Diversity, localization and (patho)physiology of mature lymphocyte populations in the bone marrow

The bone marrow (BM) is responsible for generating and maintaining lifelong output of blood and immune cells. Besides its key hematopoietic function, the BM acts as an important lymphoid organ, hosting a large variety of mature lymphocyte populations, including B-cells, T-cells, NK(T)-cells and innate lymphoid cells (ILCs). Many of these cell types are thought to only transiently visit the BM, but for others, like plasma cells and memory T-cells, the BM provides supportive niches that promote their long-term survival. Interestingly, accumulating evidence points towards an important role for mature lymphocytes in the regulation of hematopoietic stem cells (HSCs) and hematopoiesis in health and disease. In this review, we describe the diversity, migration, localization and function of mature lymphocyte populations in murine and human BM, focusing on their role in immunity and hematopoiesis. We also address how various BM lymphocyte subsets contribute to the development of aplastic anemia and immune thrombocytopenia, illustrating the complexity of these BM disorders, but also the underlying similarities and differences in their disease pathophysiology. Finally, we summarize the interactions between mature lymphocytes and BM resident cells in HSC transplantation and graft-versus-host disease. A better understanding of the mechanisms by which mature lymphocyte populations regulate BM function will likely improve future therapies for patients with benign and malignant hematological disorders.

An Unusual Case of Prolymphocytic Leukemia Transformation in a Patient With Chronic Lymphocytic Leukemia

B-cell prolymphocytic leukemia (B-PLL) is a rare leukemia characterized by rapidly increasing leukocytosis with splenomegaly and lymphadenopathy. Treatment strategies are largely based on studies of chronic lymphocytic leukemia (CLL). Antibodies against the cell surface protein CD20 are considered to be first-line therapy. A 76-year-old male with known CLL presented 2 weeks after starting chemoimmunotherapy for newly refractory CLL after failing ibrutinib therapy. White blood cell count was elevated at 226.7 × 103/µL. Fluorescent in situ hybridization analysis of a bone marrow specimen showed new development of complex cytogenetics. Flow cytometry revealed B cells appearing slightly dimmer on CD45 and brighter on CD20 compared with typical B-CLL suggestive of less mature lymphocyte forms. The patient was diagnosed with B-PLL and started on obinutuzumab and venetoclax with rapid normalization of white blood cells. This case recapitulates the challenges in diagnosing and treating B-PLL. Ibrutinib resistance is a growing area of study with several proposed mechanisms of acquired resistance. The pathogenesis of B-PLL is not completely understood, although mutations in MYC are presumed to play a role.

SIMPLE PERIPHERAL BLOOD SMEAR FINDINGS OF COVID-19 PATIENTS PROVIDE INFORMATION ABOUT THE SEVERITY OF THE DISEASE AND THE DURATION OF HOSPITAL STAY.

Backround Data about the morphological changes of Covid-19 infection in peripheral blood smear are limited and association with clinical severity of the disease are not known yet. We aimed to examine the characteristics of the cells detected in the pathological rate and / or appearance and whether these findings are related to the clinical course by evaluating the peripheral blood smear at the time of diagnosis in Covid-19 patients. Methods Clinical features, laboratory data, peripheral blood smear of fifty patients diagnosed with Covid-19 by PCR was evaluated at diagnosis. Peripheral smear samples of the patients were compared with the age and sex matched 30 healthy controls. Pictures were taken from the paitients’peripheral blood smear. Patients were divided into two groups. Early and advanced stage patient groups were compared in terms of laboratory data and peripheral smear findings. The relationship between the laboratory values of all patients and the duration of hospitalization was analyzed. Results Pseudo pelger-huet, atypical lymphocytes, vacuole monocytes and pycnotic neutrophils rates were high in the patient group. Increased pseudo pelger-huet anomaly, psodo-pelger huet/mature lymphocyte ratio, decreased number of mature lymphocytes, and eosinophils in peripheral blood smear were observed in the advanced stage patients (p <0.05). A negative correlation was observed between the duration of hospitalization and mature lymphocyte, and monocytes with vacuoles rates (p <0.05). Conclusion Peripheral smear is a cheap, easily performed, and rapid test. Increased pseudo-pelger huet anomaly/mature lymphocytes rate is suggesting advanced stage disease, while high initial monocytes with vacuoles and mature lymphocyte rates at the time of diagnosis may be an indicator of shortened duration of hospitalization.

Leaky Severe Combined Immunodeficiency in Mice Lacking Non-homologous End Joining Factors XLF and MRI

Non-homologous end-joining (NHEJ) is a DNA repair pathway required to detect, process, and ligate DNA double-stranded breaks (DSBs) throughout the cell cycle. The NHEJ pathway is necessary for V(D)J recombination in developing B and T lymphocytes. During NHEJ, Ku70 and Ku80 form a heterodimer that recognizes DSBs and promotes recruitment and function of downstream factors PAXX, MRI, DNA-PKcs, Artemis, XLF, XRCC4, and LIG4. Mutations in several known NHEJ genes result in severe combined immunodeficiency (SCID). Inactivation of Mri, Paxx or Xlf in mice results in normal or mild phenotype, while combined inactivation of Xlf/Mri, Xlf/Paxx, or Xlf/Dna-pkcs leads to late embryonic lethality. Here, we describe three new mouse models. We demonstrate that deletion of Trp53 rescues embryonic lethality in mice with combined deficiencies of Xlf and Mri. Furthermore, Xlf-/-Mri-/-Trp53+/- and Xlf-/-Paxx-/-Trp53+/- mice possess reduced body weight, severely reduced mature lymphocyte counts, and accumulation of progenitor B cells. We also report that combined inactivation of Mri/Paxx results in live-born mice with modest phenotype, and combined inactivation of Mri/Dna-pkcs results in embryonic lethality. Therefore, we conclude that XLF is functionally redundant with MRI and PAXX during lymphocyte development in vivo. Moreover, Mri genetically interacts with Dna-pkcs and Paxx.

Leaky severe combined immunodeficiency in mice lacking non-homologous end joining factors XLF and MRI

Non-homologous end-joining (NHEJ) is a DNA repair pathway required to detect, process, and ligate DNA double-stranded breaks (DSBs) throughout the cell cycle. The NHEJ pathway is necessary for V(D)J recombination in developing B and T lymphocytes. During NHEJ, Ku70 and Ku80 form a heterodimer that recognizes DSBs and promotes recruitment and function of downstream factors PAXX, MRI, DNA-PKcs, Artemis, XLF, XRCC4, and LIG4. Mutations in several known NHEJ genes result in severe combined immunodeficiency (SCID). Inactivation of Mri, Paxx or Xlf in mice results in normal or mild phenotype, while combined inactivation of Xlf/Mri, Xlf/Paxx, or Xlf/Dna-pkcs leads to late embryonic lethality. Here, we describe three new mouse models. We demonstrate that deletion of Trp53 rescues embryonic lethality in mice with combined deficiencies of Xlf and Mri. Furthermore, Xlf-/-Mri-/-Trp53+/- and Xlf-/-Paxx-/-Trp53+/- mice possess reduced body weight, severely reduced mature lymphocyte counts, and accumulation of progenitor B cells. We also report that combined inactivation of Mri/Paxx results in live-born mice with modest phenotype, and combined inactivation of Mri/Dna-pkcs results in embryonic lethality. Therefore, we conclude that XLF is functionally redundant with MRI and PAXX during lymphocyte development in vivo. Moreover, Mri genetically interacts with Dna-pkcs and Paxx.

Spontaneous Apoptosis of the Lymphocytes in Cord Blood of Late Preterm Newborns

Background/Aims: Apoptosis plays an important role in shaping the repertoire of lymphocytes and in regulating the size of the mature lymphocyte pool. The best-defined regulators of apoptosis in T cells are members of the Fas and Bcl families. Fas/FasL pathway is critically involved in the elimination of mature, self-reactive lymphocytes. Bcl-2 protein is important for lymphocyte survival in response to growth factors and various activating stimuli. We have compared the role of CD4+, CD8+, Fas, FasL and Bcl-2 in spontaneous apoptosis of T lymphocytes from cord blood and tested there is or not relation to gender. Patients and Methods: We have introduced 28 late preterm newborns to our studies, 15 females and 13 males. Blood was collected from umbilical artery immediately after cutting cord. We have determined the following parameters: the per cent of CD4+ and CD8+ lymphocytes, the Fas and FasL phenotype and Bcl-2 using flow cytometer Coulter PC 500, Krefeld, Germany. Results: We have found the increase per cent of CD4+ in females 56.10 %, in relation to males 38.15 %. The same per cent was found for CD8+ in both gender: 19.60 % females, 17.35 % males. The antigen Fas (CD95) was more expressed in females: on CD4+ 12.80 %, CD8+ 8.15 %, in comparison to males: CD4+ 9.89 %, CD8+ 6.06 %. FasL (CD95L) was lower per cent in females: both on CD4+ 6.92 %, CD8+ 5.68 %, in relation to males: CD4+ 8.94 %, CD8+ 7.04 %. Bcl-2 was a little lower in females on CD4+ 96.9 % and evidently lower on CD8+ 79.14%. In males we noted on CD4+ 97.40% and on CD8+ 93.01 %. Summarizing our findings we can underline T lymphocytes in females are more activated, what indicate Fas. Inhibitor of apoptosis Bcl-2 is less expressed in females, in comparison to males lymphocytes. Conclusion: Our preliminary results seems to indicate spontaneous apoptosis of T lymphocytes is more expressed in female newborns. Table I. Spontaneous apoptosis of the T lymphocytes in cord blood of late preterm newborns. N Gender Lymphocyte T % Fas % FasL % Bcl-2 % 15 Female CD4+ 56.10 12.80 6.92 96.90 13 Male 38.15 9.89 8.94 97.40 15 Female CD8+ 19.60 8.15 5.68 79.14 13 Male 17.35 6.06 7.04 93.01 Disclosures No relevant conflicts of interest to declare.

Putative GTPase GIMAP1 is critical for the development of mature B and T lymphocytes

The guanosine triphosphatases (GTPases) of the immunity-associated protein (GIMAP) family of putative GTPases has been implicated in the regulation of T-lymphocyte development and survival. A mouse conditional knockout allele was generated for the immune GTPase gene GIMAP1. Homozygous loss of this allele under the influence of the lymphoid-expressed hCD2-iCre recombinase transgene led to severe (> 85%) deficiency of mature T lymphocytes and, unexpectedly, of mature B lymphocytes. By contrast there was little effect of GIMAP1 deletion on immature lymphocytes in either B or T lineages, although in vitro studies showed a shortening of the survival time of both immature and mature CD4+ single-positive thymocytes. These findings show a vital requirement for GIMAP1 in mature lymphocyte development/survival and draw attention to the nonredundant roles of members of the GIMAP GTPase family in these processes.

Detection of trisomy 12 by fluorescent in situ hybridization (FISH) in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) presents a varying incidence of karyotypic abnormalities whose detection is complicated by difficulties in obtaining mitosis for analysis in this type of mature lymphocyte disorder. Since the introduction of molecular cytogenetics (FISH = fluorescent in situ hybridization), applying centromeric probes for chromosome 12 has made it possible to detect a higher percentage of trisomy 12 cases. The objective of the present study was to detect trisomy 12 by FISH (alpha satellite probe) in 13 patients with CLL whose karyotypes by G-banding were either normal or inadequate. Using this method trisomy 12 was detected in three patients in a percentage of positive cells varying from 55.5% to 79%, showing that FISH is a sensitive and highly specific method for trisomy detection and should be routinely performed when the karyotype is normal.

Lymphocyte Migration in Lymphocyte Function-associated Antigen (LFA)-1–deficient Mice

Using lymphocyte function-associated antigen (LFA)-1−/− mice, we have examined the role of LFA-1 and other integrins in the recirculation of lymphocytes. LFA-1 has a key role in migration to peripheral lymph nodes (pLNs), and influences migration into other LNs. Second, the α4 integrins, α4β7 and α4β1, have a hitherto unrecognized ability to compensate for the lack of LFA-1 in migration to pLNs. These findings are confirmed using normal mice and blocking LFA-1 and α4 monoclonal antibodies. Unexpectedly, vascular cell adhesion molecule (VCAM)-1, which is essential in inflammatory responses, serves as the ligand for the α4 integrins on pLN high endothelial venules. VCAM-1 also participates in trafficking into mesenteric LNs and Peyer's patch nodes where mucosal addressin cell adhesion molecule 1 (MAdCAM-1), the α4β7-specific ligand, dominates. Both α4β1, interacting with ligand VCAM-1, and also LFA-1 participate in substantial lymphocyte recirculation through bone marrow. These observations suggest that organ-specific adhesion receptor usage in mature lymphocyte recirculation is not as rigidly adhered to as previously considered, and that the same basic sets of adhesion receptors are used in both lymphocyte homing and inflammatory responses.