The emergence of chimeric antigen receptor (CAR) T-cell therapy has significantly revolutionized the current treatment paradigm, providing a highly promising alternative for individuals suffering from refractory or relapsed B-cell non-Hodgkin lymphoma and B-cell acute lymphoblastic leukemia (r/r B-ALL) [1, 2, 3]. However, despite the remarkable efficacy of CAR T-cell therapy, CAR T-cell recipients are highly susceptible to infection, which affects their prognosis due to many insults to immune function, including the malignancy, prior cytotoxic treatments, and the on-target-off-tumor effects of CAR T-cells [4, 5, 6]. Cytokine release syndrome (CRS) is the most common toxicity; the clinical presentations of CRS are typically characterized by fever, hypotension, and respiratory insufficiency, which are hard to distinguish from severe infection [7, 8, 9]. Clinically, rapid disease change is usually observed after CAR T-cell infusion and some of them develop seriously, especially grade 3–4 CRS, whereas the results of CAR transgene copy number require 24–48 h, which is obviously not enough to meet urgent clinical needs. Although CRS can be successfully managed with interleukin-6 (IL-6) receptor monoclonal antibodies or corticosteroids, these drugs can also increase the incidence of infection [10, 11, 12]. Thus, the early identification between CRS and infections is important for providing appropriate management and improving the clinical outcome of these patients.

Current methods to diagnose infection complications in CAR T-cell recipients, including culture and non-culture-based methods such as nucleic acid detection/metagenomic next-generation sequencing (mNGS) and antigen detection by conventional serological antibody assays, are often time-consuming and expensive [13, 14, 15]. Analysis of peripheral blood smears (PBS) is a widely accepted and reliable diagnostic method in laboratory settings, enabling the rapid preliminary diagnosis of numerous hematological disorders [16, 17, 18]. By detecting the characteristic footprints left by various infections on blood cell morphology, analysis of PBS can quickly provide valuable information on infection [19, 20, 21]. Recently, it was reported that morphology or CAR T-cells in PBS can provide important clues to assess their kinetics [22, 23]. The morphology of CAR T-cells is very important, especially the atypical characteristics of the activation state, which are similar to the non-specifically activated T cells seen in some infections. However, few data are available on the morphological characteristics of CAR T-cells after infusion in humans, especially for distinguishing infections. In this study, we investigated the characteristics of CAR T-cells in PBS when the recipients had a fever after CAR T-cell infusion, preliminarily to distinguish CRS from infection, and combined a conventional pathogen detection method with mNGS to investigate the cause of fever.

Despite the great successes of CAR T-cell therapy in hematological malignancy, infection remains a main obstacle in this field. Recipients are immunocompromised after CAR T-cell infusion, making them highly susceptible to opportunistic infections [26, 27]. A previous study showed that in patients with infection after CAR T-cell infusion, significantly higher mortality within 90 days was noted [13, 28]. Consequently, there is a justified need for studies focusing on enhancing infection prevention and treatment strategies for these high-risk patients.

In this study, we described the morphology of CD7, CD19 and CD22 CAR T-cells in detail and found that PBS morphology and atypical lymphocyte percentage were capable of reflecting the clonal kinetics of CAR T-cells. Lesesve et al. [29] examined the PBS of a patient with large cell B-cell lymphoma treated with CD19 CAR-T, and the morphological description of CAR T-cells detected under the microscope was similar to what we observed. In addition to CD19 CAR T-cells, we also observed two other types of CAR T-cells, and the results showed that different CAR T-cells had similar morphological characteristics. This finding supplements the morphological characteristics of CAR T-cells after infusion, which is conducive to the accurate and rapid identification of CAR T-cells. In a previous study, Faude et al. [30] established that the peripheral blood absolute lymphocyte count parameters can be used as a surrogate to assess CAR T-cell expansion, which was also found in our study. We further found that the percentage of atypical lymphocytes in PBS was both positively correlated with CAR transgene copy number and absolute lymphocyte count (p 0.05), suggesting that PBS could be a reliable method to assess the expansion of CAR T-cells in these recipients. Furthermore, we grouped the patients by whether there were infection events and compared the PBS morphology of these two groups. Interestingly, although they were similar in many clinical parameters, including the grade of CRS, ICANS, white blood cell count, absolute lymphocyte count, or some inflammatory markers (ferritin, CRP, PCT), the percentage of atypical lymphocytes was significantly higher in the non-infection group (p 0.05), suggesting that the recurrent fever in this group was mainly due to CAR T-cell proliferation. Regarding the morphology observed in PBS samples, atypical lymphocytes exhibited deep outer and shallow inner cytoplasm, along with loosely and unevenly distributed chromatin, indicating the activated proliferative state of CAR T-cells, which aligned with an increased CAR transgene copy number. In our observations, CAR T-cells exhibited smooth cell membranes and abundant, bright cytoplasm with purplish-red granules showing cytotoxic effects as NK cells, which could be well distinguished from infection-associated lymphocytes (disordered dark blue cytoplasm) and infectious mononucleosis cells (bright light blue cytoplasm). Therefore, it should be noted that if PBS shows a large number of atypical lymphocytes, patients with post-infusion fever should be on high alert for CRS, which is an ideal time to immediately administer appropriate corticosteroids and IL-6 receptor antibodies rather than waiting for CAR transgene number results.

We observed a similar CAR transgene copy number between infection and non-infection groups. Further analyses revealed that in the infection group, two out of nine patients experienced grade 4 CRS and showed the highest CAR transgene copy number in this group (data not shown), suggesting that in patients with infection, the fever may be caused by both pathogenic microbes and CAR T-cells expansion. In addition, IL-6 is increased in critical illnesses such as sepsis and CRS, driven by the activity of antigen-presenting cells and CAR T-cells [31, 32, 33]. In this study, while a low percentage of atypical lymphocytes was noted in patients with infection, a high level of IL-6 was observed in these patients, suggesting that a higher level of IL-6 in CAR T-cell recipients may be due to an infection state rather than CAR T-cell activation only. In this study, a high IL-6 level accompanied by a low percentage of atypical lymphocytes in PBS may have been a potential indicator for the delayed administration of IL-6 receptor antibody treatment, as infections occurred in these fever CAR T-cell recipients.

Both infection and CRS could be involved in the pathogenesis of fever after CAR T-cell infusion [34, 35]. Infection and CRS exhibit similar clinical symptoms [36]. CAR transgene copy number, flow cytometry analyses, mNGS, and conventional methods to investigate CAR T-cell proliferation and infection complications in recipients are expensive and time-consuming. PBS could be a more convenient and reliable method to assess CAR T-cell kinetics. While some patients with infection also experience severe CRS, identifying the cause of fever in such patients is complex, and examination at multiple time points to assess both CAR T-cell kinetics and microbiological tests is necessary, causing an appropriate temporal window of corticosteroids and IL-6 receptor antibody to break the CRS inflammatory activation loop and avoiding the aggravation of infection, which is very important. Therefore, we suggest the diagnosis and management flowchart for patients with recurrent fever post-CAR T-cell infusion (Fig. 6).

Fig. 6.

Flow chart of quick diagnosis and management for recurrent fever patients post CAR T-cell infusion using PBS. CRP, C-reactive protein; PCT, procalcitonin; mNGS, metagenomic next-generation sequencing; CRS, cytokine release syndrome; IL-6, Interleukin-6; TNF-$\alpha$, Tumor necrosis factor-alpha; IFN-$\gamma$, Interferon-gamma.

In summary, our results showed that PBS could be an indicator of CAR T-cell expansion and a quick and reliable method to distinguish causes of fever in the early phase of post-CAR T-cell infusion.

CD7, cluster of differentiation 7; PB, peripheral blood; BM, bone marrow; NK, natural killer; CAR, chimeric antigen receptor; PBS, peripheral blood smears; CRS, cytokine release syndrome; r/r B-ALL, refractory or relapsed B-cell acute lymphoblastic leukemia; r/r DLBCL, refractory or relapsed Diffuse large B cell lymphoma; HSCT, Hematopoietic stem cell transplant; ICANS, immune effector cell-associated neurotoxicity syndrome; HHV6-B, human herpesvirus6-B; CBCs, complete blood count; CRP, C-reactive protein; PCT, procalcitonin; mNGS, metagenomic next-generation sequencing; ddPCR, droplet digital PCR.

The datasets are available upon request.

JW and ZW designed this study, participated in the revision, and approved the final version to be released. LiaH, FM, ZG, and WT managed patients in clinical trials and provided patients’ data. SH, JP, XY, JZ, and LifH analyzed and interpreted the data. SH, JP, and XY wrote the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

The 5 ongoing clinical trials in this study were approved by the institutional review board. This retrospective study was approved by the Medical Ethics Committee of the Department of Hematology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology and Department of Hematology, Shanxi Bethune Hospital, Shanxi Academy of Medical Sciences, Tongji Shanxi Hospital, Third Hospital of Shanxi Medical University, Taiyuan, Shanxi (Medical Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology: TJ-IRB20160314/ TJ-IRB20160310/ [2020] Lun Shen Zi (S205)/ [2022] Lun Shen Zi (S031); Academic Committee of Norman Bethune Hospital (Shanxi Academy of Medical Sciences): XS-2022-9-2).

The authors would like to thank Hebei Senlang Biotechnology Inc., Ltd., Wuhan Bio-Raid Biotechnology Co., Ltd., Nanjing IASO Biotherapeutics Co., Ltd. for technical support of CAR T-cell manufacturing. The abstract of this study had been presented as a poster in the 28th European Hematology Association (EHA) conference. For more details, interested readers are encouraged to visit the conference’s official website https://ehaweb.org/

This research was funded by the National Natural Science Foundation of China, grant number 82070217 (Jia Wei), and the National High Technology Research and Development Program of China, grant number 2021YFA1101502 (Jia Wei).

The authors declare no conflict of interest.