Acute Gvhd Induce the Critical Depletion of Naïve Pool in Regulatory T Cells: Implication for Linked Pathogenesis into Chronic Gvhd

Abstract CD4+Foxp3+ regulatory T cells (Treg) play a central role in establishing immune tolerance after allogeneic hematopoietic stem cell transplantation (HSCT). We previously reported that the long-term severe lymphopenia could result in the collapse of Treg homeostasis leading to the onset of chronic GVHD (Matsuoka et al. JCI 2010). We recently found that, not only in the chronic phase but also in the acute phase, the homeostasis of Treg is more susceptible to the post-transplant environment as compared to other lymphocyte subsets (Yoshioka et al. ASH 2014). However, the impact of acute GVHD on Treg homeostasis and the pathogenesis of following chronic GVHD has not been well studied. In this study, we examined Treg reconstitution in the early phase after transplant in patients with or without acute GVHD. For the purpose, we obtained peripheral blood samples at 2, 4, 8 and 12 weeks after transplant from 52 patients who received allogeneic HSCT, and then analyzed CD4+CD25med-highCD127lowFoxp3+ Treg comparing with CD4+CD25neg-lowCD127highFoxp3- conventional T cell (Tcon) and CD8+ T cells. CD4 T cell subsets are further divided into subpopulations by the expression of CD45RA and CD31. The expressions of Helios, Ki-67 and Bcl-2 on these subsets were also examined. After transplant, total lymphocyte counts in examined patients were significantly lower than the counts before the start of conditioning (median lymphocytes 95/ul at 2 weeks and 302/ul at 4 weeks vs 600/ul before conditioning, P<0.01 and P<0.01, respectively). As we reported before, Treg showed the active proliferation without diminishing Bcl-2 levels in the severe lymphopenia, resulted in the increased %Treg of CD4 T cells at 4 weeks after transplant (%Treg of CD4 T cells; 12.2% at 4 weeks, 4.6% in healthy controls, P<0.005). 18 patients who developed acute GVHD were studied Treg homeostasis before and after the onset of GVHD more in detail. Before the onset of acute GVHD, % Ki-67+ cells in Treg and Tcon were in the equivalent levels in these patients. After the onset of acute GVHD, % Ki-67+ cells in Treg was dramatically increased from 19.1% to 61.2% (median) and this was significantly higher than % Ki-67+ cells in Tcon after acute GVHD (P<0.05). %Treg of total CD4 T cells were significantly increased after GVHD (% Treg; Median 7.2% vs 12.2%, P<0.004). Expanded Treg after acute GVHD showed a predominant Helios+CD45RA-CD31- effector/memory phenotype with the lower level of Bcl-2 expression as compared to CD45RA+ naïve Treg. As a consequence, naïve Treg pool including CD45RA+CD31+ recent thymic emigrant Treg (RTE-Treg) were critically decreased during acute GVHD (%CD45RA+ cells; 12.7% into 6.5%, P<0.004: CD45RA+CD31+ cells; 3.6% into 2.1%, P<0.003). In contrast, Tcon still retained a relatively higher level of naïve pool (%CD45RA+ cells; 20.5%, % CD45RA+CD31+ cells; 10.9%) after acute GVHD. These data indicated that Treg proliferation was rapidly promoted in face with the inflammatory condition during acute GVHD and this appears to contribute the amelioration of developing GVHD. However, the prompt reaction resulted in the depletion of naïve Treg pool which is important to maintain stable Treg homeostasis in the long period. In conclusion, our findings suggest that acute GVHD drives aggressive Treg proliferation resulting in the increased percentage of this subset but this also induce the severe alteration of Treg homeostasis by depleting naïve Treg, which may provide the linked pathogenesis of the subsequent onset of chronic GVHD. The careful monitoring of Treg from the point of view might provide important information to promote immune tolerance. Disclosures No relevant conflicts of interest to declare.

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Early Homeostatic Expansion of Regulatory T Cells with the Predominant Effector/Memory Phenotype May Stabilize Immune Recovery in the First Month after HSCT

Blood ◽

10.1182/blood.v124.21.2501.2501 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2501-2501

Author(s):

Takanori Yoshioka ◽

Yusuke Meguri ◽

Takeru Asano ◽

Taro Masunari ◽

Kumiko Kagawa ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Acute Phase ◽

Cd4 T Cells ◽

Chronic Phase ◽

T Cell Subsets ◽

Effector Memory ◽

Ki 67 ◽

Memory Phenotype ◽

Post Transplant

Abstract CD4+Foxp3+ regulatory T cells (Treg) play a central role in establishing immune tolerance after allogeneic hematopoietic stem cell transplantation (HSCT). We previously reported that the long-term severe lymphopenia could result in the collapse of Treg homeostasis leading to the onset of chronic GVHD (Matsuoka et al. JCI 2010). However, Treg homeostasis in the very early phase after HSCT has not been well studied. To address this issue, we here examined the early lymphocytes reconstitution in total 34 patients who received HSCT. Peripheral blood samples were obtained at 2, 4, 8 and 12 weeks after transplant and analyzed the reconstitution of CD4+CD25med-highCD127lowFoxp3+ Treg comparing with CD4+CD25neg-lowCD127highFoxp3- conventional T cell (Tcon) and CD8+ T cells. CD4 T cell subsets are further divided into subpopulations by the expression of CD45RA and CD31. The expressions of Helios, Ki-67, Bcl-2 and C-C chemokine receptor type 4 (CCR4) on these subsets were also examined. These patients were transplanted the grafts from various stem cell sources (7 HLA-matched PBSCT, 12 HLA-matched BMT, 6 HLA-mismatched CBT and 9 HLA-haploidentical PBSCT) and this enables us the opportunity to comparatively evaluate the early lymphocyte reconstitution among the different types of HSCT. After transplant, total lymphocyte counts were significantly lower than the counts before the start of conditioning (median lymphocytes 113/ul at 2 weeks and 223/ul at 4 weeks vs 550/ul before conditioning, P<0.01 and P<0.01, respectively). In the severely lymphopenic condition in the first month after HSCT, all T cell subsets were undergoing aggressive proliferation in this acute phase as compared to proliferation in the chronic phase, however, Treg proliferation was significantly higher than in Tcon at 4 weeks (%Ki-67+ cells; median 56.4%, 23.4%, respectively, P<0.02). %Treg of total CD4 T cells elevated and peaked at 4 weeks post-transplant. At this timepoint, %Treg of CD4 T cells showed the clear inverse correlation with %CD45RA+ of Treg (r2=0.40), suggesting the expansion of Treg in this phase appears to be a result from severe lymphopenia-driven proliferation which involves conversion from naive into memory phenotype. Elevation of %Treg was most evident in the patients who received HLA-haploidentical graft after ATG-containing conditioning (median 8.41% in haplo-HSCT, 5.25% in other groups, P<0.05), again indicating the lymphopenia is critical factor to drive Treg proliferatrion. Expanded Treg showed a predominant Helios+CD45RA-CD31- effector/memory phenotype with the lower level of Bcl-2 expression as compared to CD45RA+ naïve Treg. The elevation of Treg did not sustain and %Treg of CD4 T cells got back to the baseline level by 8 weeks. During the first 3 months after HSCT, CD45RA- Treg exhibited high level of CCR4 and the recovery of this subset was critically delayed in Adult T-cell Leukemia (ATL) patients treated with anti-CCR4 antibody in the peri-transplant period, resulting in the development of acute graft-versus-host diseases. In conclusion, our findings suggest that, not only in the chronic phase but also in the acute phase, the homeostasis of Treg is more susceptible to the post-transplant environment as compared to other lymphocyte subsets. Post-transplant lymphopenia drives aggressive Treg proliferation resulting in the increased percentage of this subset in the very acute phase which may contribute to stabilize the immune recovery. The careful monitoring of Treg from the point of view might provide important information to promote immune tolerance. Disclosures No relevant conflicts of interest to declare.

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Elevated frequencies of highly activated CD4+ T cells in HIV+ patients developing immune reconstitution inflammatory syndrome

Blood ◽

10.1182/blood-2010-05-285080 ◽

2010 ◽

Vol 116 (19) ◽

pp. 3818-3827 ◽

Cited By ~ 114

Author(s):

Lis R. V. Antonelli ◽

Yolanda Mahnke ◽

Jessica N. Hodge ◽

Brian O. Porter ◽

Daniel L. Barber ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Regulatory T Cells ◽

Immune Reconstitution Inflammatory Syndrome ◽

Cd4 T Cells ◽

Immune Reconstitution ◽

Interferon Γ ◽

Effector Memory ◽

Hiv Patients ◽

Inflammatory Syndrome

Abstract Immune reconstitution inflammatory syndrome (IRIS) is a considerable problem in the treatment of HIV-infected patients. To identify immunologic correlates of IRIS, we characterized T-cell phenotypic markers and serum cytokine levels in HIV patients with a range of different AIDS-defining illnesses, before and at regular time points after initiation of antiretroviral therapy. Patients developing IRIS episodes displayed higher frequencies of effector memory, PD-1+, HLA-DR+, and Ki67+ CD4+ T cells than patients without IRIS. Moreover, PD-1+ CD4+ T cells in IRIS patients expressed increased levels of LAG-3, CTLA-4, and ICOS and had a Th1/Th17 skewed cytokine profile upon polyclonal stimulation. Elevated PD-1 and Ki67 expression was also seen in regulatory T cells of IRIS patients. Furthermore, IRIS patients displayed higher serum interferon-γ, compared with non-IRIS patients, near the time of their IRIS events and higher serum interleukin-7 levels, suggesting that the T-cell populations are also exposed to augmented homeostatic signals. In conclusion, our findings indicate that IRIS appears to be a predominantly CD4-mediated phenomenon with reconstituting effector and regulatory T cells showing evidence of increased activation from antigenic exposure. These studies are registered online at http://clinicaltrials.gov as NCT00557570 and NCT00286767.

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

Blood ◽

10.1182/blood.v106.11.1071.1071 ◽

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

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Phenotypic and Functional Analysis of T-Cells Mobilized in HLA-Matched Sibling Donors Following Treatment with the Chemokine Antagonist AMD3100.

Blood ◽

10.1182/blood.v108.11.3001.3001 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3001-3001 ◽

Cited By ~ 1

Author(s):

Michael Rettig ◽

Steven M. Devine ◽

Julie Ritchey ◽

John F. DiPersio

Keyword(s):

T Cells ◽

T Cell ◽

Peripheral Blood ◽

Acute Gvhd ◽

T Cell Subsets ◽

Effector Memory ◽

Phenotypic Properties ◽

Functional Changes ◽

Cd8 Cells ◽

Matched Sibling

Abstract We are currently evaluating a novel method for the procurement of peripheral blood stem cells from HLA matched sibling donors using a direct antagonist of the CXCR4/SDF-1 interaction called AMD3100 (A). Donors receive a single subcutaneous injection of A and then undergo a 20 liter leukapheresis (LP) four hours later. The LP product is then cryopreserved and subsequently transplanted following ablative conditioning. To date, we have performed 15 transplants with allografts collected following A alone. In comparison to allografts collected following five days of G-CSF, A mobilized allografts contain approximately 50% less CD34+ cells but 2–3 times more CD3+ cells. Nevertheless, the kinetics of neutrophil and platelet engraftment have been virtually identical to that observed following G-mobilized allografts and grades 2–4 acute GVHD has been observed in only 20% of recipients. We sought to analyze the functional and phenotypic properties of T cells collected following A alone to understand the relatively low rates of acute GVHD despite the transplantation of higher T-cell doses. In 3 donors, extensive T cell phenotyping was performed on donor peripheral blood prior to A, 6 hours following A, and also on the LP product collected after A. Specifically, we were seeking to determine whether any alteration in CD4+ or CD8+ subsets had occurred. We analyzed T-cell subsets using well described markers for central memory, effector memory, naïve, and effector memory RA phenotypes. We also assessed expression of CD62L, CD127, CCR7, and SLAM family members (CD48, CD150, and CD244) on both CD4+ and CD8+ cells. The activation status on CD4 and CD8 cells was assessed using markers for CD25, CD30, and CD69. Finally we assessed for quantitative changes in the mobilization of regulatory T cells by assaying the proportion of CD4+CD25+FoxP3+ cells mobilized following A. In none of these analyses could we detect any significant alteration in the relative ratios of CD4 or CD8 subsets mobilized by A. Finally, the functional capacity of purified CD3+ cells collected following A was assessed using a NOD/SCID xenogeneic GVHD model we have recently developed. In that model, survival of mice transplanted with A mobilized T-cells was similar to that observed with untreated T cells, suggesting that A mobilized T cells retain their GVHD-inducing capacity. In summary, these preliminary data suggest that AMD3100 induces a “pan-mobilization” of T cell subsets without any apparent skewing toward a particular subset. These studies are in contrast to others suggesting subtle phenotypic and functional changes in donor T cells after mobilization with G-CSF. Further studies evaluating A mobilized allografts are ongoing.

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Proportions of T-Cell CD8+ Subsets Lacking CD28 Expression at Day 30 after Myeloablative Allogeneic Stem Cell Transplantation Are Predictive for Relapse and Chronic GVHD.

Blood ◽

10.1182/blood.v110.11.2979.2979 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2979-2979

Author(s):

Ibrahim Yakoub-Agha ◽

Pasquine Saule ◽

Leonardo Magro ◽

Pascale Cracco ◽

Valerie Coiteux ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Chronic Gvhd ◽

Immunosuppressive Treatment ◽

Cd8 T Cell ◽

T Cell Subsets ◽

Mixed Chimerism ◽

Effector Memory ◽

Myeloablative Conditioning ◽

Risk Of Relapse

Abstract The curative potential of allo-SCT for malignancies derives from the progressive reconstitution of the immune system and the development of effective anti-tumor immunity, but GVHD and disease relapse remain considerable obstacles to improvement in overall outcomes. Because in recipients target antigens are persisting, donor-derived T-cell responses may be expected to lead to the accumulation of a sizable proportion of differentiated T-cells, as happens following infection with persisting pathogens. A few cross-sectional studies have pointed to the preponderance of certain memory T-cell subsets associated with chronic GVHD (cGVHD), but the subset identified differed between studies. Inasmuch as qualitative T-cell recovery takes months to years to complete and there is substantial variability in time to development of GVHD or relapse, serial analysis might be more suitable to unveil early changes in T-cell subset composition attributable to transplantation-related events. From October 2003 on, 55 pts who underwent an allo-SCT after myeloablative conditioning were monitored prospectively in terms of clinical post-graft complications, including graft rejection, infections, GVHD and relapse. Blood samples were obtained on days 30±2, 60±3, 90±5, 180±10 and 365±15 post-transplant. Naive (CD45RA+CCR7+), central memory (TCM, CD45RAnegCCR7+), effector memory (TEM, CD45RAnegCCR7neg), and terminally differentiated effector (TTD, CD45RA+CCR7neg) were enumerated within the CD4+ and CD8+ pools, and the percentage of cells coexpressing CD28 was calculated within each eight subsets. The degree of donor-derived T-cell chimerism was assessed by real time PCR (sensitivity ≤ 1%). Median follow-up was 733 d (404–1251). Dynamics of CD4+ and CD8+ naive, TCM, TEM, and TTD were similar between the pts who developed cGVHD (n=15) and those who did not and between pts who relapsed and those who did not. However, costaining to detect CD28 demonstrated contrasting differences between cGVHD and relapse. At day 30, pts who subsequently relapsed (n=17) had elevated percentages of cells keeping CD28 expression within CD8+ T-cell subsets (TCM, p=.001; TCM, p=.021; and TTD, p=.007). Conversely, pts who subsequently developed cGVHD (n=15; only one relapsed) had diminished percentages of CD28+ cells within the two CD8+CCR7+ subsets at day 30 (p=.002 and p=.034, respectively). Loss of CD28 expression is known to be a hallmark of CMV infection but multivariate analysis ruled out, however, a confounding effect of CMV. Adjusted hazard ratios were 0.10 (95% CI, 0.01-0.76; p=.026) and 5.56 (95% CI, 1.16-25.00; p=.032) with CD28neg cells 16.7% of all CD8+ TCM at day 30 for relapse and cGVHD, respectively. Furthermore, pts with relapse had more often mixed chimerism at day 30 while those with cGVHD had more often full-donor chimerism (p=.042 and p=.023, respectively). CONCLUSION: This prospective study is the first to associate an early contrasting change in CD8+CD28neg T-cells with the risk of relapse and cGVHD after a myeloablative conditioning. Determination at day 30 of the proportions of CD8+ T-cell subsets expressing CD28 and of the level of T-cell chimerism could assist in predicting risk of relapse and cGVHD and help build an algorithm for the management of immunosuppressive treatment.

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CD28-Directed T Cell Costimulation Blockade with Abatacept to Prevent GvHD During High-Risk Unrelated HSCT: A First-in-Disease Feasibility Trial,

Blood ◽

10.1182/blood.v118.21.4078.4078 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4078-4078

Author(s):

Leslie S. Kean ◽

Amelia Langston ◽

Muna Qayad ◽

H. Jean Khoury ◽

Divya Tiwari ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Acute Gvhd ◽

Chronic Gvhd ◽

Hematologic Malignancies ◽

Feasibility Trial ◽

Effector Memory ◽

Unrelated Donor ◽

Gvhd Prophylaxis ◽

The Mean

Abstract Abstract 4078 Background: Acute GvHD remains the major cause of complications and death following unrelated-donor HSCT. In a non-human primate model, we have previously shown that in vivo costimulatory blockade of donor T-cells could provide effective protection against GVHD. To begin to explore its clinical utility, we are conducting a trial (Clinical Trials.Org # NCT01012492) to determine the feasibility of combining abatacept (CTLA4-Ig) with cyclosporine and methotrexate as acute GVHD prophylaxis for patients undergoing unrelated marrow and peripheral blood stem cell transplants for hematologic malignancies. Methods: Patients older than 12 with advanced hematologic malignancies, conditioned with either TBI/Cytoxan, Busulfan/Cytoxan or Fludarabine/Melphalan are eligible. Abatacept is administered IV on days −1, +5, +14, and +28 at 10 mg/kg in addition to standard GvHD prophylaxis consisting of cyclosporine (day −2 to day 100), and methotrexate (15 mg/m2 on day +1 and 10 mg/m2 on days +3, 6 and 11). Patients are then followed for clinical outcomes and immunologic reconstitution through day +365. Results: 9 patients (planned enrollment = 11 patients) have thus far been enrolled on the study of which 5 are evaluable for engraftment, toxicity and acute GvHD. The other four patients consist of 2 who are currently receiving abatacept, 1 who was discovered to have an ongoing viral infection at the start of the first abatacept infusion so was removed from the treatment regimen, and 1 who is awaiting transplant. The median age for the 5 evaluable patients is 47 years (17–74 years). 3 patients had AML and 2 had ALL. Patients were conditioned with Bu/Cy (n=1), TBI/Cy (n=2) and Flu/Melphalan (n=2). 4 donor-recipient pairs were allele matched at 9 of 10 loci (A, B, C, DRB1 and DQB1), while 1 was fully matched. Four of the 5 patients are currently alive and in remission and 1 relapsed at day +98 (and died on day +121 with refractory AML). The four other patients are surviving without relapse with a follow-up of 155–313 days. All 5 patients received the 4 scheduled abatacept doses. No infusional side effects were noted. All patients achieved neutrophil engraftment (median day +20 (11–47). 4 of 5 patients have achieved platelet engraftment (median day +27 (14–35). Donor engraftment (100% CD33 and 99–100% CD3 at Day +30) occurred in all cases. All patients have demonstrated rapid lymphocyte engraftment, with the mean ALC reconstituting to >500 cells/μL by day +21 post-transplant. At day +100, the mean CD3+ count was 673 +/− 251 cells/μL. Both CD8+ and CD4+ T cells reconstituted by day 100, with the mean CD8+ count = 384 +/− 148 cells/μL and the mean CD4+ count = 229 +/− 119 cells/μL. T cell reconstitution was accompanied by a shift away from naïve (Tn, CCR7+/CD45RA+) toward a CCR7-/CD45RA- effector memory (Tem)-predominant phenotype. Thus, the average proportion of CD4+ Tem cells in the recipient increased from 22 +/− 6% pre-transplant to 46 +/− 7% at day +100 with a concomitant loss of CD4+ Tn cells. Likewise, the proportion of CD8+ Tem also significantly increased, from an average of 15 +/− 4% pre-transplant to 32 +/− 7% at day +100, also with a reciprocal decrease in CD8+ Tn cells. One patient developed steroid responsive grade 3 acute GVHD involving the skin and the liver, followed by steroid responsive liver chronic GvHD. This patient is currently weaning corticosteroids. Another patient developed steroid responsive late-onset (day +217) acute GVHD (liver and GI) during cyclosporine weaning, which was also steroid responsive, and is also currently weaning corticosteroids. No other systemic acute or chronic GvHD has occurred. No unexpected complications or life-threatening infections were observed. 3 patients have experienced 5 episodes of CMV reactivation, all responsive to antiviral therapy. One patient developed polyclonal EBV-related PTLD (plasmacytic hyperplasia) in the absence of EBV viremia, which regressed without intervention. No other EBV-related disease has occurred. Conclusions: These preliminary data suggest that abatacept can be safely added to cyclosporine and methotrexate for GVHD prophylaxis in recipients of hematopoietic grafts from unrelated donors, with encouraging rates of acute GVHD. As such, they support the conduct of a larger, randomized phase 2 study. Disclosures: Off Label Use: Abatacept: It is an immunosuppressive agent that targets the CD28/B7 T cell costimulation pathway. It is approved for use in Rheumatoid arthritis.

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NaïVe T Cell Depletion of PBSC Grafts Results in Very Low Rates of Chronic Gvhd and High Survival

Blood ◽

10.1182/blood.v128.22.668.668 ◽

2016 ◽

Vol 128 (22) ◽

pp. 668-668

Author(s):

Marie Bleakley ◽

Ted A. Gooley ◽

Barbara Hilzinger ◽

Stanley R Riddell ◽

Warren D Shlomchik

Keyword(s):

T Cells ◽

T Cell ◽

Acute Leukemia ◽

Chronic Gvhd ◽

Hematopoietic Cell ◽

T Cell Subsets ◽

Cell Depletion ◽

Effector Memory ◽

Phase Ii Trials ◽

T Cell Depletion

Abstract Background Graft-versus-host disease (GVHD) frequently causes morbidity and mortality after allogeneic hematopoietic cell transplantation (HCT) as a result of organ damage and infections. In HLA-identical HCT, GVHD results from recognition by donor T cells of minor histocompatibility (H) antigens on recipient tissues. Complete T cell depletion (TCD) of donor hematopoietic cell products is more effective than pharmacologic immunosuppression for preventing GVHD, but is complicated by delayed immune reconstitution and consequent life-threatening infections.Approaches to HCT which preferentially deplete the T cells that primarily cause GVHD and preserve pathogen-specific T cells may improve HCT outcomes. Mature CD3+ CD8+ and CD3+ CD4+ T cells can be classified into CD45RA+ CD62L+ naïve (TN) and CD45RO+ memory (TM) subsets, the latter of which includes effector memory (TEM) and central memory (TCM) cells. Murine studies in which allogeneic TCD bone marrow (BM) is transplanted with purified T cells from individual T cell subsets to irradiated minor H antigen disparate recipients have demonstrated that the most severe GVHD results from transplanting T cells of the TN subset. Purified TCM causes mild GVHD and TEM do not cause detectable GVHD and can transfer immunity to pathogens.In vitro studies have similarly demonstrated that human donor CD8+ T cells specific for recipient minor H antigens are found predominantly within the TN cell subset, suggesting selective TN cell depletion may alter the GVHD incidence and/or severity in human HCT. Methods and results We developed an effective process for engineering human peripheral blood stem cell (PBSC) grafts that depletes CD45RA+ TN cells and retains CD34+ stem cells and functional CD45RO+ TM cells specific for a broad range of opportunistic pathogens (Bleakley BBMT 2014). We are conducting clinical trials to evaluate the selective depletion of TN cells from HLA-matched allogeneic PBSC grafts for the prevention of GVHD in patients with acute leukemia, the first of which has been published (Bleakley JCI 2015, N=35). Seventy patients have now been treated on three consecutive phase II trials. The median age was 34 years (1-56 years), 56% of patients had a diagnosis of ALL, 46% had previously relapsed or had detectable disease (MRD or relapse) at the time of HCT, and 23% had unrelated donor (URD) grafts. Intensive myeloablative, TBI-containing (13.2Gy) conditioning was used for 63 patients, whilst 7 patients received a medium intensity 'midi' preparative regimen, including 4Gy of TBI. The TN-depletion procedure was successfully performed on URD PBSC products shipped overnight from donor centers throughout the US, as well as on MRD PBSC collected at our centers. Reliable engraftment with high-level donor chimerism was observed in recipients of 'midi' as well as intensive myeloablative conditioning. The 2-year estimates of overall survival, disease-free survival, survival free of relapse and chronic GVHD (CRFS) and survival free of relapse, grade II-IV acute GVHD, and chronic GVHD (GRFS) are 79%, 73%, 69% and 63% respectively. Median follow-up among survivors is 26 months. The frequency and severity of chronic GVHD is remarkably low (5%) compared to historical rates of 40-60% chronic GVHD in HLA-matched PBSC transplantation with conventional calcineurin inhibitor-based immunosuppression. Relapse and non-relapse mortality (NRM) are acceptably low at 19% and 8%, respectively. No NRM occurred in patients <40 years. Updated results will be presented. Conclusions The outcomes of recipients of TN-depleted PBSC grafts compare very favorably to published results of HCT for patients with acute leukemia. For example, the 69% incidence of CRFS at 2 years in TN-depleted recipients compares with reported 2-year GRFS rates of 37% and 17% in recipients of allogeneic PBSC from HLA-matched related donors with or without ATG (Kroger et al. NEJM 2016). Our results suggest that TN-depletion of PBSC grafts may reduce the risk of chronic GVHD without negatively impacting other important HCT outcomes. Disclosures Riddell: Juno Therapeutics: Equity Ownership, Patents & Royalties, Research Funding; Cell Medica: Consultancy, Honoraria; Adaptive Biotechnologies: Consultancy, Honoraria.

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Glycolytic metabolism of pathogenic T cells enables early detection of GvHD by 13C-MRI

10.1101/2020.03.16.984609 ◽

2020 ◽

Author(s):

Julian C. Assmann ◽

Don E. Farthing ◽

Keita Saito ◽

Natella Maglakelidze ◽

Brittany Oliver ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Mouse Model ◽

Chronic Gvhd ◽

T Cell Subsets ◽

Effector Memory ◽

Non Invasive ◽

Glycolytic Activity ◽

Target Organs ◽

Imaging Approach

AbstractGraft-versus-host disease (GvHD) is a prominent barrier to allogeneic hematopoietic stem cell transplantation (HSCT). Definitive diagnosis of GvHD is invasive and biopsies of involved tissues pose a high risk of bleeding and infection. Our previous studies in a chronic GvHD mouse model demonstrated that alloreactive CD4+ T cells are distributed to target organs ahead of overt symptoms, meanwhile CD4+ T cell activation is tied to increased glycolysis. Thus, we hypothesized that metabolic imaging of glycolysis would allow non-invasive detection of insipient GvHD in target organs infiltrated by glycolytic effector memory CD4+ T cells. We metabolically characterized CD4+ T cell subsets on day 14 post-transplant before the onset of chronic GvHD in a pre-clinical mouse model and performed 13C hyperpolarized magnetic resonance imaging (MRI) to quantify glycolytic activity in the liver of mice over the course of the disease. Intracellular metabolic screening and ex vivo metabolic profiling of CD4+ T cell subsets at day 14 confirmed that activated CD4+ T cells were highly glycolytic. Concurrently, hyperpolarized 13C-pyruvate MRI of the liver showed high conversion of pyruvate to lactate, indicative of increased glycolytic activity, that distinguished allogeneic from syngeneic HSCT recipients prior to the development of overt chronic GvHD. Furthermore, single cell sequencing of T cells in patients undergoing allogeneic HSCT indicated that similar metabolic changes may play a role in acute GvHD, providing a rationale for testing this imaging approach in the clinical post-HSCT setting. Our imaging approach is amenable to clinical translation and may allow early, non-invasive diagnosis of GvHD.

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Interplay between IL-10, IFN-γ, IL-17A and PD-1 Expressing EBNA1-Specific CD4+ and CD8+ T Cell Responses in the Etiologic Pathway to Endemic Burkitt Lymphoma

Cancers ◽

10.3390/cancers13215375 ◽

2021 ◽

Vol 13 (21) ◽

pp. 5375

Author(s):

Catherine S. Forconi ◽

David H. Mulama ◽

Priya Saikumar Lakshmi ◽

Joslyn Foley ◽

Juliana A. Otieno ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Burkitt Lymphoma ◽

Cd4 T Cells ◽

Cd8 T Cell ◽

T Cell Response ◽

T Cell Subsets ◽

Effector Memory ◽

Healthy Children ◽

Ifn Γ

Children diagnosed with endemic Burkitt lymphoma (eBL) are deficient in interferon-γ (IFN-γ) responses to Epstein–Barr Nuclear Antigen1 (EBNA1), the viral protein that defines the latency I pattern in this B cell tumor. However, the contributions of immune-regulatory cytokines and phenotypes of the EBNA1-specific T cells have not been characterized for eBL. Using a bespoke flow cytometry assay we measured intracellular IFN-γ, IL-10, IL-17A expression and phenotyped CD4+ and CD8+ T cell effector memory subsets specific to EBNA1 for eBL patients compared to two groups of healthy children with divergent malaria exposures. In response to EBNA1 and a malaria antigen (PfSEA-1A), the three study groups exhibited strikingly different cytokine expression and T cell memory profiles. EBNA1-specific IFN-γ-producing CD4+ T cell response rates were lowest in eBL (40%) compared to children with high malaria (84%) and low malaria (66%) exposures (p < 0.0001 and p = 0.0004, respectively). However, eBL patients did not differ in CD8+ T cell response rates or the magnitude of IFN-γ expression. In contrast, eBL children were more likely to have EBNA1-specific CD4+ T cells expressing IL-10, and less likely to have polyfunctional IFN-γ+IL-10+ CD4+ T cells (p = 0.02). They were also more likely to have IFN-γ+IL-17A+, IFN-γ+ and IL-17A+ CD8+ T cell subsets compared to healthy children. Cytokine-producing T cell subsets were predominantly CD45RA+CCR7+ TNAIVE-LIKE cells, yet PD-1, a marker of persistent activation/exhaustion, was more highly expressed by the central memory (TCM) and effector memory (TEM) T cell subsets. In summary, our study suggests that IL-10 mediated immune regulation and depletion of IFN-γ+ EBNA1-specific CD4+ T cells are complementary mechanisms that contribute to impaired T cell cytotoxicity in eBL pathogenesis.

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IL-2 Ameliorates Chronic GVHD by Inducing Antigen-Specific Regulatory T Cells.

Blood ◽

10.1182/blood.v104.11.2654.2654 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2654-2654

Author(s):

Jens G. Lohr ◽

Birgit Knoechel ◽

Estelle C. Kahn ◽

Abul K. Abbas

Keyword(s):

T Cells ◽

T Cell ◽

Regulatory T Cells ◽

Cd4 T Cells ◽

Chronic Gvhd ◽

The Self ◽

T Cell Homeostasis ◽

Cell Homeostasis ◽

Peripheral Tissues ◽

Self Antigen

Abstract We have developed a mouse model in which a GvHD-like syndrome develops in response to a defined soluble self-antigen. This phenotype is caused after transfer of CD4+ T cells that have a single specificity and are reactive to the self-antigen into a lymphopenic host that expresses the cognate antigen. By using a clonotypic antibody we are able to identify these cells and can therefore follow their migration, kinetics and functional characteristics. At least two distinct phases can be identified by clinical picture and correlated with accumulation of T cells - an early phase, resembling acute GvHD, leading to wasting and death coinciding with rapid accumulation of T cells, and a late phase in which a stable number of T cells is maintained clinically reminiscent of chronic GvHD. We show here that a fraction of the naïve T cells that encounter the self-antigen after transfer develop into CD4+CD25+ regulatory T cells (Treg) in the periphery. This population controls T cell homeostasis, activation and severe immune pathology. The development of CD4+CD25+ Treg critically depends on IL-2 produced by the T cells. Therefore, in the absence of IL-2, T cell homeostasis cannot be maintained and massive accumulation of CD4+ T cells leads to severe inflammation of the skin. Importantly, only IL-2 that is produced by the T cells themselves, but not from peripheral tissues, leads to efficient generation of Treg and T cell homeostasis. We suggest that Treg-development is a differentiation step of T cells that encounter self-antigen in the periphery, and is essential for maintaining homeostasis even in the presence of self-recognition. Our data provide mechanistic insight into the re-establishment of homeostasis after cell transfer into a lymphopenic host and have important implications for the use and timing of therapeutic approaches targeting the IL-2 pathway.

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