal Effector ◽  
Homeostatic Mechanisms ◽  
Lgl Leukemia

Abstract Background: Clonal proliferation by mature Large Granular Lymphocytes is associated with LGL leukemia. This expansion of CD3− NK cells or CD3+ T cells may be the result of chronic antigen stimulation by autoantigens or viral antigens. In association with T cell lymphoproliferation, approximately 45% of patients with LGL leukemia have severe neutropenia (absolute neutrophil count <0.5×109/L) and 20% of patients have transfusion-dependent anemia. Homeostatic mechanisms normally modulate the generation of naïve and memory T cell pools and regulate the T cell repertoire; however, the pathways elicited during T memory differentiation, maintenance and expansion are not fully characterized. The goal of this work was to characterize the homeostatic mechanisms that regulate LGL leukemia. Methods: Peripheral blood mononuclear cells were isolated from patients with LGL leukemia and normal controls. We performed multiplex TCR-Vβ (CDR3) PCR on cells from 16 LGL patients to identify clonal T cell proliferation. The percentage of CD3+ T cells that expressed each of the TCR-Vβ families was determined in 20 healthy donors to establish the mean and standard deviation (S.D.) of the control population. Naïve and memory CD4 and CD8 T cell sub-populations were segregated by expression of CD45RA and CD62L expression by flow cytometry and T cell proliferation was assessed by Brdu incorporation in CD4+ and CD8+ T cells. Results: The absolute number of CD4+ T cells was reduced in LGL patients compared to normal donors and T cell clones were characterized by a CD8+ phenotype. By flow cytometry, expansion of a single Vβ clonal population occurred in 8 of 16 patients (50%), two clones were present in 4 of 16 patients (25%), and three clones in 4 of 16 patients (25%). The immunophenotype of TCR Vβ+ clonal T cells was CD8 positive, CD57 positive, CD28 negative, CD25 negative, and NKG2D (NKG2-family) positive and CD244 (2B4) positive. Three patients examined expressed Killer-Immunoglobulin-like (KIR) receptors. Further phenotype analysis showed that there were fewer than normal CD4+ naïve (CD4+/CD45RA+/CD62L+) T cells (23%±16 vs. 41%± 15, P=0.04 by a t test) in LGL patients. CD4+ T cells from patients had reduced proliferation in response to antigen stimulation. The reduction in CD4+ naïve T cells was associated with increased percentages of CD4+/CD45RA−/CD62L+ central memory T cells (P<0.05). Reduced percentage of naive CD8+ T cells in detected in LGL leukemia patients. In addition, CD4+ central memory cells were also significantly reduced in patients. CD8+ T cells were primarily characterized by a CD45RA+/CD62L− terminal effector memory phenotype that was significantly increased compared to normal donors (mean 75% ± 13 in patients vs 30% ± 13 in normal controls, P<0.0001). In the presence of a skewed repertoire and terminal effector memory cell accumulation, antigen-induced proliferation of CD8+ T cells in LGL did not differ from normal controls (13% ± 11 in patients vs. 9% ± 3 in normal controls, P=0.3). Conclusions: These results suggest that leukemic LGL represent the accumulation of CD8+ terminal effector memory cells with the capacity for increased proliferation. Our findings suggest that normal homeostatic signals are impaired in LGL leukemia that limits the terminal CD8 differentiation phase of an immune response.

Autoproliferation Contributes to Clonal Expansion and Changes In Cellular Topography In T Cell Large Granular Lymphocyte (LGL) Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1373-1373
Author(s):  
JianXiang Zou ◽  
Jeffrey S Painter ◽  
Fanqi Bai ◽  
Lubomir Sokol ◽  
Thomas P. Loughran ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
Cd8 T Cell ◽  
Effector Memory ◽  
Ki67 Expression ◽  
Terminal Effector ◽  
Lgl Leukemia

Abstract Abstract 1373 Introduction: LGL leukemia is associated with cytopenias and expansion of clonally-derived mature cytotoxic CD8+ lymphocytes. The etiology of LGL leukemia is currently unknown, however, T cell activation, loss of lymph node homing receptor L-selectin (CD62L), and increased accumulation of T cells in the bone marrow may lead to suppressed blood cell production. The broad resistance to Fas (CD95) apoptotic signals has lead to the hypothesis that amplification of clonal cells occurs through apoptosis resistance. However, the proliferative history has not been carefully studied. To define possible mechanism of LGL leukemia expansion, T cell phenotype, proliferative history, and functional-related surface marker expression were analyzed. Methods: Peripheral blood mononuclear cells (PBMCs) were obtained from 16 LGL leukemia patients that met diagnostic criteria based on the presence of clonal aβ T cells and >300 cells/ml CD3+/CD57+ T cells in the peripheral blood. Samples were obtained from 10 age-matched healthy individuals from the Southwest Florida Blood Services for comparisons. Multi-analyte flow cytometry was conducted for expression of CD3, CD4/8, CD45RA, CD62L, CD27, CD28, CD25, CD127, IL15Ra, IL21a, CCR7 (all antibodies from BD Biosciences). The proliferative index was determined by Ki67 expression in fixed and permeabilized cells (BD Biosciences) and the proliferative history in vivo was assessed by T-cell-receptor excision circle (TREC) measurement using real-time quantitative PCR (qRT-PCR) in sorted CD4+ and CD8+ T cells. TRECs are episomal fragments generated during TCR gene rearrangements that fail to transfer to daughter cells and thus diminish with each population doubling that reflects the in vivo proliferative history. Results: Compared to healthy controls, significantly fewer CD8+ naïve cells (CD45RA+/CD62L+, 8.4 ± 10.8 vs 24.48 ± 11.99, p=0.003) and higher CD8+ terminal effector memory (TEM) T cells (CD45RA+/CD62L-, 67.74 ± 28.75 vs 39.33 ± 11.32, p=0.007) were observed in the peripheral blood. In contrast, the percentage of CD4+ naïve and memory cells (naïve, central memory, effector memory, and terminal effector memory based on CD45RA and CD62L expression) was similar in patients as compared to controls. The expression of CD27 (31.32 ± 34.64 vs 71.73 ± 20.63, p=0.003) and CD28 (31.38 ± 31.91 vs 70.02 ± 22.93, p=0.002) were lower in CD8+ T cell from patients with LGL leukemia and this reduction predominated within the TEM population (17.63±24.5 vs 70.98±22.5 for CD27, p<0.0001 and 13±20.5 vs 69.43± 21.59 for CD28, p<0.0001). Loss of these markers is consistent with prior antigen activation. There was no difference in CD25 (IL2Ra, p=0.2) expression on CD4+ or CD8+ T cells, but CD127 (IL7Ra, p=0.001), IL15Ra, and IL21Ra (p=0.15) were overexpressed in TEM CD8+ T cell in patients vs controls. All of these cytokine receptors belong to the IL2Rβg-common cytokine receptor superfamily that mediates homeostatic proliferation. In CD8+ T cells in patients, the IL-21Ra was also overexpressed in naïve, central and effector memory T cells. The topography of the expanded CD8+ T cell population was therefore consistent with overexpression of activation markers and proliferation-associated cytokine receptors. Therefore, we next analyzed Ki67 expression and TREC DNA copy number to quantify actively dividing cells and determine the proliferative history, respectively. We found that LGL leukemia patients have more actively dividing CD8+ TEM T cells compared to controls (3.2 ± 3.12 in patients vs 0.44 ± 0.44 in controls, p=0.001). Moreover, the TREC copy number in CD8+ T cells was statistically higher in healthy individuals after adjusting for age (177.54 ± 232 in patients vs 1015 ± 951 in controls, p=0.019). These results show that CD8+ cells in the peripheral compartment have undergone more population doublings in vivo compared to healthy donors. In contrast, the TREC copies in CD4+ T-cells were similar between LGL patients and controls (534.4 ± 644 in patients vs 348.78 ± 248.16 in controls, p>0.05) demonstrating selective cellular proliferation within the CD8 compartment. Conclusions: CD8+ T- cells are undergoing robust cellular activation, contraction in repertoire diversity, and enhanced endogenous proliferation in patients with LGL leukemia. Collectively, these results suggest that clonal expansion is at least partially mediated through autoproliferation in T-LGL leukemia. Disclosures: No relevant conflicts of interest to declare.

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