Pneumocystis jirovecii pneumonia after CD4+ T‐cell recovery subsequent to CD19‐targeted chimeric antigen receptor T‐cell therapy: A case report and brief review of literature

Abstract Background CD19‐targeted chimeric antigen receptor (CAR)‐T cell therapy involves administration of patient‐derived T cells that target B cells, resulting in B‐cell depletion and aplasia. In immunity against Pneumocystis jirovecii (Pj), CD4+ T cells and, more recently, B cells, are generally considered important. Antigen presentation by B cells to CD4+ T cells is particularly important. Trimethoprim‐sulfamethoxazole (TMP/SMX) for Pj pneumonia (PJP) prophylaxis is generally discontinued when the CD4+ T‐cell count is >200/μL. Here we report the first case, to our knowledge, of PJP in a patient with a CD4+ T cell count of >200/μL after CAR‐T cell therapy. Case A 14‐year‐old girl developed hemophagocytic lymphohistiocytosis (HLH) after cord blood transplantation (CBT) for relapsed precursor B‐cell acute lymphoblastic leukemia (B‐ALL). Twenty‐one months after CBT, she was diagnosed with combined second relapse in the bone marrow and central nervous system. The patient was treated with CD19‐targeted CAR‐T cell therapy for the relapse. After CAR‐T cell therapy, the patient remained in remission and continued to receive TMP/SMX for PJP prophylaxis. Seven months after CAR‐T cell therapy, CD4+ T cells recovered and TMP/SMX was discontinued. The B‐cell aplasia persisted. Ten months after CAR‐T cell therapy, the patient developed PJP. The patient was also considered to have macrophage hyperactivation at the onset of PJP. Treatment with immunoglobulin, TMP/SMX, and prednisolone was initiated, and the patient's symptoms rapidly ameliorated. Conclusion The patient in the present case developed PJP despite a CD4+ T‐cell count of >200/μL after CAR‐T cell therapy, probably because of inadequate CD4+ T‐cell activation caused by B‐cell depletion after CAR‐T cell therapy and repeated abnormal macrophage immune responses after CBT. It is important to determine the duration of TMP/SMX for prophylaxis after CAR‐T cell therapy according to each case, as well as the CD4+ T‐cell count.


| INTRODUCTION
CD19-targeted chimeric antigen receptor (CAR)-T cell therapy involves administration of patient-derived T cells that target B cells, resulting in B-cell depletion and aplasia.Various infections can occur after CAR-T cell therapy.Among these, fungal infections are commonly caused by invasive aspergillosis and candidiasis; however, Pneumocystis jirovecii pneumonia (PJP) is a rare complication. 1,2Although the immunological mechanism in PJP remains unclear, CD4+ T cells are generally given importance because of their prevalence in patients with acquired immunodeficiency syndrome (AIDS), which is characterized by CD4+ T-cell depletion. 35][6][7][8][9] After CAR-T cell therapy, trimethoprim-sulfamethoxazole (TMP/SMX) for PJP prophylaxis is usually discontinued within 6 months to 1 year or when the CD4+ T-cell count is >200/μL. 10,11Here we report the first case, to our knowledge, of PJP in a patient with a CD4+ T-cell count of >200/ μL after CD19-targeted CAR-T cell therapy.This case is unique because B-cell aplasia after CAR-T cell therapy may have contributed to the development of PJP.she developed autoimmune cytopenia with the production of antineutrophil antibodies, anti-erythrocyte antibodies, and plateletassociated IgG (PA-IgG), as well as pleural effusion and ascites.She was treated with prednisolone, cyclosporine, mycophenolate mofetil, and rituximab.Twenty-one months after CBT, she presented with dyspnea, dysuria, diarrhea, and disorders of consciousness and was diagnosed with combined second relapse in the bone marrow and central nervous system.The ferritin level was 10 606 ng/mL at the second relapse.She underwent multidrug chemotherapy, intrathecal chemotherapy, and whole-brain irradiation.Subsequently, she was referred to Keio University Hospital for CD19-targeted CAR-T cell therapy in September 2021, 1 month after diagnosis of the second relapse.CD19-targeted CAR-T cell therapy was performed, and 2.2 Â 10 6 /kg/dose of CAR-T cells were administered (Table S1).

| CASE
Three to seven days after CAR-T cell therapy, the patient developed fever as grade 1 cytokine release syndrome.Pancytopenia requiring blood transfusion persisted for 2 months after CAR-T cell therapy.
The patient remained in remission after the therapy and continued to receive TMP/SMX for PJP prophylaxis.The CD4+ T-cell counts remained above 200/μL from 3 months and above 500/μL from 6 months after CAR-T cell therapy.Phytohemagglutinin (PHA)induced lymphocyte proliferation was normal.TMP/SMX therapy was discontinued 7 months after CAR-T cell therapy.CD19+ B-cell aplasia persisted, and IgG levels were maintained at 400-800 mg/dL with periodic immunoglobulin replacement therapy.Ten months after CAR-T cell therapy, she presented to our hospital with fever, cough, and dyspnea for 5 days.On admission, her body temperature was

| DISCUSSION
PJP after CAR-T cell therapy is rare, with only nine adult cases reported thus far.These included five cases with a CD4+ T-cell count of <200/μL at onset and four cases with unknown CD4+ T-cell data.
3][14] In the present case, TMP/SMX was discontinued 7 months after CAR-T cell therapy for post-CBT relapse of B-ALL under the conditions of a CD4+ T-cell count of >500/μL and normal PHA-induced lymphocyte proliferation.Three months after discontinuation, the patient developed PJP despite a sufficient CD4+ T-cell count of 771/μL.It is possible that the total number of CD4+ T cells included those derived from CAR-T cells.Although fluorescence activated cell sorter analysis was not performed in this case, it is expected that the CD4+ T cells derived from CAR-T cells were only a part of the total count at 10 months after CAR-T cell therapy. 15The role of CD4+ T cells derived from CD19-targeted CAR-T cells in immunity against PJP is not clear, but it is not expected to be significant because they are only a part of the total count.To our knowledge, this is the first case of PJP in a patient with a CD4+ T-cell count of >200/ μL after CD19-targeted CAR-T cell therapy.1][12][13][14] There are two possible reasons for the development of PJP despite a CD4+ T-cell count of >200/μL in this patient.
First is the inadequate activation of CD4+ T cells owing to B-cell depletion after CAR-T cell therapy.4][5][6][7][8][9] Antigen presentation by B cells to CD4+ T cells was found to be particularly important in PJP mouse models. 4The study showed that CD4+ T cells were not activated when B cells were depleted, even when sufficient dendritic cells were present as antigen-presenting cells.It is also shown that IgG and IgM produced by B cells are involved in immunity against Pj; however, these factors alone are not sufficient. 4Our patient had a low IgG level of 244 mg/dL on admission, which may have contributed to the onset of PJP.However, the patient was seen several days after the onset of symptoms, and it is possible that her IgG level was consumptively low as a result of Pj infection.In the immune response against Pj, B cells present antigens to CD4+ T cells, which are mutually activated.Activated CD4+ T cells act against antigens by producing inflammatory cytokines and activating alveolar macrophages.In contrast, activated B cells produce antibodies specific for the Pj antigen and promote opsonization by macrophages. 16Since CD19-targeted CAR-T cell therapy results in B-cell aplasia, the absence of antigen presentation from B cells results in the inactivation of CD4+ T cells; furthermore, no antibodies are produced by B cells.In this case, the CD4+ T cells were active because of normal PHA-induced lymphocyte proliferation, but their activity specific to Pj antigen was probably reduced because of the absence of Pj antigen presentation from B cells.
The second reason is an abnormal macrophage response after CBT.8][19] HLH is also a common complication after CBT. 20r patient developed HLH with bone marrow findings of hemophagocytic macrophages and delayed engraftment.Anti-neutrophil antibodies, anti-erythrocyte antibodies, and PA-IgG produced during pancytopenia after engraftment are associated with autoimmuneassociated hemophagocytic syndrome (AAHS). 21The patient also had pleural effusion and ascites.These findings suggested that cytokine storm and abnormal activation of macrophages were present.Dyspnea, dysuria, diarrhea, impaired consciousness, and an abnormally high ferritin level of 10 606 ng/mL at the second relapse, as well as the prolonged pancytopenia after CAR-T cell therapy, were also expected to be related to the cytokine storm and macrophage hyperactivation. 17,22,23At the onset of PJP, the patient had a high ferritin level, a high soluble IL-2R level, elevated liver enzymes, thrombocytopenia, and hypoproteinemia, which were also probable indicators of an abnormal macrophage immune response, as observed in HLH. 24though the M1/M2 phenotype of the macrophages was not examined, frequent occurrences of cytokine storm associated with abnormal macrophage activation were considered on the basis of her clinical course, high ferritin level, and high soluble IL-2R level, among other factors.Immunity against Pj requires macrophages and cytokines, which can cause lung injury at the same time. 16Previous reports have documented the association between PJP and HLH 25 as well as the association between HLH and lung injury. 26Late-onset HLH after transplants is triggered by viral and fungal infections. 27In this case, Pj infection was thought to have triggered abnormal macrophage activation and cytokine storm, resulting in lung injury and symptomatic PJP.

F
I G U R E 1 Imaging findings for a patient with Pneumocystis jirovecii pneumonia (PJP) after CD19-targeted chimeric antigen receptor T-cell therapy.(A) Chest radiography shows diffuse groundglass opacities indicated by red arrow marks in both lungs.(B) Chest computed tomography also shows diffuse ground-glass opacities indicated by red arrow marks in both lungs, especially the dorsal regions.These findings are typical radiological features of PJP.
Thus, the patient developed PJP despite a CD4+ T-cell count of >200/μL because of a predisposition to abnormal macrophage activation and cytokine storm after CBT as well as B-cell aplasia after CAR-T cell therapy combined with decreased CD4+ T-cell function specific for Pj.In patients predisposed to abnormal macrophage activation and cytokine storm, a risk of PJP complications should be considered even if CD4+ T cells recover under B-cell depletion after CD19-targeted CAR-T cell therapy.In such cases, it may be better to continue TMP/SMX for prophylaxis until B-cell recovery after CAR-T cell therapy.It is important to determine the duration of TMP/SMX for prophylaxis after CAR-T cell therapy according to each case as well as the CD4+ T-cell count.However, this conclusion is based on the findings for a single case, and further studies are needed for a better understanding of the involved immune mechanisms and optimization of prophylactic strategies for patients undergoing CAR-T cell therapy.