**4. Assessment of post-transplant immune recovery**

There are different methods to assess the immune recovery after transplant, such as estimation of absolute lymphocyte count (ALC), levels of immune cell subsets (NK cells, B cells, and T cells), and antibody titers to assays for T- and B-cell repertoires [91].

ALC levels have been reported in association with overall survival and rate of relapse. ALC >500 cells/μL on day 15 is linked with better OS and lower relapse after autologous as well as allogeneic transplantation [92, 93]. An increase in the levels of CD16+ monocytes has been associated with aGvhD [94].

Early recovery of CD4+ T cells is associated with overall survival, nonrelapse mortality, and risk of infections [95, 96]. Admiral et al. [97] reported the time taken by circulating CD4+ T cells to reach 0.5× 109/L as a strong marker for probability of relapse. In myeloablative allogeneic HSCT, higher levels of CD3+, CD8+ T cells, regulatory T cells, and myeloid dendritic cells are correlated with relapse-free survival [98].

Recently, flow cytometric analysis has been used to differentiate between the T, B, and NK-cell subpopulations. Low levels of NK cells within the first few weeks after transplant have been associated with poor transplant outcomes like lower overall survival and higher risk of infection [99, 100]. Surface markers such as CD45RA, CD28, CD27, CD62L, and CCR7 can be used to differentiate naïve, effector, effector memory, and central memory CD4+ and CD8+ subsets [101, 102]. The surface markers expressed by naïve T cells are CD45RA+CCR7+; central memory T cells are CD45RA-CCR7+; effector memory T cells are CD45RA-CCR7–; and effector T cells are CD45RA+CCR7– [91]. CD4+ T cells also include regulatory T cells (CD25+FoxP3+) and Th17 cells [103, 104]. The expression of CD27, IgM, and IgD helps in distinguishing between naïve B cells (CD27-IgD+), memory B cells (CD27+IgD+), and isotype switched memory B cells (CD27+IgD-) [105]. Myeloid and plasmacytoid dendritic cells can be distinguished based on the expression of CD123 and CD11c: CD123low CD11c+(myeloid) and CD123bright CD11c- (plasmacytoid) [106].

TRECs have been suggested as a marker for reconstitution of naïve T cells (CD4+CD45RA+) derived from the thymus. TRECs, however, remain low up to 6 months after HSCT [107]. Due to thymic atrophy with age, older patients have T cells with low TCR repertoire, which leads to higher risk of infections leading to lower transplant outcomes [108, 109]. Thymopoiesis can also be evaluated by measuring the number of TRECs by real-time quantitative in purified CD4+ and CD8+ T cells [110]. Lewin et al. [111] reported faster recovery of TRECs in younger patients and patients who received conventional grafts as compared to T-cell depleted grafts. Lower levels of TRECs are associated with GvHD and opportunistic infections [77, 112].

Certain cytokines can also be used as predictive markers for transplant outcomes. One such marker is IL7, which can be used to evaluate successful T-cell recovery. Increased IL7 is associated with delayed reconstitution and increased mortality and aGvHD [113]. High levels of IL6, GCSF, and IL2α have also been indicated in association with risk of aGvHD [96, 114]. For assessing chronic GvHD, high levels of IL8 and low levels of IL17A have been suggested [103, 115]. Min et al. [104] have also correlated high levels of IL6 and IL10 with poor transplant-related outcomes.

*Cells of the Immune System*

receiving HLA matched HSCT.

normal individuals [86].

**Cell type and numbers Bone** 

*Reconstitution of different immune cells depending on the graft source.*

regimen in case of T-cell depleted transplant. Naïve T cells/T-cell receptor excision circles (TRECs) are lower for approximately 10–30 years after transplant [71, 72]. Reconstitution of functional T cells as observed by their ability to secrete interferon gamma and interleukin-4 to normal levels returns in 30 days after haploidentical HSCT for patients in whom acute GvHD is not observed [73]. Recipients of T-cell depleted haploidentical HSCT show higher CD31+ naïve CD4+ T cells than their donors at approximately 4–6 years [74]. Homeostatic peripheral expansion is induced by various homeostatic cytokines such as IL7 and IL15, inflammatory cytokines, and viral exposure. Peripheral homeostatic expansion leads to an inverse CD4/CD8 ratio in patients for several months after transplant. CD4 counts are considered as the best predictive marker for the recovery of immune competence after HSCT, and its recovery has also been associated with lower risk of infections and improved transplant outcomes [1]. CD4+ T-cell counts are as low as <200 cells/μL in the first 3 months and reach levels of 450 cells/μL at about 5 years after transplant [55, 75]. CD8+ T-cell counts increase rapidly during the first 3 months after transplant possibly due to the expansion of herpesvirus-specific CD8 T cells [55, 76]. GvHD reduces the number of CD4+ T cells by inhibiting the thymic output, whereas CD8+ cells increase in number during GvHD or CMV reactivation [77, 78]. The reconstituting CD4+ T cells have a higher expression of CD11a, CD29, CD45RO, and HLA-DR and a lower expression of CD28, CD45RA, and CD62L than normal individuals [79, 80]. The early reconstituting CD8+ T cells are mostly memory or effector cells. Naïve or TREC+CD8+ T cells recover at a slower rate [77, 81]. The number of regulatory T cells (Tregs) is much higher after transplant than normal individuals and may contribute to remission [82, 83]. A Treg:CD4+ T cell ratio of less than 9% has been associated with higher risk of aGvHD [84]. Chang et al. [16] reported lower CD4+ T cells, dendritic cells, and higher CD28 expression on CD4+ and CD8+ T cells in patients receiving haploidentical HSCT than patients

B-cell reconstitution is also delayed after HSCT: ~6 months for autologous and ~9 months after allogeneic transplantation and is mainly due to GvHD or its treatment. In the first 2 months after transplant, B-cell counts are low but rise higher than the normal levels in approximately 1–2 years [55, 85]. Since restoration of full humoral immune functioning requires both naïve and memory B cells, all patients who have undergone HSCT remain susceptible to infections for at least a year after transplant [1]. The reconstituted B cells express higher levels of CD1c, CD38, CD5, membrane IgM, and membrane IgD and lower levels of CD25 and CD26L than

A number of studies have reported comparisons between reconstitution of different immune cells depending on the graft source. Faster reconstitution of

Neutrophils (>0.5 × 109/L) 16 days 15 days 19 days [87] Natural killer cells (>0.1 × 109/L) 1.5 months 4 months 4 months [16, 87] T cells (>0.5 × 109/L) CD4 2–3 months 6 months 3 months [28, 88] Naïve T cells (>0.5 × 109/L) 9 months 24 months 12 months [87, 89] Cytotoxic T cells (>0.25 × 109/L 3 months 9 months 8 months [65, 90] T helper cells (>0.2 × 109/L) 4 months 10 months 1 months [65, 90]

**Peripheral blood**

**Unrelated cord blood**

**Reference**

**marrow**

**196**

**Table 2.**

Further, T- and B-cell receptor repertoire gene arrangements can be evaluated by molecular techniques such as next generation sequencing [116, 117]. Michalek et al. [118] have demonstrated β chain sequencing of the T-cell receptor in order to identify the T-cell clones that could mediate either graft-versus-host disease or graft-versus-leukemia effect. Brink et al. [9] reported higher diversity in CD4+ T cells than CD8+ T cells following allogeneic HSCT. Greater diversity was observed in cord blood grafts, followed by unmanipulated grafts and T-cell depleted grafts.

#### **5. Strategies to improve immune reconstitution**

Many strategies, such as administration of recombinant cytokines, adoptive cell therapy, and hormone-based therapies, have recently been used to improve immune reconstitution after transplantation.

IL7 cytokine has been shown to effectively enhance reconstitution of T and B lymphoid cells by enabling thymopoiesis [105, 119]. It has been demonstrated that IL7 increased the CD3+, CD4+, and CD8+ T-cell levels to more than four folds and also leads to increase in functional and diverse T cells [120]. Administering IL-7 predominantly increases the naïve CD8+ T cells. The timing of administering is, however, important, as administering early after transplant aggravates GvHD [116, 121], whereas administering it at a later stage after HSCT results in lower risk of GvHD. This is contributed by the activation of alloreactive T cells that express lower IL-7Rα levels [32, 38]. Other cytokines that enable immune reconstitution are insulin-like growth factor 1(IGF-1), IL22, IL15, and IL12 [122–124]. IL15 has been shown to significantly increase the reconstitution of CD8+ T cells and NK cells and improve the GvL effect in haploidentical murine models [125]. Sauter et al. [126] reported better lymphocyte reconstitution after IL-15 administration in T-cell depleted allogeneic HSCT; however, it has been shown to worsen GvHD.

Recently, it has been suggested that modulating the function of dendritic cells could reduce GvHD while maximizing GvL [127]. Studies on reconstitution of dendritic cells after HSCT have been contradictory. Maraskovsky et al. [128] have shown that treatment with Flt3-L can expand DC subsets; however, when administered after HSCT, it can worsen GvHD [38]. Gauthier et al. [38] have demonstrated that SDF-1α therapy can expand the DC1 subsets and lower the severity of GvHD. Because of their immunosuppressive properties, mesenchymal stem cells have recently been used for suppressing GvHD [129–131]. Mesenchymal stem cells release cytokines such as IL-7, which improve T-cell survival and promote reconstitution of dendritic cells by secreting SDF-1α [132].

NK-cell immunotherapy is one of the novel strategies underway to reduce GvHD and enhance graft-versus-leukemia effect in a KIR-HLA mismatched haploidentical HSCT [133–135].

### **6. Future directions**

Recently, few studies have identified the association of reconstitution of certain immune subsets with predicting post-HSCT outcomes. However, these studies are often limited by small sample size, lack of detailed immune reconstitution, and secretome profile, which could be used as biomarkers to predict immune reconstitution. Prospective studies involving a large number of patients should be conducted to determine which immune factors and tests to detect the same could have prognostic value and understand the impact of such predictive risk factors on transplant outcomes. This is most beneficial, especially for recipients of

**199**

India

**Author details**

Meenakshi Singh1

\*, Selma Z. D'Silva1

\*Address all correspondence to: meenakshisingha@gmail.com

provided the original work is properly cited.

†Meenakshi Singh and Selma Z. D'Silva share the first authorship.

and Abhishweta Saxena<sup>2</sup>

1 HLA and Immunogenetics Laboratory, Tata Memorial Hospital, Mumbai, India

2 Department of Transfusion Medicine, Homi Bhabha Cancer Hospital, Varanasi,

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

*Assessment of Immune Reconstitution Following Hematopoietic Stem Cell Transplantation*

haploidentical HSCT, in which a routine strategy could be adopted to result in faster immune reconstitution and hence lower probability of poor transplant outcomes,

*DOI: http://dx.doi.org/10.5772/intechopen.89198*

The authors declare no conflict of interest.

such as TRM, relapse, and GvHD.

**Conflict of interest**

*Assessment of Immune Reconstitution Following Hematopoietic Stem Cell Transplantation DOI: http://dx.doi.org/10.5772/intechopen.89198*

haploidentical HSCT, in which a routine strategy could be adopted to result in faster immune reconstitution and hence lower probability of poor transplant outcomes, such as TRM, relapse, and GvHD.
