Chemokine-mediated tissue recruitment of CXCR3+ CD4+ T cells plays a major role in the pathogenesis of chronic GVHD

Abstract Chemokines regulate the migration of hemopoietic cells and play an important role in the pathogenesis of many immune-mediated diseases. Intradermal recruitment of CD8+ T cells by CXCL10 is a central feature of the pathogenesis of cutaneous acute GVHD (aGVHD), but very little is known about the pathogenesis of chronic GVHD (cGVHD). Serum concentrations of the 3 CXCR3-binding chemokines, CXCL9, CXCL10, and CXCL11, were found to be markedly increased in patients with active cGVHD of the skin (n = 8). An 80% decrease in CD4+ cells expressing CXCR3 was seen in the blood of these patients (n = 5), whereas CD4+ cells were increased in tissue biopsies and were clustered around the central arterioles of the dermis. The well-documented increase in expression of CXCL10 in aGVHD therefore diversifies in cGVHD to include additional members of the CXCR3-binding family and leads to preferential recruitment of CD4+ T cells. These observations reveal a central role for chemokine-mediated recruitment of CXCR3+ T cells in cGVHD.

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Recovery of Functional EBV Specific T Cells Is Not Impaired In Patients with Chronic Gvhd.

Blood ◽

10.1182/blood.v116.21.1286.1286 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1286-1286

Author(s):

Mette Hoegh-Petersen ◽

Yiping Liu ◽

Stephanie Liu ◽

Alejandra Ugarte-Torres ◽

Kevin Fonseca ◽

...

Keyword(s):

T Cells ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Epstein Barr Virus ◽

Acute Gvhd ◽

De Novo ◽

Chronic Gvhd ◽

Cd8 Cells ◽

Barr Virus ◽

Epstein Barr

Abstract Abstract 1286 Introduction: T cell expression of PD-1, a marker of functional exhaustion manifested by inability to produce cytokines upon stimulation, is upregulated in patients with acute GHVD. This is thought to explain at least in part why patients with acute GVHD have frequent infections (Gallez-Hawkins et al, BBMT15:872, 2009). Here we wished to evaluate whether this is true also for chronic GVHD. Patients and Methods: We studied 17 allogeneic HCT recipients for AML who have not developed acute or chronic GVHD by day 84. Their blood was drawn on day 84, 180 and 365. Between day 84 and 365, 7 patients did and 10 patients did not develop chronic GVHD needing systemic immunosuppressive therapy (de novo, ie, without preceding acute GVHD). Onset of the chronic GVHD was on median day 103 (range, 90–147). We studied total CD4 and CD8 T cells as well as Epstein-Barr virus (EBV)-specific CD4 and CD8 T cells, as patients with chronic GVHD are at risk of EBV disease (Landgren et al, Blood,14:4992, 2009). Blood mononuclear cells were stimulated with Epstein-Barr virus (EBV) lysate, EBNA3A+B+C overlapping peptides, no or irrelevant stimulus as negative control, or Staphylococcal enterotoxin B as positive control. After overnight incubation, expression of IFNγ, TNFα, IL2 and PD-1 on CD3+CD4+CD8- or CD3+CD4-CD8+ cells was determined by flow cytometry. Cells expressing IFNγ, TNFα, IL2 or their combinations were enumerated. PD-1 expression was quantified using beads coated with anti-mouse antibody (Quantum Simply Cellular, Bangs Laboratories) and expressed as antibody binding capacity units (ABC) (dynamic range, approximately 300 to 500,000 ABC units per cell). Results: PD-1 expression on total, EBV lysate-specific or EBNA3-specific CD4 or CD8 T cells was not significantly higher among patients who did vs did not develop chronic GVHD. On the contrary, there was a trend toward lower PD-1 expression on EBV lysate-specific CD4 and CD8 T cells and EBNA3-specific CD4 T cells in patients who developed chronic GVHD. This was significant (p<.05, Mann-Whitney test) for EBV lysate-specific CD4 T cells on day 84, EBV lysate-specific CD8 T cells on day 180, EBV lysate-specific CD4 and CD8 T cells on day 365, EBNA3-specific CD4 T cells on day 84 and EBNA3-specific CD4 T cells on day 365. Consistent with that, absolute counts of total, EBV lysate-specific or EBNA3-specific T cells were not significantly lower in patients who did vs did not develop chronic GVHD. On the contrary, there was a trend toward higher EBV lysate-specific and EBNA3-specific CD4 or CD8 T cell counts in patients who developed chronic GVHD. This was significant on day 84 for total EBV lysate-specific CD4 and CD8 cells, EBV lysate-specific CD4+IFNγ+ cells and CD8+IFNγ+ cells, and total EBNA3-specific CD4 cells, EBNA3-specific CD4+IFNγ+ cells, CD4+IL2+ cells, CD4+IFNγ+TNFα+IL2+ cells and CD8+IFNγ+ cells. Conclusion: De novo chronic GvHD and its treatment do not adversely affect the counts of functional EBV specific T cells. Disclosures: No relevant conflicts of interest to declare.

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Bone Marrow Derived, De Novo Generated CD4+ T Cells Are Previously Unsuspected Participants in CD8+ T Cell-Mediated Graft-Versus-Host Disease.

Blood ◽

10.1182/blood.v106.11.581.581 ◽

2005 ◽

Vol 106 (11) ◽

pp. 581-581

Author(s):

Yi Zhang ◽

Elizabeth Hexner ◽

Dale Frank ◽

Joe Gerard ◽

Frank Kung ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Acute Gvhd ◽

De Novo ◽

Memory T Cells ◽

Tissue Injury ◽

Chronic Gvhd ◽

Host Tissue

Abstract Although mature CD8+ T cells are known to be major effectors of acute GVHD, patients receiving T cell-depleted allografts remain at high risk for chronic GVHD. To what extent CD8+, CD4+ or both T cell subsets contribute to this chronic immunopathology is not known. We have recently demonstrated that alloreactive memory T cells develop in mice with acute GVHD and account for the persistence of host tissue injury (Journal of Immunology, 2005;174:3051). Based on these findings, we now ask whether de novo generated donor T cells from engrafted T-BM themselves contribute to persistent host tissue injury in GVHD. Confirming previous observations, we found that transplantation of lethally irradiated C57BL/6SJL (B6, CD45.1) mice with highly purified C3H.SW (CD45.2) CD4+ naïve T cells did not cause GVHD, but mice receiving highly purified CD8+ naïve T cells together with C3H.SW T-BM, suffered severe acute GVHD. Surprisingly, in these mice receiving only CD8+ T cells, a substantial number of donor CD4+ T cells as well as CD8+ T cells were detected in GVHD target tissues, indicating that these infiltrating CD4+ T cells had arisen de novo from the transplanted T-BM. Donor CD4+ T cells recovered from GVHD mice expressed surface markers of activated effector/effector memory T cells, including CD25, CD69, CXCR3, and CD44hiCD62Llo. In response to host DCs, purified GVHD CD4+ T cells proliferated and expanded 4-5X more, and produced 10X higher levels of IFN-γ than did CD4+ T cells derived from B6 mice receiving C3H.SW T-BM alone. Furthermore, adoptive transfer of these in vivo generated GVHD CD4+ T cells, without CD8+ T cells, into secondary irradiated B6 recipients induced clinical GVHD characterized by delayed onset, weight loss, diarrhea, and lymphopenia, but without cutaneous inflammation. Histologic examination demonstrated chronic inflammation in the liver and intestinal tract, including epithelial apoptosis. Thymic pathology was dramatic in secondary B6 recipients of GVHD CD4+ T cells, including thymic atrophy, loss of thymic cortex, and infiltration of large amount of tingible macrophages. Taken together, these results demonstrate that donor bone marrow derived, de novo generated CD4+ T cells also contribute to GVHD together with transferred mature CD8+ T cells. Moreover, they suggest that these CD4+ T cells, in concert with alloreactive memory CD8+ T cells that develop during the evolution of GVHD, cause the persistence of acute GVHD and its subsequent progression into chronic GVHD. Thus, donor BM-derived, de novo generated CD4+ T cells are the “Hidden Dragon” of CD8+ T cell-mediated GVHD. Understanding how these CD4+ T cells are generated and regulated will prove to be critical to the prevention and treatment of both acute and chronic GVHD.

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Stat3 Regulates Alloreactive T Cell Susceptibility Towards PD-L1-Mediated Tolerance Signaling, Gvhd and GVL Activity

Blood ◽

10.1182/blood-2018-99-119474 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3323-3323

Author(s):

Shijie Yang ◽

Xiaoqi Wang ◽

Yuankun Zhang ◽

Deye Zeng ◽

Qingxiao Song ◽

...

Keyword(s):

T Cells ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Acute Gvhd ◽

Chronic Gvhd ◽

B Cell Lymphoma ◽

Tissue Expression ◽

Lymphoid Tissues ◽

Target Tissue ◽

Donor T Cells

Abstract Prevention of graft versus host disease (GVHD) while preserving graft-versus-leukemia/lymphoma (GVL) activity of alloreactive donor T cells remains an elusive and long-sought goal. We and others reported that Stat3 deficiency in donor T cells results in expansion of Foxp3+ Treg cells and reduction of pathogenic Th17 cells, leading to prevention of chronic GVHD. We have now tested whether Stat3 deficiency in donor T cells can prevent acute GVHD while preserving GVL activity. We transplanted wild-type (WT) or Stat3-/- T cells from C57BL/6 (H-2b) donors into irradiated WT BALB/c recipients bearing B cell lymphoma line BCL1 or aggressive acute lymphocytic leukemia (ALL) cells. WT donor T cells caused acute GVHD, as expected. Stat3-/- donor T cells did not cause acute GVHD but still had strong GVL activity. This separation of GVL activity from GVHD was associated with augmented expansion and impaired tolerance by Stat3-/- T cells in lymphoid tissues such as the spleen. In contrast, tolerance by Stat3-/- T cells was enhanced in GVHD target tissues by different mechanisms. The yield of Stat3-/- T cells in gut tissues was lower than the yield of WT T cells due to the markedly higher apoptosis of Stat3-/- T cells compared to WT T cells. The yield of Stat3-/- T cells in the lung and liver was not lower than the yield of WT T cells, but expression of anergy/exhaustion markers such as PD-1 and KLRG1 was markedly higher in Stat3-/- T cells than in WT T cells. We recently reported that GVHD tissue expression of PD-L1 can tolerize donor CD8+ T cells in the absence of donor CD4+ T cells, but not when CD4+ and CD8+ T cells were given together. In contrast, PD-L1 expression in recipient GVHD target tissues tolerized Stat3-/- donor T cells when both CD4+ T cells and CD8+ T cells were given together. The induction of tolerance in Stat3-/- T cells by GVHD target tissue expression of PD-L1 depended on PD-L1/PD-1 interaction, which led to marked reduction of glycolysis in Stat3-/- T cells but not in WT T cells. These observations fit with previous findings that enhanced glycolysis was associated with enhanced GVHD activity of alloreactive T cells, as reported by Xue-zhong Yu's group. Our results indicate that Stat3 deficiency in donor T cells can augment their susceptibility to tissue PD-L1-mediated tolerance mechanisms such as down-regulation of glycolysis. Current experiments are testing whether Stat3 knockdown in mature T cells in the graft can prevent GVHD while preserving GVL activity. (Grant support: R01AI066008 to Zeng) Disclosures No relevant conflicts of interest to declare.

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Anti-GvHD Effect of Antithymocyte Globulin Is Mediated by Antibodies Binding to T Cells and Regulatory NK Cells, and Less So or Not at All by Antibodies Binding to Other Mononuclear Cell Subsets

Blood ◽

10.1182/blood.v116.21.2346.2346 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2346-2346

Author(s):

Mette Hoegh-Petersen ◽

Minaa Amin ◽

Yiping Liu ◽

Alejandra Ugarte-Torres ◽

Tyler S Williamson ◽

...

Keyword(s):

T Cells ◽

Dendritic Cells ◽

B Cells ◽

Nk Cells ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Antithymocyte Globulin ◽

Chronic Gvhd ◽

Wilcoxon Test ◽

Effector Memory

Abstract Abstract 2346 Introduction: Polyclonal rabbit-anti-human T cell globulin may decrease the likelihood of graft-vs-host disease (GVHD) without increasing the likelihood of relapse. We have recently shown that high levels of antithymocyte globulin (ATG) capable of binding to total lymphocytes are associated with a low likelihood of acute GVHD grade 2–4 (aGVHD) as well as chronic GVHD needing systemic therapy (cGVHD) but not increased likelihood of relapse (Podgorny PJ et al, BBMT 16:915, 2010). ATG is polyclonal, composed of antibodies for antigens expressed on multiple cell subsets, including T cells, B cells, NK cells, monocytes and dendritic cells. These cell subsets may play a role in the pathogenesis of GVHD. The anti-GVHD effect of ATG may be mediated through killing/inhibition of one or several of these cell subsets (eg, T cells) or their subsets (eg, naïve T cells as based on mouse experiments naïve T cells are thought to play a major role in the pathogenesis of GVHD). To better understand the mechanism of action of ATG on GVHD, we set out to determine levels of which ATG fraction (capable of binding to which cell subset) are associated with subsequent development of GVHD. Patients and Methods: A total of 121 patients were studied, whose myeloablative conditioning included 4.5 mg/kg ATG (Thymoglobulin). Serum was collected on day 7. Using flow cytometry, levels of the following ATG fractions were determined: capable of binding to 1. naïve B cells, 2. memory B cells, 3. naïve CD4 T cells, 4. central memory (CM) CD4 T cells, 5. effector memory (EM) CD4 T cells, 6. naïve CD8 T cells, 7. CM CD8 T cells, 8. EM CD8 T cells not expressing CD45RA (EMRA-), 9. EM CD8 T cells expressing CD45RA (EMRA+), 10. cytolytic (CD16+CD56+) NK cells, 11. regulatory (CD16-CD56high) NK cells, 12. CD16+CD56− NK cells, 13. monocytes and 14. dendritic cells/dendritic cell precursors (DCs). For each ATG fraction, levels in patients with versus without aGVHD or cGVHD were compared using Mann-Whitney-Wilcoxon test. For each fraction for which the levels appeared to be significantly different (p<0.05), we determined whether patients with high fraction level had a significantly lower likelihood of aGVHD or cGVHD than patients with low fraction level (high/low cutoff level was determined from ROC curve, using the point with maximum sum of sensitivity and specificity). This was done using log-binomial regression models, ie, multivariate analysis adjusting for recipient age (continuous), stem cell source (marrow or cord blood versus blood stem cells), donor type (HLA-matched sibling versus other), donor/recipient sex (M/M versus other) and days of follow up (continuous). Results: In univariate analyses, patients developing aGVHD had significantly lower levels of the following ATG fractions: binding to naïve CD4 T cells, EM CD4 T cells, naïve CD8 T cells and regulatory NK cells. Patients developing cGVHD had significantly lower levels of the following ATG fractions: capable of binding to naïve CD4 T cells, CM CD4 T cells, EM CD4 T cells, naïve CD8 T cells and regulatory NK cells. Patients who did vs did not develop relapse had similar levels of all ATG fractions. In multivariate analyses, high levels of the following ATG fractions were significantly associated with a low likelihood of aGVHD: capable of binding to naïve CD4 T cells (relative risk=.33, p=.001), EM CD4 T cells (RR=.30, p<.001), naïve CD8 T cells (RR=.33, p=.002) and regulatory NK cells (RR=.36, p=.001). High levels of the following ATG fractions were significantly associated with a low likelihood of cGVHD: capable of binding to naïve CD4 T cells (RR=.59, p=.028), CM CD4 T cells (RR=.49, p=.009), EM CD4 T cells (RR=.51, p=.006), naïve CD8 T cells (RR=.46, p=.005) and regulatory NK cells (RR=.55, p=.036). Conclusion: For both aGVHD and cGVHD, the anti-GVHD effect with relapse-neutral effect of ATG appears to be mediated by antibodies to antigens expressed on naïve T cells (both CD4 and CD8), EM CD4 T cells and regulatory NK cells, and to a lesser degree or not at all by antibodies binding to antigens expressed on B cells, cytolytic NK cells, monocytes or DCs. This is the first step towards identifying the antibody(ies) within ATG important for the anti-GVHD effect without impacting relapse. If such antibody(ies) is (are) found in the future, it should be explored whether such antibody(ies) alone or ATG enriched for such antibody(ies) could further decrease GVHD without impacting relapse. Disclosures: No relevant conflicts of interest to declare.

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Rapamycin Is Highly Effective in a Mouse Model of Immune-Mediated Bone Marrow Failure By Mechanisms Distinct from Cyclosporine

Blood ◽

10.1182/blood.v126.23.1202.1202 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1202-1202

Author(s):

Xingmin Feng ◽

Zenghua Lin ◽

Marie Desierto ◽

Keyvan Keyvanfar ◽

Daniela Malide ◽

...

Keyword(s):

T Cells ◽

Lymph Node ◽

T Cell ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Th2 Cytokines ◽

Failure Model ◽

Murine Models ◽

Immune Mediated ◽

Lymph Node Cells

Abstract Acquired aplastic anemia (AA) is bone marrow (BM) failure characterized by pancytopenia and marrow hypocellularity, in most patients due to immune attack by T cells that target hematopoietic stem and progenitor cells. Most patients respond to immunosuppressive therapy, but relapse, especially on withdrawal of cyclosporine A (CsA), occurs frequently (Scheinberg P, Am J Hematol., 2014). Rapamycin has been successful in some human autoimmune diseases and in mouse models of autoimmunity; rapamycin also appears to induce tolerance, as for example in the organ transplant setting. We have developed murine models of BM failure; animals can be salvaged by biologics and drugs that are effective in humans with AA. One purpose of these models is to test potential new therapies. We have compared rapamycin with customary immunosuppression by CsA. Infusion of lymph node cells from C57BL6 (B6) donor mice into CByB6F1 (F1) recipient mice (MHC-mismatched) induced massive BM destruction by activated T cells. Treatment with rapamycin (2 mg/kg/day, starting 1 hour post lymphocyte injection and continued for 2 weeks, n=9) effectively ameliorated pancytopenia and improved BM cellularity, better than did maximal dosing with CsA (50 mg/kg/day, starting 1 hour post lymphocyte injection, continued for 5 days, n=8) (Fig 1A). Rapamycin eliminated most BM-infiltrating CD8+ T cells, while CsA had less effect on CD8+ T cells than did rapamycin. Elimination of BM infiltrated T cells and restoration of megakaryocytes by rapamycin was visualized by confocal microscopy using whole-mounts of sternum, for which donor B6 lymph node cells were replaced with B6-DsRed lymph node cells. Plasma cytokines were measured by Luminex: IFNg, TNFa, IL-2, MIP1b, RANTES, sCD137 (all p < 0.001) were increased in BM failure mice compared with the control animals, indicating an inflammatory environment in AA. Rapamycin reduced these cytokines (p < 0.001) but increased Th2 cytokines such as IL-4 and IL-10 (p < 0.001) levels. CsA only decreased sCD137, reversely it even increased IFNg levels. Transcriptome analysis using pooled FACS-sorted CD4+ and CD8+ T cells from BM focusing on genes related to T cell functions revealed that rapamycin suppressed expression of Icam1, and Tnfsf14 in CD8+ T cells, and Cd27, Lgals3, Il10ra, Itga1, Tbx21, Gzmb, Tnfsf14 and Cd70 in CD4+ T cells, but increased Il-4, Il-2ra, and Tnfrsf8 expression in CD4+ T cells compared with AA mice. CsA suppressed Lgals3 in CD8+ T cells and Cd70 in CD4+ T cells, suggesting differential mechanisms of action by these two immunosuppressive drugs. All untreated AA mice (n=6) died within 3 weeks post lymphocyte infusion, while all mice treated with rapamycin for 2 weeks (n=8) survived until study termination at 7 weeks; similar results were obtained when we tested delayed treatment with rapamycin (starting 3 days post lymphocyte injection and continued for 10 days, n=8) in BM failure mice; but brief exposure to rapamycin, for only 5 days from 1 hour post lymphocyte infusion (n=8), could not rescue mice, suggesting a requirement for sustained administration. In contrast, all animals treated with CsA (n=6) died within 5 weeks (Fig 1B). We also tested the effect of rapamycin on antigen-specific T cells in another BM failure model induced by infusion of lymphocytes from B6 donor mice into C.B10-H2b /LilMcd recipient mice (MHC-matched but minor antigen-mismatched, n=10), in which BM destruction is mediated by H60-specific cytotoxic T cells (CTL) (Chen J, JI, 2007). Similar results were observed. Flow cytometry revealed massive expansion of H60-specific CTL in BM of untreated AA mice, rapamycin eliminated BM CD8+ T cell infiltration. CsA decreased BM CD8+ T cells, but had much weaker effect on H60 CTLs (Fig 1C). In summary, rapamycin is effective in treatment of AA murine models, which holds implications in the application in immune-mediated pathophysiologies in the laboratory and in the clinic. Compared with CsA, rapamycin suppressed expression of T cell activation genes more broadly, increased Th2 cytokines, eliminated antigen-specific T cells, and had better survival rate in animal BM failure model, supporting a clinical trial of rapamycin to prevent relapse and induce tolerance in patients with AA, many of whom are dependent on CsA administration for support of blood counts but at risk of CsA nephrotoxicity. Disclosures No relevant conflicts of interest to declare.

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The Anti-CD25 Antibody Daclizumab Delays Treg Reconstitution, Promotes CD4 Memory, and Does Not Prevent Acute or Chronic Gvhd After Allogeneic Stem Cell Transplantation

Blood ◽

10.1182/blood.v120.21.4195.4195 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4195-4195 ◽

Cited By ~ 1

Author(s):

Frederick L Locke ◽

Joseph Pidala ◽

Barry Storer ◽

Paul J. Martin ◽

Michael A Pulsipher ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Binding Sites ◽

Acute Gvhd ◽

Disease Risk ◽

Chronic Gvhd ◽

Central Memory ◽

Cd4 Cells ◽

Grade Ii

Abstract Abstract 4195 Background: Daclizumab, a humanized monoclonal antibody, binds CD25 and blocks formation of the high affinity IL-2 receptor on T cells. A multi-center, randomized, double-blind clinical trial using daclizumab to perturb IL-2 signaling as a way of reducing T cell mediated graft versus host disease (GVHD) after allogeneic unrelated bone marrow transplantation (BMT) was completed 18 years ago. We report long term outcomes and biological correlates. Methods and Patients: Between April, 1993 and December, 1994, 209 adult or pediatric patients receiving marrow from an unrelated donor for hematologic malignancies or severe aplastic anemia at 12 sites in Europe and North America were randomized to one of three arms: 5 weekly doses of placebo (arm A, n=64); 0.3 mg/kg daclizumab (arm B, n=69); or 1.2 mg/kg daclizumab (arm C, n=76) to a maximum of 100 mg beginning on the day before BMT. Conditioning included total body irradiation (TBI) (1200–1440 cGy). All patients received methotrexate (15 mg/m2 day 1; 10 mg/m2 days 3, 6, 11) plus cyclosporine for at least 180 days. The study was designed to provide 80% power to detect a reduction in the incidence of acute GVHD (grade II-IV, requiring treatment with corticosteroids to day +100) from 80% to 55%. Randomization was stratified by donor HLA (matched vs. mismatched) and age (<20 vs. >=20). Arms were well balanced for HLA matching, age, disease risk, TBI dose, and diagnosis. Diagnoses were 31 ALL, 30 AML, 118 CML, 16 MDS, and 14 other. Median age was 31 years (range 1–54); 61% were male; 45% had high risk disease (14% AML with active disease or beyond 2nd remission, 20% CML beyond first chronic phase); 29% had a HLA-A, -B or -DR mismatched donor. T cells from a subset of patients (n=107) were collected at days 11–35, days 36–80, and days 81–100 and analyzed by flow cytometry for expression of total CD25, daclizumab binding, and free CD25 binding sites. Samples from arm A and arm C, including those from 1 year long term follow up (LTFU) were also evaluated for T cell phenotype. Results: The incidence of grade II-IV acute GVHD for arms A, B, and C were 34%, 42%, and 45%, and results did not differ between arm A vs. arm B (p=0.60) or arm C (p=0.38). Since other clinical outcomes did not differ significantly between arm B and arm C, the two daclizumab dose arms were combined for post-hoc exploratory analysis. There was a suggestion that the daclizumab arms showed decreased risk of relapse (HR 0.57, 95% CI: 0.32–1.00, p=0.06) and increased risk of chronic GVHD (HR 1.49, 95% CI: 0.96–2.31, p=0.07) compared to the placebo arm. Daclizumab did not increase OS (HR 0.89, 95% CI: 0.61–1.29, p=0.54) compared to placebo, and there was no interaction between disease risk and OS. Daclizumab administration reduced the total numbers of T cells expressing CD25 at days 11–35 (arm A=28% vs. arm B=16%, p<0.0001; or arm C=18%, p=0.0002); at days 36–80 (arm A=26% vs. arm B=19%, p=0.03; or arm C=19%, p=0.03); figures were equivalent at days 81–101. Daclizumab was bound to CD25 in vivo, at days 11–35 (arm A=3% vs. arm B=74%, p<0.0001; or arm C=83%, p<0.0001) and persisted at days 81–101 (arm A=4% vs. arm B=17%, p=0.05; or arm C=39%, p<0.0001). Daclizumab administration decreased the numbers of cells with free CD25 binding sites at days 11–35 (arm A=45% vs. arm B=7%, p<0.0001; or arm C=3%, p<0.0001); at days 36–80 (arm A=49% vs. arm B=25%, p<0.0001; or arm C=11%, p<0.0001); and was equivalent at days 81–101. Available samples from arm A (n=18) and arm C (n=40) were tested for Treg (CD4+ CD127- CD25+ FOXP3+) and central memory (CD4+ CD45RA- CCR7+) phenotype. Compared to placebo (arm A), daclizumab administration (arm C) decreased the proportion of CD4 cells that were Tregs at days 11–35 (12% arm A vs. 7% arm C; p=0.008), but not at days 81–101 and LTFU. Daclizumab increased the proportion of CD4 cells exhibiting central memory phenotype at LTFU (10% arm A vs. 21% arm C, p=0.02). Conclusion: This randomized phase II/III study shows that daclizumab does not prevent GVHD. There was suggestion for increased chronic GVHD and decreased relapse. Daclizumab delayed Treg reconstitution while increasing CD4 central memory at LTFU. Based on these results, further trials should test whether anti-CD25 therapy can promote anti-tumor immunity by impairing Treg reconstitution after transplant, in the appropriate clinical setting. Disclosures: Off Label Use: Daclizumab administration following hematopoietic cell transplantation. Walker:Baxter Corporation: Research Funding. Light:Bristol Myers Squibb: Employment.

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Prophylactic transfer of CD8-depleted donor lymphocytes after T-cell–depleted reduced-intensity transplantation

Blood ◽

10.1182/blood-2006-03-005769 ◽

2006 ◽

Vol 109 (1) ◽

pp. 374-382 ◽

Cited By ~ 64

Author(s):

Ralf G. Meyer ◽

Cedrik M. Britten ◽

Daniela Wehler ◽

Klaus Bender ◽

Georg Hess ◽

...

Keyword(s):

T Cells ◽

Cd8 T Cells ◽

Immune Reconstitution ◽

Acute Gvhd ◽

Chronic Gvhd ◽

Hematologic Malignancies ◽

Good Manufacturing Practice ◽

Hematopoietic Stem ◽

Log Reduction ◽

Reduced Intensity

Abstract Allogeneic hematopoietic stem cell transplantation (SCT) regimens incorporating the lymphocytotoxic CD52 antibody alemtuzumab demonstrate efficient engraftment and reduced graft-versus-host disease (GVHD). However, these protocols substantially impair posttransplantation antiviral and antitumor immunity. To accelerate immune reconstitution after alemtuzumab-based reduced-intensity SCT, we administered prophylactic CD8-depleted donor lymphocyte infusions (DLIs) starting on days 60 and 120 after transplantation. DLIs were processed in an immunomagnetic good manufacturing practice depletion procedure resulting in a 2.5- to 6-log reduction in CD8 T cells. Of 23 high-risk patients with hematologic malignancies, 11 received a total of 21 CD8-depleted DLIs. Five patients developed transient grade I acute GVHD following transfer. Only 2 patients with HLA-C–mismatched donors showed grade II and III acute GVHD and subsequently progressed to limited chronic GVHD. Following DLIs, 4 patients with declining hematopoietic donor chimerism converted to full chimeras. A 2.1-fold median increase of circulating CD4 T cells was observed within 2 weeks after infusion. Non-DLI patients did not show a comparable rise in CD4 counts. Four patients demonstrated enhanced frequencies of cytomegalovirus-specific CD4 and CD8 T cells following transfer. Our results suggest that prophylactic CD8-depleted DLIs accelerate immune reconstitution after lymphodepleted HLA-matched SCT and carry a low risk of inducing severe GVHD.

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A Critical Role for Donor-Derived IL-22 in Cutaneous Chronic Gvhd

Blood ◽

10.1182/blood.v130.suppl_1.69.69 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 69-69

Author(s):

Kate H Gartlan ◽

Hemamalini Bommiasamy ◽

Katelyn Paz ◽

Andrew Wilkinson ◽

Mary Owen ◽

...

Keyword(s):

T Cells ◽

Cd4 T Cells ◽

Acute Gvhd ◽

Chronic Gvhd ◽

Protein Detection ◽

Cytokine Gene Expression ◽

Cell Transplant ◽

Cutaneous Manifestations ◽

Post Transplant

Abstract Graft-versus-host disease (GVHD) is the major cause of non-relapse morbidity and mortality after allogeneic stem cell transplant (allo-SCT). Chronic cutaneous manifestations of GVHD are common late after allo-SCT with limited treatment options beyond protracted steroid therapy. Cytokines are critical mediators of inflammatory processes during GVHD and both IL-17A and IL-22 have been found to have dual pathogenic and protective roles, which are largely dependent upon their cellular source. We have demonstrated in mice that whilst recipient-derived IL-17A and IL-17R signaling suppresses inflammatory responses and prevents gut dysbiosis, donor T cell-derived IL-17A is pathogenic and drives GVHD in both lung and skin. Similarly, contrasting roles have also been described for IL-22 in mice, such that recipient-derived IL-22 helps maintain gut epithelial integrity after allo-SCT, but donor IL-22 can exacerbate acute GVHD pathology. To date, investigation of the effects of donor-derived IL-22 in allo-SCT has focused on acute GVHD, with no data regarding the role of IL-22 in chronic GVHD. Given the complexities and the potential competing risks of targeting IL-22 early post-transplant, we examined IL-22 deficiency in murine models of chronic GVHD following allo-SCT. In the absence of donor-derived IL-22, we observed a significant decrease in skin GVHD clinical scores and chronic GVHD skin pathology (histopathology scores in WT donors: 10.2±2.1, vs. IL-22-/- donors: 2.6±0.9, p <0.01), which is in line with the pathogenic role of IL-22 in autoimmune skin diseases. Intriguingly, lung manifestations of chronic GVHD (i.e. bronchiolitis obliterans) were not IL-22-dependent (histopathology scores in WT donors: 1.6±0.5, vs. IL-22-/- donors: 1.5±0.3, p >0.05), despite the fact we have recently defined their IL-17-dependency. These data demonstrate a relationship between IL-22 and IL-17 and the distribution of chronic GVHD. We identified CD4+ T cells as the major source of donor IL-22 by both direct protein detection and cytokine reporter systems, where we observed co-expression of IL-22 with a range of pro-inflammatory cytokines including IL-17A, IFNγ, GM-CSF and TNF. We identified IL-22+IL-17A+ and IL-22+IL-17A- CD4+ T cells as two distinct sources of donor-derived IL-22 post-transplant (2.2±0.1% and 6.5±0.3 % of LN CD4+ T respectively), both of which were highly dependent upon IL-6 for their development (IL-22+IL-17A+: IgG 1.3±0.2% vs. anti-IL-6Rα 0.08±0.01% of splenic CD4+ T, p < 0.001; IL-22+IL-17A-:IgG 3.4±0.2 vs. anti-IL-6Rα: 0.6±0. 1% of splenic CD4+ T, p < 0.001). Since we have previously identified significant cytokine plasticity within IL-17A+ T cells after allo-SCT, we performed lineage assessment to determine if these two populations arose independently. Using IL-17creRosa26YFP fate-mapping mice that permanent label any cell that has expressed IL-17A, we found that IL-22+IL-17A- CD4+ T cells did not have a history of IL-17A production and were therefore identified as a definitive Th22 population after allo-SCT. Since IL-22 and IL-17A have been reported to induce synergistic responses in the skin, we also explored the possibility of interdependence between Th17 and Th22 development. Using both IL-17RC-/- mice and an IL-17creRosa26YFP/iDTR reporter-deleter system we demonstrate that IL-17 signaling to donor Th22 directly promotes their development in allo-SCT (WT donors: 6.5±0.4 %, Th/Tc17 deleter donors: 3.6±0.2, IL-17RC-/- donors: 3.7±0.3 % Th22 in LN CD4+ T, p <0.001). Finally, we observed a similar pro-inflammatory cytokine gene expression signature (Il22, Il17, Ifng) in the skin of patients who developed cutaneous GVHD > 100 days following allo-SCT but not in those after autologous SCT. These data demonstrate a key role for donor-derived IL-22 in chronic skin GVHD and confirm parallel but symbiotic developmental pathways of Th22 and Th17 differentiation. Together these findings suggest that IL-22 intervention late post-transplant may reduce cutaneous chronic GVHD, whilst maintaining the protective effects of IL-22 in the gut early after transplant. Disclosures Serody: Merck: Research Funding.

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A role for CD4 helper cells in HIV control and progression

10.1101/2021.12.20.473599 ◽

2021 ◽

Author(s):

Igor M Rouzine

Keyword(s):

T Cells ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Alternative Explanation ◽

Arms Race ◽

Cd4 Cells ◽

Viral Dissemination ◽

Small Minority ◽

Helper Cells ◽

Untreated Individuals

It remains unclear why HIV persists in most untreated individuals, and why a small minority of individuals can control the virus, either spontaneously or after an early treatment. The present work motivated by the striking differences in the functional avidity of CD4 T cells discovered between patient cohorts in a recent study [1] offers an experimentally–testable mathematical model that explains the diverse outcome of infection. The model predicts an arms race between viral dissemination and the proliferation of HIV-specific CD4 helper cells leading to one of two states: a low-viremia state or a high-viremia state. Helper CD4 cells with a higher avidity favor virus control. The parameter segregating spontaneous and post-treatment controllers is the infectivity asymmetry between activated and resting CD4 T cells. The predictions are found to be consistent with the data from [1] and with data on the avidity CD8 T cells [2]. I also analyze the alternative explanation of T cell exhaustion previously proposed to explain the diverse patient cohorts and demonstrate that it does not explain these and some other experimental data.

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The Role of CMV Serostatus of the Donor in the Graft Lymphoid Composition and Prediction of Gvhd and CMV Reactivation

Blood ◽

10.1182/blood.v124.21.2422.2422 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2422-2422

Author(s):

Anna Kreutzman ◽

Itxaso Portero Sainz ◽

Valle Gomez Garcia De Soria ◽

Carlos Fernandez ◽

Royg Mercedes ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Chronic Gvhd ◽

Cell Subpopulation ◽

Cell Phenotype ◽

Number Of Patients ◽

Cmv Reactivation ◽

Graft Composition

Abstract Background. Cytomegalovirus (CMV) is a widespread persistent β–herpesvirus, which can cause severe complications during primary infection or reactivation in immunocompromised patients, such as after allogeneic stem cell transplantation (alloSCT). Another major complication associated with alloSCT is graft-versus-host disease (GVHD). The pathogenesis of GVHD involves migration of the transplanted donor naïve T-cells into the secondary lymphoid organs in the recipient, which is mainly steered by CD62L and CCR7. As these homing molecules have been associated with both acute GVHD (aGVHD) and chronic GVHD (cGVHD), we studied whether the CMV serostatus of the donor affects the lymphoid composition of the graft product and whether this phenotype can predict CMV reactivation and GVHD. Methods. This single-center study included 77 donor-recipient pairs who underwent alloSCT. 64 pairs were HLA identical, 12 had 1 mismatch and 3 had 2 mismatch. 36 donors were related to their recipients. All recipients were followed at least for 100 (aGVHD) or 360 days (cGVHD) after transplantation. 43 donors were CMV-seropositive (CMVpos) and 34 were CMV-seronegative (CMVneg). 62 recipients were CMVpos, and 32 of them developed CMV reactivation, 25 aGVHD and 30 cGVHD. Samples from the graft product (donor) were phenotyped by flow cytometry (CD45, CD3, CD8, CD4, CD62L, CCR7) and both frequency (freq) and absolute number (abs) of each T-cell subpopulation were analyzed. Results. When the donors were divided based on their CMV serostatus, we observed that the grafts from CMVpos donors had a lower freq of naïve (CCR7+CD62L+) CD4+ T-cells (of lymphocytes p=0.06, of CD3 p=0.06, of CD4 p=0.07) and naïve CD8+ T-cells (of leukocytes p=0.03, of lymphocytes p=0.041, of CD3 p=0.011, of CD8 p=0.012) compared to CMVneg donors. Further, the abs of transplanted naïve CD8+ T-cells was significantly lower in the grafts from CMVpos donors (p=0.048). No differences were observed in T-cells (CD3+, CD4+, CD8+). We next studied if the CMV-serostatus and T-cell phenotype of the graft associates with GVHD. CMVpos donors whose recipients developed aGVHD had higher abs (p=0.05) and freq of naïve CD8+ T-cells (of lymphocytes p=0.08, of CD3 p=0.08, of CD8 p=0.11) compared to those without aGVHD. The same trend was observed with abs (p=0.11) and freq of CCR7+CD4+ T-cells (of leukocytes p=0.15). Similarly, those CMVpos donors whose recipients developed cGVHD had higher abs (p=0.05) and freq of CCR7+CD8+ T-cells (of leukocytes p=0.03, of lymphocytes p=0.06). Further, cGVHD patients who received the transplant from CMVpos donors were infused with a higher freq of CD3+ (of leukocytes p=0.03) and CD4+ T cells (of leukocytes p=0.04) than patients who received a graft from CMVpos donors but did not develop cGVHD. In contrast, CMVneg donors who developed aGVHD had a higher freq of CD3+ (p=0.018) and CD4+ T-cells (p=0.09), whereas no differences were seen in the T-cell subpopulations. Conversely, the abs (p=0.08) and freq of CCR7+CD4+ T-cells (of leukocytes p=0.11) were higher in those who later developed cGVHD. To study whether the graft lymphoid composition can be used to predict CMV reactivation, we analyzed the lymphoid composition in the graft product of those donors (both CMVpos and CMVneg) whose recipients were CMV seropositive but did not develop any form of GVHD (to avoid the influence of GVHD in the reactivation of CMV). Despite the low number of patients (CMV reactivation n=9, and no CMV reactivation n=13), we observed trends of higher portion of CD4+ T-cells (p=0.09 of lymphocytes, of CD3 p=0.20) and CCR7+CD4+ T-cells (of lymphocytes p=0.18, of CD4 p=0.16) in those grafts that were transplanted into CMV seropositive recipients who did not reactivate CMV. Conclusions. CMVpos donors whose recipient developed either aGVHD or cGVHD had a higher abs and freq of naïve CD8+ T-cells, which was not seen with CMVneg donors. This suggests that seropositivity sets the abs and freq of CD8 subpopulations near to a decisive cutoff for the development of GVHD. Conversely, other factors influences the development of GVHD in those patients whose donors were seronegative. In other words, seropositivity of the donor affects the graft composition and thus the risk of GVHD. Finally, our data indicate that a higher proportion of naïve or central memory CCR7+ CD4+ T cells in the donor graft could prevent CMV reactivation suggesting that graft composition affects also CMV reactivation. Disclosures No relevant conflicts of interest to declare.

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