HIV-Specific CD8 T Cells Producing CCL-4 Are Associated With Worse Immune Reconstitution During Chronic Infection

ABSTRACT To exert their cytotoxic function, cytotoxic T-lymphocytes (CTL) must be recruited into infected lymphoid tissue where the majority of human immunodeficiency virus (HIV) replication occurs. Normally, effector T cells exit lymph nodes (LNs) and home to peripheral sites of infection. How HIV-specific CTL migrate into lymphoid tissue from which they are normally excluded is unknown. We investigated which chemokines and receptors mediate this reverse homing and whether impairment of this homing could contribute to CTL dysfunction as HIV infection progresses. Analysis of CTL chemokine receptor expression in the blood and LNs of untreated HIV-infected individuals with stable, chronic infection or advanced disease demonstrated that LNs were enriched for CXCR3+ CD8 T cells in all subjects, suggesting a key role for this receptor in CTL homing to infected lymphoid tissue. Compared to subjects with chronic infection, however, subjects with advanced disease had fewer CXCR3+ CD8 T cells in blood and LNs. CXCR3 expression on bulk and HIV-specific CD8 T cells correlated positively with CD4 count and negatively with viral load. In advanced infection, there was an accumulation of HIV-specific CD8 T cells at the effector memory stage; however, decreased numbers of CXCR3+ CD8 T cells were seen across all maturation subsets. Plasma CXCL9 and CXCL10 were elevated in both infected groups in comparison to the levels in uninfected controls, whereas lower mRNA levels of CXCR3 ligands and CD8 in LNs were seen in advanced infection. These data suggest that both CXCR3+ CD8 T cells and LN CXCR3 ligands decrease as HIV infection progresses, resulting in reduced homing of CTL into LNs and contributing to immune dysfunction.

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Prophylactic Amotosalen-Treated Donor T-Cells Prevent Late CMV Infection in Allogeneic Bone Marrow Transplantation.

Blood ◽

10.1182/blood.v106.11.3024.3024 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3024-3024

Author(s):

Mohammad S. Hossain ◽

John D. Robak ◽

Edmund K. Waller

Keyword(s):

Bone Marrow ◽

T Cells ◽

Viral Infection ◽

Cd8 T Cells ◽

Immune Reconstitution ◽

Chronic Gvhd ◽

Mcmv Infection ◽

Tnf Α ◽

Donor T Cells ◽

Ifn Γ

Abstract A major problem in allogeneic BMT is post transplant immunodeficiency leading to opportunistic infection and relapse. Previously we showed that amotosalen-treated allogeneic donor T cells given at the time of BMT and experimental murine cytomegalovirus (MCMV) infection could prevent lethal MCMV disease without producing GvHD. In this study we have focused on a more clinically applicable model where prophylactic amotosalen-treated allogeneic donor splenocytes are given at the time of BMT, followed by MCMV infection 100 days later. We observed that amotosalen-treated donor T-cells significantly expanded and responded well in presence of viral infection without inducing any GvHD, protected recipients against viral disease, and were associated with significantly improved hematopoietic engraftment and immune reconstitution. Methods: Using a parent to F1 mouse BMT model, splenocytes (3x106 untreated or 10x106 amotosalen-treated) from MCMV immunized C57BL/6 donors were transplanted along with 5x106 T-cell depleted bone marrow (TCD BM) from naïve congeneic mice into lethally irradiated (11Gy) CB6F1 recipients (C57BL/6 x Balb/C). Recipient mice were infected i.p. with a sublethal dose (5x104 pfu per mouse) of MCMV 100 days or more after transplant. Clinical chronic GvHD was monitored by weight loss, hair loss, ruffled fur, diarrhea, and decreased activity. Flow cytometry was used to quantitate T cell chimerism (in recipient PBMC, spleen, liver and thymus) and MCMV-peptide specific CD8+ T-cells (tetramer+ and IFN-γ producing). Serum IFN-γ and TNF-α were determined by ELISA. Liver and spleen viral loads were determined by counting PFU in tissue homogenates plated onto 3T3 confluent monolayers. Results: Recipients of untreated control donor splenocytes suffered from chronic GvHD within 100 days of transplant, while those that received amotosalen-treated splenocytes experienced no GvHD. In response to MCMV infection at 100 days post transplant, residual amotosalen-treated donor T-cells rapidly expanded over 25-fold within 10 days, but did not cause lethality or detectable GvHD. Expanded amotosalen-treated T-cells showed activated anti-viral responses and developed a memory phenotype at late phases of viral infection. PBMC, spleen and liver showed elevated levels of MCMV specific tetramer+, IFN-γ+, and TNF-α+ CD8+ T-cells that were associated with accelerated viral clearance within day 3 after viral infection. While expansion and generation of amotosalen-treated donor T-cells mostly occurred in the liver, the generation of donor bone marrow-derived new T-cells occurred through both the thymus and the liver. In contrast, recipients of untreated donor splenocytes had reduced thymic function, resulting in severely impaired immune reconstitution and decreased anti-viral immunity. Conclusion: Prophylactically administered amotosalen-treated allogeneic donor T cells 1) were almost completely devoid of GvHD activity, 2) promoted hematopoietic engraftment and improved immune reconstitution, and 3) persisted long-term (>100 days) and successfully protected recipients from sublethal MCMV infection. Thus, infusion of amotosalen-treated donor T-cells at the time of transplantation is a clinically-attractive approach to adoptive anti-viral immunotherapy without chronic GvHD following hematopoietic progenitor cell transplantation.

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Natural Killer Cell Immune Reconstitution Predicts Outcomes for Patients with Chronic Lymphocytic Leukemia Undergoing Allogeneic Stem Cell Transplantation

Blood ◽

10.1182/blood.v112.11.3300.3300 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3300-3300

Author(s):

Don Benson ◽

Leslie Andritsos ◽

Mehdi Hamadani ◽

Thomas Lin ◽

Joseph Flynn ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Nk Cells ◽

Cd8 T Cells ◽

Immune Reconstitution ◽

Nk Cell ◽

Disease Status ◽

Lymphocytic Leukemia ◽

T Cell Subsets ◽

Cell Recovery

Abstract Introduction: Chronic lymphocytic leukemia (CLL), the most common form of leukemia in the Western hemisphere, is associated with severe innate, adaptive and humoral immune dysregulation. CLL remains essentially incurable, with the potential exception of allogeneic stem cell transplantation (ASCT). Natural killer (NK) cells are CD56(+), CD3(−) large granular lymphocytes that comprise a key cellular subset of the innate immune system. Preliminary in vitro data suggest an NK cell versus CLL effect exists, similar to that observed in acute myeloid leukemia (AML) and other blood cancers. Novel immune therapies for CLL (e.g., rituximab, alemtuzumab) likely exert anti-tumor effect, in part, through NK cells, in fact. Although NK cells contribute to the graft-versus-tumor effect following ASCT for other blood cancers, little is known regarding the potential role NK cells may play in the clinical allogeneic transplant setting for CLL. Herein, we provide, to our knowledge, the first report regarding NK cell immune reconstitution following ASCT for CLL. Methods: 27 CLL patients underwent reduced intensity conditioning (RIC) with ASCT. Median age was 52 years (43–69), median number of prior therapies was 3 (2–11). 55% had chemotherapy-refractory disease, and 55% had “high-risk” cytogenetics by FISH (deletion 17p or 11q22-23 abnormality). 14 patients had sibling donors, 15 had volunteerunrelated donors. Conditioning regimens included Fludarabine/TBI/Alemtuzumab (n=8), Fludarabine/Busulfan with (n=9) or without ATG (n=6), and Fludarabine/Cyclophosphamide (n=4). GVHD prophylaxis consisted of tacrolimus/MMF (n=8) or tacrolimus/methotrexate (n=19). Patients underwent bone marrow assessment prior to day +75 following ASCT. Marrow was studied for engraftment, donor chimerism, and disease status as well as lymphoid immune reconstitution by percentage of total lymphocytes and absolute lymphocyte counts by multi-color flow cytometry. Results: NK cell immune reconstitution was predicted by disease status at transplantation. Patients in complete or partial remission at the time of ASCT had more robust NK cell recovery (mean = 45% of total lymphocytes +/− SEM 5%) as compared to patients entering transplant with refractory disease (16% +/− 1, p < 0.01). No differences were observed in CD4(+) or CD8(+) T cells and no lymphocyte subset recovery was associated with CD34(+) or CD3(+) cell dosage. Achieving complete donor chimerism by day +60 was associated with robust NK cell recovery (55% +/− 1 versus 7% +/−1, p = 0.02), recovery of CD4 and CD8 T cells was not associated with chimerism status, however. Patients who went onto exhibit a complete response to ASCT had greater early NK cell reconstitution (31% +/− 3) as compared to those who had no response (8% +/− 1, p = 0.01). No differences in T cell subsets were associated with response. Patients who ultimately achieved complete remission following transplant had a lower CLL:NK cell ratio in marrow (0.35 +/− 0.07) than those who did not (8.1 +/− 1, p = 0.01). However, differences in CLL:CD4(+) and CLL:CD8(+) T cells were not predictive of response. Trends to improvement in progression free survival and overall survival were observed for patients with NK cell reconstitution above the median for the group as compared to those below; no such trends were observed regarding T cell subsets. Greater NK cell reconstitution trended towards ultimate eradication of minimal residual disease following ASCT, but no such trends were observed for T cell subsets. Conclusions: Early NK cell recovery predicts survival following autologous and allogeneic SCT in a number of hematologic malignancies; however, little is known regarding this phenomenon in CLL. To our knowledge, these are the first findings to implicate a potentially important therapeutic role for early NK cell compartment recovery in CLL following ASCT. Further research into restoring and augmenting NK cell function following RIC/ASCT for CLL is warranted.

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Releasing CD8+ Treg Mediated Suppresssion Enhances Anti-Viral Immune Response

Blood ◽

10.1182/blood.v122.21.2280.2280 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2280-2280

Author(s):

Tobias A.W. Holderried ◽

Hye-Jung Kim ◽

Philipp A Lang ◽

Harvey Cantor

Keyword(s):

Immune Response ◽

T Cells ◽

Viral Infection ◽

Cd8 T Cells ◽

Chronic Infection ◽

Cd8 T Cell ◽

Chronic Viral Infection ◽

Target Cells ◽

Wild Type ◽

Self Tolerance

Abstract Recent findings have shown that a small subset of IL-15 dependent CD8+ regulatory T cells is essential for maintenance of self- tolerance and prevention of autoimmune disease in mice (Kim et al., Nature 2010). These CD8+ T cells target CD4+ follicular T helper (TFH) cells through recognition of the murine class Ib MHC molecule Qa-1 (HLA-E in man), resulting in perforin-dependent elimination of target cells and diminished antibody production in the steady state and during disease. This analysis was based on generation of Qa-1 knock-in mice (D227K mice) that harbor a single Qa-1 D→K amino acid exchange point mutation at position 227 that abrogates binding of Qa-1/peptide to the CD8/TCR complex. B6.Qa-1 D227K mutant mice develop severe autoimmune disease marked by generation of autoantibodies to multiple tissues, lymphocyte infiltration into non-lymphoid tissues and lethal glomerulonephritis. Qa-1-restricted CD8+ Treg are characterized by the CD44+CD122+Ly49+ phenotype (Kim et al., PNAS 2011). Here, we analyzed the contribution of CD8+ Treg to modulation of the anti-viral immune response. Virus-specific CD8+ cytotoxic T cells are of central importance for successful control of the Lymphocytic Choriomeningitis Virus (LCMV). LCMV clone 13, however, a genetic variant of LCMV Armstrong, persists in the host and chronic antigen exposure leads to exhaustion of CD8+ T cells and continuous tissue inflammation. The contribution of CD8+ Treg in the anti-viral immune response to acute and chronic viral infection remained elusive so far. We found that CD8+ Treg not only control self-tolerance but also diminish the immune response to viral infection. By comparing wild-type and D227K mutant mice after infection with LCMV Armstrong or LCMV clone 13, we observed in both cases reduced effector CD8+ T cell responses. This was true for polyclonal CD44+CD62L– CD8+ T cells as well as LCMV-specific gp33+ effector CD8+ T cells. During acute infection KLRG1+CD127-CD44+CD62L- cells (short-lived effector CD8+ cells) (Joshi et al., Immunity 2007) were particularly diminished as well as effector cytokines in wild-type mice compared to D227K mice. In contrast, increased effector responses in D227K mice resulted in enhanced control of virus and reduced inflammation of tissues. During chronic infection with LCMV, wild-type mice become severely ill and present with a pronounced clinical phenotype. Increased effector CD8+ T cell immune responses in D227K mice resulted in dramatic alleviation of disease. During late stage of chronic infection, D227K mice showed enhanced virus control and reduced tissue pathology compared with wild-type mice. Interestingly, expression of inhibitory receptors such as PD-1, 2B4 and LAG3 were increased in wild-type mice whereas activating receptors such as NKG2D and KLRG1 were increased in D227K mice, resulting in a memory phenotype in D227K mice compared with exhausted CD8+ T cells in wild-type mice. Adoptive transfer experiments revealed that CD8+ Treg directly suppress CD8+ target cells and thereby inhibit induction of a robust anti-viral response. Taken together, we show that Qa-1-restricted CD8+ Treg have a direct inhibitory effect on effector CD8+ T cells during acute and chronic viral infection, resulting in a more violent disease and diminished recovery. These data suggest that depletion or inactivation of CD8+ Treg represents a potentially effective strategy to enhance anti-viral immunity. Disclosures: No relevant conflicts of interest to declare.

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Lymphocyte reconstitution in multiple myeloma patients after allogeneic G-CSF-mobilized hematopoietic progenitor cells transplantation from HLA-identical siblings

Journal of Clinical Oncology ◽

10.1200/jco.2009.27.15_suppl.7098 ◽

2009 ◽

Vol 27 (15_suppl) ◽

pp. 7098-7098

Author(s):

M. Martino ◽

R. Fedele ◽

G. Irrera ◽

G. Messina ◽

M. Cuzzola ◽

...

Keyword(s):

Multiple Myeloma ◽

T Cells ◽

T Cell ◽

Progenitor Cells ◽

Cd8 T Cells ◽

Immune Reconstitution ◽

Median Number ◽

Hematopoietic Progenitor Cells ◽

Immune Recovery ◽

Hematopoietic Progenitor

7098 Background: Allogeneic transplantation of G-CSF-mobilized hematopoietic progenitor cells (HPC) results in rapid and complete engraftment in a large proportion of patients and in relatively fast immune recovery. Methods : We have analyzed by flow cytometry the immune reconstitution in 19 patients (pts) affected by multiple myeloma undergone to allogeneic HPC transplant from HLA-identical related donors after nonmyeloablative conditioning regimen with fludarabine 90 mg/m2 and cyclophosphamide 900 mg/m2. In each patient a comparable number of mononuclear cells, CD3+ T lymphocytes and CD34+ progenitor cells was infused. To evaluate the kinetics of the immune reconstitution, the overall number of total lymphocytes, T, B and NK cells of each patient were assessed before and 1, 2, 3, 6, 12, 18, 24, 30, 36 months after allogeneic HPC transplant. Results: Overall T cell reconstitution was in all the pts at 3 months, since at that time the CD3+ T cell median number was 880 cells/microl (r. 589–1,357). However, in all pts high numbers of CD3+ T cells were achieved at 12 months after transplant (median 1,326 cells/microl, r. 850–2,309). The CD4+ T cell median number was 281 cells/microl (r. 185–433) at 6 months, 391 cells/microl (r. 303–505) at 12 months, 603 cells/microl (r. 433–736) at 18 months. The CD8+ T cell median number was increased from the transplant to 18 months in which it was 1,489 cells/microl (r. 760–1,976). The decrease of CD8+ T cells with the normalization of CD4+/CD8+ ratio was observed at 30 months when CD4+ T cells were 650 cells/microl (r. 370–989) and CD8+ T cells were 690 cells/microl (r. 445–1,743). B cells recovery was observed at 18 months with a median number of 194 cells/microl (r. 40–404). The faster reconstitution was documented for NK cells with a median number of 314 cells/microl (r. 61–647) at 2 months. Conclusions: the complete immune reconstitution in our pts was achieved at 30 months after transplant. Our objective is to evaluate if this slow immune recovery is associated with a high incidence of infectious diseases and a low incidence of chronic GVHD. No significant financial relationships to disclose.

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