Natural killer (NK) cells mediate anti-AML responses and previously published clinical trials of adoptive allogeneic NK cell therapy provide proof-of-principle that NK cells may eliminate leukemia ...cells in patients. However, complete remissions occur in 30-50% of patients with active AML and are typically of limited duration. Thus, improvements are needed for this promising cellular immunotherapy strategy. Following paradigm-shifting studies in mice, it was established that human NK cells exhibit an innate 'memory-like' responses following a brief, combined pre-activation with IL-12, -15, and -18 (Romee R et. al., Blood, 2012). These long-lived memory-like NK cells have an enhanced ability to produce IFN-g in response to restimulation with cytokines or activating receptor ligation, even following extensive proliferation. We hypothesized that memory-like NK cells exhibit enhanced responses to myeloid leukemia.
Compared to control NK cells from the same donor, IL-12/15/18-induced memory-like NK cells produced significantly increased IFN-g upon co-culture with primary AML blasts in vitro (P<0.001), following 7 days of rest in low dose IL-15 vitro. In addition, memory-like NK cells had increased granzyme B expression (P<0.01), and enhanced killing of K562 leukemia targets in vitro (P<0.05). Utilizing an in vivo xenograft model of human NK cells in NSG mice (Leong J et. al., BBMT, 2014), IL-12/15/18-induced memory-like NK cells that differentiated in NSG mice for 7 days exhibited increased IFN-g responses after ex vivo re-stimulation with K562 leukemia, confirming their memory-like functionality (P<0.05). To test in vivo responses to human leukemia in this model, luciferase-expressing K562 cells were engrafted into NSG mice (1x106/mouse, IV), and on day 3, groups of mice were injected with IL-12/15/18-pre-activated or control NK cells from the same donor (4x106/mouse). Mice treated with a single dose of memory-like NK cells exhibited significantly improved in vivo leukemia control measured by whole mouse bioluminescent imaging (P=0.03), as well as overall survival (P<0.05), compared to mice treated with control or no NK cells.
Based on these pre-clinical findings, we initiated a first-in-human clinical trial of HLA-haploidentical IL-12/15/18-induced memory-like NK cells in patients with AML (NCT01898793). Relapsed/refractory (rel/ref) AML patients receive lymphodepleting non-myeloablative flu/cy conditioning, infusion of a single dose of CD56+CD3- memory-like donor NK cells, followed by two weeks of low dose rhIL-2. Three patients were treated at dose level 1 (0.5x106 cells/kg) and two patients treated at dose level 2 (1.0x106/kg) with no DLTs observed, and accrual continues. Correlative analyses utilizing donor-specific HLA mAbs allow tracking of donor memory-like NK cell frequency and function following adoptive transfer. Donor memory-like NK cells were detectable in the PB and BM of all tested patients with informative HLA (4/5), peak in frequency at 7-8 days post-infusion, and contract after 14-21 days as expected following recipient T cell recovery (Figure). Memory-like NK cells exhibit significantly increased Ki67%+ as a marker of proliferation at day 7 97.8+1.0% (donor) vs. 21.6+5.5% (recipient), mean+SEM, P<0.001. Moreover, functional analyses of NK cells at days 7-8 post-infusion reveal increased numbers of donor IFN-g+ NK cells following restimulation with K562 leukemia cells in the same blood 1009+590 (donor) vs. 8+3 (recipient) IFN-g+ NK cells or BM 686+423 (donor) vs. 4+2 (recipient) IFN-g+ NK cells samples. Two of four evaluable patients treated with memory-like NK cells had leukemia free BM and PB at days 14 post-therapy, which correlated with BM NK cell frequency and IFN-g production (Figure). CIML007 had rel/ref AML with 48% BM blasts pre-therapy, and had no evidence of leukemia on day 14, 28, and 100 BM biopsies, and has an ongoing complete remission more than 100 days after this therapy. CIML009 had 80% BM blasts pre-therapy, and had no evidence of leukemia on day 14 BM biopsy post-infusion. Thus, human IL-12/15/18-induced memory-like NK cells expand and have enhanced anti-AML function following adoptive transfer in patients, thereby constituting a promising translational innovation for immunotherapy of AML.
Display omitted
Fehniger:Celgene: Research Funding.
Natural killer (NK) cells are innate lymphoid cells that mediate anti-tumor responses via cytotoxicity and effector cytokine production. Human NK cells are divided into two subsets based on relative ...expression of CD56 (CD56bright and CD56dim) that classically participate in distinct functions. Cytotoxic CD56dim NK cells respond to tumor targets without prior stimulation, resulting in target cell death and transient secretion of effector cytokines (e.g. IFN-γ). In contrast, immunoregulatory CD56bright NK cells secrete abundant IFN-γ and other cytokines in response to cytokine receptor stimulation, but respond minimally to tumor target-based triggering. As a result of this dichotomy, translational strategies to enhance NK cell function for cancer immunotherapy have focused exclusively on the CD56dim subset.
Based upon studies in mouse NK cells, we hypothesized IL-15 priming would enhance CD56bright anti-tumor functionality. Primary human NK cells from healthy donors were purified (>95% CD56+CD3-), cultured overnight in medium alone (control) or medium with 5 ng/mL rhIL-15 (primed), washed, and assayed for anti-tumor responses. IL-15 priming significantly enhanced multiple CD56bright NK cell functional responses to the prototypical AML target cell line K562 (CD107a+: control 20% vs. primed 59%, p<0.001; IFN-γ+: 3% vs. 27%, p<0.001; TNF: 3% vs. 20%, p<0.001), as well as primary AML blasts (N=3 unique AML sample: CD107a+,7% vs. 30%, p<0.001; IFN-γ 2% vs. 14%, p<0.001; TNF: 2% vs. 22%, p<0.001). IL-15-priming of CD56bright NK cells was evident after 1 hour, and peaked after only 6 hours. In addition, flow-sorted IL-15-primed CD56bright NK cells exhibited markedly increased killing of leukemia target cells. Similar results for functional comparisons were observed using primary NK cells from AML patients. Unexpectedly, IL-15-primed CD56bright NK cell anti-leukemia responses significantly exceeded those of IL-15-primed CD56dim NK cells. Multidimensional CyTOF analyses revealed that the maturity status of CD56dim NK cells did not determine the extent to which they could be primed by IL-15.
In response to IL-15, we observed selective activation of the PI3K/Akt/mTOR (4.2 fold increase CD56bright NK cells, 1.2 CD56dim NK cells, p<0.001) and Ras/Raf/MEK/ERK (1.9 fold increase CD56bright NK cells, 1.2 CD56dim NK cells, p<0.001) pathways in CD56bright NK cells. The Jak/STAT pathway was strongly activated in both CD56bright and CD56dim subsets. These data suggested a signaling mechanism for preferential CD56bright NK cell priming by IL-15. Indeed, small molecule inhibitors of PI3K/Akt/mTOR and Ras/Raf/MEK/ERK pathways abrogated the anti-tumor responses of IL-15-primed CD56bright NK cells, supporting this idea. Several NK cell effector mechanisms were enhanced in IL-15-primed CD56bright NK cells, likely contributing to their augmented anti-tumor responsiveness. These included increased cytotoxic effector protein levels (granzyme B and perforin), as well as improved conjugate formation with tumor targets. Furthermore, blocking experiments demonstrated that IL-15-primed CD56bright NK cell anti-tumor responses depended on LFA-1, CD2, and NKG2D receptor interactions with target cells. Finally, since IL-15-based therapeutics are being investigated in clinical trials, we tested whether the IL-15 super-agonist complex ALT-803 primes CD56bright NK cells in vivo in cancer patients. There was an increase in leukemia target cell-triggered degranulation (CD107a+ unprimed 7% vs. primed 30%, p<0.001) and cytokine production (IFN-γ+ 6% vs. 31%, p<0.01; TNF+ 2% vs. 20%, p<0.05) by CD56bright NK cells 24 hours post-ALT-803 administration to multiple myeloma patients, compared to unprimed, pre-therapy values.
Collectively, these results suggest that CD56bright NK cells play an under-appreciated anti-tumor role in settings of abundant IL-15, such as following lymphodepleting chemotherapy, during preparation for stem cell transplantation, at sites of inflammation, or after exogenous IL-15 administration. Since CD56bright NK cells have different in vivo tissue localization (secondary lymphoid organs), distinct inhibitory, activating, and chemokine receptor expression compared to CD56dim NK cells, and are thought to be the most abundant NK cell subset when considering all human tissues, this study identifies a promising NK cell subset to harness for cancer immunotherapy.
No relevant conflicts of interest to declare.
NK cells, lymphocytes of the innate immune system, are important for defense against infectious pathogens and cancer. Classically, the CD56dim NK cell subset is thought to mediate antitumor ...responses, whereas the CD56b,ightsubset is involved in immunomodulation. Here, we challenge this paradigm by demonstrating that brief priming with IL-15 markedly enhanced the antitumor response of CD56b'ight NK cells. Priming improved multiple CD56bright cell functions: degranulation, cytotoxicity, and cytokine production. Primed CD56b'ight cells from leukemia patients demonstrated enhanced responses to autologous blasts in vitro, and primed CD56bright cells controlled leukemia cells in vivo in a murine xenograft model. Primed CD56b'ight cells from multiple myeloma (MM) patients displayed superior responses to autologous myeloma targets, and furthermore, CD56b'ight NK cells from MM patients primed with the IL-15 receptor agonist ALT-803 in vivo displayed enhanced ex vivo functional responses to MM targets. Effector mechanisms contributing to IL-15-based priming included improved cytotoxic protein expression, target cell conjugation, and LFA-1-, CD2-, and NKG2D-dependent activation of NK cells. Finally, IL-15 robustly stimulated the PI3K/Akt/mTOR and MEK/ERK pathways in CD56b'ightcompared with CD56dim NK cells, and blockade of these pathways attenuated antitumor responses. These findings identify CD56bright NK cells as potent antitumor effectors that warrant further investigation as a cancer immunotherapy.
NK cells, lymphocytes of the innate immune system, are important for defense against infectious pathogens and cancer. Classically, the CD56.sup.dim NK cell subset is thought to mediate antitumor ...responses, whereas the CD56.sup.bright subset is involved in immunomodulation. Here, we challenge this paradigm by demonstrating that brief priming with IL-15 markedly enhanced the antitumor response of CD56.sup.bright NK cells. Priming improved multiple CD56.sup.bright cell functions: degranulation, cytotoxicity, and cytokine production. Primed CD56.sup.bright cells from leukemia patients demonstrated enhanced responses to autologous blasts in vitro, and primed CD56.sup.bright cells controlled leukemia cells in vivo in a murine xenograft model. Primed CD56.sup.bright cells from multiple myeloma (MM) patients displayed superior responses to autologous myeloma targets, and furthermore, CD56b'igl,t NK cells from MM patients primed with the IL-15 receptor agonist ALT-803 in vivo displayed enhanced ex vivo functional responses to MM targets. Effector mechanisms contributing to IL-15- based priming included improved cytotoxic protein expression, target cell conjugation, and LFA-1-, CD2-, and NKG2D- dependent activation of NK cells. Finally, IL-15 robustly stimulated the PI3K/Akt/mTOR and MEK/ERK pathways in CD56.sup.bright compared with CD56.sup.dim NK cells, and blockade of these pathways attenuated antitumor responses. These findings identify CD56.sup.bright NK cells as potent antitumor effectors that warrant further investigation as a cancer immunotherapy.
Herein the descriptive label-free mass spectrometry (LC-MS/MS) proteome of Erythroxylum pungens (roots, barks, leaves) harvested from its natural habitat is described. Processes related to defense ...were most important in roots. Main protective proteins differed among organs, suggesting part-specific strategies of environmental adaptation.
The influence of a SARS-CoV-2 infection on inflammatory bowel disease (IBD) has not yet been well characterized and it is unclear whether this requires an adaptation of the immunosuppressive therapy.
...A national register was established for the retrospective documentation of clinical parameters and changes in immunosuppressive therapy in SARS-CoV-2 infected IBD patients.
In total, only 3 of 185 IBD patients (1.6 %) were tested for SARS-CoV-2 infection because of abdominal symptoms. In the course of COVID-19 disease, 43.5 % developed diarrhea, abdominal pain or hematochezia (risk of hospitalization with vs. without abdominal symptoms: 20.0 % vs. 10.6 %, p < 0.01). With active IBD at the time of SARS-CoV-2 detection, there was an increased risk of hospitalization (remission 11.2 %, active IBD 23.3 % p < 0.05). IBD-specific therapy remained unchanged in 115 patients (71.4 %); the most common change was an interruption of systemic therapy (16.2 %).
New abdominal symptoms often appeared in SARS-CoV-2 infected IBD patients. However, these only rarely led to SARS-CoV-2 testing. A high IBD activity at the time of SARS-CoV-2 detection was associated with an increased risk of hospitalization.
To handle the large scale of whole slide images in computational pathology,
most approaches first tessellate the images into smaller patches, extract
features from these patches, and finally ...aggregate the feature vectors with
weakly-supervised learning. The performance of this workflow strongly depends
on the quality of the extracted features. Recently, foundation models in
computer vision showed that leveraging huge amounts of data through supervised
or self-supervised learning improves feature quality and generalizability for a
variety of tasks. In this study, we benchmark the most popular vision
foundation models as feature extractors for histopathology data. We evaluate
the models in two settings: slide-level classification and patch-level
classification. We show that foundation models are a strong baseline. Our
experiments demonstrate that by finetuning a foundation model on a single GPU
for only two hours or three days depending on the dataset, we can match or
outperform state-of-the-art feature extractors for computational pathology.
These findings imply that even with little resources one can finetune a feature
extractor tailored towards a specific downstream task and dataset. This is a
considerable shift from the current state, where only few institutions with
large amounts of resources and datasets are able to train a feature extractor.
We publish all code used for training and evaluation as well as the finetuned
models.
To assess the effect of tubal sterilization on the risk of hysterectomy, we studied 7414 women aged 20 to 49 years who had had a tubal sterilization at a health maintenance organization between ...January 1, 1968, and December 31, 1983. Compared with a population-based cohort of nonsterilized women, women sterilized while 20 to 29 years old were 3.4 times more likely to have had a subsequent hysterectomy (95% confidence interval, 2.4 to 4.7). Adjustment for the effects of potential confounders with a subset of 276 women did not appreciably alter this association. For multivariate comparisons with 5323 wives of vasectomized men, there was no significant elevation in the risk of hysterectomy following sterilization among women sterilized while 20 to 29 years old. Tubal sterilization was not associated with hysterectomy for married women who underwent tubal sterilization at age 30 or older. These results do not support a biological basis for the relationship between tubal sterilization and hysterectomy.
Cytokine-induced memory-like natural killer (NK) cells differentiate following short-term pre-activation with interleukin (IL)-12, IL-15, and IL-18 and display enhanced effector function in response ...to cytokines or tumor targets for weeks following the initial pre-activation. Conventional NK cell function is dependent upon a licensing signal, classically delivered by an inhibitory receptor engaging its cognate MHC class I ligand. How licensing status integrates with cytokine-induced memory-like NK cell responses is unknown. We investigated this interaction using killer immunoglobulin-like receptor (KIR)- and human leukocyte antigen (HLA)-genotyped primary human NK cells. Memory-like differentiation resulted in enhanced IFN-γ production triggered by leukemia targets or FcγRIIIa ligation within licensed NK cells, which exhibited the highest functionality of the NK cell subsets interrogated. IFN-γ production by unlicensed memory-like NK cells was also enhanced, to a level comparable to that of licensed control NK cells. Mechanistically, differences in responses to FcγRIIIa-based triggering were not explained by alterations in key signaling intermediates, indicating that the underlying biology of memory-like NK cells is distinct from that of adaptive NK cells in HCMV
+
individuals. Additionally, memory-like NK cells responded robustly to cytokine receptor re-stimulation with no impact of licensing status. These results demonstrate that both licensed and unlicensed memory-like NK cell populations have enhanced functionality, which may be translated to improve leukemia immunotherapy.
PDF
